PROSTHESIS FOR THE LUNG AND THE USE THEREOF

Abstract

The invention relates to prosthesis for treating pulmonary fibrosis, in particular, idiopathic pulmonary fibrosis (IPF), and a method for treating pulmonary fibrosis, in particular, idiopathic pulmonary fibrosis (IPF).

Claims

1. A prosthesis for treating pulmonary fibrosis, wherein the prosthesis is designed to be placed under the pulmonary lobe, as long as a level of mechanical tension on alveolar epithelium is reduced at a end of normal inspiratory.

2. The prosthesis of claim 1, wherein the prosthesis is designed to be anchored under the lobes and above a diaphragm, and is designed to match with the bottom of the lobes, as long as the mechanical tension on the alveolar epithelium is reduced at the end of normal inspiratory.

3. The prosthesis of claim 1, wherein the prosthesis occupies at least ⅛ of the space surrounded by the bottom of the lobes, diaphragm and the wall of pleural cavity at the end of normal inspiratory.

4. The prosthesis of claim 3, wherein the prosthesis occupies at least ¼, at least half, at least ¾, or the whole of a space surrounded by a basal of the lobes, the diaphragm and the wall of pleural cavity at the end of normal inspiratory.

5-7. (canceled)

8. The prosthesis of claim 3, wherein the prosthesis is surrounded by pleural fluid.

9. The prosthesis of claim 1, wherein the area of the prosthesis projected to the bottom of the lower lung lobe accounts for at least ⅛, at least ¼, at least half, at least ¾, or the whole of the area of the bottom of the lower lung lobe.

10-13. (canceled)

14. The prosthesis of claim 2, wherein the prosthesis comprises a body having an upper side, a lower side and a lateral side extending between the upper side and the lower side.

15. The prosthesis of claim 14, wherein the upper side of the body of the prosthesis is matched with the bottom of the lobes, wherein the lower side of the body of the prosthesis is matched with the diaphragm, wherein an outer lateral side of the prosthesis is matched with a wall of pleural cavity between a bottom of the lobes and the diaphragm far from a heart, and/or wherein an inner lateral side of the prosthesis is matched with an outer wall of a lower lung lobe.

16-18. (canceled)

19. The prosthesis of claim 14, wherein the prosthesis is in a shape of plate, ellipse, irregular U, arc, conical, scapula, or irregular, and so on, so as to occupy the space under the lung lobes.

20. The prosthesis of claim 14, wherein the prosthesis has a smooth curved profile without edges and corners so as to reduce the discomfort and avoid injury to the pleura.

21. The prosthesis of claim 14, wherein the prosthesis comprises a body having an upper side, a lower side, a lateral side extending between the upper side and the lower side, and a medial border approaching the heart, wherein the lateral side is opposite to the medial border.

22-25. (canceled)

26. The prosthesis of claim 21, wherein the prosthesis upper side tapers toward the medial border, and the prosthesis lower side tapers toward the medial border.

27. (canceled)

28. The prosthesis of claim 14, wherein the lateral side is at a height of 0.5 m-8 cm.

29. (canceled)

30. The prosthesis of claim 14, wherein the thickness of the wall of the prosthesis is between 0.1-4 cm.

31. (canceled)

32. The prosthesis of claim 1, wherein the prosthesis is in a shape of arc, and the arc is matched with the lower edge of the lower lung lobe.

33. The prosthesis of claim 1, wherein the arch is matched with at least ⅛, at least ¼, or at least ½ of the lower edge of the lower lung lobe far from a heart, or matched with the overall length of the lower edge of the lower lung lobe.

34-36. (canceled)

37. The prosthesis of claim 33, wherein the prosthesis is at a height of 1 cm-4 cm.

38. (canceled)

39. The prosthesis of claim 1, wherein the prosthesis is designed to be placed in the pleural cavity, in particular, the prosthesis is placed at the lower side of the pleural cavity, so as to reduce the mechanical tension on the alveolar epithelium at the end of normal inspiratory.

40. The prosthesis of claim 1, wherein the prosthesis is fixed in the pleural cavity, in particular, the prosthesis is placed at the lower side of the pleural cavity.

41. The prosthesis of claim 40, wherein the prosthesis is sutured to the pleural cavity wall, particularly, the lower portion of the pleural cavity wall, using surgical sutures.

42. The prosthesis of claim 40, wherein the prosthesis is sutured to of the wall of pleural cavity through the pinholes on the lateral side.

43. The prosthesis of claim 1, wherein the prosthesis is made of soft spongy latex, foam latex, 380 micron hollow fiber, gelatin foam, material, plastic sponge (lvalon), polythene bag filled with fiberglass, rubber, silicone rubber, silicone gel, carbon materials involving carbon nanotube, grapheme, ultra-light porous carbon, hollow porous carbon, carbon fiber, or carbon titanium alloy.

44. The prosthesis of claim 1, wherein the prosthesis is solid, or the prosthesis is a pocket or a sack, filled with fluid or jelly.

45. (canceled)

46. A method for treating pulmonary fibrosis, comprising a step of reducing the mechanical tension on the alveolar epithelium at the end of normal inspiratory, by placing the prosthesis of claim 1 in the pleural cavity so as to reduce the level of mechanical tension on the alveolar epithelium at the end of normal inspiratory.

47. The method of claim 46, wherein placing the prosthesis in the lower part of the pleural cavity.

48. The method of claim 46, wherein the prosthesis is fixed in the pleural cavity, and the prosthesis is placed at the lower part of the pleural cavity.

49. The method of claim 48, wherein the prosthesis is sutured to the wall of pleural cavity, and the lower part of the chest wall.

50. The method of claim 46, wherein the prosthesis is designed to be placed under the pulmonary lobe, as long as the level of mechanical tension on the alveolar epithelium is reduced the end of normal inspiratory.

51. The method of claim 46, wherein the prosthesis is designed to be anchored under the lobes and above the diaphragm, and is designed to be matched with the basal of the lobes, as long as the level of mechanical tension on the alveolar epithelium is reduced at the end of normal inspiratory.

52. The prosthesis of claim 1, wherein the pulmonary fibrosis is idiopathic pulmonary fibrosis.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0052] FIGS. 1A-1F show six perspectives of a prosthesis manufactured in accordance with the present invention.

[0053] FIGS. 2A-2D show four perspectives of a prosthesis manufactured in accordance with the present invention.

[0054] FIGS. 3A-3D show four perspectives of a prosthesis manufactured in accordance with the present invention.

[0055] FIGS. 4A-4F show six perspectives of a prosthesis manufactured in accordance with the present invention.

[0056] FIGS. 5A-5E show five perspectives of a prosthesis manufactured in accordance with the present invention.

[0057] FIGS. 6A-6F show six perspectives of a prosthesis manufactured in accordance with the present invention.

[0058] FIGS. 7A-7C show three perspectives of a prosthesis manufactured in accordance with the present invention.

[0059] FIGS. 8A-8C show three perspectives of a prosthesis manufactured in accordance with the present invention.

[0060] FIGS. 9A-9C show three perspectives of a prosthesis manufactured in accordance with the present invention.

[0061] FIGS. 10A-10C show three perspectives of a prosthesis manufactured in accordance with the present invention.

[0062] FIGS. 11A-11C show three perspectives of a prosthesis manufactured in accordance with the present invention.

[0063] FIGS. 12A-12C show three perspectives of a prosthesis manufactured in accordance with the present invention.

[0064] FIGS. 13A-13D show four perspectives of a prosthesis manufactured in accordance with the present invention.

[0065] FIGS. 14A-14D show four perspectives of a prosthesis manufactured in accordance with the present invention.

[0066] FIGS. 15A-15D show four perspectives of a prosthesis manufactured in accordance with the present invention.

[0067] FIGS. 16A-16E show four perspectives of a prosthesis manufactured in accordance with the present invention.

[0068] FIG. 17 illustrates a prosthesis of the present invention in position in a person's chest.

[0069] FIG. 18 shows that a mouse line in which Cdc42 gene is specifically deleted in AT2 cells is generated.

[0070] FIG. 19 shows that loss of Cdc42 gene in AT2 cells impairs the differentiation of AT2 cells during post-PNX alveolar regeneration or alveolar homeostasis.

[0071] FIG. 20 shows that loss of Cdc42 gene in AT2 cells leads to progressive lung fibrosis in PNX-treated mice.

[0072] FIG. 21 shows that loss of Cdc42 gene in AT2 cells leads to progressive lung fibrosis in non-PNX-treated aged mice.

[0073] FIG. 22 shows the development of α-SMA.sup.+ fibroblastic foci in the lungs of Cdc42 AT2 null mice.

[0074] FIG. 23 shows that the expression level of CDC42-GTP increases significantly at post-PNX day 7.

[0075] FIG. 24 shows that reducing mechanical tension on alveoli attenuates progressive lung fibrosis.

[0076] FIG. 25 shows the fragments of Cdc42 DNA sequence before or after deleting the exon2 of the Cdc42 gene.

DESCRIPTION OF PARTICULAR EMBODIMENTS OF THE INVENTION

[0077] The descriptions of particular embodiments and examples are provided by way of illustration and not by way of limitation. Those skilled in the art will readily recognize a variety of noncritical parameters that could be changed or modified to yield essentially similar results.

[0078] The pleural cavity is the thin fluid-filled space between the two pulmonary pleurae (known as visceral and parietal) of each lung. A pleura is a serous membrane which folds back onto itself to form a two-layered membranous pleural sac. The outer pleura (parietal pleura) is attached to the chest wall, but is separated from it by the endothoracic fascia. The inner pleura (visceral pleura) covers the lung and adjacent structures, including blood vessels, bronchi and nerves. The pleural cavity can be viewed as a potential space because the two pleurae adhere to each other (through the thin film of serous liquid) under all normal conditions. The pleural cavity is surrounded by the rib cage, and it surrounds the lungs. A small amount of fluid is located in the potential space between the two layers of the pleura.

[0079] Pleural fluid is serous fluid produced by the serous membrane covering normal pleurae. Most fluid is produced by the parietal circulation (intercostal arteries) via bulk flow and reabsorbed by the lymphatic system. Thus, pleural fluid is produced and reabsorbed continuously.

[0080] The lungs are located in the chest on both sides of the heart in the rib cage. They are conical in shape with a narrow rounded apex at the top, and a broad concave base that rests on the convex surface of the diaphragm. The lungs are surrounded by the pulmonary pleurae. The pleurae are two serous membranes; the outer parietal pleura lines on the inner wall of the rib cage and the inner visceral pleura directly lines on the surface of the lungs. Between the pleurae is a potential space called the pleural cavity, which contains a thin layer of lubricating pleural fluid. Each lung is divided into lobes by the infoldings of the pleura as fissures. The fissures are double folds of pleura that separates the lungs and helps the lung to expand. The right lung has three lobes, namely, the superior (upper), middle and inferior (lower) lobes, and there are only two lobes in the left lung: the superior (upper) and inferior (lower) lung lobes.

[0081] The term “mechanical tension” in the present invention means “physical tension”, “stretch”, “distension”, “stress”, or “strain”, for example, “strain” involves “compressive strain”, “tensile strain”, and an angular distortion is a “shear strain”.

[0082] The idiopathic pulmonary fibrosis (IPF) is a type of chronic lung disease characterized by progressive and irreversible decline in lung function. Symptoms typically include gradual shortness of breath and a dry cough. Other changes may include feeling tired and nail clubbing. Complications may include pulmonary hypertension, heart failure, pneumonia, or pulmonary embolism.

[0083] Prosthesis is an artificial device that replaces a body part and is intended to restore the normal functions of the body part. Prostheses can be made by hand or with CAD (Computer-Aided Design), a software interface that helps creators to visualize the creation in a 3D form. The prosthetics are made in lightweight to facilitate the subject, and some of the materials include: plastics (polyethylene, polypropylene, acrylics, polyurethane), lightweight metals (titanium, aluminum), composite (carbon fibre), and silicone rubber.

[0084] Surgical suture is used to hold body tissues together after an injury or surgery. Surgical suture is made from numerous materials. The original suture is made from biological materials, such as catgut suture and silk, and synthetic materials, such as absorbable polyglycolic acid, polylactic acid, monocryl and polydioxanone as well as the non-absorbable nylon, polyester, PVDF and polypropylene. The polymer materials are based on one or more of five cyclic monomers: glycolide, 1-lactide, p-dioxanone, trimethylene carbonate and c-caprolactone.

[0085] Silicone rubber is an elastomer composed of silicone, a polymer, containing silicon together with carbon, hydrogen, and oxygen. Silicone rubber offers properties such as good resistance to extreme temperatures, elongation, creep, cyclic flexing, tear strength, compression set, dielectric strength (at high voltage), thermal conductivity, fire resistance and in some cases tensile strength can be—at extreme temperatures—far superior to organic rubbers in general.

[0086] The persons skilled in the art will anticipate that any drugs or prosthesis that reducing the mechanical tension of alveolar epithelium will be involved in the scope of the invention, since the inventors of the present invention firstly establish a direct linkage between the mechanical tension and the IPF, that is to say, reducing the mechanical tension of alveolar epithelium will be effective.

EXAMPLES

Methods

[0087] Mice and Survival Curve Record

[0088] Rosa26-CAG-mTmG (Rosa26-mTmG).sup.20 and Cdc42.sup.flox/flox mice.sup.21 have been described previously. All experiments were performed in accordance with the recommendations in the Guide for Care and Use of Laboratory Animals of the National Institute of Biological Sciences. To monitor the survival of mice, both the Control and the Cdc42 AT2 null mice were weighed every week after the PNX treatment. Once the mice reached the pre-defined criteria for endpoints, the mice were sacrificed. We define the endpoints according to the pre-defined criteria.sup.22,23.

[0089] Generating Spc-CreER Knock-in Allele

[0090] The CreERT2, p2a, and rtTA element were enzyme-linked and inserted into the mouse endogenous Sftpc gene. The insertion site is the stop codon of the endogenous Sftpc gene, then a new stop codon was created at the 3′ end of rtTA. The CRISPR/Cas9 technology was used to insert the CreERT2-p2a-rtTA fragment into the genome.

[0091] Pneumonectomy (PNX) and Prosthesis Implantation

[0092] The male mice of 8 weeks old were injected with tamoxifen (dosage: 75 mg/kg) every other day for 4 times. The mice were anesthetized and connected to a ventilator (Kent Scientific, Topo) from 14th day after the final dose of tamoxifen injection. The chest wall was incised at the fourth intercostal ribs and the left lung lobe was removed. For prosthesis implantation, a soft silicone prosthesis with a similar size and shape of the left lung lobe was inserted into the empty left lung cavity.

[0093] Pulmonary Function Test

[0094] Lung function parameters were measured using the invasive pulmonary function testing system (DSI Buxco® PFT Controller). Mice were first anesthetized before inserting an endotracheal cannula into their trachea. The dynamic compliance results were obtained from the Resistance & Compliance Test.

[0095] Hematoxylin and Eosin (H&E) Staining and Immunostaining

[0096] Lungs were inflated with 4% paraformaldehyde (PFA) and were continually fixed in 4% PFA at 4° C. for 24 hours. Then the lungs were cryoprotected in 30% sucrose and embedded in OCT (Tissue Tek).

[0097] The H&E staining experiment followed the standard H&E protocol. Briefly, slides were washed by water to remove the OCT. The nuclei were stained by hemotoxylin (Abcam, ab150678) for 2 minutes and the cytoplasm was stained by eosin (Sigma, HT110280) for 3 minutes. Slices were sealed with neutral resin after the dehydration and clearing steps.

[0098] The immunofluorescence staining experiments followed the protocol previously described.sup.24. In brief, after removing the OCT, the lung slices were blocked with 3%BSA/0.1%TritonX-100/PBS for 1 hour, then slides were incubated with primary antibodies at 4° C. for overnight. After washing the slides with 0.1%TritonX-100/PBS for 3 times, the slices were incubated with secondary antibodies for 2 hours at room temperature.

[0099] The primary antibodies used in the paper are listed below:

TABLE-US-00001 Name Company and catalog number Dilution Chicken anti-GFP Abcam, ab13970-100 1:500 Rabbit anti-Collagen I Abcam, ab34710 1:300 Mouse anti α-SMA Sigma, C6198 1:300 Hamster anti-Pdpn Developmental Studies Hybridoma 1:100 Bank, clone8.1.1 Rat anti-Ki67 Bioscience, 514-5698-82 1:300

[0100] The secondary antibodies used in the paper are listed below:

TABLE-US-00002 Name Company and catalog number Dilution Alexa Fluor 488 Donkey 703-545-155, Jackson Immuno 1:500 anti-Chicken Research Alexa Fluor 488 Donkey 715-545-150, Jackson Immuno 1:500 anti-mouse Research Alexa Fluor 568 Donkey A11057, Invitrogen 1:500 anti-rabbit Alexa Fluor 647 Goat A-21451, Invitrogen 1:500 anti-hamster Biotin Donkey Anti- 711-065-152, Jackson Immuno Rabbit Research

[0101] Statistical Analysis

[0102] All data are presented as mean±s.e.m. (as indicated in figure legends). The data presented in the figures were collected from multiple independent experiments that were performed on different days using different mice. Unless otherwise mentioned, most of the data presented in figure panels are based on at least three independent experiments. The inferential statistical significance of differences between sample means was evaluated using two-tailed unpaired Student's t-tests.

[0103] Isolating Mouse AT2 Cells

[0104] After 4 doses of tamoxifen injection, the lungs of Spc-CreER, Rosa26-mTmG mice were dissociated as previously described.sup.19,44. Briefly, anesthetized mice were inflated with neutral protease (Worthington-Biochem, LS02111) and DNase I (Roche, 10104159001). AT2 cells were directly sorted based on the GFP fluorescence using the single-cell-select-mode in BD FACS Aria II and III appliances.

[0105] Quantitative RT-PCR (qPCR)

[0106] Total RNA was isolated from either whole lung or primary AT2 cells using Zymo Research RNA Mini Prep Kits (R2050). Reverse transcription reactions were performed with a two-step cDNA synthesis Kit (Takara, Cat. #6210A/B) according to the manufacturer's recommendations. qPCR was done with a CFX96 Touch™ Real-Time PCR Detection System. The mRNA levels of target genes were normalized to the Gapdh mRNA level.

[0107] Primers used for qPCR are listed below.

TABLE-US-00003 Forward Reverse Gapdh AAGGTCGGTGTGAACGGATTTGG CGTTGAATTTGCCGTGAGTGGAG (SEQ ID NO: 1) (SEQ ID NO: 2) Sftpc TTGTCGTGGTGATTGTAGGG TGGAAAAGGTAGCGATGGTG (SEQ ID NO: 3) (SEQ ID NO: 4) Scd1 GCAAGCTCTACACCTGCCTCTT CGTGCCTTGTAAGTTCTGTGGC (SEQ ID NO: 5) (SEQ ID NO: 6) Lyz2 TGCCAGAACTCTGAAAAGGAATGG CAGTGCTTTGGTCTCCACGGTT (SEQ ID NO: 7) (SEQ ID NO: 8) Cbr2 CATGGGCAAGAAAGTCTCTGCAG ACTGGTAGAGGCACTTCTGTCG (SEQ ID NO: 9) (SEQ ID NO: 10) Sftpa1 ACCTGGATGAGGAGCTTCAGAC CTGACTGCCCATTGGTGGAAAAG (SEQ ID NO: 11) (SEQ ID NO: 12) Actb CATTGCTGACAGGATGCAGAAGG TGCTGGAAGGTGGACAGTGAGG (SEQ ID NO: 13) (SEQ ID NO: 14) Actn1 TCGCCAAGTGTCAACGCTCGTT GGTCGATGGTTTCCAGCAGCTT (SEQ ID NO: 15) (SEQ ID NO: 16) Pfn1 CATCGTAGGCTACAAGGACTCG CCAAGTGTCAGCCCATTGACGA (SEQ ID NO: 17) (SEQ ID NO: 18) Ezr ATCGAGGTGCAGCAGATGAAGG CGGAGCATCTGCTCCTTTTCTC (SEQ ID NO: 19) (SEQ ID NO: 120) Gsn GGCTTTGAGTCGTCCACCTTCT GTCCTTTGACCTGGAAGAGCCT (SEQ ID NO: 21) (SEQ ID NO: 22)

[0108] 3D Alveolar Reconstruction

[0109] For vibratome sections, lungs were gently inflated to full capacity with 2% low-melting agarose. Then lungs were fixed in 4% PFA for overnight at 4° C. Thick vibratome sections were sliced at a thicknesses of 200 μm using the vibrating microtome (Leica VT100S). Immunostaining experiments were performed as the standard wholemount staining protocol. Z stack images were taken by Leica LSI macro confocal microscope and/or A1-R inverted confocal microscope.

[0110] CDC42-GTP Assay

[0111] The GTP-CDC42 level is determined using the CDC42 activation assay biochem kit (cytoskeleton, #BK127) according to the provided manufacturer's recommendations. Briefly, the whole lung lobes were grinded in liquid nitrogen, then lysed using the cell lysis buffer (applied in the kit). Then the cell lysates were added into the microplate wells applied. After the reaction, the absorbance at 490 nm was measured.

Example 1

[0112] As shown in FIGS. 1A-1F, the prosthesis is in a shape of arc and occupies about ¾ of the edge of the lower part of the lung lobe. The lateral side has a height of about 1.5 cm. The prosthesis has a thickness of about 2 mm. The bottom of the prosthesis has a width of 1 cm. The prosthesis may be sewed onto the wall of pleural cavity. The sides of the prosthesis are round and smooth as far as possible.

[0113] The opening is toward the heart. At the end of normal inspiratory, the prosthesis presses the lower part of the lung lobe and the mechanical tension of alveolar epithelium is reduced.

Example 2

[0114] As shown in FIGS. 2A-2D, the prosthesis is in a shape of arc and occupies about ⅛ of the edge of the lower part of the lung lobe. The lateral side has a height of about 3 cm. The prosthesis has a thickness of about 7 mm. The bottom of the prosthesis has a width of 2 cm. The prosthesis may be sewed onto the side wall of pleural cavity far from the heart. The sides of the prosthesis are round and smooth as far as possible.

[0115] At the end of normal inspiratory, the prosthesis presses the lower part of the lung lobe and the mechanical tension of alveolar epithelium is reduced.

Example 3

[0116] As shown in FIGS. 3A-3D, the prosthesis is in a shape of arc and occupies about ¼ of the edge of the lower part of the lung lobe. The lateral side has a height of about 3 cm. The prosthesis has a thickness of about 6 mm. The bottom of the prosthesis has a width of 1.3 cm. The prosthesis may be sewed onto the side wall of pleural cavity far from the heart. The sides of the prosthesis are round and smooth as far as possible.

[0117] At the end of normal inspiratory, the prosthesis presses the lower part of the lung lobe and the mechanical tension of alveolar epithelium is reduced.

Example 4

[0118] As shown in FIGS. 4A-4F, the prosthesis is in a shape of plate and matches the whole area of the basal of the lower lung lobe. The lateral side has a height of about 2.5 cm. The prosthesis has a thickness of about 2 mm. The prosthesis may be sewed onto the side wall of pleural cavity. The sides of the prosthesis are round and smooth as far as possible.

[0119] At the end of normal inspiratory, the prosthesis presses the lower part of the lung lobe and the level of mechanical tension of alveolar epithelium is reduced.

Example 5

[0120] As shown in FIGS. 5A-5E, the prosthesis is in a shape of shell-like and matches the whole area of the bottom of the lower lung lobe. The prosthesis has depression in the middle and tapers to the side wall of pleural cavity. The prosthesis also has a depression on the side of heart to hold the heart. The prosthesis has a thickness of about 4 mm. The sides of the prosthesis are round and smooth as far as possible.

[0121] At the end of normal inspiratory, the prosthesis presses the lower part of the lung lobe and the mechanical tension of alveolar epithelium is reduced.

Example 6

[0122] As shown in FIGS. 6A-6F, the prosthesis is in a shape of U-like and matches more than ¾ of the edge of the lower lung lobe. The section of the prosthesis is close to triangle. The side touching the edge of the lower lung lobe is a concave arc. The outer side of the prosthesis has height of 1.0 cm. The sides of the prosthesis are round and smooth as far as possible.

[0123] The prosthesis may be sewed onto the side wall of pleural cavity. At the end of normal inspiratory, the prosthesis presses the lower part of the lung lobe and the mechanical tension of alveolar epithelium is reduced.

Example 7

[0124] As shown in FIGS. 7A-7C, the prosthesis is in a shape of arc and matches more than ¼ of the edge of the lower lung lobe. The section of the prosthesis is close to triangle. The side touching the edge of the lower lung lobe is a concave arc. The outer side of the prosthesis has a height of 1.6 cm. The sides of the prosthesis are round and smooth as far as possible.

[0125] The prosthesis may be sewed onto the side wall of pleural cavity far from the heart. At the end of normal inspiratory, the prosthesis presses the lower part of the lung lobe and the mechanical tension of alveolar epithelium is reduced.

Example 8

[0126] As shown in FIGS. 8A-8C, the prosthesis is in a shape of arc and matches more than ¼ of the edge of the lower lung lobe. The prosthesis has a smaller radian than the prosthesis in Example 7. The section of the prosthesis is close to triangle. The side touching the edge of the lower lung lobe is a concave arc. The outer side of the prosthesis has a height of 2.2 cm. The sides of the prosthesis are round and smooth as far as possible.

[0127] The prosthesis may be sewed onto the side wall of pleural cavity far from the heart. At the end of normal inspiratory, the prosthesis presses the lower part of the lung lobe and the mechanical tension of alveolar epithelium is reduced.

Example 9

[0128] As shown in FIGS. 9A-9C, the prosthesis is in a shape of arc and matches more than ⅛ of the edge of the lower lung lobe. The prosthesis has a smaller radian than the prosthesis in Example 7. The section of the prosthesis is close to triangle. The side touching the edge of the lower lung lobe is a concave arc. The outer side of the prosthesis has a height of 3.5 cm. The sides of the prosthesis are round and smooth as far as possible.

[0129] The prosthesis may be sewed onto the side wall of pleural cavity far from the heart. At the end of normal inspiratory, the prosthesis presses the lower part of the lung lobe and the mechanical tension of alveolar epithelium is reduced.

Example 10

[0130] As shown in FIGS. 10A-10C, the prosthesis is in a shape of circular arc and matches more than ¾ of the edge of the lower lung lobe. The section of the prosthesis is close to circular. The radius of the section is about 0.5 cm.

[0131] At the end of normal inspiratory, the prosthesis presses the lower part of the lung lobe and the mechanical tension of alveolar epithelium is reduced.

Example 11

[0132] As shown in FIGS. 11A-11C, the prosthesis is in a shape of arc and matches more than ¼ of the edge of the lower lung lobe. The section of the prosthesis is close to circular. The radius of the section is about 0.8 cm.

[0133] The prosthesis may be sewed onto the side wall of pleural cavity far from the heart. At the end of normal inspiratory, the prosthesis presses the lower part of the lung lobe and the mechanical tension of alveolar epithelium is reduced.

Example 12

[0134] As shown in FIGS. 12A-12C, the prosthesis is in a shape of circular arc and matches more than ⅛ of the edge the lower lung lobe. The section of the prosthesis is close to circular. The radius of the section is about 1.0 cm.

[0135] The prosthesis may be sewed onto the side wall of pleural cavity far from the heart. At the end of normal inspiratory, the prosthesis presses the lower part of the lung lobe and the mechanical tension of alveolar epithelium is reduced.

Example 13

[0136] As shown in FIGS. 13A-13D, the prosthesis is in a shape of plate and matches more than ⅓ area of the basal of the lower lung lobe. The lateral side has a height of about 1.0 cm. The prosthesis has a thickness of about 0.4 cm. The prosthesis has a concave bottom. The prosthesis may be sewed onto the side wall of pleural cavity. The sides of the prosthesis are round and smooth as far as possible.

[0137] At the end of normal inspiratory, the prosthesis presses the lower part of the lung lobe and the mechanical tension of alveolar epithelium is reduced.

Example 14

[0138] As shown in FIGS. 14A-14D, the prosthesis is in a shape of plate and matches more than ⅓ area of the basal of the lower lung lobe. The lateral side has a height of about 4 cm. The prosthesis has a thickness of about 1.0 cm. The prosthesis may be sewed onto the side wall of pleural cavity. The sides of the prosthesis are round and smooth as far as possible.

[0139] At the end of normal inspiratory, the prosthesis presses the lower part of the lung lobe and the mechanical tension of alveolar epithelium is reduced.

Example 15

[0140] As shown in FIGS. 15A-15D, the prosthesis is in a shape of half plate and matches more than ⅓ area of the basal of the lower lung lobe. The lateral side has a height of about 5 cm. The prosthesis has a thickness of about 0.5 cm. The prosthesis may be sewed onto the side wall of pleural cavity. The sides of the prosthesis are round and smooth as far as possible.

[0141] At the end of normal inspiratory, the prosthesis presses the lower part of the lung lobe and the mechanical tension of alveolar epithelium is reduced.

Example 16

[0142] As shown in FIGS. 16A-16E, the prosthesis is in a shape of half plate and matches more than ⅓ area of the basal of the lower lung lobe. The lateral side has a height of about 3 cm. The prosthesis has a thickness of about 0.3 cm. The prosthesis may be sewed onto the side wall of pleural cavity. The sides of the prosthesis are round and smooth as far as possible.

[0143] At the end of normal inspiratory, the prosthesis presses the lower part of the lung lobe and the mechanical tension of alveolar epithelium is reduced.

Example 17

[0144] As shown in FIG. 17, a prosthesis of Example 4 is positioned in a persons's chest. At the end of normal inspiratory, the prosthesis presses the lower part of the lung lobe and the mechanical tension of alveolar epithelium is reduced.

Example 18

[0145] Generation and Characterization of Cdc42 AT2 Null Mice

[0146] In order to construct a progressive lung fibrosis animal model, Cdc42 AT2 null mice are generated by knocking out Cdc42 gene specifically in alveolar type II epithelial cells (AT2 cells).

[0147] In order to specifically delete Cdc42 gene in AT2 cells, mice carrying a Spc-CreER knock-in allele are crossed with the Cdc42 floxed (Cdc42.sup.flox/flox) mice (FIG. 18A). In Cdc42.sup.flox/flox mice, the exon 2 of Cdc42 gene, which contains the translation initiation exon of Cdc42 gene, is flanked by two loxp sites. In Spc-CreER; Cdc42.sup.flox/flox mice, the exon 2 of Cdc42 gene is specifically deleted in AT2 cells by Cre/loxp-mediated recombination after tamoxifen treatment (FIG. 18B). Spc-CreER; Cdc42.sup.flox/flox mice are named as Cdc42 AT2 null mice.

[0148] The Spc-CreER, Cdc42.sup.flox/− mice were performed genome purification and PCR amplification. Then the fox and null bands of Cdc42 were purified and sequenced using the primers as below: CTGCCAACCATGACAACCTAA (SEQ ID NO:23);

TABLE-US-00004 (SEQ ID NO: 24) AGACAAAACAACAAGGTCCAG.

[0149] The fragments of Cdc42 DNA sequence before or after deleting the exon2 of the Cdc42 gene are shown in FIG. 25.

[0150] As shown in (FIG. 19A), 200 μm lung sections of Control and Cdc42 AT2 null mice are immunostained with antibodies against GFP, Pdpn, and Prospc. At post-PNX day 21, many newly differentiated AT1 cells and newly formed alveoli are observed in Control lungs (FIG. 19B). However, in Cdc42 AT2 null lungs, few AT2 cells have differentiated into AT1 cells, and no new alveoli are formed at post-PNX day 21 (FIG. 19B). It is observed that the alveoli in peripheral region of PNX-treated Cdc42 AT2 null lungs are profoundly overstretched (FIG. 19B).

[0151] During alveolar homeostasis, AT2 cells slowly differentiate into AT1 cells and build new alveoli. When 12-month Cdc42 AT2 null mice that had not undergone PNX are examined, enlarged alveoli with lacking any new AT1 cell formation are observed; and in contrast, the lungs of 12-month Control mice exhibit formation of many new alveoli (FIGS. 19C and 19D).

[0152] PNX-treated Cdc42 AT2 null and Control mice are observed for a longer period of time after PNX treatment (FIG. 20A). Some Cdc42 AT2 null mice show significant weight loss and increased respiration rates at post-PNX day 30. Indeed, fully 50% of PNX-treated Cdc42 AT2 null mice reach the predefined health-status criteria for endpoint euthanization by post-PNX day 60 (FIG. 20B), and about 80% of PNX-treated Cdc42 AT2 null mice reach their endpoints by post-PNX day 180 (FIG. 20B).

[0153] H&E staining of PNX-treated Control and Cdc42 AT2 null mice reveals severe fibrosis in the lungs of Cdc42 AT2 null mice at their endpoints (FIGS. 20C-20D). In order to determine the point at which Cdc42 AT2 null mice begin to develop lung fibrosis following PNX, the lungs of Cdc42 AT2 null mice are analyzed at various time points after PNX using H&E staining (FIG. 20D). The subpleural regions of some Cdc42 AT2 null lungs exhibit signs of tissue thickening by post-PNX day 21 (FIG. 20D compared with control lung in FIG. 20C). By the end-point, the dense fibrosis has progressed to the centre of most Cdc42 AT2 null lungs.

[0154] In addition to detecting strong immunofluorescence signals for Collagen I in these dense fibrotic regions (FIG. 20E), we observe the proportion of Collagen I expressing area per lobe gradually increased in Cdc42 AT2 null mice after PNX (FIG. 20F). Our qPCR analysis also shows that the Collagen I mRNA expression levels increased gradually from post-PNX day 21 (FIG. 20G). Moreover, gradually decreased lung compliance is observed in PNX-treated Cdc42 AT2 null mice from post-PNX day 21 as compared to their PNX-treated Control mice (FIG. 20H), an intriguing finding given that decreased lung compliance is known to occur frequently as lungs become fibrotic.sup.20-25.

[0155] Control and Cdc42 AT2 null mice were exposed to 4 doses of tamoxifen 14 days starting at age of 2 months. Lungs of Control and Cdc42 AT2 null mice without PNX treatment were collected at 10, 12, 16, or 24 months (FIG. 21A). The lungs of Control and Cdc42 AT2 null mice without PNX treatment were analyzed and found no significant fibrotic changes before the Cdc42 AT2 null mice reached 10-months of age (FIGS. 21B and 21C). By 12 months, fibrosis had obviously begun to develop in the subpleural regions of Cdc42 AT2 null lungs and to progress toward the center of the lung (FIG. 21C). Thus, the loss of Cdc42 in AT2 cells leads to progressive lung fibrosis in no-PNX-treated Cdc42 AT2 null mice starting from around 12 months of age.

[0156] Fibroblastic foci are considered a relevant morphologic marker of progressive pulmonary fibrosis and are recognized as sites where fibrotic responses are initiated and/or perpetuated in progressive pulmonary fibrosis. The fibroblastic foci contain proliferating α-SMA.sup.+ fibroblasts. Lungs of Cdc42 AT2 null mice at post-PNX day 21 are stained with antibodies against α-SMA (FIG. 22A). Some α-SMA.sup.+ fibroblasts accumulating next to a cluster of AT2 cells (GFP.sup.+ cells) in the relative normal alveolar regions of Cdc42 AT2 null lungs are observed (area 1, FIG. 22A). And the dense fibrosis region of the lungs is filled with α-SMA.sup.+ fibroblasts (area 2, FIG. 22A). In addition, the cell proliferation of α-SMA.sup.+ cells is increased dramatically in the lungs of Cdc42 AT2 null mice at post-PNX day 21 by immunostaining using antibodies against both α-SMA and proliferation marker, Ki67, indicating that the proliferating α-SMA.sup.+ fibroblasts contribute to the development of lung fibrosis (FIG. 22B).

Example 19

[0157] Elevated Mechanical Tension Caused by Impaired Alveolar Regeneration Leads to Progressive Lung Fibrosis

[0158] The fact that lung fibrosis in Cdc42 AT2 null mice is greatly accelerated by the PNX treatment (FIG. 20) suggests a close link between lung fibrosis and mechanical tension-induced alveolar regeneration.

[0159] The loss of alveoli resulting from PNX substantially increases mechanical tension exerted upon the alveolar epithelium. The subsequent efficient regeneration of alveoli that occurs in normal mice eventually reduces the intensity of the mechanical tension to pre-PNX levels; however, as Cdc42 null AT2 cells are unable to differentiate into AT1 cells and thus cannot regenerate new alveoli (FIG. 19B), the alveolar epithelium of Cdc42 AT2 null mice continue to experience elevated mechanical tension (FIG. 19B), which results in the progressive development of fibrosis (FIG. 20D).

Example 20

[0160] Progressive Lung Fibrosis Can be Prevented by the Prosthesis Implantation

[0161] By measuring the expression levels of CDC42-GTP, the GTP-bound state of CDC42, in the post-PNX lungs, it is found that the activity of CDC42-GTP increased significantly at post-PNX day 7 (FIG. 23A-23B). Such increased expression of CDC42-GTP can be inhibited by implanting a prosthesis in the chest (FIGS. 23A-23B).

[0162] As shown in FIGS. 24A-24H, reducing mechanical tension on alveoli attenuate progressive lung fibrosis. Control and Cdc42 AT2 null mice were exposed to 4 doses of tamoxifen 14 days prior to PNX. A prosthesis was implanted at post-PNX day 14 (FIG. 24A). Lungs of prosthesis-implanted Control and Cdc42 AT2 null mice were collected at post-PNX day 21. Images show the maximum intensity of a 200 μm Z-projection of lung sections that were stained with antibodies against GFP, Pdpn, and Prospc (FIG. 24B). The average alveolar size of prosthesis-implanted Control and Cdc42 AT2 null mice is not significantly different at post-PNX day 21 (mean±S.E.M., n=3), indicating the enlarged alveoli phenotype of Cdc42 AT2 null lungs can be greatly rescued by the implantation of the prosthesis (FIGS. 24B-24C). Recalling moderately increased actin-cytoskeleton regulatory genes and decreased expression levels of surfactant-associate genes were observed in IPF patient lungs. By qPCR, we compared the expression levels of actin-cytoskeleton regulatory genes and AT2 biomarker genes in AT2 cells of non-prosthesis and prosthesis implanted Cdc42 AT2 null mice (mean±S.E.M., n=3). In non-prosthesis implanted Cdc42 null AT2 cells, the expression levels of actin-cytoskeleton regulatory genes are increased and the expression levels of AT2 biomarker genes are decreased (FIGS. 24D-24E). The prosthetic implantation not only suppressed the increased expression of actin-cytoskeleton regulatory genes (FIG. 24D), but also greatly rescued the decreased expression of AT2 biomarker genes (FIG. 24E). Our results support the conclusion that the increased mechanical tension in the overstretched alveoli of Cdc42 AT2 null lungs positively regulates actin-cytoskeleton regulatory genes and negatively regulates the expression of AT2 biomarker genes. The percentage of survivors among the no-prosthesis-implanted and prosthesis-implanted Cdc42 AT2 null mice were calculated at post-PNX day 180. Strikingly, by post-PNX 180 days, none of the Cdc42 AT2 null mice (n=10) that were implanted with a prosthesis died while fully 70% of the no-prosthesis-implanted Cdc42 AT2 null mice (n=19) were dead by this time point (FIG. 24F). The levels of hydroxyproline in the lungs of no-prosthesis-implanted and prosthesis-implanted Cdc42 AT2 null mice when no-prosthesis-implanted Cdc42 AT2 null mice reached their end points (mean±S.E.M., n=5) (FIG. 24G). H&E staining revealed that little to no lung fibrosis was evident in prosthesis-implanted Cdc42 AT2 null mice by post-PNX day 180, whereas modest to severe lung fibrosis was observed in all of the surviving Cdc42 AT2 null mice that were not given a prosthesis following PNX (FIG. 24H). Together, these results demonstrate an essential regulatory role for the mechanical tension in driving the development of lung fibrosis in Cdc42 AT2 null mice. *P<0.05, **P<0.01, ***P<0.001; ****P<0.0001, NS, not significant, Student's t test. Scale bar: 20 μm (B); 2 mm (H).

[0163] As shown in FIG. 20-FIG. 22, the loss of Cdc42 in AT2 cells leads to progressive lung fibrosis following lung injury. The progressive development of lung fibrosis that we observed here is apparently similar to the pathological process that occurs in IPF patients, in which fibrosis initially starts at peripheral regions of the lung before slowly proceeding inwards, eventually affecting entire lung lobes. We show that the loss of alveoli resulting from PNX substantially increases mechanical tension exerted upon the alveolar epithelium. The subsequent efficient regeneration of alveoli that occurs in normal mice eventually reduces the intensity of the mechanical tension to pre-PNX levels; however, as Cdc42 null AT2 cells are unable to differentiate into AT1 cells and thus cannot regenerate new alveoli, the alveolar epithelium of Cdc42 null mice continue to experience elevated mechanical tension.

[0164] And further, the present invention provides a whole new and inventive method for treating IPF, the progression of which could not be reversed or even slowed down up to now. The present method uses pulmonary prosthesis implanted in the pleural cavity of human beings and is an efficient method for treating IPF without using any drugs.

REFERENCES

[0165] 1 Wynn, T. Cellular and molecular mechanisms of fibrosis. The Journal of Pathology: A Journal of the Pathological Society of Great Britain and Ireland 214, 199-210 (2008). [0166] 2 Wynn, T. A. & Ramalingam, T. R. Mechanisms of fibrosis: therapeutic translation for fibrotic disease. Nature medicine 18, 1028 (2012). [0167] 3 Mehal, W. Z., Iredale, J. & Friedman, S. L. Scraping fibrosis: expressway to the core of fibrosis. Nature medicine 17, 552 (2011). [0168] 4 Barkauskas, C. E. & Noble, P. W. Cellular mechanisms of tissue fibrosis. 7. New insights into the cellular mechanisms of pulmonary fibrosis. American Journal of Physiology-Cell Physiology 306, C987-C996 (2014). [0169] 5 Rock, J. R. et al. Multiple stromal populations contribute to pulmonary fibrosis without evidence for epithelial to mesenchymal transition. Proceedings of the National Academy of Sciences 108, E1475-E1483 (2011). [0170] 6 Gross, T. J. & Hunninghake, G. W. Idiopathic pulmonary fibrosis. New England Journal of Medicine 345, 517-525 (2001). [0171] 7 Vyalov, S. L., Gabbiani, G. & Kapanci, Y. Rat alveolar myofibroblasts acquire alpha-smooth muscle actin expression during bleomycin-induced pulmonary fibrosis. The American journal of pathology 143, 1754 (1993). [0172] 8 King Jr, T. E., Pardo, A. & Selman, M. Idiopathic pulmonary fibrosis. The Lancet 378, 1949-1961 (2011). [0173] 9 Plantier, L. et al. Ectopic respiratory epithelial cell differentiation in bronchiolised distal airspaces in idiopathic pulmonary fibrosis. Thorax 66, 651-657 (2011). [0174] 10 Steele, M. P. & Schwartz, D. A. Molecular mechanisms in progressive idiopathic pulmonary fibrosis. Annual review of medicine 64, 265-276 (2013). [0175] 11 Nogee, L. M. et al. A mutation in the surfactant protein C gene associated with familial interstitial lung disease. New England Journal of Medicine 344, 573-579 (2001). [0176] 12 Seibold, M. A. et al. A common MUC5B promoter polymorphism and pulmonary fibrosis. New England Journal of Medicine 364, 1503-1512 (2011). [0177] 13 Wang, Y. et al. Genetic defects in surfactant protein A2 are associated with pulmonary fibrosis and lung cancer. The American Journal of Human Genetics 84, 52-59 (2009). [0178] 14 Barkauskas, C. E. et al. Type 2 alveolar cells are stem cells in adult lung. The Journal of clinical investigation 123, 3025-3036 (2013). [0179] 15 Desai, T. J., Brownfield, D. G. & Krasnow, M. A. Alveolar progenitor and stem cells in lung development, renewal and cancer. Nature 507, 190 (2014). [0180] 16 Haies, D. M., Gil, J. & Weibel, E. R. Morphometric study of rat lung cells: I. Numerical and dimensional characteristics of parenchymal cell population. American Review of Respiratory Disease 123, 533-541 (1981). [0181] 17 Selman, M. & Pardo, A. Idiopathic pulmonary fibrosis: an epithelial/fibroblastic cross-talk disorder. Respiratory research 3, 3 (2001). [0182] 18 Kropski, J. A., Blackwell, T. S. & Loyd, J. E. The genetic basis of idiopathic pulmonary fibrosis. European Respiratory Journal 45, 1717-1727 (2015). [0183] 19 Goodwin, A. T. & Jenkins, G. Molecular endotyping of pulmonary fibrosis. Chest 149, 228-237 (2016). [0184] 20 Meltzer, E. B. & Noble, P. W. Idiopathic pulmonary fibrosis. Orphanet journal of rare diseases 3, 8, doi:10.1186/1750-1172-3-8 (2008). [0185] 21 Richeldi, L., Collard, H. R. & Jones, M. G. Idiopathic pulmonary fibrosis. The Lancet 389, 1941-1952, doi:10.1016/s0140-6736(17)30866-8 (2017). [0186] 22 T E, J. K. et al. Idiopathic pulmonary fibrosis. American journal of respiratory and critical care medicine 161, 646-664 (2000). [0187] 23 Lynch, D. A. et al. High-resolution computed tomography in idiopathic pulmonary fibrosis: diagnosis and prognosis. American journal of respiratory and critical care medicine 172, 488-493 (2005). [0188] 24 Noble, P. W. et al. Pirfenidone in patients with idiopathic pulmonary fibrosis (CAPACITY): two randomised trials. The Lancet 377, 1760-1769 (2011). [0189] 25 Raghu, G. et al. An official ATS/ERS/JRS/ALAT statement: idiopathic pulmonary fibrosis: evidence-based guidelines for diagnosis and management. American journal of respiratory and critical care medicine 183, 788-824 (2011).