Abstract
The present disclosure relates to a composition for various applications, which is capable of effectively preventing, ameliorating or treating lupus using a secretome derived from mesenchymal stem cells.
The secretome derived from mesenchymal stem cells according to the present disclosure may significantly decrease mortality and the amount of proteinuria, and may increase body weight, decrease the expression of serum creatinine, and inhibit glomerular, coronary and vascular damage in kidney tissue. Furthermore, the secretome may reduce the size of an enlarged spleen and reduce the number of splenocytes and CD4-positive T cells. In addition, the secretome may increase the expression of the anti-inflammatory cytokines IL-10 and TGF-1 in serum, and decrease the expression of anti-dsDNA antibody. In the mechanism thereof, the secretome may effectively prevent, ameliorate or treat lupus nephritis and, furthermore, lupus, by increasing the activity of Treg cells and inhibiting the activity of the inflammatory cells Th1 and Th2 cells, B cells, dendritic cells and inflammatory macrophages.
Claims
1-18. (canceled)
19. A method for preventing or treating lupus, comprising a step of administering a target subject a secretome derived from mesenchymal stem cells in order to prevent or treat lupus.
20. The method of claim 19, wherein the secretome is isolated from a culture obtained by culturing the mesenchymal stem cells.
21. The method of claim 20, wherein the culturing of the mesenchymal stem cells is performed by culturing the mesenchymal stem cells in a mesenchymal stem cell culture medium for 24 to 96 hours, and then culturing the mesenchymal stem cells in a serum-free medium for 24 to 72 hours.
22. The method of claim 21, wherein the mesenchymal stem cell culture medium is any one selected from the group consisting of a Dulbecco's modified Eagle's medium (DMEM) containing 5 to 15 wt % of fetal bovine serum (FBS) and 0.05 to 0.2 mM of mercaptoethanol, RPMI-1640 medium, StemPro medium, MSCGro medium, MesenCult medium, and Nutri Stem medium.
23. The method of claim 20, wherein the secretome is a concentrate obtained after centrifuging the culture of the mesenchymal stem cells at 500 to 1,500 xg and recovering a supernatant of the centrifuged culture.
24. The method of claim 23, wherein the concentrate is obtained by a step of filtering the supernatant through a 0.1 to 0.3 m filter, and a step of filtering molecules of 3 kDa or less in size.
25. The method of claim 24, wherein the filtering of the molecules of 3 kDa or less in size is performed by diafiltration using a tangential flow filtration (TFF) system.
26. The method of claim 23, wherein the concentrate is obtained by reacting the recovered supernatant with a polar alcohol solvent.
27. The method of claim 26, wherein the reaction of the supernatant with the polar alcohol solvent is performed at a temperature of 30 to 0 C. for 5 to 500 minutes.
28. The method of claim 26, wherein the polar alcohol solvent is mixed with the supernatant in an amount of 2 to 5 times the weight of the supernatant.
29. The method of claim 23, wherein the concentrate is a freeze-dried concentrate.
30. A method for preventing or ameliorating lupus, comprising a step of administering a target subject a secretome derived from mesenchymal stem cells in order to prevent or ameliorate lupus.
31. The method of claim 30, wherein the secretome is isolated from a culture obtained by culturing the mesenchymal stem cells.
32. The method of claim 31, wherein the culturing of the mesenchymal stem cells is performed by culturing the mesenchymal stem cells in a mesenchymal stem cell culture medium for 24 to 96 hours, and then culturing the mesenchymal stem cells in a serum-free medium for 24 to 72 hours.
33. The method of claim 32, wherein the mesenchymal stem cell culture medium is any one selected from the group consisting of a Dulbecco's modified Eagle's medium (DMEM) containing 5 to 15 wt % of fetal bovine serum (FBS) and 0.05 to 0.2 mM of mercaptoethanol, RPMI-1640 medium, StemPro medium, MSCGro medium, MesenCult medium, and NutriStem medium.
34. The method of claim 31, wherein the secretome is a concentrate obtained after centrifuging the culture of the mesenchymal stem cells at 500 to 1,500 xg and recovering a supernatant of the centrifuged culture.
35. The method of claim 34, wherein the concentrate is obtained by a step of filtering the supernatant through a 0.1 to 0.3 m filter, and a step of filtering molecules of 3 kDa or less in size.
36. The method of claim 35, wherein the filtering of the molecules of 3 kDa or less in size is performed by diafiltration using a tangential flow filtration (TFF) system.
Description
DESCRIPTION OF DRAWINGS
[0054] FIG. 1 schematically shows an experimental design in which lupus nephritis mouse models are treated with either a secretome, isolated and concentrated according to one embodiment of the present disclosure, or adipose-derived mesenchymal stem cells (MSCs) as a control, in Example 2.
[0055] FIG. 2 graphically shows the results of comparing the survival rate of mice after treating lupus nephritis mouse models with each of a secretome, isolated and concentrated according to one embodiment of the present disclosure, and adipose-derived mesenchymal stem cells (AD-MSCs) or methylprednisolone as a positive treatment control, in Example 2. The mortality of lupus nephritis mouse models in the group treated with the secretome decreased compared to that in the untreated group.
[0056] FIG. 3 graphically shows the results of measuring proteinuria after treating lupus nephritis mouse models with each of a secretome, isolated and concentrated according to one embodiment of the present disclosure, and adipose-derived mesenchymal stem cells (AD-MSCs) or methylprednisolone as a positive treatment control, in Example 2. The amount of proteinuria in the group treated with the secretome significantly decreased compared to that in the untreated group, and the level of decrease in proteinuria in the group treated with the secretome was similar to that in the group treated with methylprednisolone (*,p<0.05; **,p<0.01; ***,p<0.001).
[0057] FIG. 4 graphically shows the results of measuring serum creatinine after treating lupus nephritis mouse models with each of a secretome, isolated and concentrated according to one embodiment of the present disclosure, and methylprednisolone as a positive treatment control, in Example 2. The expression of serum creatinine in the group treated with the secretome significantly decreased compared to that in the untreated group (*,p<0.05; ***,p<0.001).
[0058] FIG. 5 shows images of PAS-stained kidney tissue, obtained after treating lupus nephritis mouse models with each of a secretome, isolated and concentrated according to one embodiment of the present disclosure, and adipose-derived mesenchymal stem cells (AD-MSCs) or methylprednisolone as a positive treatment control, in Example 3.
[0059] FIG. 6 graphically shows the results of evaluating the extent of kidney glomerular damage after treating lupus nephritis mouse models with each of a secretome, isolated and concentrated according to one embodiment of the present disclosure, and adipose-derived mesenchymal stem cells (AD-MSCs) or methylprednisolone as a positive treatment control, in Example 3. Glomerular damage in the group treated with the secretome was significantly inhibited compared to that in the untreated group (***,p<0.001).
[0060] FIG. 7 graphically shows the results of evaluating the extent of kidney tubular damage after treating lupus nephritis mouse models with each of a secretome, isolated and concentrated according to one embodiment of the present disclosure, and adipose-derived mesenchymal stem cells (AD-MSCs) or methylprednisolone as a positive treatment control, in Example 3. Kidney tubular damage in the group treated with the secretome was significantly inhibited compared to that in the untreated group (***p<0.001).
[0061] FIG. 8 graphically shows the results of evaluating the extent of kidney vascular damage after treating lupus nephritis mouse models with each of a secretome, isolated and concentrated according to one embodiment of the present disclosure, and adipose-derived mesenchymal stem cells (AD-MSCs) or methylprednisolone as a positive treatment control, in Example 3. Kidney vascular damage in the group treated with the secretome was significantly inhibited compared to that in the untreated group (*, p<0.05).
[0062] FIG. 9 graphically shows the results of measuring the expression levels of IgG and C3 in kidney tissue after treating lupus nephritis mouse models with each of a secretome, isolated and concentrated according to one embodiment of the present disclosure, and adipose-derived mesenchymal stem cells (AD-MSCs) or methylprednisolone as a positive treatment control, in Example 3. The fluorescence intensities of IgG and C3 in the group treated with the secretome significantly decreased compared to those in the untreated group (***,p<0.001).
[0063] FIG. 10 depicts images showing the size of spleen after treating lupus nephritis mouse models with each of a secretome, isolated and concentrated according to one embodiment of the present disclosure, and adipose-derived mesenchymal stem cells (AD-MSCs) or methylprednisolone as a positive treatment control, in Example 4.
[0064] FIG. 11 graphically shows the results of measuring changes in the weight of spleen after treating lupus nephritis mouse models with each of a secretome, isolated and concentrated according to one embodiment of the present disclosure, and adipose-derived mesenchymal stem cells (AD-MSCs) or methylprednisolone as a positive treatment control, in Example 4.
[0065] FIG. 12 graphically shows the results of measuring changes in the number of splenocytes after treating lupus nephritis mouse models with each of a secretome, isolated and concentrated according to one embodiment of the present disclosure, and adipose-derived mesenchymal stem cells (AD-MSCs) or methylprednisolone as a positive treatment control, in Example 4. The number of splenocytes in the group treated with the secretome significantly decreased compared to that in the untreated group (*,p<0.05; **,p<0.01).
[0066] FIG. 13 graphically shows the results of measuring changes in the number of CD4+ T cells in spleen tissue after treating lupus nephritis mouse models with each of a secretome, isolated and concentrated according to one embodiment of the present disclosure, and methylprednisolone as a positive treatment control, in Example 5. The number of CD4+ T cells in the group treated with the secretome significantly decreased compared to that in the untreated group (*,p<0.05; **,p<0.01).
[0067] FIG. 14 graphically shows the results of analyzing changes in the expression level of CD4+Foxp3+ cells (regulatory T cells) among mouse splenocytes after treating lupus nephritis mouse models with each of a secretome, isolated and concentrated according to one embodiment of the present disclosure, and adipose-derived mesenchymal stem cells (AD-MSCs) or methylprednisolone as a positive treatment control, in Example 6. The expression level of CD4+Foxp3+ cells in the group treated with the secretome significantly increased compared to that in the untreated group (*,p<0.05).
[0068] FIG. 15 graphically shows the results of analyzing changes in the expression level of CD4+CD25+Foxp3+PD-1+ cells (regulatory T cells) among mouse splenocytes after treating lupus nephritis mouse models with each of a secretome, isolated and concentrated according to one embodiment of the present disclosure, and adipose-derived mesenchymal stem cells (AD-MSCs) or methylprednisolone as a positive treatment control, in Example 6. The expression level of CD4+CD25+Foxp3+PD-1+ cells in the group treated with the secretome significantly increased compared to that in the untreated group (*,p<0.05; **,p<0.01).
[0069] FIG. 16 graphically shows the results of analyzing changes in the expression level of CD4+IFN-+ cells (Th1 cells) among mouse splenocytes after treating lupus nephritis mouse models with each of a secretome, isolated and concentrated according to one embodiment of the present disclosure, and adipose-derived mesenchymal stem cells (AD-MSCs) or methylprednisolone as a positive treatment control, in Example 6.
[0070] FIG. 17 graphically shows the results of analyzing changes in the expression level of CD4+IL-4+ cells (Th2 cells) among mouse splenocytes after treating lupus nephritis mouse models with each of a secretome, isolated and concentrated according to one embodiment of the present disclosure, and adipose-derived mesenchymal stem cells (AD-MSCs) or methylprednisolone as a positive treatment control, in Example 6. The expression level of CD4+IL-4+ cells in the group treated with the secretome significantly decreased compared to that in the untreated group (*,p<0.05).
[0071] FIG. 18 graphically shows the results of analyzing changes in the expression level of CD4+IL-17A+ cells (Th17) cells among mouse splenocytes after treating lupus nephritis mouse models with each of a secretome, isolated and concentrated according to one embodiment of the present disclosure, and methylprednisolone as a positive treatment control, in Example 6.
[0072] FIG. 19 graphically shows the results of analyzing changes in the expression level of CD19+CD138+ cells (B cells) among mouse splenocytes after treating lupus nephritis mouse models with each of a secretome, isolated and concentrated according to one embodiment of the present disclosure, and adipose-derived mesenchymal stem cells (AD-MSCs) or methylprednisolone as a positive treatment control, in Example 7. The expression level of CD19+CD138+ B cells in the group treated with the secretome significantly decreased compared to that in the untreated group (***,p<0.001).
[0073] FIG. 20 graphically shows the results of analyzing changes in the expression level of CD11c+CD86+ cells (dendritic cells) among mouse splenocytes after treating lupus nephritis mouse models with each of a secretome, isolated and concentrated according to one embodiment of the present disclosure, and adipose-derived mesenchymal stem cells (AD-MSCs) or methylprednisolone as a positive treatment control, in Example 8. The expression level of CD11c+CD86+ dendritic cells in the group treated with the secretome significantly decreased compared to that in the untreated group (*,p<0.05; ***,p<0.001).
[0074] FIG. 21 graphically shows the results of analyzing changes in the expression level of CD11c+MHCII+ cells (dendritic cells) among mouse splenocytes after treating lupus nephritis mouse models with each of a secretome, isolated and concentrated according to one embodiment of the present disclosure, and adipose-derived mesenchymal stem cells (AD-MSCs) or methylprednisolone as a positive treatment control, in Example 8. The expression level of CD11c+MHCII+ dendritic cells in the group treated with the secretome significantly decreased compared to that in the untreated group (**, p<0.01; ***,p<0.001).
[0075] FIG. 22 graphically shows the results of analyzing changes in the expression level of F4/80+CD86+ cells (inflammatory macrophages) among mouse splenocytes after treating lupus nephritis mouse models with each of a secretome, isolated and concentrated according to one embodiment of the present disclosure, and methylprednisolone as a positive treatment control, in Example 9. The expression level ofF4/80+CD86+ macrophages in the group treated with the secretome significantly decreased compared to that in the untreated group (**,p<0.01).
[0076] FIG. 23 graphically shows the results of measuring the number of total lymphocytes in kidney tissue after treating lupus nephritis mouse models with each of a secretome, isolated and concentrated according to one embodiment of the present disclosure, and methylprednisolone as a positive treatment control, in Example 10. The number of total lymphocytes in kidney tissue in the group treated with the secretome significantly decreased compared to that in the untreated group (***,p<0.001).
[0077] FIG. 24 graphically shows the results of analyzing changes in the expression level of IL-17A in mouse serum after treating lupus nephritis mouse models with each of a secretome, isolated and concentrated according to one embodiment of the present disclosure, and adipose-derived mesenchymal stem cells (AD-MSCs) or methylprednisolone as a positive treatment control, in Example 11. The expression level of IL-17A in serum in the group treated with the secretome significantly decreased compared to that in the untreated group (*,p<0.05).
[0078] FIG. 25 graphically shows the results of analyzing changes in the expression level of IL-6 in mouse serum after treating lupus nephritis mouse models with each of a secretome, isolated and concentrated according to one embodiment of the present disclosure, and adipose-derived mesenchymal stem cells (AD-MSCs) or methylprednisolone as a positive treatment control, in Example 11. The expression level of IL-6 in serum in the group treated with the secretome significantly increased compared to that in the untreated group (**,p<0.01).
[0079] FIG. 26 graphically shows the results of analyzing changes in the expression level of IL-10 in mouse serum after treating lupus nephritis mouse models with each of a secretome, isolated and concentrated according to one embodiment of the present disclosure, and adipose-derived mesenchymal stem cells (AD-MSCs) or methylprednisolone as a positive treatment control, in Example 11. The expression level of IL-10 in serum in the group treated with the secretome significantly increased compared to that in the untreated group (**,p<0.05).
[0080] FIG. 27 graphically shows the results of analyzing changes in the expression level of TGF-1 in mouse serum after treating lupus nephritis mouse models with each of a secretome, isolated and concentrated according to one embodiment of the present disclosure, and adipose-derived mesenchymal stem cells (AD-MSCs) or methylprednisolone as a positive treatment control, in Example 11. The expression level of TGF-1 in serum in the group treated with the secretome significantly increased compared to that in the untreated group (*,p<0.05).
[0081] FIG. 28 graphically shows the results of analyzing changes in the expression level of anti-dsDNA in mouse serum after treating lupus nephritis mouse models with each of a secretome, isolated and concentrated according to one embodiment of the present disclosure, and adipose-derived mesenchymal stem cells (AD-MSCs) or methylprednisolone as a positive treatment control, in Example 12. The expression level of anti-dsDNA in serum in the group treated with the secretome significantly decreased compared to that in the untreated group (*,p<0.05).
[0082] FIG. 29 graphically shows the results of analyzing the changes in mouse body weight depending on the treatment period after treating lupus nephritis mouse models with each of a secretome, isolated and concentrated according to one embodiment of the present disclosure, and adipose-derived mesenchymal stem cells (AD-MSCs) or methylprednisolone as a positive treatment control, in Example 13. The mouse body weight in the group treated with the secretome increased as the treatment period increased.
BEST MODE
[0083] One embodiment of the present disclosure is directed to a pharmaceutical composition for preventing or treating lupus, containing a secretome derived from mesenchymal stem cells.
Mode for Disclosure
[0084] Hereinafter, the present disclosure will be described in more detail with reference to Examples. These Examples are merely to illustrate the present disclosure in detail, and it will be obvious to those skilled in the art that the scope of the present disclosure according to the subject matter of the present disclosure is not limited by these Examples.
EXAMPLES
[Example 1] Preparation of Secretome Derived from Mesenchymal Stem Cells
[0085] 1. Reagents and Chemical Products
[0086] DMEM (Dulbecco modified Eagle's medium-low glucose), FBS (fetal bovine serum), penicillin/streptomycin and 2-mercaptoethanol (X1000) were purchased from Invitrogen Corp.
[0087] 2. Obtaining of Mesenchymal Stem Cells from Human Adipocytes
[0088] Adipose-derived human mesenchymal stem cells at an early passage were obtained from the Cell Therapy Center of Yonsei University that complies with the Korean Food and Drug Administration guidelines [GMP (Pharmaceutical Manufacturing Quality Control Standards)], and the cells were cultured. Mesenchymal stem cell culture medium (DMEM low glucose supplemented with 10% FBS and 0.1 mM mercaptoethanol) was placed in a culture dish under human mesenchymal stem cell culture conditions clinically approved by the FDA, and the cells were cultured in the medium for 72 to 86 hours. The medium was replaced every 2 to 3 days, and the cells were passaged to a confluence of 70 to 85%. The cells at passage 5 were used in the study.
[0089] 3. Culturing of Mesenchymal Stem Cells
[0090] Mesenchymal stem cells were cultured to a confluence of 80% and repeatedly washed four times or more with PBS buffer to remove protein components such as fetal bovine serum. Next, the cells were cultured with a serum-free medium (DMEM-low glucose) not containing antibiotic and fetal bovine serum for 48 hours, and then the cell culture was recovered.
[0091] 4. Isolation and Concentration of Secretome from Culture of Mesenchymal Stem Cells
[0092] The culture obtained by culturing the mesenchymal stem cells in large scale as described above was centrifuged once at 1000g to remove the cell residue. Thereafter, large particles such as cell debris were filtered out through a 0.2 m filter, and molecules of 3 kDa or less in size were filtered by a diafiltration system using tangential flow filtration (TFF) capsules (PALL, Minimate TFF capsules). During the filtration, the culture was concentrated at 4 C. while it was continuously replaced and diluted with water for injection (saline solution or Ringer's solution) by a peristaltic tubing pump. The concentration of protein in the concentrated culture was determined by refractometer measurement and Bradford reagent, and the concentrated culture was stored at 80 C. until the experiment was started.
[Example 2] Survival Rate of Lupus-Induced Mouse Models, Change in Proteinuria, and Change in Serum Creatinine Concentration
[0093] In order to evaluate the lupus nephritis therapeutic effect of the secretome isolated and concentrated in Example 1 above, an experiment was performed as shown in FIG. 1. Specifically, the secretome isolated and concentrated in Example 1 was injected intraperitoneally into lupus nephritis mouse ((NZB/NZW) F1) models (23 weeks of age)) three times a week at a dose of 200 g/mouse. Then, the survival rate of the mice, proteinuria and the concentration of creatinine in the serum were measured, and the results of the measurement are graphically shown in FIGS. 2 to 4.
[0094] Proteinuria was measured twice a week during the experimental period in the spot urine collected from each mouse using an albumin reagent strip (URiSCA; Yeongdong Pharm., Korea). Proteinuria was expressed semi-quantitatively: 0=none or trace; 1+=100 mg/dL or less; 2+=300 mg/dL or less; 3+=2,000 mg/dL or less; and 4+=2,000 mg/dL or more.
[0095] In addition, the concentration of creatinine in the serum was measured using a BioAssay Systems QuantiChrom creatinine assay kit by adding a mixed reagent to 30 l of the serum, and then immediately measuring the OD value, and after 5 minutes, measuring the OD value once more. Thereafter, the concentration of creatinine in the serum was calculated using the following equation:
ODsample 5-OD sample 0/OD STD 5-ODSTD 0STD (mg/dL).
[0096] However, in order to compare the therapeutic effect of the secretome according to the present disclosure, the mice of the negative control group were untreated, and as a positive treatment control, Solumedrol (methylprednisolone), which has been used as a lupus treatment drug, was injected, or 510.sup.6 human adipose-derived stem cells contained in 100 l of PBS were injected into the tail veins of the lupus nephritis mouse models (23 weeks old).
[0097] As shown in FIG. 2, the mortality of the lupus nephritis mouse models of the group treated with the secretome according to the present disclosure decreased compared to that of the untreated group.
[0098] In addition, as shown in FIG. 3, proteinuria increased in the untreated group, but significantly decreased in the group treated with the secretome according to the present disclosure, and the extent of the decrease was similar to that in the group treated with the mesenchymal stem cells or the group treated with the standard lupus treatment drug methylprednisolone.
[0099] In addition, as shown in FIG. 4, proteinuria in the group treated with the secretome according to the present disclosure decreased to an extent similar to that in the group treated with methylprednisolone. Thus, it could be seen that the secretome had an anti-inflammatory effect.
[0100] The concentration of creatinine in the serum represents kidney function. As shown in FIG. 5, it could be confirmed that the content of creatinine in the group treated with the secretome according to the present disclosure significantly decreased compared to that in the untreated group.
[Example 3] Effect of Protection Against Kidney Tissue Damage in Lupus-Induced Mouse Models
[0101] After performing the experiment in the same manner as in Example 2 above, the lupus nephritis mouse models were euthanized, and then the kidney tissues were fixed in formalin, embedded in paraffin, sectioned thinly, and then subjected to PAS staining. The results of the staining are shown in FIG. 5. In addition, the extents of glomerular damage, tubular damage and vascular damage in the kidney tissue of each treatment group were evaluated, and the results of the evaluation are shown in FIGS. 6 to 8, respectively.
[0102] The expression levels of IgG and C3, which are deposited in kidney tissue at the onset of lupus nephritis, were analyzed by fluorescence staining. The kidney tissues, treated with an OCT compound and stored at 20 C., were sectioned thinly, and then treated with anti-mouse IgG and anti-mouse C3 antibodies and additionally treated with secondary fluorescent antibodies. Next, the sections were imaged under confocal microscopy and the fluorescence intensities thereof were quantitatively analyzed. The results of the analysis are shown in FIG. 9.
[0103] As shown in FIGS. 5 to 8, it could be confirmed that when the mice were treated with the secretome according to the present disclosure, the kidney tissue was more protected from damage than when the mice were treated with methylprednisolone or mesenchymal stem cells.
[0104] As shown in FIG. 9, it could be confirmed that the expression levels of IgG and C3 in the kidney tissue of the group treated with the secretome according to the present disclosure significantly decreased compared to those in the untreated group and the expression levels decreased compared to those in the group treated with mesenchymal stem cells.
[Example 4] Changes in Size of Spleen and Number of Splenocytes in Lupus-Induced Mouse Models
[0105] After performing the experiment in the same manner as Example 2 above, the lupus nephritis mouse models were euthanized, and then the spleen tissues were imaged. The results of the imaging are shown in FIG. 10. The weight of the spleen in each treatment group was measured, and the results of the measurement are shown in FIG. 11. The number of splenocytes in each treatment group was measured, and the results of the measurement are shown in FIG. 12.
[0106] As shown in FIGS. 10 and 11, it could be confirmed that the size of the enlarged spleen in the lupus nephritis mouse models significantly decreased when treated with the secretome according to the present disclosure. As shown in FIG. 12, it could be confirmed that the increased number of splenocytes in the lupus nephritis mouse models was also more decreased when treated with the secretome according to the present disclosure than when treated with methylprednisolone or mesenchymal stem cells.
[Example 5] Change in Expression Level of CD4+ T Cells in Lupus-Induced Mouse Models
[0107] After performing the experiment in the same manner as Example 2 above, the expression level of CD4+ T cells in the splenocytes of the lupus nephritis mouse models was measured using a flow cytometer, and the results of the measurement are shown in FIG. 13.
[0108] As shown in FIG. 13, it could be confirmed that the increased expression level of CD4+ T cells in the lupus nephritis mouse models significantly decreased when treated with the secretome according to the present disclosure. Thus, it could be seen that the secretome had an anti-inflammatory effect.
[Example 6] Analysis of T Cells in Lupus-Induced Mouse Models
[0109] After performing the experiment in the same manner as Example 2 above, the expression levels of CD4+Foxp3+ cells (corresponding to regulatory T (Treg) cells), CD4+CD25+Foxp3+PD-1+ cells, CD4+IFN-+ cells (corresponding to Th1 cells), CD4+IL-4+ cells (corresponding to Th2 cells) and CD4+IL-17+ cells (corresponding to Th17 cells) in the splenocytes of the lupus nephritis mouse models were analyzed using a flow cytometer, and the results of the analysis are shown in FIGS. 14 to 18.
[0110] As shown in FIGS. 14 to 18, the expression levels of the Th1 and Th2 cells significantly decreased when the lupus nephritis mouse models were treated with the secretome according to the present disclosure compared to when untreated or treated with methylprednisolone, and the expression levels of the Treg cells (CD4+Foxp3+ cells and CD4+CD25+Foxp3+PD-1+ cells significantly increased when the lupus nephritis mouse models were treated with the secretome according to the present disclosure. That is, it could be confirmed that the secretome according to the present disclosure controls the function of inflammatory cells, Th1 cells and Th2 cells, but induces the function of Treg cells that control inflammatory cells.
[0111] Meanwhile, the expression level of Th17 cells in the group treated with the secretome according to the present disclosure was maintained at the same level as that in the untreated group, but decreased in the group treated with the mesenchymal stem cells. Thus, it could be seen that the secretome according to the present disclosure and the mesenchymal stem cells acted by different mechanisms.
[Example 7] Analysis of B Cells in Lupus-Induced Mouse Models
[0112] After performing the experiment in the same manner as Example 2 above, the expression level of CD19+CD138+ cells (corresponding to B cells and plasma B cells) in the splenocytes of the lupus nephritis mouse models was analyzed using a flow cytometer, and the results of the analysis are shown in FIG. 19.
[0113] As shown in FIG. 19, it could be confirmed that when the lupus nephritis mouse models were treated with the secretome according to the present disclosure, the expression level of B cells significantly decreased compared to when untreated or treated with methylprednisolone, and more greatly decreased than when treated with the mesenchymal stem cells.
[Example 8] Analysis of Dendritic Cells and M1 Cells in Lupus-Induced Mouse Models
[0114] After performing the experiment in the same manner as Example 2 above, the expression levels of CD11c+CD86+ cells and CD11c+MHCII+ cells (corresponding to dendritic cells) in the splenocytes of the lupus nephritis mouse models were analyzed using a flow cytometer, and the results of the analysis are shown in FIGS. 20 and 21.
[0115] The CD86 and MHCII are markers indicating the activity of dendritic cells. As shown in FIGS. 20 and 21, it could be confirmed that when the lupus nephritis mouse models were treated with the secretome according to the present disclosure, the activity of dendritic cells significantly decreased when untreated or treated with methylprednisolone, and the activity more decreased than when the lupus nephritis mouse models were treated with the mesenchymal stem cells.
[Example 9] Analysis of Macrophages in Lupus-Induced Mouse Models
[0116] After performing the experiment in the same manner as Example 2 above, the expression level of F4/80+CD86+ cells (inflammatory macrophages) in the splenocytes of the lupus nephritis mouse models was analyzed using a flow cytometer, and the results of the analysis are shown in FIG. 22.
[0117] As shown in FIG. 22, it could be confirmed that when the lupus nephritis mouse models were treated with the secretome according to the present disclosure, the activity of the inflammatory macrophages significantly decreased compared to when untreated or treated with methylprednisolone.
[Example 10] Analysis of Number of Lymphocytes in Lupus-Induced Mouse Models
[0118] After performing the experiment in the same manner as Example 2 above, the number of total lymphocytes in the kidney tissues of the lupus nephritis mouse models was measured using a flow cytometer, and the results of the measurement are shown in FIG. 23.
[0119] As shown in FIG. 23, it could be confirmed that when the lupus nephritis mouse models were treated with methylprednisolone, the number of lymphocytes was almost similar to that in the untreated group, but when the lupus nephritis mouse models were treated with the secretome according to the present disclosure, the number of lymphocytes significantly decreased.
[Example 11] Analysis of Expression Levels of Cytokines in Serum in Lupus-Induced Mouse Models
[0120] After performing the experiment in the same manner as Example 2 above, the expression levels of the cytokines IL-17, IL-6, IL-10 and TGF-1 in the sera of the lupus nephritis mouse models were measured by an FT ISA assay, and the results of the measurement are shown in FIGS. 24 to 27, respectively.
[0121] As shown in FIGS. 24 to 27, it could be confirmed that when the lupus nephritis mouse models were treated with the secretome according to the present disclosure, the expression levels of IL-17 in the serum decreased compared to when untreated or treated with the mesenchymal stem cells, and the expression levels of IL-6, IL-10, and TGF-1 increased.
[Example 12] Analysis of Double-Stranded DNA in Serum in Lupus-Induced Mouse Models
[0122] After performing the experiment in the same manner as Example 2 above, the expression level the autoantibody anti-dsDNA, which is expressed at the onset of lupus, in the sera of the lupus nephritis mouse models, was measured by an FT ISA assay, and the results of the measurement are shown in FIG. 28.
[0123] As shown in FIG. 28, it could be confirmed that when the lupus nephritis mouse models were treated with the secretome according to the present disclosure, the expression level of anti-dsDNA in the serum significantly decreased compared to when untreated.
[Example 13] Analysis of Change in Body Weight in Lupus-Induced Mouse Models
[0124] According to the same method as Example 2 above, the lupus nephritis mouse models were treated with each of the secretome, isolated and concentrated according to one embodiment of the present disclosure, and adipose-derived mesenchymal stem cells (AD-MSCs) or methylprednisolone as a positive treatment control. Then, changes in the body weights of the mouse models were measured, and the results of the measurement are shown in FIG. 29.
[0125] As shown in FIG. 29, it could be confirmed that when the lupus nephritis mouse models were untreated or treated with methylprednisolone, the body weight decreased, but when the lupus nephritis mouse models were treated with the secretome according to the present disclosure, the body weight increased as the treatment period increased.
[0126] In general, when mesenchymal stem cells are administered, not only a sufficient supply thereof is difficult, but also when these cells are transplanted in vivo, the possibility of allogeneic transplantation rejection and tumorigenesis may become problematic. However, the secretome derived from mesenchymal stem cells according to the present disclosure may be produced and synthesized in large scale from a cell line, and has no immunogenicity. In addition, from the in vivo experiment as described above, it can be seen that the secretome has a therapeutic effect equivalent to or greater than mesenchymal stem cells against lupus, particularly lupus nephritis. Furthermore, from the results that the regulation of expression level of Th17 cells in the spleen and expression of IL-6 in the serum differs between the mesenchymal stem cell-derived secretome and the mesenchymal stem cells, it can be seen that the mechanisms of the therapeutic effects of the mesenchymal stem cell-derived secretome and the mesenchymal stem cells differ from each other.
INDUSTRIAL APPLICABILITY
[0127] The present disclosure is directed to a medicament capable of effectively preventing, ameliorating or treating lupus using a secretome derived from mesenchymal stem cells.
