METHOD FOR SCREENING THERAPEUTIC TARGET OF ACUTE GASTROINTESTINAL SYNDROME AND USE OF TIGAR TARGET IN PREPARATION OF MEDICINE FOR TREATING RADIATION-INDUCED GASTROINTESTINAL SYNDROME

Abstract

The invention discloses a method for screening a therapeutic target of acute radiation-induced gastrointestinal syndrome and use of TIGAR target in the preparation of a medicine for treating radiation-induced gastrointestinal syndrome. The CreERT-loxP transgenic mouse model is used, in which quiescent intestinal crypt stem cells are effectively promoted to proliferate after exposure to high-dose ionizing radiation, to screen a therapeutic target that still has a therapeutic effect for radiation-induced gastrointestinal syndrome 18-24 h after ionizing radiation. Gene splicing occurs in particular cells in the CreERT-loxP transgenic mice only after the injection of tamoxifen, thereby regulating gene expression. The actual situation of initial exposure and then treatment after a nuclear accident is well simulated, so the invention is of great practical significance. The screened therapeutic target is developed into a medicine for treatment after nuclear accidents, to save precious time for the treatment after nuclear accidents.

Claims

1. A method for screening a therapeutic target of acute radiation-induced gastrointestinal syndrome, comprising: exposing a CreERT-loxP transgenic mouse model having a candidate therapeutic target gene to ionizing radiation at 15-18 Gy, injecting an estrogen analog after ionizing radiation to induce the candidate therapeutic target gene to express, and screening a therapeutic target promoting the proliferation of quiescent intestinal crypt stem cells.

2. The method according to claim 1, wherein the CreERT-loxP transgenic mouse model comprises a Bmi1-CreERT-loxP transgenic mouse model.

3. The method according to claim 1, comprising specifically: S1: inserting the candidate therapeutic target gene into the downstream of the loxP-STOP-loxP sequence, and inserting the constructed sequence into the mouse genome, to construct loxP mice with a candidate therapeutic target gene; S2: co-breeding the loxP mice having the candidate therapeutic target gene with CreERT mice, screening the CreERT-loxP transgenic mice having the candidate therapeutic target gene for use as the mice in the experimental group, and using the loxP mice having the candidate therapeutic target gene as the mice in the control group; S3: exposing the mice in the experimental group and the mice in the control group to ionizing radiation at 15-18 Gy; and S4: immediately after ionizing radiation, injecting an estrogen analog to induce the overexpression of the target gene, and screening a therapeutic target that promotes the proliferation of quiescent intestinal crypt stem cells by evaluating the therapeutic effect against radiation.

4. The method according to claim 3, wherein in Step S1, the constructed sequence is inserted into the H11 or ROSA26 locus of the mouse genome.

5. The method according to claim 3, wherein the dose rate of the ionizing radiation is 0.5-10 Gy/min, and the range of exposure is whole-abdomen exposure.

6. The method according to claim 3, wherein the estrogen analog is tamoxifen.

7. The method according to claim 6, wherein tamoxifen is injected at a dose of 4-5 mg/20 g body weight of mouse.

8. The method according to claim 3, wherein the therapeutic effect against radiation is evaluated by the proliferation of quiescent intestinal crypt stem cells and the survival rate of mice.

9. The method according to claim 8, wherein the proliferation of quiescent intestinal crypt stem cells is the proliferation of quiescent intestinal crypt stem cells 3-5 days after ionizing radiation.

10. The method according to claim 8, wherein the survival rate of mice is the survival rate of mice in 30 days after ionizing radiation.

11. The method according to claim 1, wherein the therapeutic target promoting the proliferation of quiescent intestinal crypt stem cells comprises TIGAR gene or protein.

12. Use of the TIGAR gene or protein in the preparation of a medicine for treating radiation-induced gastrointestinal syndrome.

13. The use according to claim 12, wherein the medicine for treating radiation-induced gastrointestinal syndrome is a medicine promoting the proliferation of quiescent intestinal crypt stem cells.

14. The use according to claim 12, wherein the medicine for treating radiation-induced gastrointestinal syndrome is TIGAR protein or a medicine for inducing the overexpression of TIGAR protein.

15. The use according to claim 14, wherein the TIGAR protein is used to scavenge destructive ROS and retain the proliferation-related ROS signal in the quiescent intestinal crypt stem cells.

16. The use according to claim 12, wherein the medicine for treating radiation-induced gastrointestinal syndrome is in a dosage form of injections, capsules, tablets, oral preparations or microcapsules.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0042] FIG. 1 shows the principle of the Cre-loxP transgenic animal model;

[0043] FIG. 2 shows a mouse model overexpressing TIGAR specific to quiescent crypt stem cells;

[0044] FIG. 3 shows the whole-abdomen exposure of mice;

[0045] FIG. 4 shows the overexpression of TIGAR in quiescent crypt stem cells induced after ionizing radiation;

[0046] FIG. 5 shows that TIGAR overexpression in quiescent intestinal crypt stem cells promotes the survival of exposed mice;

[0047] FIG. 6 shows that TIGAR overexpression in quiescent intestinal crypt stem cells promotes the reconstruction of intestinal crypts in exposed mice; and

[0048] FIG. 7 shows that TIGAR has a better ability to promote the proliferation of quiescent crypt stem cells than NAC.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0049] The present invention will be further described below with reference to the accompanying drawings and specific examples, so that those skilled in the art can better understand and implement the present invention; however, the present invention is not limited thereto.

[0050] FIG. 1 shows the principle of the Cre-loxP transgenic animal model. In FIG. 1, the estrogen receptor (ER or ERT) that is closely related to the induction of expression is not indicated. When the Cre recombinase binds to the estrogen receptor, it cannot enter the nucleus to complete the cleavage. Only when a drug such as tamoxifen is injected, the Cre recombinase is unbound from the estrogen receptor, thereby completing the gene splicing. Only by using the above characteristic, gene induction and regulation can be achieved after ionizing radiation.

[0051] In the examples of the present invention, the Bmi1-CreERT;loxP transgenic mice are used as an example. The gene encoding the recombinase Cre is inserted in the downstream of a specific promoter (such as Bmi1) of quiescent intestinal crypt stem cells, to specifically regulate the gene in quiescent intestinal crypt stem cells, and simulate the treatment intervention after accidental exposure to the greatest extent in terms of temporal and spatial specificity, so as to effectively screen the genes that promote the proliferation of quiescent intestinal crypt stem cells after radiation damage.

Example 1: Construction of CreERT-loxP Transgenic Mice

[0052] To effectively promote the proliferation of quiescent crypt stem cells, Bmi1-CreERT;loxP transgenic mice were used in this technical solution, and genetic intervention was performed on quiescent crypt stem cells in mice. TIGAR was used as a target gene, and TIGAR was induced to express in quiescent crypt stem cells by tamoxifen, as shown in FIG. 2.

[0053] In mice with the above-mentioned gene phenotype, TIGAR was allowed to be overexpressed only in quiescent crypt stem cells by Bmi1, a promoter specific to quiescent crypt stem cells.

[0054] The mice were designated as Bmi1-creERT;H11-Tigar. Specifically, Bmi1 is a specific promoter of quiescent crypt stem cells. Cre is a gene encoding recombinase, which can be translated into recombinase to cleave a specific gene sequence. ERT encodes the estrogen receptor. When creERT is translated as a whole, the recombinase binds to the estrogen receptor and cannot enter the nucleus to complete DNA splicing. Therefore, the estrogen receptor needs to be disassociated from the recombinase by injecting tamoxifen. After dissociation, the recombinase can enter the nucleus to splice a specific DNA sequence (loxP sequence). As a result of splicing, the gene sequence between two loxP sites is removed from the DNA sequence, and the remaining two truncated ends are spliced to form a new complete DNA sequence. For Bmi1-creERT;H11-Tigar mice, the result of splicing is that the STOP site (polyA) previously located upstream of Tigar is removed from the DNA sequence, and the Tigar gene that previously cannot be transcribed (due to the upstream STOP site) is allowed to be transcribed and translated after splicing, resulting in increased expression of Tigar protein. It takes 18-24 h from the intraperitoneal injection of tamoxifen to the achievement of overexpression of TIGAR protein.

[0055] The EGFP gene downstream of Tigar can be translated into green fluorescent protein to serve as a tracer. The 2A between Tigar and EGFP is a linker to ensure that TIGAR and EGFP will not fuse with each other after translation, causing the destruction of the spatial structure and loss of functions of the protein. Due to the presence of Bmi1, a specific promoter of quiescent intestinal crypt stem cells, the entire cleavage process mentioned above only occurs in quiescent intestinal crypt stem cells. In summary, by means of the above-mentioned animal model, the overexpression of TIGAR protein in quiescent crypt stem cells can be achieved 18-24 h after exposure. (tamoxifen is injected immediately after exposure)

[0056] Once the Tigar gene sequence is replaced by other genes with potential therapeutic value, the therapeutic targets of acute radiation-induced gastrointestinal syndrome can be screened. Of course, the above therapeutic target is proposed with respect to quiescent intestinal crypt stem cells.

Example 2: Induction of Expression of Target Gene after Ionizing Radiation

[0057] Since it takes a certain period of time from drug injection to overexpression of TIGAR in quiescent crypt stem cells (usually 18-24 h for CreERT-loxP animal model), the drug was injected intraperitoneally (tamoxifen, single injection, 4.5 mg/20 g body weight of mouse) immediately after whole-abdomen exposure by X-rays at 15 Gy was received by the mice (FIG. 3).

[0058] On days 1, 3, and 5 after the mice were exposed, the mice were sacrificed and the intestinal tissues were made into frozen sections to observe the expression of TIGAR protein in quiescent crypt stem cells, as shown in FIG. 4. Since TIGAR and enhanced green fluorescent protein (EGFP) are expressed simultaneously during the design and construction of transgenic mice, the expression level of enhanced green fluorescent protein can be used to indicate the expression level of TIGAR. On day 1 after exposure, only 1-2 green cells are observed in the crypts, that is, quiescent crypt stem cells have been successfully overexpressed. Because quiescent crypt stem cells have the ability to divide and proliferate, a large number of progeny cells can be formed 3 and 5 days after exposure. The progeny cells of quiescent crypt stem cells also have green fluorescence, so the green fluorescence not only reflects the overexpression of TIGAR protein, but also reflect the progeny cells of quiescent crypt stem cells. The DAPI fluorescence in the figure is used to indicate the nucleus, facilitating better location and counting of the cells. It can be seen that 3-5 days after exposure, the reconstructed crypts consist essentially of the progeny cells of quiescent crypt stem cells, indicating that TIGAR overexpression promotes the proliferation of quiescent crypt stem cells and accelerates the reconstruction of crypts after exposure.

Example 3: Evaluation of Therapeutic Effect Against Radiation

[0059] The therapeutic effect of TIGAR overexpression against radiation was evaluated by the survival rate of mice and HE staining of intestinal tissue sections. In the survival rate test, mice in the control group (where the Tigar gene was inserted downstream of the loxP-STOP-loxP sequence, to obtain the loxP-STOP-loxP-Tigar sequence, which was inserted into the H11 locus of the mouse genome to obtain H11-Tigar small mice) and mice with TIGAR overexpressed in quiescent intestinal crypt stem cells received whole-abdomen exposure by X-rays at 15 Gy (FIG. 3), and tamoxifen was injected intraperitoneally immediately after exposure (single injection, 4.5 mg/20 g body weight of mouse). After the injection, the mice were continuously bred to observe the survival of mice, as shown in FIG. 5.

[0060] It can be seen that mice in the control group (H11-Tigar mice, WT) all die of radiation-induced gastrointestinal syndrome (survival rate 0%) 7 days after exposure, and mice with TIGAR overexpressed in quiescent intestinal crypt stem cells (Bmi1-creERT;H11-Tigar) still have a survival rate of close to 40% 30 days after the exposure. Accordingly, the therapeutic effect is obvious (p<0.01).

[0061] In the HE staining of intestinal tissue sections (as shown in FIG. 6), the mice were sacrificed 1, 3, and 5 days after exposure, and the intestinal tissues were collected and prepared into tissue sections for HE staining. It can be seen that with the proliferation of quiescent intestinal crypt stem cells after exposure, the number of intestinal crypts and the size of crypts in the mice with TIGAR overexpressed in quiescent intestinal crypt stem cells are significantly larger than those in the control group 3 and 5 days after exposure, suggesting that the TIGAR overexpression in quiescent intestinal crypt stem cells definitely promotes the proliferation and reconstruction of intestinal crypts after exposure. As the source of intestinal villi renewal, the timely reconstruction of intestinal crypts is of decisive significance for the treatment of radiation-induced gastrointestinal syndrome.

[0062] It is particularly to be noted here that although tamoxifen is injected intraperitoneally immediately after ionizing radiation in the experiment, TIGAR can only be considered to start to exert an effect 24 h after induction considering the time (18-24 h) needed for tamoxifen to induce TIGAR expression in quiescent intestinal crypt stem cells. Therefore, the idea mentioned in the object of the invention is confirmed that TIGAR can still promote the proliferation of intestinal crypt stem cells and promote the survival of mice 24 hrs after radiation exposure.

Example 4: Evaluation of Effect on Promoting Proliferation of Quiescent Intestinal Crypt Stem Cells

[0063] To better reflect the effect of TIGAR on promoting the proliferation of quiescent crypt stem cells, the intestinal crypt organoid model cultured in vitro was used in the experiment. Intestinal crypt organoids were extracted from the small intestine of living mice. They had similar cell composition and proliferation kinetics to intestinal crypts in mice, and also had quiescent intestinal crypt stem cells.

[0064] The green fluorescence in Bmi1-creERT;Rosa26-mTmG mice was used to indicate the proliferation of quiescent intestinal crypt stem cells. The TIGAR was compared with the traditional reducing agent N-acetylcysteine (NAC) in promoting the proliferation of quiescent intestinal crypt stem cells, as shown in FIG. 7. It can be seen that TIGAR overexpression (transfected in adenovirus) can significantly increase the proliferation ability of quiescent crypt stem cells in the radiated crypt organoids, and the proliferation ability of quiescent crypt stem cells in the NAC treatment group is only promoted to a certain extent, which is, however, much lower than in the TIGAR treatment group (the number of green fluorescence-positive crypts and the count of green fluorescence-positive cells are significantly less than those in the TIGAR overexpressing group).

[0065] The traditional reducing agent NAC can remove both the destructive ROS and proliferation-related ROS signal in the cell, and TIGAR is reported to have the ability to scavenge only the destructive ROS, but retain the proliferation-related ROS. Therefore, the above experimental results show that TIGAR promotes the proliferation of quiescent intestinal crypt stem cells after exposure by specifically scavenging the destructive ROS and retaining the proliferation-related ROS in quiescent intestinal crypt stem cells.

[0066] The above-described embodiments are merely preferred embodiments for the purpose of fully illustrating the present invention, and the scope of the present invention is not limited thereto. Equivalent substitutions or modifications can be made by those skilled in the art based on the present invention, which are within the scope of the present invention as defined by the claims.