Application of FGF5 gene inhibitor in preparation of anti-tumor angiogenesis drugs

Abstract

The invention provides application of an FGF5 gene inhibitor in preparation of anti-tumor angiogenesis drugs, and belongs to the technical field of biological medicines. The invention provides application of a reagent for inhibiting expression of an FGF5 coding gene or a reagent for inhibiting expression or functions of FGF5 in preparation of anti-tumor angiogenesis drugs. In the invention, tumor angiogenesis can be inhibited by inhibiting expression of FGF5. Results of the embodiment of the invention show that overexpression of the FGF5 protein and the coding gene thereof can effectively promote migration and invasion of human umbilical veins and tubule formation, and knock-down of expression of the FGF5 inhibits migration and invasion ability of the human umbilical veins and tubule formation ability. A subcutaneous tumor generation experiment further verifies that the FGF5 can promote the growth of a subcutaneous tumor HGC27 of a nude mouse, and promote tumor angiogenesis and tumor endothelial cell proliferation and migration.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of an FGF5 gene inhibitor in preparation of an anti-tumor angiogenesis drug. BACKGROUND

[0002] Fibroblast Growth Factor 5 (FGF5) is a member of the Fibroblast Growth Factor (FGF) family, which is a kind of key protein for regulating cell proliferation, differentiation and tissue repair. FGF5 was first cloned from mice in 1989 and became a research hotspot due to its important role in the hair follicle cycle. Studies have shown that FGF5 is mainly expressed in the outer root sheath and dermal papilla cells of the hair follicle, and activates the MAPK / ERK signaling pathway by binding to the FGFR receptor, thereby promoting the hair follicle to enter the catagen phase from the anagen phase, thereby inhibiting hair growth.

[0003] FGF5 gene deletion or functional inhibition can cause excessive hair growth, such as the long hair phenotype of Angora rabbits and some dog breeds due to natural FGF5 mutations. Human genetics research has also found that FGF5 gene polymorphism is associated with androgenetic alopecia (AGA) and hypertension, making it a potential target for hair loss treatment. Currently, there is little research on FGF5 in tumor angiogenesis. SUMMARY

[0004] In view of this, the application provides a reagent for inhibiting the expression of an FGF5 coding gene or a reagent for inhibiting the expression or function of FGF5 in the preparation of an anti-tumor angiogenesis drug, which can effectively inhibit tumor angiogenesis by inhibiting the expression of fibroblast growth factor 5.

[0005] To achieve the above-mentioned purpose, the application provides the following technical solutions:

[0006] The application provides a reagent for inhibiting the expression of an FGF5 coding gene or a reagent for inhibiting the expression or function of FGF5 in the preparation of an anti-tumor angiogenesis drug.

[0007] Preferably, the anti-tumor angiogenesis includes at least one of inhibiting the proliferation of human umbilical vein endothelial cells, inhibiting the migration of human umbilical vein endothelial cells, and reducing the tube formation ability of human umbilical vein endothelial cells.

[0008] Preferably, the reagent for inhibiting the expression of an FGF5 coding gene or the reagent for inhibiting the expression or function of FGF5 includes a reagent for knocking out or silencing the FGF5 coding gene.

[0009] Preferably, the reagent for knocking out or silencing the fibroblast growth factor 5 encoding gene includes siRNA targeting the fibroblast growth factor 5 encoding gene.

[0010] The nucleotide sequence of the siRNA is shown in SEQ ID NO:1.

[0011] Preferably, the reagent for knocking out or silencing the fibroblast growth factor 5 encoding gene further includes the gene derivative of the siRNA;

[0012] The gene-derived products of the siRNA include at least one of the following products containing the siRNA: recombinant vectors, recombinant viral strains, and drug delivery systems.

[0013] This invention provides the application of fibroblast growth factor 5, the fibroblast growth factor 5 encoding gene, or reagents that promote fibroblast growth factor 5 expression in the construction of tumor animal models.

[0014] Preferably, the tumor includes gastric cancer.

[0015] This invention provides a method for constructing an animal model of gastric cancer, which involves inoculating gastric cancer cells into an animal and then injecting the animal with fibroblast growth factor 5.

[0016] Preferably, the gastric cancer cells include HGC27.

[0017] Preferably, the injection dose of fibroblast growth factor 5 injection is 80–120 μl;

[0018] The concentration of fibroblast growth factor 5 in the fibroblast growth factor 5 injection is 45–55 ng / ml.

[0019] Compared with the prior art, the present invention has the following advantages:

[0020] This invention provides the application of a reagent for inhibiting the expression of the FGF5 encoding gene or a reagent for inhibiting FGF5 expression or function in the preparation of anti-tumor angiogenesis drugs. Inhibiting the expression of FGF5 or its encoding gene can suppress the migration and invasion of umbilical vein endothelial cells, effectively reducing tumor angiogenesis and thus inhibiting tumor formation and growth. In this invention, the effects of FGF5 protein on the migration and invasion capabilities and tubule formation capabilities of human umbilical vein endothelial cells were analyzed by knocking down or overexpressing FGF5 protein and its encoding gene. The results showed that overexpression of FGF5 protein and its encoding gene effectively promoted human umbilical vein migration, invasion, and tubule formation, while knocking down FGF5 expression inhibited these capabilities. This invention further verified through subcutaneous tumorigenesis experiments that FGF5 can promote the growth of HGC27 subcutaneous tumors in nude mice and promote tumor angiogenesis. Attached Figure Description

[0021] Figure 1 The graph shows the correlation between FGF5 and gastric cancer. In the graph, A shows the relationship between FGF5 expression and the survival of gastric cancer patients, and B shows the results of FGF5 expression level detection in the blood of gastric cancer patients.

[0022] Figure 2 The diagram shows the protein structure of FGF5 and the results of the human umbilical vein endothelial cell proliferation assay. A is the protein structure of FGF5, and B is the results of the human umbilical vein endothelial cell proliferation assay.

[0023] Figure 3 The results of inhibiting FGF5 to suppress vascular endothelial cell migration and tube formation are shown in Figure 1. In Figure 2, HUVEC cells stimulated by tumor supernatant transfection with siFGF5 and siNC, respectively, are shown. The knockdown efficiency was detected by qPCR and Western blot, respectively. In Figure 3, HUVEC cells stimulated by tumor supernatant transfection with siFGF5 and siNC, respectively, are shown. The migration was detected by Transwell assay and the tube formation was detected by tube formation assay.

[0024] Figure 4 The diagram shows the results of HUVEC cells promoted by FGF5 overexpression in terms of migration and tube formation. In the diagram, A and B are HUVEC cells stimulated by tumor supernatant after transfection with AdFGF5 and AdNC, respectively, and the knockdown efficiency was detected by qPCR and Western blot, respectively. In the diagram, C is HUVEC cells stimulated by tumor supernatant after transfection with AdFGF5 and AdNC, respectively, and the migration was detected by Transwell assay and the tube formation assay was detected by tube formation assay.

[0025] Figure 5The image shows the experimental results of subcutaneous tumor development; where A and B represent tumor volume and weight, and C and D represent immunofluorescence staining results. Detailed Implementation

[0026] This invention provides the application of a reagent for inhibiting the expression of the gene encoding fibroblast growth factor 5 or a reagent for inhibiting the expression or function of fibroblast growth factor 5 in the preparation of anti-tumor angiogenesis drugs.

[0027] In this invention, the reagent for inhibiting the expression of the fibroblast growth factor 5 (GF5) encoding gene, or the reagent for inhibiting GF5 expression or function, preferably includes a reagent for knocking out or silencing GF5. The reagent for knocking out or silencing the GF5 encoding gene includes siRNA targeting the GF5 encoding gene. The nucleotide sequence of the siRNA is preferably as shown in SEQ ID NO:1 (UAUACUUGUACAUCCUUGUAGCGAATT). In this embodiment, the siRNA is commercially available under the name SiFGF5, catalog number 2213105, and is purchased from Shanghai Bioengineering Co., Ltd. The reagent for knocking out or silencing the GF5 encoding gene further includes a gene-derived product of the siRNA; the gene-derived product of the siRNA includes at least one of the following products containing the siRNA: recombinant vector, recombinant viral strain, and drug delivery system.

[0028] In this invention, the anti-tumor angiogenesis includes at least one of the following: inhibiting the proliferation of human umbilical vein endothelial cells, inhibiting the migration of human umbilical vein endothelial cells, and reducing the tube-forming ability of human umbilical vein endothelial cells.

[0029] In this invention, FGF5 is a cell-secreting protein with the amino acid sequence shown in SEQ ID NO:2 (MSKKGKLHASAKFTDDCKFRERFQENSYNTYASAIHRTEKTGREWYVALN KRGKAKRGCSPRVKPQHISTHFLPRFKQSEQPELSFTVTVPEKKKPPSPIKPKIP LSAPRKNTNSVKYRLKFRFG), and its NCBI accession number is NP_001278741.1, with the full name fibroblast growth factor 5isoform 3 [Homo sapiens]. Inhibiting the expression of FGF5 or its encoding gene can suppress the migration and invasion of umbilical vein endothelial cells, effectively reducing tumor angiogenesis and thus inhibiting tumor formation and growth. This invention analyzed the effects of FGF5 protein on the migration and invasion capabilities and tubule formation capabilities of human umbilical vein endothelial cells by knocking down or overexpressing FGF5 protein and its encoding gene. The results showed that overexpression of FGF5 protein and its encoding gene effectively promoted human umbilical vein migration and invasion and tubule formation, while knockdown of FGF5 expression inhibited these capabilities. Furthermore, this invention verified through subcutaneous tumorigenesis experiments that FGF5 can promote the growth of HGC27 subcutaneous tumors in nude mice, and promote tumor angiogenesis, tumor endothelial cell proliferation, and migration.

[0030] This invention provides the application of fibroblast growth factor 5, the fibroblast growth factor 5 encoding gene, or reagents that promote fibroblast growth factor 5 expression in the construction of tumor animal models.

[0031] In this invention, the tumor includes gastric cancer.

[0032] This invention provides a method for constructing a subcutaneous gastric cancer tumor model in nude mice. After inoculating gastric cancer cells subcutaneously into nude mice, the nude mice are injected with fibroblast growth factor 5.

[0033] In this invention, the gastric cancer cells preferably include HGC27 gastric cancer cells. The mice preferably include nude mice. The fibroblast growth factor 5 injection is prepared by mixing physiological saline with a fibroblast growth factor 5 solution with a concentration of 256 ng / ml at a volume ratio of 4:1. The preferred method of injection of the fibroblast growth factor 5 injection is intraperitoneal injection, once daily for at least 15 days, more preferably 20-40 days, and most preferably 28 days. The preferred concentration of fibroblast growth factor 5 in the fibroblast growth factor 5 injection is 45-55 ng / ml, more preferably 51.2 ng / ml. The preferred injection dose is 80-120 μl, more preferably 100 μl. This invention does not impose any special limitation on the source of the fibroblast growth factor 5, which can be purchased through conventional commercial channels. In this embodiment of the invention, the fibroblast growth factor 5 was purchased from Abcam (Abcam, AB50229).

[0034] One embodiment of the present invention examined the effect of continuous injection of fibroblast growth factor 5 (FGF5) into mice after inoculation with gastric cancer cells on the tumor. The results showed that, compared with the control group, FGF5 injection could promote the growth of HGC27 subcutaneous tumors in nude mice, and promote tumor angiogenesis, tumor endothelial cell proliferation and migration.

[0035] To further illustrate the present invention, the solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0036] Take 1 μl of FGF5 stock tube (10 μg) and add it to 1 ml of serum-free culture medium. Take 100 μl of the stock tube and add it to 900 μl of culture medium to make 1 ml, thus obtaining an FGF5 solution with a concentration of 256 ng / ml.

[0037] Example 1

[0038] 1. High expression of fibroblast growth factor (FGF5) in gastric cancer tissues is associated with poor prognosis, and its expression is also high in the blood of gastric cancer patients. The results of GEPIA online analysis of the relationship between FGF5 expression and survival in gastric cancer patients are shown below. Figure 1 In study A, blood samples were collected from gastric cancer patients, and the expression of FGF5 in the blood was detected and analyzed using an FGF5 ELISA kit. The results were statistically analyzed and are shown in [the table below]. Figure 1 B.

[0039] 2. FGF5-induced proliferation of human umbilical vein endothelial cells (HUVECs)

[0040] The structure of the FGF5 protein was retrieved from the UCSC database. FGF5 protein structure detection... Figure 2 A.

[0041] HUVEC cell lines in logarithmic growth phase were subjected to serum-free starvation for 8 h, followed by stimulation with 51.2 ng / mL FGF5 solution for 12 h or control PBS. The stimulated HUVEC cells were then digested, centrifuged, and counted. Cells were resuspended in serum-containing medium to obtain a single-cell suspension. 100 μL of cell suspension (3000 cells per well) was added to each well of a 96-well plate. PBS solution was added around the perimeter of the 96-well plate to prevent errors caused by drying and evaporation. The plates were incubated at 37°C, 5% CO2, and 90% humidity. After cell attachment, 10 μL of cck8 was added to 90 μL of serum-free and antibiotic-free medium, taking care not to introduce air bubbles into the wells as this would interfere with OD readings. The absorbance at 490 nm was measured using a microplate reader after 45 min. Cell viability was assessed at 0 h, 12 h, 24 h, 48 h, 72 h, 96 h, and 110 h, and cell growth curves were recorded.

[0042] The results show that ( Figure 2 (B) Fibroblast growth factor FGF5 promotes HUVEC cell proliferation.

[0043] Example 2

[0044] Experiments to reduce the ability of siFGF5 to knock down cell tube formation and migration

[0045] SiFGF5 and SiNC, catalog number 2213105, were purchased from Shanghai Bioengineering Co., Ltd.

[0046] (1) Methods for establishing FGF5 knockdown cell lines

[0047] Day 1: Plate 6-well plates and culture HUVEC cells in high-glucose DMEM medium with 10% FBS until the cell density reaches about 30% to 50%, with good growth and no mycoplasma contamination;

[0048] Day 2: Before transfection, the serum-free medium was replaced. HUVEC cells were divided into two groups: one group was transfected with SiNC, and the other group was transfected with SiFGF5. Transfection was performed using the RFectPrime nucleic acid transfection kit (purchased from Baidai Biotechnology Co., Ltd.). The prepared transfection complex was added dropwise to the culture dish, gently shaken, and the culture dish was immediately placed in a 37°C incubator. After 8 hours, the medium was replaced with serum-containing medium and cultured for 48–72 hours. RNA and protein were extracted from the cells using a cell scraper for q-PCR and protein imprinting detection.

[0049] q-PCR detection method for knocking down FGF5 cell lines: RNA was extracted using the Shanghai Feijie Total RNA Rapid Extraction Kit, followed by reverse transcription to obtain cDNA, which was then detected by q-PCR. Reverse transcription reagent: Aikerui Biotechnology, catalog number: AG11728; qPCR reagent: Vazyme 2xChamQUnivesalSYBR; q-PCR primers: forward primer: 5′-GCTGCAGAACCAGAAGACCA-3′ (SEQ ID NO:3), reverse primer: 5′-TCCAGGTCAGGTTCAGGTTC-3′ (SEQ ID NO:4).

[0050] Western blotting protein imprinting method for FGF5 knockdown cell lines: After treatment, cells were quickly scraped off with a cell scraper. An appropriate amount of RIRA lysis buffer was added, and protease inhibitors and phosphatase inhibitors were added at a 1:100 ratio. Cell lysis was performed using a cell sonicator (the cells were kept on ice throughout the protein extraction process). Total cell protein was prepared, and protein quantification was performed using the BCA method. One-fifth of the protein supernatant was added to the supernatant, and the sample was boiled to denature it. Protein samples were loaded according to the concentration determined by BCA and subjected to electrophoresis. The samples were transferred to a PVDF membrane using the wet transfer method (aligning each addition to ensure no air bubbles). Blocking was performed for 2 hours (using 5% skim milk powder), followed by washing and incubation with primary antibody FGF5ACTIN (overnight on a shaker at 4°C). After washing, secondary antibody incubation was performed for 45 minutes, followed by washing and exposure using HRP staining. β-actin was used as an internal control. The antibodies used in the Western blotting experiment were FGF5 (Invitrogen, PA580630) and Actin (Affinity, AF7018).

[0051] The results of q-PCR and Western Blot assays are shown below. Figure 3 The results showed that the levels of FGF5 protein and mRNA in the siFGF5 group were significantly downregulated compared to the control cells in the siNC group, indicating that the siFGF5 group FGF5 knockdown cell line was successfully constructed. Figure 3 (A and B in the middle).

[0052] (2) Transwell assay to detect cell migration and invasion ability

[0053] Select stable siNC control cells and siFGF5 knockdown cells. Wash the cells in PBS, centrifuge, discard the supernatant, and resuspend the pellet in serum-free medium to obtain 8 × 10⁸ cells. 5Single-cell suspensions of 100 μL were added to the upper chamber of the invasion chamber, and 600 μL of complete culture medium was added to the lower chamber. The cells were incubated at 37°C with 5% CO2 for 24 h. After incubation, the chambers were removed and washed three times with PBS. 200 μL of 4% paraformaldehyde fixative was added to the upper chamber, and 600 μL of 4% paraformaldehyde fixative was added to the lower chamber. Cells were fixed for 30 min, then washed three times with PBS. 100 μL of 0.1% crystal violet was added to the upper chamber, and 600 μL of 0.1% crystal violet was added to the lower chamber for staining. Cells were stained for 20 min. After staining, the cells were washed with PBS until the color lightened. Cells inside the chambers were wiped with a cotton swab moistened with tap water and photographed using a 4× and 10× microscope. Figure 3 (C)

[0054] (3) Tube formation experiment

[0055] Select siNC control cells and siFGF5 knockdown cell lines that are stable and treat them with serum-free starvation for 10 h. One day in advance, move the matrix gel from -20 degrees to 4 degrees and pre-cool the 96-well plate and pipette tip in the -20 degree freezer. Add 50 μL of matrix gel to the 96-well plate, taking care not to generate air bubbles, and transfer it to the incubator for 30 min to allow the matrix gel to solidify.

[0056] HUVEC cells that reached 60% confluence after starvation were digested, centrifuged, and counted. The cells were then resuspended in serum-free medium to prepare a concentration of 4 × 10⁻⁶ cells / mL. 5 Single-cell suspension of 40,000 cells / mL. Add 100 μL of cell suspension (40,000 cells per well) to each well of a 96-well plate. Add PBS solution around the perimeter of the 96-well plate to effectively prevent errors caused by drying and evaporation.

[0057] After incubating the well plates at 37°C, 5% CO2, and 90% humidity for 4–6 hours, the culture medium was discarded, the plates were washed once with PBS, and 50 μL of calcein (1:800 PBS) was applied to each well. The plates were then placed in a cell culture incubator for 15 minutes. The formation of the tubes was observed under a microscope, and the results were photographed and recorded.

[0058] The results of the Transwell migration experiment and the Matrigel tube formation experiment are shown in […]. Figure 3 The results showed that, compared to siNC control cells, siFGF5 knockdown reduced the ability of cells to form tubes and migrate, indicating that fibroblast growth factor FGF5 knockdown inhibits the ability of cells to form tubes and migrate. Figure 3 (C)

[0059] Example 3

[0060] Experiment on the tube formation and migration ability of AdFGF5-overexpressing cells

[0061] Please provide the lentiviruses AdNC and AdFGF5 that were purchased from Jiangsu Kaiji Biotechnology Co., Ltd., under the trade name LV-FGF5-OE.

[0062] (1) Establishment of ADFGF5 overexpression cell lines: HUVEC cells were divided into two groups, one group transfected with AdNC lentivirus and the other group transfected with AdFGF5 lentivirus. The viral stock solution (1x10^8 TU / ml) was diluted 100 times and 2 mL of serum-free medium containing polybrene was added. The original culture medium was aspirated and the viral mixture was added. After culturing at 37℃ for 6-8 hours, complete culture medium (containing serum) was added. The cells were cultured for another 48-72 hours to observe reporter gene (GFP) expression. GFP fluorescence was observed after 72 hours. Cell function experiments were performed after successful transfection.

[0063] The expression level of the FGF5-encoding gene in the FGF5-overexpressing cell line was detected by q-PCR. The RNA extraction and reverse transcription methods were the same as in Example 2, and the primers used for detection were the same as in Example 2.

[0064] FGF5 was detected in FGF5-overexpressing cell lines using Western blotting, following the same procedure as in Example 2.

[0065] The results of q-PCR and Western Blot assays are shown below. Figure 4 The results showed that the levels of FGF5 protein and mRNA in the AdFGF5 group were significantly upregulated compared to those in the AdNC group, indicating that the AdFGF5 overexpression cell line was successfully constructed. Lentivirally transfected HUVEC cells were designated as AdNC control cells, and lentivirus-transfected HUVEC cells were designated as AdFGF5 overexpressing cells. Figure 4 (A and B in the middle).

[0066] (2) Transwell assay to detect cell migration and invasion capabilities:

[0067] Transwell assays were performed on stable AdNC control cells and AdFGF5 overexpressing cells, using the same methods as in Example 2. Figure 4 (C)

[0068] (3) Tube formation experiment

[0069] Tube formation experiments were performed on stable AdNC control cells and AdFGF5 overexpressing cells, using the same experimental methods as in Example 2.

[0070] The results of the Transwell migration experiment and the Matrigel tube formation experiment are shown in […]. Figure 4The results showed that, compared with AdNC control cells, AdFGF5 overexpressing cells exhibited enhanced tube formation and migration abilities, indicating that fibroblast growth factor FGF5 overexpression promotes cell migration and tube formation. Figure 4 (C)

[0071] Example 4

[0072] Subcutaneous tumor formation experiment

[0073] 1×10 7 Human gastric cancer cells (HGC27) were inoculated subcutaneously into 3-4 week old nude mice. The mice were then divided into a control group and an FGF5 group, with 6 mice in each group. The FGF5 group received an intraperitoneal injection of FGF5 (51.2 ng / ml, 100 μl) once daily, while the control group received an equal volume of PBS. Tumors were isolated and weighed after 3 weeks, and statistical analysis was performed. Tumors were paraffin-embedded, and sections were stained with immunofluorescence. The staining area of ​​CD31 antibody was detected, and the fluorescence area was measured and statistically analyzed using ImageJ software. The fluorescence area reflects angiogenesis.

[0074] Results of subcutaneous tumor development experiments are shown in Figure 5 The results showed that, compared with the control group, the FGF5 group had significantly increased tumor volume and weight. Figure 5 In A and B), the staining area of ​​CD31 antibody was significantly increased. Figure 5 (Neutralization D). This indicates that FGF5 has a positive regulatory effect on the growth of HGC27 subcutaneous tumors in nude mice and promotes tumor angiogenesis.

[0075] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. Other embodiments can be obtained based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The application of reagents that inhibit the expression of the gene encoding fibroblast growth factor 5 or reagents that inhibit the expression or function of fibroblast growth factor 5 in the preparation of antitumor angiogenesis drugs.

2. The application according to claim 1, characterized in that, The anti-tumor angiogenesis includes at least one of the following: inhibiting the proliferation of human umbilical vein endothelial cells, inhibiting the migration of human umbilical vein endothelial cells, and reducing the tube-forming ability of human umbilical vein endothelial cells.

3. The application according to claim 1, characterized in that, The reagents that inhibit the expression of the fibroblast growth factor 5 encoding gene or the reagents that inhibit the expression or function of fibroblast growth factor 5 include reagents that knock out or silence the fibroblast growth factor 5 encoding gene.

4. The application according to claim 3, characterized in that, The reagents used to knock out or silence the fibroblast growth factor 5 encoding gene include siRNA that targets the fibroblast growth factor 5 encoding gene. The nucleotide sequence of the siRNA is shown in SEQ ID NO:

1.

5. The application according to claim 4, characterized in that, The reagent for knocking out or silencing the fibroblast growth factor 5 encoding gene also includes the gene derivative of the siRNA. The gene-derived products of the siRNA include at least one of the following products containing the siRNA: recombinant vectors, recombinant viral strains, and drug delivery systems.

6. Application of fibroblast growth factor 5, fibroblast growth factor 5 encoding genes or reagents that promote fibroblast growth factor 5 expression in the construction of tumor animal models.

7. The application according to claim 6, characterized in that, The tumors include stomach cancer.

8. A method for constructing an animal model of gastric cancer, characterized in that, After inoculating animals with gastric cancer cells, the animals were then injected with fibroblast growth factor 5.

9. The method according to claim 7, characterized in that, The gastric cancer cells include HGC27.

10. The method according to claim 8 or 9, characterized in that, The injection dose of fibroblast growth factor 5 is 80–120 μl; The concentration of fibroblast growth factor 5 in the fibroblast growth factor 5 injection is 45–55 ng / ml.

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