CN103622975B - Glipizide application in preparation tumor - Google Patents

Abstract

The invention discloses glipizide application in preparation tumor. Present invention demonstrates that glipizide has the effect suppressing tumor-blood-vessel growth and tumor proliferation, thus suppressing growth and the transfer of tumor, reach the effect for the treatment of tumor, and glipizide is being clinically used for treatment type-II diabetes, safe and reliable, it is the medicine of a kind for the treatment of tumor having DEVELOPMENT PROSPECT.

Description

Application of Glipizide in the Preparation of Drugs for Treating Tumors

technical field

The invention relates to a new application of glipizide, in particular to the application of glipizide in the preparation of drugs for treating tumors.

Background technique

Glipizide, the chemical name is 1-cyclohexyl-3-{4-[2-(5-methylpyrazine-2-amide)-ethyl]benzenesulfonyl}urea, and its structural formula is: . Glipizide is a disclosed compound, mainly used for the treatment of non-insulin-dependent (Ⅱ) diabetes mellitus, and it is a second-generation sulfonylurea oral hypoglycemic agent.

Sulfonylureas have been clinically used to treat type Ⅱ diabetes since the 1950s. They bind to the sulfonylurea receptors on the surface of pancreatic β cells, trigger the closure of potassium ion channels to prevent the outflow of potassium ions from the cytoplasm, and open voltage-sensitive calcium ion channels at the same time, increasing the influx of extracellular calcium ions and promoting insulin It is secreted and released to the outside of the cell, which increases the sensitivity of peripheral tissues to insulin and reduces the degradation of glycogen in the liver. Sulfonylureas and glinide derivatives tolbutamide, chlorpropamide, etc., all have the effect of stimulating islet β cells to secrete insulin. However, the hyperglycemia and abnormal glucose metabolism associated with diabetes are also indirectly related to the formation of cancer. For example, pyruvate and lactate produced by sugar catabolism can induce the expression of hypoxia-inducible factor 1 (HIF-1), Upregulate vascular endothelial growth factor (VEGF) to induce angiogenesis; glycerol triphosphate dehydrogenase (GAPDH) in glucose metabolism is also related to tumor formation; hyperglycemia can activate insulin-like growth factor and NF-kB transcription factor family; Aerobic glycolysis produces a variety of growth factors, oncogenes, and signaling molecules, inhibits the action of tumor suppressors, and more. However, so far there is no clear information to confirm whether elevated blood sugar can induce cancer.

At present, there is no report about the anti-angiogenic effect of glipizide, let alone the application of this compound in antitumor drugs.

Malignant tumors are the main diseases that threaten human health. According to the statistics of the World Health Organization (WHO), 3/5 of the people in the world die from the four major diseases of cancer, diabetes, cardiovascular disease, and chronic respiratory disease, and cancer is the most important. One of the leading causes of death. Tumor growth, invasion and metastasis are closely related to tumor angiogenesis. Research on inhibiting tumor angiogenesis, cutting off the nutrient supply of tumor cells and metastasis pathway has gradually become the focus of tumor treatment in recent years. Then, targeting tumor angiogenesis and developing drugs that effectively inhibit tumor angiogenesis is one of the effective means for treating tumors.

Contents of the invention

The purpose of the present invention is to provide the application of glipizide in the preparation of drugs for treating tumors.

The technical scheme that the present invention takes is:

Application of glipizide in the preparation of drugs for treating tumors.

The beneficial effects of the present invention are:

The present invention proves that glipizide has the effect of inhibiting tumor angiogenesis and tumor proliferation, thereby inhibiting tumor growth and metastasis, and achieving the effect of treating tumors, and glipizide has been clinically used to treat type 2 diabetes, which is safe and reliable. It is a promising drug for treating tumors.

Description of drawings

Figure 1 is a diagram of glipizide (Glipizide) inhibiting CAM and YSM angiogenesis, A represents the angiogenesis of CAM under the treatment of DMSO, 50 μg/mL, 100 μg/mL, and 200 μg/mL glipizide respectively, B is Quantitative statistics of the number of CAM blood vessels under various treatments in A, C indicates the angiogenesis of YSM under the treatment of DMSO and different concentrations of glipizide, D is the angiogenesis of YSM under the treatment of different concentrations of glipizide Vessel density is the percentage under DMSO treatment, and E is the statistics of the blood vessel number of YSM after 24 hours of treatment with DMSO and different concentrations of glipizide;

Figure 2 is a graph showing that glipizide (Glipizide) inhibits the growth and metastasis of spontaneous breast cancer in MMTV-PyMT transgenic mice. A, B, and C are the volume, weight and weight of breast cancer under the treatment of glipizide and control DMSO, respectively. The number of lung metastatic nodules, the blood vessel density map of breast cancer tissue in D mice treated with glipizide and control DMSO respectively, E is the quantitative statistics of the blood vessel density of breast cancer tissue in D, F is the MMTV-PyMT mouse respectively Tumor cells under the treatment of glipizide and control DMSO, G is the quantitative statistics of the tumor cells in F;

Figure 3 is a graph showing that Glipizide inhibits the growth and metastasis of breast cancer cell 4T1 subcutaneous xenograft tumors. A, B, and C are the volume, weight, and lung metastasis of 4T1 tumors under the treatment of glipizide and control DMSO, respectively The number of nodules, D is the blood vessel density map of mouse 4T1 tumor tissue treated with glipizide and control DMSO respectively, E is the quantitative statistics of the blood vessel density of 4T1 tumor tissue in D, F is the 4T1 tumor tissue treated with glipizide Tumor cells under oxazine and control DMSO treatment, G is the quantitative statistics of tumor cells in F.

detailed description

The following will illustrate the inhibitory effect of glipizide on tumor angiogenesis, tumor growth and metastasis in conjunction with specific experiments. Using the most commonly used in vivo assay models for the study of angiogenesis—chicken chorioallantoic membrane (CAM) and yolk sac membrane (YSM) models, firstly, using these two models to preliminarily detect the effect of glipizide on angiogenesis , and then through subsequent experiments to verify that glipizide has an inhibitory effect on tumor angiogenesis and cell proliferation, thereby inhibiting tumor growth and metastasis. Glipizide described in the experiment was purchased from Sigma Company.

1. Glipizide inhibits angiogenesis in chick chorioallantoic membrane (CAM)

1) Treatment of chicken embryos: Clean the surface of chicken embryos (purchased from the breeder factory of South China Agricultural University) that have been developing normally for 8 days, and then wipe them with 1:1000 chlorhydrinate. Egg trays were placed in an ordinary incubator at 45° for incubation at a temperature of 37.8±0.5°C and a humidity of 60%. The embryos were rotated under light and observed 3 times a day.

2) Opening the window: On the ninth day of incubation, find and mark the air cell of the chick embryo under the canister as the position for opening the window. After disinfecting with 75% alcohol, use a slow-speed dental drill to drill a small hole at the mark of the air cell. Then use ophthalmic curved tweezers to carefully peel off a small part of the eggshell to form a gap with a diameter of about 1 cm. Use ophthalmic tweezers to gently peel off the egg membrane to expose the chorioallantoic membrane of the chicken embryo.

3) Drug preparation: first prepare a stock solution of glipizide with a concentration of 50 mg/mL, and dissolve it in DMSO. Then it was diluted with normal saline to form glipizide solutions of 50 μg/mL, 100 μg/mL, and 200 μg/mL; a DMSO solution with a concentration of 1‰ was prepared as a control solution.

4) Drug treatment: The chicken embryos after opening the window were randomly divided into an experimental group and a control group, with 8 chicken embryos in each group. 50 μL of glipizide solutions of 50 μg/mL, 100 μg/mL and 200 μg/mL were added to the experimental group, and the same volume of control solution was added to the control group. Carefully add the drug to the allantoic membrane from the opening, seal it with medical tape, and incubate for 48 hours in an incubator at 37.8°C and 50% to 60% temperature.

5) Observation of results: After 48 hours of incubation with the drug, cut the eggshell from the midline, remove the contents in the eggshell, directly observe the blood vessels on the allantoic membrane on the side of the fenestration with the naked eye, take pictures to record the phenomenon, and count the blood vessels number of developments.

6) Results and conclusions: Centering on the administration point, in the control group that only added DMSO, it can be clearly observed that the main blood vessels bend and approach the central disk, that is, there is an obvious phenomenon of vascular attraction, and the surrounding blood vessels of the main blood vessels are in the same shape. Radial growth towards the center, that is, obvious vascular convergence phenomenon, and the vascular attraction and vascular convergence phenomenon in the experimental group gradually weakened with the increase of the dose of glipizide. Compared with the control group, the blood vessels in the experimental group were thinner and more numerous. Significantly reduced, especially under the treatment of high-concentration 200 μg/mL glipizide solution, even obvious avascular areas appeared, and the vascular density was only 55% of that of the control group, as shown in Figure 1A and B.

The above results indicate that the sulfonylurea drug glipizide can inhibit the growth and development of CAM blood vessels in a dose-dependent manner.

2. Glipizide inhibits yolk sac membrane (YSM) angiogenesis

1) Egg treatment: Clean the surface of the eggs (purchased from the Breeder Factory of South China Agricultural University), soak in 1:1000 Promoteril solution for 3 minutes, wipe the air chamber upwards, and place the egg tray in a normal Incubate in an incubator at a temperature of 37.8±0.5°C and a humidity of 60%. Rotate the chicken embryos under light and observe 3 times a day.

2) Drug preparation: first prepare a stock solution of glipizide with a concentration of 50 mg/mL, and dissolve it in DMSO. Then it was diluted with normal saline to form glipizide solutions of 50 μg/mL, 100 μg/mL, and 200 μg/mL; a DMSO solution with a concentration of 1‰ was prepared as a control solution.

3) Drug treatment: Take chicken embryos incubated for 2.5 days, pour the egg contents into sterile plates, divide them into experimental group and control group randomly, place red and black marked silicone rings of the same size on each chicken Cover the same position with fewer blood vessels on the embryonic YSM vascular network, cover the plate, and put it in an incubator at 37-38°C for 3-4 hours. Select chicken embryos with undisplaced silica gel rings, complete yolk sac membrane, well-developed vascular network and blastodisc, add 50 μg/mL, 100 μg/mL, and 200 μg/mL glipizide solutions to the red marked silica gel rings respectively, Add the same dose of control solution to the black marked rubber ring, and at least 8 chick embryos in each group, continue to be placed in the incubator for incubation.

4) Observation of results: Take pictures of chicken embryos incubated for 0h, 12h, and 24h after drug treatment, respectively, and take pictures under a stereomicroscope to observe the density and direction of YSM blood vessels in and around the silicone ring. The Image-ProPlus6.0 image analysis system was used to analyze the blood vessels in the entire silicone ring in the shooting experimental area, and the changes in blood vessel area and blood vessel density were used as analysis indicators.

5) Results and conclusions: At 12h and 24h, the growth of YSM blood vessels treated with 50μg/mL, 100μg/mL, and 200μg/mL glipizide was significantly slower than that of the DMSO group, especially the treatment with 200μg/mL glipizide After treating YSM with oxazine for 24 hours, it was found that the growth of blood vessels appeared concave, and only accounted for 2/3 of the apron, while the blood vessels in the DMSO group had completely exceeded the apron (see Figure 1C). The statistical results also showed that after YSM was treated with 50 μg/mL, 100 μg/mL, and 200 μg/mL glipizide, the density of blood vessels was significantly lower than that of the DMSO group, especially after YSM was treated with 200 μg/mL glipizide for 24 hours. The density was less than 50% of that of the DMSO group (see Figure 1D). And at 24h, the number of blood vessels in YSM was significantly less than that in the DMSO group (see Figure 1E).

The above results indicate that the sulfonylurea drug glipizide can inhibit the growth and development of YSM blood vessels in a dose-dependent manner.

3. Glipizide inhibits the growth and metastasis of spontaneous breast cancer in transgenic mice

Sixteen 9-week-old female breast cancer MMTV-PyMT transgenic mice were randomly divided into the experimental group and the control group. Each group was treated by intraperitoneal injection. The experimental group was injected with glipizide solution, in which glipizide The concentration of the stock solution is 50mg/mL, dissolved in DMSO, diluted with normal saline to a concentration of 500μg/mL when treating mice, and the treatment dose is 100μg/20g mice, and the control group is injected with the same volume of 1% DMSO solution, every Treatment three times a week for three weeks. Then check the following indicators:

1) Tumor volume measurement: Measure the long diameter and short diameter of the tumor with a vernier caliper every ⁴ days, and calculate the volume of the tumor with the formula: 0.52×long diameter×short diameter2. On day 20 of treatment, after mice were euthanized, tumor tissues were isolated and weighed.

Results and conclusion: The measurement data showed that the tumor volume of the glipizide treatment group was significantly smaller than that of the DMSO group (as shown in Figure 2A), and the tumor weight of the treatment group was also significantly lighter than that of the DMSO group (as shown in Figure 2B) . It shows that glipizide can inhibit tumor growth in MMTV-PyMT mice.

2) Detection of the number of lung metastatic nodules in mice: On the 20th day of treatment, after the mice were euthanized, the lung tissue of each mouse was separated, and the number of tumor nodules on the lung surface of the mice was counted.

RESULTS AND CONCLUSION: The data showed that the number of metastatic tumor nodules on the lung surface of the glipizide treatment group was significantly less than that of the DMSO group (as shown in Figure 2C), indicating that glipizide has the ability to inhibit tumor lung metastasis in MMTV-PyMT mice role.

3) Detection of tumor vessel density:

①Material collection: 4 mice in each of the experimental group and the control group were randomly selected after treatment, and the mice were killed by dislocation, and the tumor tissues without necrosis were collected and frozen.

② CD31 antibody immunohistochemical staining:

The tissue slices were rewarmed at room temperature, hydrated in double distilled water for 5 minutes, and washed 3 times with PBS, 5 minutes each time. Then put the tissue slices into 3% H ₂ O ₂ methanol solution (preheated for 30 minutes in advance), 37°C, 30 minutes, take out and wash with PBS three times, 5 minutes each time. Microwave repair for 10min. Wash three times with PBS, 5min each time. Dry the solution on the slide, add 10% BSA to the tissue, subject to cover the tissue, place in a closed box, put an appropriate amount of double distilled water in the box, 37 ° C, 1h.

Antibody incubation: suck off 10% BSA directly with a micropipette, add primary antibody, goat anti-CD31 antibody, and place at 4°C overnight. The tissue slices were taken out of the closed box and washed three times with PBS, 5 min each time. Wipe dry, add secondary antibody on the tissue, anti-goat IgG, place in a closed box, 37°C, 40min. Take out the slides from the closed box and wash with PBS three times, 5min each time.

DAB color development and hematoxylin counterstaining: dry the slides washed with PBS, add DABSubstrateKit solution (prepared for current use) on the tissue, let it stand at room temperature for about 5 minutes, and place it in water to stop the reaction. Then stain with hematoxylin for 40s, wash away the staining agent with running water, be careful not to wash away the tissue.

Dehydration and sealing: soak in 75% ethanol, 85% ethanol, 95% ethanol, 100% ethanol, and xylene in turn for 10 minutes each, and seal the slides with a mounting agent.

③Observation and counting: Each slice was photographed under a 20× microscope, and the number of blood vessels in each field of view was counted. The statistical data are shown in 2E, and the photos shown in Figure 2D are photos taken under a 100× microscope.

④Results and conclusions: As can be seen from Figure 2D and Figure 2E, the microvessel density in the glipizide treatment group was significantly lower than that in the DMSO group, indicating that glipizide can inhibit tumor angiogenesis in MMTV-PyMT mice.

4) Detection of tumor cell proliferation

①Materials: Randomly select 4 mice in the experimental group and control group after treatment, and inject BrdU (20 mg/ml, 2 mg BrdU per 20 g body weight) into the intraperitoneal cavity. After 1 hour, the mice were sacrificed by dislocation, and the tumor tissues without necrosis area were collected.

②Preparation of paraffin tissue sections: kill the mice by dislocation, and take the required tissues. Immediately place the tissue in Bouin's solution, fix it overnight, and change the fixation solution once in the middle; then dehydrate: immediately after fixation, soak the tissue half and half in 75% ethanol, 80% ethanol, 85% ethanol, 90% ethanol, and 95% ethanol hours, and finally soaked in 100% ethanol for one hour; then de-alcoholized: soak the dehydrated tissue in a mixture of 50% (v/v) xylene and 50% (v/v) ethanol for 15 minutes, and then soak in xylene Soak for 8 minutes, then soak in new xylene for 15 minutes; soak in wax: put the dehydrated tissue in a mixture of 50% (v/v) xylene and 50% (v/v) paraffin, paraffin, and new In paraffin wax, put it in a constant temperature box at 60°C for 1 hour each; Embedding wax: Put the paraffin wax at 65°C and continue to heat it to keep it in a liquid state. Use hard paper to fold into a wax box, first pour a layer of hot liquid paraffin into the box, quickly put the specimen, and then pour a layer of liquid paraffin. Then place immediately on top of cold water to cool quickly.

③ Slicing: Turn on the slicer and adjust its temperature at 45°C. When slicing, place the paraffin tissue block on ice or cool it to 0-4°C, which will harden the paraffin and make it easy to cut into thin slices. Lock the handwheel of the microtome so that the sectioning knife leaves the track before loading the paraffin block and place the paraffin block on the holder. According to the situation, the edge of the paraffin block can be smoothed to ensure that the blade is flush with the upper and lower edges of the tissue block, otherwise thin slices cannot be cut; use the adjusting screw to push the slicer knife within 1mm from the tissue block, and fine-tune the position of the slicer or tissue block , so that the section of the tissue block is flush with the path of the blade; set the thickness of the required section, and the maximum lower limit of the thickness of most paraffin-embedded tissue sections is 3 μm; trim the tissue block to form a smooth plane, and then prepare the desired thickness The tissue section belt; carefully pick up the tissue section belt with tweezers, and make it float on the surface of the 45°C water bath. Tweezers can be used to tidy up the floating tissue section belt, and pull the section belt to eliminate wrinkles according to the situation; put a clean glass slide (which has been coated with adhesive) under the liquid surface of the water bath, and let one side of the tissue section Or align the marked end (frosted side) of the slide on multiple sides. Slowly pull the slide out of the water at an angle of about 45° while holding the tissue section on the slide.

④ Mouse anti-BrdU specific immunostaining:

Dewax and rehydrate the tissue sections: Place the slides in xylene, fresh xylene, a mixture of 50% (v/v) xylene and 50% (v/v) ethanol in sequence, Soak in 100% ethanol, 95% ethanol, 5% ethanol, and 75% ethanol for 10 minutes each, and finally place in double distilled water and shake for 30 seconds, then put the tissue slices into methanol solution of 3% H ₂ O ₂ (preheated for 30 minutes in advance) medium, 37°C, 30min, take out and wash with PBS three times, 5min each time.

Pepsin repair: dry the slides washed with PBS, add 0.4% pepsin on the tissue, 37°C, 30min. Wash three times with PBS, 5min each time. Dry the solution on the slide, add 10% BSA to the tissue, subject to cover the tissue, place in a closed box, put an appropriate amount of double distilled water in the box, 37°C, 1h.

Antibody incubation: suck off 10% BSA directly with a micropipette, add primary antibody mouse anti-BrdU, and place at 4°C overnight. The tissue slices were taken out of the closed box and washed three times with PBS, 5 min each time. Wipe dry, add secondary antibody goat anti-mouse IgG on the tissue, place in a closed box, 37°C, 40min. Take out the slides from the closed box and wash with PBS three times, 5min each time.

DAB color development and hematoxylin counterstaining: dry the slides washed with PBS, add DABSubstrateKit solution (prepared for current use) on the tissue, let it stand at room temperature for about 5 minutes, and place it in water to stop the reaction. Then stain with hematoxylin for 40s, wash away the staining agent with running water, be careful not to wash away the tissue.

Dehydration and sealing: soak in 75% ethanol, 85% ethanol, 95% ethanol, 100% ethanol, and xylene in turn for 10 minutes each, and seal the slides with a mounting agent.

⑤ Observation and counting: Each slice was photographed under a 40× microscope and the percentage of tumor cells in each slice was counted. The statistical data are shown in 2G, and the photos shown in Figure 2F are photos taken under a 100× microscope.

⑥Results and conclusions: As can be seen from Figure 2F and Figure 2G, the number of tumor cell proliferation in the glipizide treatment group was significantly less than that in the control group, indicating that glipizide has the ability to inhibit tumor cell proliferation in MMTV-PyMT mice effect.

4. Glipizide inhibits the growth and metastasis of breast cancer cell line 4T1 subcutaneously transplanted tumor

Establishment of subcutaneous xenograft tumor model and treatment plan: Take 24 BalB/C mice, female, 6-8 weeks old; take 4T1 breast cancer cells in logarithmic growth phase, digest with 2.5g/L trypsin and reweigh with serum-free medium The cells were collected by suspension, and the cell density was adjusted with sterile saline. Trypan blue staining was performed to confirm that the number of viable cells was above 95%, and 1×10 ⁵ (0.2 mL) cells were inoculated subcutaneously in the armpit of the right upper limb of each mouse. The inoculated mice were randomly divided into two groups, the glipizide experimental group and the DMSO control group, 12 mice/group, and each group was treated by intraperitoneal injection. The experimental group was injected with glipizide solution, glipizide The stock solution concentration of the drug was 50 mg/ml, dissolved in DMSO. When treating mice, dilute it with normal saline to a concentration of 500μg/ml, and the therapeutic dose is 100μg/20g mice. The control group is injected with the same volume of 1% DMSO solution. The experimental animals in each group are administered by intraperitoneal injection, once a day , Stop treatment until the tumor volume reaches about 2cm ³ , observe the tumor volume and make records before each treatment. Then check the following indicators:

1) Tumor volume and weight measurement: Measure the long and short diameter of the tumor with a vernier caliper every ³ days, and calculate the volume of the tumor with the formula: 0.52×long diameter×short diameter2. The treatment was stopped when the tumor volume reached about 2 cm ³ , and the mice were euthanized, and the tumor tissues were separated and weighed.

Results and conclusion: The measurement data showed that the tumor volume of the glipizide treatment group was significantly smaller than that of the DMSO group (as shown in Figure 3A), and the tumor weight of the treatment group was also significantly lighter than that of the DMSO group (as shown in Figure 3B) . It shows that glipizide can inhibit the growth of 4T1 subcutaneous xenograft tumor.

2) Detection of the number of lung metastatic nodules in mice: Stop treatment when the tumor volume reaches about 2cm ³ , after the mice are euthanized, separate the lung tissue of each mouse, and count the number of tumor nodules on the lung surface of the mice.

RESULTS AND CONCLUSION: The data showed that the number of metastatic tumor nodules on the lung surface of the glipizide treatment group was significantly less than that of the DMSO group (as shown in Figure 3C), indicating that glipizide has the ability to inhibit the lung metastasis of the mouse model 4T1 cell lung. transfer function.

3) Detection of tumor vessel density:

①Material collection: 4 mice in each of the experimental group and the control group were randomly selected after treatment, and the mice were killed by dislocation, and the tumor tissues without necrosis were collected and frozen.

② CD31 antibody immunohistochemical staining:

The tissue slices were rewarmed at room temperature, hydrated in double distilled water for 5 minutes, and washed 3 times with PBS, 5 minutes each time. Then put the tissue slices into 3% H ₂ O ₂ methanol solution (preheated for 30 minutes in advance), 37°C, 30 minutes, take out and wash with PBS three times, 5 minutes each time. Microwave repair for 10min. Wash three times with PBS, 5min each time. Dry the solution on the slide, add 10% BSA to the tissue, subject to cover the tissue, place in a closed box, put an appropriate amount of double distilled water in the box, 37°C, 1h.

Antibody incubation: suck off 10% BSA directly with a micropipette, add primary antibody, goat anti-CD31 antibody, and place at 4°C overnight. The tissue slices were taken out of the closed box and washed three times with PBS, 5 min each time. Wipe dry, add secondary antibody on the tissue, anti-goat IgG, place in a closed box, 37°C, 40min. Take out the slides from the closed box and wash with PBS three times, 5min each time.

DAB color development and hematoxylin counterstaining: dry the slides washed with PBS, add DABSubstrateKit solution (prepared for current use) on the tissue, let it stand at room temperature for about 5 minutes, and place it in water to stop the reaction. Then stain with hematoxylin for 40s, wash away the staining agent with running water, be careful not to wash away the tissue.

Dehydration and sealing: soak in 75% ethanol, 85% ethanol, 95% ethanol, 100% ethanol, and xylene in turn for 10 minutes each, and seal the slides with a mounting agent.

③ Observation and counting: Each slice was photographed under a 20× microscope, and the number of blood vessels in each field of view was counted. The statistical data are shown in 3E, and the photos shown in Figure 3D are photos taken under a 100× microscope.

④ Results and conclusion: From Figure 3D and Figure 3E, it can be seen that the microvessel density in the glipizide treatment group was significantly lower than that in the DMSO group, indicating that glipizide can inhibit the angiogenesis of 4T1 cell subcutaneous xenografts.

4) Detection of tumor cell proliferation

①Materials: Randomly select 4 mice in the experimental group and control group after treatment, and inject BrdU (20 mg/ml, 2 mg BrdU per 20 g body weight) into the intraperitoneal cavity. After 1 hour, the mice were sacrificed by dislocation, and the tumor tissues without necrosis area were collected.

②Preparation of paraffin tissue sections: kill the mice by dislocation, and take the required tissues. Immediately place the tissue in Bouin's solution, fix it overnight, and change the fixation solution once in the middle; then dehydrate: immediately after fixation, soak the tissue half and half in 75% ethanol, 80% ethanol, 85% ethanol, 90% ethanol, and 95% ethanol hours, and finally soaked in 100% ethanol for one hour; then de-alcoholized: soak the dehydrated tissue in a mixture of 50% (v/v) xylene and 50% (v/v) ethanol for 15 minutes, and then soak in xylene Soak for 8 minutes, then soak in new xylene for 15 minutes; soak in wax: put the dehydrated tissue in a mixture of 50% (v/v) xylene and 50% (v/v) paraffin, paraffin, and new In paraffin wax, put it in a constant temperature box at 60°C for 1 hour each; Embedding wax: Put the paraffin wax at 65°C and continue to heat it to keep it in a liquid state. Use hard paper to fold into a wax box, first pour a layer of hot liquid paraffin into the box, quickly put the specimen, and then pour a layer of liquid paraffin. Then place immediately on top of cold water to cool quickly.

③ Slicing: Turn on the slicer and adjust its temperature at 45°C. When slicing, place the paraffin tissue block on ice or cool it to 0-4°C, which will harden the paraffin and make it easy to cut into thin slices. Lock the handwheel of the microtome so that the sectioning knife leaves the track before loading the paraffin block and place the paraffin block on the holder. According to the situation, the edge of the paraffin block can be smoothed to ensure that the blade is flush with the upper and lower edges of the tissue block, otherwise thin slices cannot be cut; use the adjusting screw to push the slicer knife within 1mm from the tissue block, and fine-tune the position of the slicer or tissue block , so that the section of the tissue block is flush with the path of the blade; set the thickness of the required slice, and the maximum lower limit of the thickness of most paraffin-embedded tissue slices is 3 μm; trim the tissue block to form a smooth plane, and then prepare the desired thickness The tissue section belt; carefully pick up the tissue section belt with tweezers, and make it float on the surface of the 45°C water bath. Tweezers can be used to tidy up the floating tissue section belt, and pull the section belt to eliminate wrinkles according to the situation; put a clean glass slide (which has been coated with adhesive) under the liquid surface of the water bath, and let one side of the tissue section Or align the marked end (frosted side) of the slide on multiple sides. Slowly pull the slide out of the water at an angle of about 45° while holding the tissue section on the slide.

④ Mouse anti-BrdU specific immunostaining:

Dewax and rehydrate the tissue sections: Place the slides in xylene, fresh xylene, a mixture of 50% (v/v) xylene and 50% (v/v) ethanol in sequence, Soak in 100% ethanol, 95% ethanol, 5% ethanol, and 75% ethanol for 10 minutes each, and finally place in double distilled water and shake for 30 seconds, then put the tissue slices into methanol solution of 3% H ₂ O ₂ (preheated for 30 minutes in advance) medium, 37°C, 30min, take out and wash with PBS three times, 5min each time.

Pepsin repair: dry the slides washed with PBS, add 0.4% pepsin on the tissue, 37°C, 30min. Wash three times with PBS, 5min each time. Dry the solution on the slide, add 10% BSA to the tissue, subject to cover the tissue, place in a closed box, put an appropriate amount of double distilled water in the box, 37°C, 1h.

Antibody incubation: suck off 10% BSA directly with a micropipette, add primary antibody mouse anti-BrdU, and place at 4°C overnight. The tissue slices were taken out of the closed box and washed three times with PBS, 5 min each time. Wipe dry, add secondary antibody goat anti-mouse IgG on the tissue, place in a closed box, 37°C, 40min. Take out the slides from the closed box and wash with PBS three times, 5min each time.

DAB color development and hematoxylin counterstaining: dry the slides washed with PBS, add DABSubstrateKit solution (prepared for current use) on the tissue, let it stand at room temperature for about 5 minutes, and place it in water to stop the reaction. Then stain with hematoxylin for 40s, wash away the staining agent with running water, be careful not to wash away the tissue.

Dehydration and sealing: soak in 75% ethanol, 85% ethanol, 95% ethanol, 100% ethanol, and xylene in turn for 10 minutes each, and seal the slides with a mounting agent.

⑤ Observation and counting: Each slice was photographed under a 40× microscope and the percentage of tumor cells in each slice was counted. The statistical data are shown in Figure 3G, and the photos shown in Figure 3F are photos taken under a 100× microscope.

⑥Results and conclusions: As can be seen from Figure 3F and Figure 3G, the number of tumor cell proliferation in the glipizide treatment group was significantly less than that in the control group, indicating that glipizide can inhibit the proliferation of 4T1 subcutaneously transplanted tumor cells .

Claims (2)

1. glipizide and medicinal derivative application in preparation tumor thereof; The medicinal derivative of described glipizide is the salt of glipizide;

Described tumor is breast carcinoma.

2. application according to claim 1, it is characterised in that: the medicinal derivative of described glipizide is the hydrochlorate of glipizide.