阅读说明：本技术 一种抑制胰腺癌的药物组合物及其制药用途 (Pharmaceutical composition for inhibiting pancreatic cancer and pharmaceutical application thereof ) 是由 张涛涛 何莉华 彭严 于 2019-08-26 设计创作，主要内容包括：本发明公开了一种抑制胰腺癌的药物组合物及其制药用途,该药物组合物中活性成分含有斑蝥酸钠或斑蝥酸钠和吉西他滨。本发明的药物组合物具有协同抗胰腺癌作用,能促进PANC-1细胞的凋亡作用,从而达到治疗胰腺癌的目的；且相同疗效的情况下,给药剂量低,降低了耐药性的产生,同时毒副反应降低,提高了患者的依从性和用药安全性。(The invention discloses a pharmaceutical composition for inhibiting pancreatic cancer and pharmaceutical application thereof, wherein the active ingredients in the pharmaceutical composition comprise sodium cantharidate or sodium cantharidate and gemcitabine. The pharmaceutical composition has a synergistic anti-pancreatic cancer effect, and can promote the apoptosis of PANC-1 cells, thereby achieving the purpose of treating pancreatic cancer; and under the condition of the same curative effect, the administration dosage is low, the generation of drug resistance is reduced, the toxic and side effects are reduced, and the compliance and the medication safety of patients are improved.)

1. A pharmaceutical composition for inhibiting pancreatic cancer, comprising: the medicine composition contains sodium cantharidate.

2. The pharmaceutical composition for inhibiting pancreatic cancer according to claim 1, wherein: the pharmaceutical composition also contains gemcitabine.

3. The pharmaceutical composition for inhibiting pancreatic cancer according to claim 2, wherein: the molar ratio of sodium cantharidinate to gemcitabine in the pharmaceutical composition is 1: 0.006-2.

4. The pharmaceutical composition for inhibiting pancreatic cancer according to claim 3, wherein: the molar ratio of sodium cantharidinate to gemcitabine in the pharmaceutical composition is 1: 0.5-2.

5. The pharmaceutical composition for inhibiting pancreatic cancer according to claim 4, wherein: the molar ratio of sodium cantharidinate to gemcitabine in the pharmaceutical composition is 1: 2.

6. the pharmaceutical composition according to any one of claims 1 to 5, wherein: the pharmaceutical composition is a preparation prepared by adding pharmaceutically acceptable auxiliary materials or auxiliary components into sodium cantharidinate or sodium cantharidinate and gemcitabine which are used as active components.

7. The pharmaceutical composition of claim 6, wherein: the preparation is an oral preparation or an injection.

8. The pharmaceutical composition of claim 7, wherein: the preparation is injection, powder injection, tablet, capsule and granule.

9. Application of sodium cantharidinate in preparing medicine for treating pancreatic cancer is provided.

10. The application of a pharmaceutical composition consisting of sodium cantharidinate and gemcitabine as an active ingredient in the preparation of anti-pancreatic cancer drugs.

Technical Field

The invention relates to the technical field of medicines, in particular to a medicinal composition for inhibiting pancreatic cancer and pharmaceutical application thereof.

Background

Pancreatic cancer is a malignant tumor of the digestive tract that is highly malignant and difficult to diagnose and treat, and about 90% of pancreatic cancers are ductal adenocarcinomas originating from the epithelium of the glandular duct. The morbidity and mortality of the cancer cells are obviously increased in recent years, and the 5-year survival rate is less than 1 percent, which is one of the worst malignant tumors in prognosis. In the treatment of pancreatic cancer, despite the enormous efforts in both basic and clinical studies, there are still huge therapeutic differences between preclinical and clinical stages. Even if patients are treated by surgery at an early stage, they often suffer from relapse and metastasis. Therefore, new therapeutic approaches are urgently needed for this very aggressive tumor.

Gemcitabine is a common first-line drug therapy in the treatment of pancreatic cancer. However, median survival of advanced pancreatic cancer patients treated with gemcitabine is still only 5-6 months. In order to improve the prognosis of patients, low dose gemcitabine treatment has been used in combination with radiation or other chemotherapeutic agents, but there are no reports of improving overall survival in advanced pancreatic patients.

The sodium cantharidinate is prepared by chemical synthesis of cantharidin extracted from traditional Chinese medicine cantharidin, the sodium cantharidinate in the prior art is mainly applied to clinical treatment of liver cancer and lung cancer, and the research on the treatment of sodium cantharidinate on pancreatic cancer resistance is fresh at home and abroad.

Chinese patent with application number CN201510869264.5 discloses a pharmaceutical composition for inhibiting pancreatic cancer and a pharmaceutical application thereof, wherein the pharmaceutical composition comprises cantharidin and derivatives thereof and anti-angiogenesis drugs. Research shows that cantharidin may have the effect of promoting angiogenesis, provides rich blood supply for tumor and makes tumor cell obtain rich nutrients to counteract the inhibition of cantharidin on tumor cell, so that cantharidin combined with anti-angiogenesis medicine (bevacizumab) can inhibit the growth of pancreatic cancer obviously and has clinical application foreground. However, cantharidin has great hepatorenal toxicity when used, so that cantharidin is not directly applied to clinic.

Therefore, a new drug for treating pancreatic cancer is urgently needed.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a pharmaceutical composition for inhibiting pancreatic cancer and pharmaceutical application thereof, and discloses application of sodium cantharidinate or combination of sodium cantharidinate and gemcitabine in resisting pancreatic cancer.

The invention is mainly realized by the following technical scheme:

a pharmaceutical composition for inhibiting pancreatic cancer comprises sodium cantharidate.

The pharmaceutical composition for inhibiting pancreatic cancer preferably further comprises gemcitabine in addition to sodium cantharidate.

Preferably, the molar ratio of sodium cantharidinate to gemcitabine in the pharmaceutical composition for inhibiting pancreatic cancer is 1: 0.006-2.

As a further optimization of the dosage ratio, the molar ratio of sodium cantharidinate to gemcitabine in the pharmaceutical composition for inhibiting pancreatic cancer is 1: 0.5-2.

As a further optimization of the dosage ratio, the molar ratio of sodium cantharidinate to gemcitabine in the pharmaceutical composition for inhibiting pancreatic cancer is 1: 2.

the pharmaceutical composition is a preparation prepared by adding pharmaceutically acceptable auxiliary materials or auxiliary components into sodium cantharidinate or sodium cantharidinate and gemcitabine which serve as active ingredients.

Preferably, the preparation is an oral preparation or an injection.

More preferably, the preparation is injection, powder injection, tablet, capsule and granule.

The invention aims to provide the application of one or two active ingredients in preparing an anti-pancreatic cancer medicament, namely: the application of sodium cantharidinate in preparing anti-pancreatic cancer medicine; or the application of the pharmaceutical composition consisting of sodium cantharidinate and gemcitabine as an active ingredient in the preparation of anti-pancreatic cancer drugs.

Compared with the prior art, the invention has the following beneficial effects:

(1) has less toxic and side effects;

(2) has synergistic pancreatic cancer resisting effect, and can promote apoptosis of PANC-1cell, thereby achieving the purpose of treating pancreatic cancer;

(3) under the condition of the same curative effect, the administration dosage is low, the generation of drug resistance is reduced, the toxic and side effects are reduced, and the compliance and the medication safety of patients are improved.

Drawings

FIG. 1: after PANC-1 cells are treated by sodium cantharidate with different concentrations for 72 hours, MTT test is used for detecting the cell proliferation condition;

FIG. 2: after the PANC-1 cells are treated by gemcitabine with different concentrations for 72 hours, the cell proliferation condition is detected by an MTT test;

FIG. 3: tumor size at the end of the control group, gemcitabine, sodium cantharidate and combination test;

FIG. 4: mean tumor weights ± SD (6 mice) at the end of the control, gemcitabine, sodium cantharidate and combination test;

FIG. 5: tumor size growth curves (recorded every 3 days) in control, gemcitabine, sodium cantharidate and combination tests;

FIG. 6: in control group, gemcitabine, sodium cantharidate and combination test, the mouse weight curve (recorded every 3 days);

FIG. 7: h & E staining of tumor tissue under blank control conditions;

FIG. 8: h & E staining of tumor tissue under gemcitabine conditions;

FIG. 9: h & E staining results of tumor tissues under sodium cantharidate conditions;

FIG. 10: h & E staining results of tumor tissues under the condition of combination of sodium cantharidate and gemcitabine.

Detailed Description

In order to better understand the essence of the present invention, the following examples and test examples of the pharmaceutical composition of the pharmacological action results to illustrate the superiority of the present invention, but not to limit the invention.

Example 1: preparation of pharmaceutical compositions

The method comprises the following steps: respectively taking sodium cantharidate 5 μ M, 10 μ M and 15 μ M, adding pharmaceutically common adjuvants or auxiliary components, and making into injection, powder for injection, tablet, capsule and granule by conventional method.

The method 2 comprises the following steps: mixing sodium cantharidinate 15 μ M and gemcitabine 0.1 μ M, adding pharmaceutically-acceptable adjuvants or adjuvant components, and making into injection, powder for injection, tablet, capsule, and granule by conventional method.

The method 3 comprises the following steps: mixing sodium cantharidinate 10 μ M and gemcitabine 5 μ M, adding pharmaceutically-acceptable adjuvants or adjuvant ingredients, and making into injection, powder for injection, tablet, capsule, and granule by conventional method.

The method 4 comprises the following steps: mixing sodium cantharidinate 5 μ M and gemcitabine 10 μ M, adding pharmaceutically-acceptable adjuvants or adjuvant ingredients, and making into injection, powder for injection, tablet, capsule, and granule by conventional method.

The inventor finds that sodium cantharidinate has an anti-pancreatic cancer effect in research, and the combination of the sodium cantharidinate and gemcitabine can play a synergistic effect in resisting pancreatic cancer.

The following are examples of the efficacy test of the pharmaceutical composition according to the present invention, and the test procedures are as follows:

test example 1: in vitro and in vivo pancreatic cancer resistance test of sodium cantharidinate and sodium cantharidinate combined with gemcitabine

1.1 test materials

1.1.1 test drugs

Sodium cantharidate is provided by Guizhou Bai Qian Yao Co.

1.1.2 test reagents

Gemcitabine (Gemcitabine) (Sigma, st. louis, MO, USA); fetal Bovine Serum (Fetal bone Serum, FBS) (GIBCO, Invitrogen Corporation, NY, USA); DMEM medium or RPMI-1640 medium or MEM medium (GIBCO, Invitrogen Corporation, NY, USA); penicillin (Sigma, st. louis, MO, USA); streptomycin (Sigma, st. louis, MO, USA); trypsin (Gibco, Grand Island, NY, USA); MTT reagent, bio-technical institute in cloudband.

1.1.3 test apparatus

A FORMA 700 model ultra-low temperature refrigerator, Thermo corporation; YC-300L type medicine storage cabinet, Mitsubishi low temperature science and technology, Inc., of China; Direct-Q with pump type ultrapure water instrument, Millopore corporation; SW-CJ-2FD type superclean bench: low temperature high speed centrifuge model 3K15, Sigma company; forma 3111 type water jacket CO₂An incubator: thermo Electron company; YXO-LS-50SII type vertical pressure steam sterilizer: shanghai Bocheng medical facility; BS224 type electronic balance: beijing Sidolis Instrument systems, Inc.; b iserthold LB941 microplate-type multifunctional microplate reader, Berthold Inc.

1.1.4 test cell lines

PANC-1cell line was purchased from the American Tissue Culture Collection (ATCC, Rockville, Md., USA).

1.1.5 test animals

Source, species, strain: BALB/c nude mice, purchased from Nanjing university under the certification number SCXK (Su) 2015-0001. The age in days: day 28-56, body weight 16-19g, sex: and (4) male.

1.2 test methods

1.2.1 test Subdivision and drug concentration selection

Cell experiments: the concentration of the compound sodium cantharidate is selected as follows: 0. mu.M, 2. mu.M, 5. mu.M, 10. mu.M, 20. mu.M, 40. mu.M, 80. mu.M.

Animal experiments: negative control group (control group) 6 mice were administered i.p. with PBS (1 ×); 6 positive drug groups (Gemcitabine), Gemcitabine was administered intraperitoneally; experimental group (sodium cantharidate): 6, compound sodium cantharidate was administered intraperitoneally; group of combinations (combined): the compounds sodium cantharidinate and gemcitabine were administered intraperitoneally at 6.

Table 1: test grouping and administration

1	Control group (PBS)	1×/everyday	i.p.
				2	Gemcitabine	25mg/kg/twice a week	i.p.
3	Sodium cantharidate	0.3mg/kg/everyday	i.p.
				4	Combination drug	(Gemcitabine + sodium cantharidate)	i.p.

1.2.2 cell culture conditions

The cells were subcultured for 10-15 passages in DMEM medium containing penicillin (final concentration of 100U/mL), streptomycin (final concentration of 100. mu.g/mL) and 10% FBS under conditions such that the cells fused to 90%, the old medium was discarded, the cells were washed with 2mL of PBS for 2 times, 2mL of 0.25% (w/v) Trypsin-0.53mM EDTA mixed digest was added after the PBS was discarded, the mixture was observed under a microscope for about 30 seconds, and when the cells became round, 2mL of complete medium was rapidly added to terminate the digestion, gently pipetting and collecting the cells. Centrifuging at 800rpm and 4 deg.C for 5min, discarding supernatant, suspending cells with complete culture medium, culturing in bottles, and changing the culture medium every other day.

1.2.3 cell MTT assay

Cells in logarithmic growth phase were grown at 1X 10⁴cells were plated in 96-well plates at 37 ℃ with 5% CO₂The cells were cultured under the condition until the cells are 90% confluent, and then the cells were synchronized by incubating for 2h with serum-free DMEM medium. Subsequently, the supernatant was discarded, and sodium cantharidate complete medium was added at a concentration of (0. mu.M, 2. mu.M, 5. mu.M, 10. mu.M, 20. mu.M, 40. mu.M, 80. mu.M), respectively, and incubated for 72 hours. 4h before the end of incubation, 20. mu.L of MTT solution (5mg/mL) was added to each well. After incubation is finished, discarding supernatant of each well, adding 150 mu L DMSO into each well, oscillating on a cell oscillator for 10min, and measuring OD (optical density) by using an enzyme-labeling instrument after crystals are fully dissolved₅₇₀。

In the same manner, MTT assay was performed using gemcitabine (0. mu.M, 0.01. mu.M, 0.1. mu.M, 1. mu.M, 10. mu.M, 100. mu.M, 1000. mu.M) and gemcitabine (0. mu.M, 0.1. mu.M, 1. mu.M, 10. mu.M) in combination with sodium cantharidate (0. mu.M, 5. mu.M, 10. mu.M, 15. mu.M).

Inhibition rate (control OD value-experiment OD value)/control OD value X100%

1.2.4 establishment of transplantable tumor model and drug therapy

PANC-1 cells are subcultured for 10-12 generations under the condition of DMEM medium containing penicillin (final concentration of 100U/mL), streptomycin (final concentration of 100 mu g/mL) and 10% FBS, when the cells are fused to 90%, the old medium is discarded, the cells are washed for 1 time by 5mL of PBS, 1mL of trypsin-EDTA digestive solution is added after the PBS is discarded, the cells are placed under a microscope for observation for about 1min, 2mL of complete medium is rapidly added after the cells are rounded to stop digestion, the cells are gently blown and beaten, and the cells are collected. Centrifuging at 4 deg.C for 3min at 500rpm, discarding supernatant, resuspending cells with complete culture medium, culturing in culture dish, and changing culture medium every other day.

Selecting 24 healthy experimental mice, feeding for one week to obtain a body weight of 27g, injecting 0.2ml of cell suspension into right axilla of each mouse, and making the cell concentration be 2 × 10⁶/tomor. Measuring the diameter of the transplanted tumor of the mouse by using a vernier caliper until the tumor grows to 100mm³After left and right, animals were randomized into groups as day 0 and then started to be dosed for 21 days according to the dosing method in the group. The test compounds are dynamically evaluated for in vivo anti-tumor activity using a method for measuring tumor size. Tumor diameter was measured every 3 days. The administration volume was different for each group. After 21 days, the mice were sacrificed and the tumor mass was surgically removed and weighed. The Tumor Volume (TV) is calculated as:

TV＝0.52×a×b²

wherein a and b represent the length and width, respectively.

1.2.5 data analysis

After data were obtained, GraphPad Prism 6 was fitted to obtain IC₅₀。

1.2.6

Statistical differences between data groups were determined to be significant using the one-way ANOVA test with P values less than 0.05.

1.3 test results

Table 2: growth inhibition of PANC-1cell line by Gemcitabine and sodium cantharidinate after 72h incubation ((IC)₅₀Values inμM)。

Group of	IC50(μM)
		Gemcitabine (Gemcitabine)	37.54
Sodium cantharidate (sodium. C)	13.24

In addition, the inhibition of PANC-1cell growth by sodium cantharidate and gemcitabine at different concentrations is shown in FIG. 1 and FIG. 2.

As is clear from the data in Table 2, FIGS. 1 and 2, the IC of sodium cantharidinate on PANC-1 cells in the MTT assay₅₀Gemcitabine IC on PANC-1 cells at 13.24. mu.M₅₀37.54 mu M, and the tumor inhibition effect of the sodium cantharidate on pancreatic cancer cells is obviously stronger than that of gemcitabine.

Table 3: in MTT test, sodium cantharidinate with different concentrations and gemcitabine are mixed and incubated to inhibit the growth of PANC-1cell line.

As can be seen from Table 3, sodium cantharidinate in combination with gemcitabine gave a significant synergistic effect in inhibiting the proliferation of PANC-1 cells.

In order to detect the in-vivo anti-tumor combined effect of the sodium cantharidinate and the gemcitabine, the gemcitabine and the sodium cantharidinate of tumor-bearing mice are treated independently and mixed, the tumor size and the weight of the tumor-bearing mice are detected every 3 days, and tumor tissues are separated, photographed and weighed after the experiment is finished. The results are shown in FIGS. 3 to 6. (wherein p < 0.05, p < 0.01, p < 0.001,) indicates that gemcitabine and sodium cantharidinate, when used alone, significantly inhibited tumor growth, and that the combination of gemcitabine and sodium cantharidinate was more effective than gemcitabine alone.

In addition, the present inventors also performed H & E staining on tumor tissues, and the results are shown in fig. 7 to fig. 10. As can be seen from FIGS. 7-10, the cell number of tumor tissues was significantly reduced when sodium cantharidinate and gemcitabine were used alone, and the combination of sodium cantharidinate and gemcitabine was more effective than gemcitabine alone.

Test example 2: anti-pancreatic cancer activity test of sodium cantharidinate in primary cells

2.1 test materials

2.1.1 test drugs

Sodium cantharidate is provided by Guizhou Bai Qian Yao Co.

2.1.2 test reagents

Gemcitabine (Gemcitabine) (Sigma, st. louis, MO, USA); fetal Bovine Serum (Fetal bone Serum, FBS) (GIBCO, Invitrogen Corporation, NY, USA); RPMI-1640 medium (GIBCO, Invitrogen Corporation, NY, USA); penicillin (Sigma, st. louis, MO, USA); streptomycin (Sigma, st. louis, MO, USA); trypsin (Gibco, Grand Island, NY, USA); MTT reagent, bio-technical institute in cloudband.

2.1.3 Experimental instruments

A FORMA 700 model ultra-low temperature refrigerator, Thermo corporation; YC-300L type medicine storage cabinet, Mitsubishi low temperature science and technology, Inc., of China; Direct-Q with pump type ultrapure water instrument, Millopore corporation; SW-CJ-2FD type superclean bench: low temperature high speed centrifuge model 3K15, Sigma company; forma 3111 type water jacket CO₂An incubator: thermo Electron company; YXO-LS-50SII type vertical pressure steam sterilizer: shanghai Bocheng medical facility; BS224 type electricityA sub-balance: beijing Sidolis Instrument systems, Inc.; berthold LB941 microplate type multifunctional microplate reader, Berthold Co.

2.1.4 test cell lines

Human primary pancreatic cancer cells and Human primary paracancerous cells (primary cells of the paracancerous tissue of Human pancreatic cancer).

2.2 test methods

2.2.1 test Subdivision and drug concentration selection

Test grouping

The concentration of the compound sodium cantharidate is selected as follows: 0. mu.M, 1. mu.M, 5. mu.M, 10. mu.M, 50. mu.M, 100. mu.M.

All compounds treated cells for 72 h.

2.2.2 isolation of Primary cells of human pancreatic cancer or of Primary cells of tissue beside human pancreatic cancer

Taking human pancreatic cancer tissues and tissues beside the human pancreatic cancer, disinfecting with 75% ethanol solution, taking the cancer tissues or tissues beside the cancer in a sterile table, shearing, adding 0.125% trypsin and 1% collagenase mixed solution, digesting for 10min in a 37 ℃ water bath oscillator, stopping digestion with RPMI 1640 containing 10% Fetal Bovine Serum (FBS), and sucking the mixed solution. Repeating digestion for 5 times, collecting digestive juice, filtering with 200 mesh sieve, centrifuging the filtrate (800rpm, 10min), discarding supernatant, adding appropriate amount of culture solution, suspending and precipitating, and inoculating to 25cm²Culturing in a culture flask at 37 deg.C and 5% CO₂Culturing in an incubator.

The culture conditions were RPMI-1640 medium containing penicillin (final concentration of 100U/mL), streptomycin (final concentration of 100. mu.g/mL), 10% FBS, when the cells fused to 90%, the old medium was discarded, the cells were washed with 2mL of PBS 2 times, after discarding PBS, 2mL of 0.25% (w/v) Trypsin-0.53Mm EDTA mixed digest was added, the mixture was observed under a microscope for about 30s, when the cells became round, 2mL of complete medium was rapidly added to stop the digestion, gently pipetting, and the cells were collected. Centrifuging at 800rpm and 4 deg.C for 5min, discarding supernatant, suspending cells with complete culture medium, culturing in bottles, and changing the culture medium every other day.

2.2.3 cell MTT assay

Cells in logarithmic growth phase were grown at 1X 10⁵cells were plated in 96-well plates at 37 ℃ with 5% CO₂The cells were synchronized by incubation in serum-free RPMI-1640 medium for 2h after 90% cell confluence. Subsequently, the supernatant was discarded and compounds were added according to the protocol described in the protocol for assay 1.4.1 and 1.4.2 groups and incubated for 72 h. 4h before the end of incubation, 20. mu.L of MTT solution (5mg/mL) was added to each well. After incubation is finished, discarding supernatant of each well, adding 150 mu L DMSO into each well, oscillating on a cell oscillator for 10min, and measuring OD (optical density) by using an enzyme-labeling instrument after crystals are fully dissolved₅₇₀。

Inhibition rate (control OD value-experiment OD value)/control OD value X100%

2.2.4 Collection of protein samples

After treatment of the cells under the treatment conditions grouped according to experiment 1.4.3, the supernatant was discarded, washed once with pre-cooled PBS, the cells were scraped off with a cell scraper, and the cells were blown off with a pipette. Cells were collected by centrifugation and the supernatant was aspirated off, leaving the cell pellet for use. The cell is prevented from being digested by pancreatin to prevent the pancreatin from degrading the target protein to be extracted. 200. mu.L of the cytoplasmic protein extraction reagent A added with PMSF was added to each 20. mu.L of the cell pellet. The cell pellet was vortexed vigorously at the highest speed for 5 seconds to completely suspend and disperse the cell pellet. Water bath for 10-15min, and cell plasma protein extracting reagent B10 μ L. Vortex vigorously at highest speed for 5s, water bath 1 min. The highest speed vortex was vigorously carried out for 5s, and centrifugation was carried out at 12000-16000g at 4 ℃ for 5 min. Immediately sucking the supernatant into a precooled Eppendorf tube to obtain the extracted cytoplasm protein, and training in winter and keeping at-80 ℃ for later use. For the precipitation, the supernatant was completely aspirated, and 50. mu.L of a nuclear protein extraction reagent containing PMSF was added. The cell pellet was vortexed vigorously at the highest speed for 15-30s to completely suspend and disperse the cell pellet. Then placing the mixture back into the ice bath, carrying out high-speed vigorous vortex for 15-30s every 1-2min for 30min, and centrifuging for 10min at 12000-16000g at 4 ℃. And immediately sucking the supernatant into a precooled Eppendorf tube to obtain the extracted cell nuclear protein, mixing the cell plasma protein and the nuclear protein, and then completing the preparation of the sample at-20 ℃.

2.2.5 Collection of RNA samples

After the cells were treated under the treatment conditions according to test 1.4.3, the supernatant was discarded, 0.5ml of Trizol was added, the mixture was repeatedly pipetted to clarify the cells and transferred to an EP tube, and the tube was left to stand for 5min, 0.1ml of chloroform (1/5 in volume of Trizol) was added to each tube, followed by vigorous shaking, standing for 10min, centrifugation at 12000rpm4 ℃ for 15min, the supernatant was taken (without hitting the middle layer), about 200. mu.L of isopropanol was added thereto, followed by vigorous shaking, standing at-20 ℃ for 30min, RNA precipitation, centrifugation at 12000rpm4 ℃ for 10min, and the supernatant was discarded. Adding 75% ethanol (DEPC water in situ), washing, centrifuging at 7500rpm4 deg.C for 5min, removing supernatant, and naturally drying for 5-10 min. Dissolving RNA in 10 μ L DEPC water, blowing, beating, and mixing to obtain total RNA solution, and storing at-20 deg.C.

2.2.6 data analysis

Results are expressed as mean ± SD, after which IC is fitted by GraphPad Prism 6 according to inhibition rate₅₀。

2.3 test results

Table 4: growth inhibition rates (IC) of different cell lines after treatment of cells with sodium cantharidinate and gemcitabine for 72h₅₀Values inμM)。

As shown in table 4, the inhibitory effect of the compound gemcitabine or sodium cantharidinate on primary cells of human pancreatic cancer and primary cells of paracancerous tissues of human pancreatic cancer after incubation was examined in MTT assay. The result shows that the sodium cantharidinate has IC of the primary cells of the human pancreatic cancer and the primary cells of the paracarcinoma tissues of the human pancreatic cancer₅₀20.63 +/-2.26 and 85.47 +/-5.32 mu M; IC of positive compound gemcitabine on human pancreatic cancer primary cells and human pancreatic cancer paracancerous tissue primary cells₅₀13.90 +/-1.35 and 221.00 +/-6.39, which shows that sodium cantharidinate has the function of resisting pancreatic cancer and the effect is superior to that of gemcitabine, a first-line medicament for resisting pancreatic cancer.

TABLE 5 IC of sodium cantharidinate in combination with gemcitabine at different ratios₅₀And drug combination index CI values.

As shown in Table 5, we examined the inhibitory effect of gemcitabine compound and sodium cantharidinate in different ratios on primary cells of human pancreatic cancer in MTT assay. The results show that the compound gemcitabine and sodium cantharidate in different proportions are combined with human-derived pancreatic cancer primary cell IC₅₀15.28. + -. 1.04. mu.M (2:1), 10.26. + -. 0.93. mu.M (1:1), 8.52. + -. 0.51. mu.M (1: 2). The results show that the CI value is minimal when the combined administration ratio of sodium cantharidinate and gemcitabine is 1: 2. Shows that the combination of sodium cantharidinate and gemcitabine in the ratio of 1:2 has the best effect.

In conclusion, the sodium cantharidinate can effectively treat pancreatic cancer, and when the sodium cantharidinate is combined with gemcitabine, the anti-pancreatic cancer effect is obviously stronger than that of the gemcitabine or the sodium cantharidinate which is singly used.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.