| Pancreatic cancer with high degree of malignancy is one of the cancer with high mortality, and its incidence has increased recently. The Incidence of pancreatic cancer ranks No. 4 in Europe. The mortality of pancreatic cancer ranks No. 9 in China. It is reported that after the initial diagnosis of pancreatic cancer, one year mortality rate was 80%, 3 years was 95%. The survival time of patients with matastasis of pancreatic cancer was 3-6 months, 6-10 months of patients with locally advanced tumor. Early diagnosis and early treatment of pancreatic cancer treatment are major factors on slight improvement over the previous.Improvements of surgical technique did not significantly improve the prognosis of patients.Currently, it .is considered that combination with chemotherapy and other comprehensive treatment can improves the quality of life and prolong survival time after operations.Gemcitabine is still the first choice for treatment of pancreatic cancer, although there is no proposal for gemcitabine chemotherapy. Effect of chemotherapy for pancreatic cancer is. still not very satisfactory. Although the effect of gemcitabine for pancreatic cancer is sure, but the efficiency of it is only 25%. Multidrug resistance (MDR) is one of the main causes of failure of chemotherapy. The mechanism of MDR is not clear, but studies have shown that abnormalexpression of P53 and Aurora may cause gemcitabine-resistance of pancreatic cancer.Compared with the characteristics of large toxicity of chemotherapy drugs,herbs have unique advantages. With the deepening of research on herbal medicine, many herbs ownning anti-tumor effects have been found. Sophora is a traditional Chinese medicine. Modern medicine showed Sophora alkaloids not only have high antitumor activity and immunomodulatory effects, but also have the effect of reversing tumor resistance to chemotherapeutic drugs.In order to evaluate the anti-tumor effect of matrine to pancreatic cancer, explore the relationship between Aurora-A expression and gemcitabine resistance of pancreatic cancer and indentify the effects of matrine on gemcitabine-resistance of pancreatic cancer and regulation the expresssion of Aurora-A, three parts of the subject were applied as follows.Part oneA Recycle-screening Technique of Establishing Gemcitabine-resistant Models for Pancreatic CarcinomaObjective: To establish new cell and animal gemcitabine-resistant models for pancreatic carcinoma, .which mimic the pathologic processes of gemcitabine-resistance of pancreatic carcinoma in clinic.Methods:The pancreatic cancer cell line PANC-1 cells were cultured in vitro, which were injected to subskin of nude mice. After formation of tumor mass, the mass was transplanted to tail of pancreas of nude mice to establish the animal models of pancreatic cancer. The mice were dealed with gemcitabine intraperitoneal chemotherapy, the mice with the largest tumor were selected and then digested and separated to gain tumor cells. The cells were used to establish the animal models of pancreatic cancer. The progresses above were recycled by two times to obtain resistant cell lines and animal models. Body and in vitro tests were applied to verify resistance of models to gemcitabine.Sequencing to identify drug-resistant models and parental cells by homologous microsatellites.Results:The cell and animal gemcitabine-resistant models of pancreatic carcinoma were established successfully. In the first cycle, the successful rate of pancreatic carcinoma was 73.3% (11/15) , mortality after gemcitabine intraperitoneal chemotherapy was 18.2% (2/11).The mean weight of five largest tumor was 1.76 ± 0.87g,and the distal metastatic rate was 11.1% (1/9) . In the seconde cycle, the successful rate of pancreatic carcinoma was 80%(12/15), mortality after gemcitabine intraperitoneal chemotherapy was 8.3%( 1/12). The mean weight of five largest tumor was2.16±1 .12g, and the distal metastatic rate was 36.3% (4/11).The weight of five largest tumor and distal metastatic rate between two groups were significantly different (P<0.05). The cell line which was screened by two cycles was named PANC-1/R2. The inhibition rates of PANC-1 and PANC-1/R2 were 21.8%, 37.1%, 46.1% and 12.6%, 25.3%, 36.8% indifidully under effection of gemcitabine on 5ug/dl, lOug/dl and 20ug/dl concentration respectively. Subcutaneous tumor of PANC-1 and PANC-1/R2 were formed for three weeks, gemcitabine 240mg/kg intraperitoneal injection per week and for 4 times,after 35 days,the tumor size were 1.87 ± 0.69g and 2.09 ± 0.71 g,the difference was statistically significant (P <0.05). The results of human genome microsatellite D14568,D18569, D205199 showed that parental pancreatic cancer cells, gemcitabine-resistant pancreatic cancer cells in three microsatellite loci amplified fragments of the same size, and pancreatic tissue of nude mice did not amplify the corresponding banding patterns.Conclusions: The recycle-screening technique of establishing gemcitabine-resistant models for pancreatic carcinoma is feasible. The cells screened by the technique obtain ignificantly enhanced resistance, compared with the primary cells. The models mimic the process of the body’s resistance with influence of multi-factor complex environment. And the cell models were constructed with the same parent cell genetic loci and genetic homology. But the process is complicated and takes a long time. Skilled and careful operation benefits the success rate. Increasing the times of recycles may enhance the resistant characteristics.Part twoThe Relationship of Aurora-A Expression with Gemcitabine-resistance, Metastatic Rate and Way of Metastasis of Pancreatic CancerObjeve: To assess the relationship of Aurora-A expression with gemcitabine-resistance,metastatic rate and way metastasis of pancreatic cancer..Methods: The expression of Aurora-A mRNA of PANC-1 and PANC-1/R2 cells were tested by qPCR technique. The animal models of pancxreatic cancer were estblised by PANC-1 and PANC-1/R2 cells. After 35days, mice were killed, and the tumor tissues were collected. The Aurora-A protein expression of the two groups were deteched by Immunohistochemistry. The correlation of Aurora-A protein positive expression in the two groups with metastatic rate and way of metastasis of pancreatic cancer was analysed as well.Results:.In the PANC-1 group, the metastasis of tumor on the five weeks was 36.8%(7/19), in which lymph node metastasis, peritoneal dissemination, liver metastasis, intestinal metastasis were 36.8% (7/19), 15.8% (3/19), 10.5% (2/19), 5.3% (1/19), the mean weight of tumor tissues was 0.453±0.110 g,and weight of mice was 23.2± 1.41 g. While the metastasis of tumor on the five weeks was 50% ( 9/18 ) , in which lymph node metastasis, peritoneal dissemination, liver metastasis, intestinal metastasis were 50% (9/18 ), 33.3% (6/18 ), 22.2%(4/18 ),11.1% ( 2/18 ),the mean weight of tumor tissues was0.564±0.203g,and weight of mice was 22.91±1.13g in PANC-1/R2 group. The metastatic rate and weight of tumor were with significant differences (P<0.05). The expression of Aurora-A mRNA of PANC-1 cells was 1.002 ± 0.040; while it was 1.845 ± 0.069 in PANC-1/R2, it was significant difference between the two groups (P <0.05). A 15 of 18 cases positive expression of Aurora-A protein were detected in PANC-1/R2 group, while it is 11 of 19 cases in PANC-1 group. The significant difference was found between the two groups (P <0.05). Aurora-A positive expression was associated with the total rate of me tastasis, lymphtic metastasis, liver metastasis and intestinal metastasis in PANC-1 group (P <0.05), inversely, Aurora-A positive expression has no associasion with way and rate of metastasis (P > 0.05).Conclusions: High expression of Aurora-A gene is associated with gemcitabine-resistance of pancreatic cancer cell lines. PANC-1/R2 cell was successful as a cell model of gemcitabine-resistance of pancreatic cancer. In vivo studies have shown that differences in the expression of Aurora-A was associated with gemcitabine-resistance of pancreatic cancer, but no significant relationship with rate and way of metastasis.Part threeThe Effect of Matrine On Inducing Apoptosis And Reversal of Gemcitabine-resistance Of Pancreatic Cancer And Its ImpactOn Aurora-A ExpressionObjective: To evaluate the effect of matrine on inducing apoptosis of pancreatic cancer cells, and regulating the expressopn of Aurora-A and reversing gemcitabine-resistance of pancreatic cancer.Methods: Detection of cytotoxicity of different concentrations of matrine on PANC-1 cells by MTT test. Safe range of concentrations of matrine was choosen to measure the inhibition rate of PANC-1, PANC-1/R1 and PANC-1/R2 after treatments of different concentrations of matrine and gemcitabine. Apoptosis of PANC-1 and PANC-1/R2 cells were analyzed by Flow Cytometry after treatment of matrine and gemcitabine. Aurora-A gene expression of PANC-1 / and PANC-1/R2 were analyzed by qPCR after treatment of matrine and gemcitabine.Results: Cytotoxicity of matrine increased as increase of concentration, but the growth rate of PANC-1 cells had little change between 2 ug / ml and 64 ug / ml of matrine. On the 32 ug / ml concentration of matrine, the inhibition rate of the tree groups all increased after treatment of 0-40ul gemcitabine (P <0.05), but inhibition rate of the three group had no significant changes on 64 ug / ml of matrine than that of 32 ug / ml (P> 0.05). At the same concentration effect of matrine, inhibition rates decreased as the rank of PANC-1, PANC-1/R1 and PANC-1/R2. The outcome of Flow cytometry shows that apoptosis rates of pancreatic cells had no significant difference in groups but significant difference between the PANC-1 and PANC-1/R2 groups. The same phenomenon was found of Aurora-A mRNA expression in the two groups.Conclusions:The combination of matrine andh gemcitabine can induce apoptosis of pancreatic cancer cells t, but can not reverse the effects of gemcitabine-resistance of pancreatic cancer, can not increase sensitivity to chemotherapy, also has no impact on expression of Aurora-A. The results indicates that matrine can play the a certain role of anti-pancreatic cancer by inducing apoptosis. |
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