| Objective:It is well known that gastric cancer is the common malignant tumor of digestive tract. The mortality and incidence of gastric cancer is increasing. According to statistics, the five years of survival rate of domestic advanced gastric carcinoma is less than 30% mainly due to gastric carcinoma cells show great drug resistance to clinical used chemotherapy drugs, which shows poor treatment. The root cause is the multi-drug resistance (MDR) of cancer cells. With the development of the modern pharmaceutical technology, it is discovered that P-glycoprotein inhibitors can reverse the drug resistance of tumor tolerance at home and abroad, Tetrandrine (Tet) can reverse MDR, but the mechanism of action still need further research. Fluorouracil Sustained Release for Implant (FSRI) can increase the local concentration of chemotherapy to increase the effect of chemotherapy thus reduce the occurrence of adverse reaction.In this study,Fluorescence quantitative PT-PCR method and immunocytochemistry technique will be utilized to observe the anticancer effect of Tet combined with FSRI on tumor cell of mice orthotopic transplanted from gastric carcinoma cell in order to provide relative experiment and theoretical foundation for clinical gastric cancer treatment and MDR reversal of tumor cell.Methods:1 The method of orthotopic transplantation of gastric carcinoma cell is used. The fine growth state ones from Anabiosis MFC cell and in serial subcultivation are chosen to subcutaneously inoculate in 615 mice back. After six generations, MFC solid tumor blocks are inoculated in the stomach of 615 mice, thereby the model of 50 orthotopic implantation of gastric carcinoma on 615 mice is established.2 After that the 50 615 mice are randomly divided into five groups with 10 in each group. Then experimental drug intervention is carried out. The control group, physiological saline intraperitoneal injection; for others, one group is processed with experimental drug 5-fluorouracil with a dose of 50 mg/Kg by intraperitoneal injection every 6 days (Group 5-FU); one group is processed with Tet with a dose of 15 mg/Kg by intraperitoneal injection every 3 days (Group Tet); one group is processed with FSRI with a dose of 6 mg for each mouse by peritumoral into small incisions for once (Group FSRI); one group is processed with Tet and FSRI with a dose of Tet 15 mg/Kg by intraperitoneal injection every 3 days, at the same time 6 mg FSRI for each mouse by peritumoral into small incisions for once (Group Tet+FSRI). Three weeks later the tumor nodules are weighted. Rate of inhibition of tumor weight=(average tumor weigh of control group tumor-average tumor weigh of experimental group tumor)/average tumor weigh of control group tumor x 100%.3 Fluorescence quantitative PT-PCR method is adopted to detect gastric cancer tissue MDR1, Survivin mRNA expression of each group.4 Gastric cancer tissue P-gp protein and Survivin protein expression of each group is detected by SP immunohistochemical method.5 All the data are statistically analyzed with SPSS17.0. Measurement data are represented with mean and standard deviation (x±s), first for homogeneity test of variance between groups, then for one-way analysis of variance, pairwise comparison performed with SNK method, immunohistochemistry positive rate is checked by x2 test with P<0.05 being statistical significant.Results: 1 Copy 615 mice subcutaneous transplantation tumor and orthotopic transplantation tumor modelMouse gastric cancer cell line MFC is subcutaneous transplantated on the first generation 2 615 mice, whose back is inoculated of MFC cells suspension. When the tumor increased gradually to 10 mm in diameter, go down to next generation. Take subcutaneous transplantation tumor tissue block after six generations to fulfill mice orthotopic transplantation. About 8～9 days later, the mice model’s upper left abdomen can be felt about scleroma of about 4 mm in diameter which means orthotopic transplantation tumor building is successful. All 50 mice tumor building are thus successful.2 Comparison of groups of 615 mice behavior differences under the influence of gastric cancer cells in orthotopic transplantationWith transplanted tumor modules gradually increasing,615 mice in the control group are getting worse and worse, including eating less, drinking less, losing weight gradually, less activity, being unresponsive all of which shows dyscrasia. Group 5-FU, Group Tet and Group FSRI all show that mice lose appetite and weight, are less active and unresponsive to different degrees. While Group Tet+FSRI show that mice are relatively good, normally eating and dringking, accompanied by mild vomiting, but no obvious change in body weight, and reaction is more sensitive, etc.3 Volume comparisons of groups of orthotopic transplantation tumors It is found that the volume of orthotopic transplantation tumors in control group, Group 5-FU, Group Tet, Group FSRI, Group Tet+FSRI are respectively 881.50±59.57mm3,701.80±87.18mm3,399.40±67.53mm3, 342.60±50.45 mm3,89.60±13.11mm3, which shows statistical significance (P <0.05). The average tumor volumes of Group 5-FU, Group Tet, and Group FSRI are significantly less than the control group (P<0.05). The average tumor volumes of Group Tet and Group FSRI have no obvious difference, but are significantly lower than Group 5-FU (P<0.05). The average tumor volume of Group Tet+FSRI is the smallest one (P<0.05).4 The morphological characteristics of transplanted tumorThe transplanted tumor is presented in entity with uneven surface, rough envelope, and unclear surrounding tissue boundaries. The cut surfaces of the tumors in the control group are grey white without obvious necrosis of central tumors; under the light microscope the mottled necrotic lesions are observed. The cut surfaces of the tumors in Group 5-FU, Group Tet, Group FSRI, Group Tet+FSRI also are grey white without burnish, scattered necrosis lesions of the central tumors and residual tumor in edge parts observed. Under the optical microscope, large cell necrosis around the central tumors can be found, which still exist not fully necrosis of tumor cells. Cells volume are different and more nuclei abnormal change is found.5 Comparisons of tumor weight and the tumor weight inhibition rate in different groupsAfter the experiment, tumor weight in the control group, Group 5-FU, Group Tet, Group FSRI, and Group Tet+FSRI are respectively 2.16±0.12g, 1.93±0.35g,1.86±0.24g,1.84±0.17g,1.27±0.21g; tumor weight inhibition rate in the Group 5-FU, Group Tet, Group FSRI, Group Tet+FSRI are respectively 10.64%,13.88%,14.81%,41.20%; the average tumor weight of Group 5-FU, Group Tet and Group FSRI are obviously less than the control group (P< 0.05), but the average tumor weight of Group Tet and Group FSRI have no obvious differences, but are significantly lower than Group 5-FU (P< 0.05).The average tumor weight of Group Tet+FSRI are the smallest one (P< 0.05).6 Expression levels of MDR1 gene mRNA in 5 groupsIt is found that expression levels of MDR1 gene mRNA in 5 groups are obviously different. The relative expression level of MDR1 mRNA in the control group is 1.0404±0.0562; 3.4404±0.6827 in Group 5-FU; 0.5414±0.0270 in Group Tet; 2.3210±0.1982 in Group FSRI; 1.8454±0.3006 in Group Tet+FSRI. The comparison of expression levels of MDR1 gene mRNA in 5 groups is statistically significant (P< 0.05). The order of expression level of MDR1 mRNA in 5 groups from strong to weak is Group 5-FU, Group FSRI, Group Tet+FSRI, the control group, and Group Tet.7 Expression levels of Survivin gene mRNA in 5 groupsIt is found that expression levels of Survivin gene mRNA in 5 groups are obviously different. The relative expression level of Survivin mRNA in the control group is 1.0544±0.1442; 3.3746±0.4746 in Group 5-FU; 0.5360±0.0623 in Group Tet; 2.4096±0.1164 in Group FSRI; 1.868±0.3814 in Group Tet+FSRI. The comparison of expression levels of Survivin mRNA in 5 groups is statistically significant (P< 0.05). The order of expression level of Survivin mRNA in 5 groups from strong to weak is Group 5-FU, Group FSRI, Group Tet+FSRI, the control group, and Group Tet.8 Expression levels of P-gp protein in 5 groupsIt is found that expression levels of P-gp protein in 5 groups are obviously different. The relative P-gp protein expression rate in the control group is 1.0404±0.0562; 3.4404±0.6827 of the positive expression rate of P-gp protein in Group 5-FU; 0.5414±0.0270 of the positive expression rate of P-gp protein in Group Tet; 2.3210±0.1982 of the positive expression rate in Group FSRI; 1.8454±0.3006 of the positive expression rate in Group Tet+FSRI. The comparison of expression levels of P-gp protein in 5 groups is statistically significant (P<0.05). The order of P-gp protein expression levels from strong to weak in 5 groups is Group 5-FU, Group FSRI, Group Tet+FSRI, the control group, and Group Tet.9 Expression levels of Survivin protein in 5 groupsIt is found that expression levels of Survivin protein in 5 groups are obviously different. The positive expression rate of Survivin protein in the control group is 1.0544±0.1442; 3.3746±0.4746 in Group 5-FU; 0.5360±0.0623 in Group Tet; 2.4096±0.1164 in Group FSRI; 1.868±0.3814 in Group Tet+FSRI. The comparison of positive expression levels of Survivin protein in 5 groups is statistically significant (P<0.05). The order of Survivin protein expression levels from strong to weak in 5 groups is Group 5-FU, Group FSRI, Group Tet+FSRI, the control group, and Group Tet.Conclusion:1 Tet combining with FSRI could effectively improve physical state of mice of gastric carcinoma (including spirit, appetite, body weight, activities and the body’s reaction etc.).2 Tet combining with FSRI could obviously inhibit the growth of tumor cells.3 Tet combining with FSRI could effectively decrease the expression level of MDR1/P-gp and Survivin mRNA.4 Tet combining with FSRI could effectively decrease the positive expression rate of P-gp protein and Survivin protein, thus enhance the apoptosis of gastric carcinoma and inverse MDR. |
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