| BACKGROUNDAccording to the Zhongshan University analyzed the current incidence of ovarian cancer in women of China’s high rate of6.1V10million, accounting for about5%of ovarian tumors. At present our country incidence of ovarian cancer is lower than that of developed countries in Europe, North America, but the statistical analysis, Shanghai and Hongkong in China and Guangzhou city was significantly higher than that of center city, the incidence of female life area rate is high, and the amount of fat in your diet, especially the animal fat content is high, so we and the developed countries the gap between the incidence rate decreased.90%-95%ovarian cancer in primary ovarian cancer, in addition to other parts of the5%-10%primary carcinoma metastatic to the ovary. Because the early symptoms of ovarian cancer is less, the lack of specificity, screening is limited, so it is difficult to diagnosis early, when seeing a doctor about70%have advanced. Therefore, although the incidence of ovarian cancer is lower than that of cervical cancer and endometrial cancer in third, malignant tumor, but the mortality rate is more than cervical cancer and endometrial cancer and, at the first maximum disease gynecological cancer, is a serious threat to women’s health. Therefore, early detection is the key for ovarian cancer. So how to early discovery, early sensitive marker of ovarian cancer, and the use of biological treatment is the focus of current research. Ovarian cancer is a complex process involving multiple genes, stage. The tumor gene activation and inactivation of tumor suppressor genes is the deciding factor of tumor growth of cancer gene. ARHI gene is one of tumor suppressor gene discovered in recent years, in the normal breast, ovarian, pancreatic tissues showed high expression. Our early2006test results showed down-regulated expression in ovarian tumor in ovarian cancer, the loss rate as high as67%. These tests show that ARHI participates in the genesis and development of ovarian cancer, we want to a further research on ARHI growth foundation increased the expression of ARHI on the assumption that can inhibit ovarian cancer cell.At present international unity,the treatment of ovarian cancer is the cytoreductive surgery, combined with chemotherapy. But for advanced ovarian cancer operation, often less than satisfactory, even can not be treated with operation, need to rely on postoperative adjuvant chemotherapy. With the wide application of chemotherapy resistance in ovarian cancer treatment, become a stumbling block. More study on resistance to chemotherapy in recent years, but many of them are limited to basic research or clinical medicine or other chemotherapy, radiotherapy sensitization. Therefore, a deeper understanding of the resistance, and the use of other biological genetic therapy of drug resistant ovarian cancer cell become a problem to be solved.ObjectiveThrough the establishment of ARHI gene transfection and stable expression in ovarian cancer cell line, detect the expression of ARHI gene, while exploring the growth test in nude mice ovarian cancer tumor before and after ARHI gene transfection; further study on impact resistance of ovarian cancer cell line cell cycle and invasion before and after transfection of ARHI gene.The study from the following three aspects.Part1.The expression of ARHI in ovarian cancer cell line A2780Method:The conventional culture of human epithelial ovarian cancer cell lines A2780and A2780/DDP, the expression of ARHI gene and protein in the two cell lines were determined using RT-PCR and Western blot method.Construct eukaryotic expression plasmid of pEGFP-C1-ARHI ARHI gene, liposome mediated human ovarian cancer cell line A2780transfected with A2780/DDP, culture in vitro, and transfection of pEGFP-Cl plasmid, untransfected cells as controls(transfection were named as ARHI/A2780group, ARHI/A2780/DDP group, pEGFP-C1/A2780/DDP group and A2780/DDP group. Application of RT-PCR, Western blot method to analyze the expression of ARHI gene and its protein.Using the paired T test A2780and A2780/DDP two cells ARHI gene difference. Analysis of A2780and A2780/DDP before and after contrast transfection gene difference by variance, and horizontal comparison of transfected ARHI/A2780group, ARHI/A2780/DDP group gene difference. So the results are less than0.05as the critical value of P.Result:1. The expression of ARHI mRNA and protein in A2780/DDP and A2780cell linesIn contrast to our earlier on the expression of ARHI in normal ovarian tissue and benign ovarian tumor tissues, in A2780and A2780/DDP two cell lines, ARHI has low expression and deletion. RT-PCR detection of A2780cells can be found with weak expression of ARHI, the average expression level was0.027±0.01, the loss of A2780/DDP expression, and preliminary experimental results are identical. Wsestern blot detected the expression of of A2780protein is deficiency in A2780/DDP group.2. The expression of ARHI mRNA in ARHI/A2780/DDP and ARHI/A2780ARHI gene transfection and stable expression of A2780, A2780/DDP cell lines, there were ARHI gene mRNA and protein expression by RT-PCR and Western blot detection of ARHI/A2780group, ARHI/A2780/DDP group, the average levels were0.086±0.01,0.081±0.01; and empty plasmid transfection group and no expression of ARHI gene and protein.For A2780/DDP group, ARHI expression was not detected, unable to T testing, so that the two groups of cells ARHI expression has significant difference. Paired T test was used to detect the A2780group before and after transfection mRNA gene and protein expression, the difference between the two groups was significant (p<0.05). Because no ARHI gene expression in A2780/DDP group was compared with that before transfection, with significant difference. Comparison of A2780and A2780/DDP cell lines transfected with stable, no significant difference after transfection (P>0.05).Conclusion:1.Ovarian cancer cell lines have low expression or deletion of ARHI gene in A2780, A2780/DDP. The deletion of ARHI gene is associated with cancer cell resistance.2.Using liposome transfection of eukaryotic ARHI plasmid, stably transfected cell clones were transfected successfully, which could increase the expression of target gene.Prat2. ARHI gene therapy combined with chemotherapy on the growth of ovarian cancer cell tumor A2780/DDP in nude mice Method:The first step in stably transfected ARHI/A2780/DDP group, pEGFP-Cl/A2780/DDP group and A2780/DDP group of three groups of cell lines in nude mice subcutaneously at the base, the right forelimb (axillary) injection of planting; each cell xenograft were randomly two small block A, B. The A packet for the detection of ARHI on ovarian cancer cells in nude mice tumorigenicity.The first step in the B group, low dose chemotherapy using cisplatin intervention tumor xenograft group, cisplatin pathways were divided into two groups by intraperitoneal injection, intravenous injection. Comparison of the effects of cisplatin on the proliferation ability of each cell line.Result:1. Establishment of ovarian cancer transplanted tumor animal modelGroup ARHI/A2780/DDP, group pEGFP-Cl/A2780/DDP and group A2780/DDP cells in nude mice subcutaneous injection, axillary, inoculation of10-12days; pEGFP-C1/A2780/DDP (n=8) group and A2780/DDP (n=8) group were all nude mice tumor formation, tumor formation rate was100%, tumor volume about control in about100mm3, the growth speed of tumor body. ARHI/A2780/DDP (n=8) group within12days after inoculation, only1nude mice tumor formation under your arms, but the tumor volume of about30mm3,30days have3nude mice tumor formation, early tumor increased to50mm3, the late two tumor volume is small, the naked eye observation is difficult. But no obvious tumor formation.2. The differences of ARHI expression between vector and ARHI gene transfection in vivo implantation tumorThe nude mice were randomly divided into A, B two groups. The disposal of group A nude mice, detection of ARHI/A2780/DDP group showed ARHI expression by the tumor immune group, other two groups of ARHI low expression or absence. There was statistical significance test of ARHI/A2780/DDP and the difference between the two groups (p<0.05). There is no obvious difference between the other two groups.3Compared the tumor growth after cisplatin therapyGroup B after each cell line in nude mice were randomly divided into two groups, one group received intraperitoneal injection of cisplatin treatment, a dose of1.35mg, single dose. A group of intravenous cisplatin injection, the dose of0.45mg, continuous use, a total of three times.7days later, the nude mice were killed, the nude mice were dissected axillary and abdominal cavity. The results showed, in ARHI/A2780/DDP group, pEGFP-Cl/A2780/DDP group and A2780/DDP group, the tumor had different degrees of degeneration and necrosis, volume and weight of tumor volume and weight is reduced. ARHI/A2780/DDP group and the other two groups showed significant differences (p<0.05). Three groups of nude mice were sacrificed after showed no obvious abdominal metastasis, no obvious ascites formation. There was no significant difference between the intraperitoneal injection group and the intravenous injection of group two (p>0.05).Conclusion:1.Stable transfection of the ARHI gene can inhibit the in vivo ovarian cancer formation and growth ability.2. Cisplatin combined with ARHI gene transfection can improve the inhibition of epithelial ovarian carcinoma in nude mice formed, transfection of ARHI gene can enhance the sensitivity of cancer cells to cisplatin. That the inactivation of tumor suppressor genes and resistance in ovarian cancer cells have a certain correlation. Part3. Exploration of ovarian cancer cell line cell A2780/DDP cycle signaling pathway and the mechanism of the invasion by ARHIMethod:In the first step of the three groups of stable transfection cell line ARHI/A2780/DDP group, pEGFP-Cl/A2780/DDP group and A2780/DDP basis, passage, according to the growth and invasion mechanism is as follows:1. PI flow cytometry staining method for the detection of ARHI gene and the empty vector on cell cycle of human ovarian cancer cell lines.2.the use of MTT and growth curve analysis of the effect of ARHI gene on the proliferation of ovarian cancer cells.3.Flow cytometry and PI effect of ARHI gene on apoptosis of ovarian cancer cell staining.4.The use of cell scratch test for detection of ARHI gene on migration of human ovarian cancer cells.5.Using the Wwstern blot method to detect the ARHI gene of PI3K/AKT signal transduction pathway and death associated protein kinase1(DAPK1) effect.Result:1.The effect of ARHI on A2780/DDP cell cycle Use the PI method,detect group ARHI/A2780/DDP, group pEGFP-Cl/A2780/DDP and group A2780/DDP cells, the result found:Gl cells by A2780/DDP51%, pEGFP-C1/A2780/DDP55%rose to69%in the ARHI group. Statistical analysis of ARHI group and the other significant difference between the two groups (p<0.05), no significant difference between two groups (p>0.05).2.The effect of ARHI on A2780/DDP cell hyperplasia MTT detected ARHI can obviously inhibit the growth of ovarian cancer cells. On the5day of the detection of A2780/DDP group and empty vector group cell growth of5-6times, group ARHI growth of only about3times. Analysis of the ARHI group and the other two groups had significant difference between A2780/DDP group statistics, empty vector group. 3. The effect of ARHI on A2780/DDP cell apoptosis PI staining was used to detect the apoptosis rate in the A2780/DDP group, empty vector group were11.1%,11.7%,29.9%in ARHI group. ARHI transfection significantly increased tumor cell apoptosis rate (p<0.05).4. The effect of ARHI on A2780/DDP cell migration Cell scratch test, cell migration rate in the A2780/DDP group, no significant difference between the empty vector group, but ARHI group cell migration rate decreased significantly (p>0.05).5. The effect of ARHI on pi3k/AKT and DAPK1Use the wester blot detect the effect,the result found ARHI group, DAPK1protein expression increase than in the A2780/DDP group, empty vector group, while P-AKT protein expression. Statistical analysis of differencesConclusion:1. ARHI gene can influence the cell cycle, preventing cell transformation from G1phase to S phase, thus blocking cell proliferation. ARHI gene leads to down-regulation of cellular proliferation, apoptosis increased;as while it lead to cell migration rate slows, invasive slowed tumor cells.Then.2. ARHI gene leads to down-regulate the expression of P-AKT protein and the up-regulation of DAPK1protein in cells, blocking PI3K/AKT signal transduction pathway.Innovation point1. The difference of ARHI gene in A2780/DDP and A2780cells is significantly.2. ARHI gene can inhibit the growth of A2780/DDP xenografts in nude mice through transfected ARHI gene by lipid eukaryotic cells.3. ARHI gene by blocking PI3K/AKT signal transduction pathway, inhibition of A2780/DDP cell proliferation and invasion.ARHI gene transfection can improve the tumor cell expression of tumor suppressor gene, inhibit tumor cell activity and tumorigenic ability. |
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