| Ovarian cancer due to its early symptoms are not obvious, is not easy to find, causing clinically found in advanced, and a secondary resistance to chemotherapy with paclitaxel and carboplatin have often occurred in the course of chemotherapy, the 5 year survival of patients with ovarian cancer is short. In the case of first-line chemotherapy drug resistance, how to effectively predict and reduce the sensitivity of drug resistant and sensitive chemotherapy has become an urgent problem to be solved.How to accurately determine the individual to first-line chemotherapy of primary and secondary drug resistance, to provide a personalized treatment program for different patients, increase the sensitivity to chemotherapy, improve the effectiveness of treatment, researchers have done a lot of work. Epithelial ovarian cancer in ovarian cancer accounted for more than 50%, but due to the occurrence and development of epithelial ovarian cancer is a multi gene, multi factors and form after a long time of complex process, therefore, the present research results and no incidence of ovarian cancer, to explain the formation of good theory of development and drug resistance. In the study, more and more evidences show that the change of cell apoptosis is closely related to the drug resistance of platinum drugs. P53 and mitochondria play an important role in the process of apoptosis. There are a number of studies show that the change of p53 gene with resistance to platinum drugs is closely related to the mitochondrial energy metabolism plays an important role in regulation of p53 objects and cells, mitochondrial D-loop region is the most prone to mutation region, but the p53 and D-loop region of mitochondrial gene mutation and the occurrence and development of ovarian cancer, drug resistance, drug sensitivity the relationship between the prognosis and the research is still less. This study was to study the relationship between p53 gene polymorphisms and the occurrence, development, drug resistance, drug sensitivity and prognosis of epithelial ovarian cancer in the D-loop gene and mitochondrial genome.In epithelial ovarian cancer chemotherapy drug choice, although many auxiliary drug to enhance the chemotherapeutic effect was found, but most clinical effect remains to be the test of time. In this study, although the sensitivity of ovarian cancer chemotherapy was controversial but in the clinical safety of 20 years of cheap, safe drug metformin as a sensitive agent to study. Many retrospective studies and in vitro studies showed that cells of metformin on breast cancer, pancreatic cancer, ovarian cancer and other tumor cell growth inhibition, but also some scholars to clinical statistics to draw the opposite conclusion. The emergence of this situation may be related to the inclusion criteria and the number of specimens, but also may be related to ethnic, geographical differences, the selection of cell lines. Therefore, it is necessary to study whether metformin can play a role in enhancing the sensitivity to chemotherapy of epithelial ovarian cancer in different regions, different individuals at the cellular level, and provide theoretical basis for clinical.In the experimental design, especially considering the ovarian carcinogenesis of ovarian tissue sampling and often difficult, almost impossible in two repeated sampling; therefore, the first hospital of epithelial ovarian cancer and non cancer patients with preserved ovarian tissue paraffin section as the research object, through the amplification and sequencing analysis the polymorphism of P53 and mitochondrial D-loop region and mutation; based on the collection of epithelial ovarian cancer and non cancerous ovarian tissues, clinical patients in paraffin, patients in the peripheral blood of free DNA, the difference of the three gene polymorphism in P53 and mitochondrial D-loop region.In the experimental design, taking into account the various commercial cell lines are often used in conventional research, its biological characteristics compared with in vivo primary cells changed greatly; therefore, the study of fresh tissue collected were cultured and passaged and artificial domestication of limited resistance, observation of different resistance mutations the situation, in order to help improve the clinical assessment of individual epithelial ovarian cancer treatment and prognosis. Study on the relationship between p53 and mitochondrial gene mutation by using the third passage cells in the treatment of metformin. And on this basis, the establishment of third generation of transplanted tumor cells in nude mice to observe the effects of passage notes, metformin for individual sources of transplanted tumor and provide a new theoretical basis for the further understanding of the relationship between P53 and mitochondrial D-loop gene polymorphism and ovarian cancer occurrence, development, prognosis, and provide a reference for the application of metformin in different gene polymorphism of epithelial ovarian cancer.Part 1 The relationship between p53, mitochondrial DNA mutations and polymorphisms and ovarian cancerObjectiveComparison of mutation by ovarian tissue to complete and clear clinical data of hospital in Henan area of normal ovarian tissues and ovarian cancer patients in cancer p53, D-loop DNA and polymorphism, according to the sequencing results combined with clinical secondary drug resistance, obtain the relationship among the population in Henan area p53, D-loop polymorphisms and mutation and ovarian cancer, drug resistance the obtained potential indicator of ovarian cancer and detection of cancer drug resistance detection index, provide a basis and reference for the clinical and follow-up tests.Method1.Differences in human ovarian tissue sections p53, mitochondrial genesIn The First Affiliated Hospital of Zhengzhou University(hereinafter referred to as the "the 1st Affiliated Hospital of Zhengzhou University") was selected to be the research object in the study of ovarian tissue sections of the patients who had lived in Henan for more than twenty years. The paraffin samples of 60 epithelial ovarian cancer tissues were collected for the first time, and the control group, 61 cases of ovarian cancer were non cancerous.1.1 Through the fixed slice thickness and area, obtain the equivalent ovarian tissue paraffin embedding, extraction and identification of these tissues DNA, comparison of paraffin embedded tissue storage time on the quality and quantity of the extracted DNA.1.2 For the realization of large-scale PCR sequencing and identification, optimization, traditional PCR amplification, sequencing identification, recovery, optimization of fluorescent quantitative PCR real-time monitoring with a fluorescent dye, reduce operation steps, avoid mistakes and pollution.1.3 The use of DNAMAN software to PCR product PCR product sequence, sequence comparison of epithelial ovarian cancer normal tissue specimens, if the nucleotide sequence of epithelial ovarian cancer tissues and normal tissues are diverse, as if only showed polymorphism; nucleotide sequence of epithelial ovarian cancer tissue and normal tissue specimen for consistency, mutation.2. Study on the differences of p53 and mitochondrial genes in peripheral blood, passage cell, ovary tissue and section2.1 The primary culture and subculture by red blood cell lysis of red blood cells, followed by 0.3% collagenase or combined with trypsin in 37 DEG C water bath in the reaction of 15min-30 min, then with 1640 containing 10% fetal bovine serum culture medium cell suspension cells and add the bottle. By using the characteristics of the fiber cell wall, the difference of the time of the adherent cells in vitro can be separated from the epithelial cells and fibroblasts. Select suitable low serum culture M199 in cultured epithelial cells(SLM) as a medium follow-up training, and add in ovarian epithelial cell medium growth promoting factor and prevent bacterial growth mycillin double anti culture. The cells grew to around 70% at the bottom with collagenase digestion and passage.2.2 Gene polymorphism:The patients with peripheral blood, the third generation of ovarian epithelial cells, fresh ovarian tissues and tissue sections of four samples were extracted from DNA, fluorescence quantitative PCR amplification and identification of the evacuation of Shanghai Shenggong sequencing using dye method, the sequencing results with DNAMAN software comparison.2.2.1 The relationship between peripheral blood DNA and epithelial ovarian cancer was studied by comparing the free DNA in peripheral blood of patients with epithelial ovarian cancer and non cancer before operation.2.2.2 The difference of gene polymorphism in peripheral blood of patients with free DNA, comparing each fresh ovarian tissues and ovarian tissue sections of the detection of peripheral blood free DNA instead of the possibility of detection and evaluation of tissue sections and two human ovarian tissue gene polymorphism and sensitivity differences.2.2.3 Through the comparison of the third passage cells and the gene polymorphism of the original cells, the changes of the related gene loci in the passage to the third generation were understood..Results1.Differences in human ovarian tissue sections p53, mitochondrial genesA total of 121 clinical ovarian tissue sections of DNA extraction, using real-time fluorescence quantitative PCR combined with melting curve to optimize and monitor the p53 gene and D-loop region PCR.1.1The extracted OD260/OD280 DNA were mostly in 1.7-2.0, and the extraction amount decreased with the increase of the storage time of the paraffin embedded tissue. There was no significant difference in the content of DNA in the normal human tissue section and the DNA content in the tissue sections of the tumor patients.1.2After several rounds of optimization, experimental verification, the optimum conditions of D-loop gene of different fragments of p53 gene and mitochondrial DNA amplification in 20 u L fluorescence quantitative PCR system to determine.By sequencing, the p53 gene was found to be 9, and 127 polymorphic loci were found in the gene of ovarian p53 in non cancer patients. Compared with the D-loop gene in the ovary of non cancer patients, the D-loop gene was found to be 11, and 165 were polymorphic.Exon 7 p53 in exon 44 codon mutation by C A can be detected in 41 positive samples, the detection rate was 68.3%; D-loop codon 73 mutation by A G were positive in 52 cases, the detection rate was 86.7%, the combined detection of 60 positive specimens of the whole detection.For sequence comparison and statistical analysis of recurrence within 1 years of patient samples for treatment of 60 cases of epithelial ovarian cancer patients, results showed that Exon4-72 G and D-loop309 T as recurrence specimens were detected if the detection rate is 17/21. If the Exon6-198 G, Exon8-67 A, D-loop309 T, D-loop446 A joint detection, the detection rate of recurrence of 21/21, but the non recurrence of specimens have 5 copies of the sentence is positive.2 the difference of p53 and mitochondrial genes in peripheral blood, passage cell, ovary tissue and tissue section2.1 normal cells in the first generation of cells filled with the bottom of the culture bottle at the bottom of the 21 area average of 70% days, when the average of 18 days of tumor cells. From the second generation to the third generation, normal ovarian cells covered the bottom of the culture area of 70% took an average of 15 days, the average time of 12 days of tumor cells. DAB staining was used to identify the epithelial cell specific surface antigen CK19 in the third cells. The results showed that the cells were positive. In 30 cases of ovarian cancer in 17 cases of ovarian epithelial cells cultured in vitro successfully and spread to the fifth generation, 15 cases of normal ovarian cells were cultured successfully in 7 cases and spread to the fifth generation.2.2 Results show :2.2.1 Patients peripheral blood free DNA content was 7.7 + 2.6ng/mL, in 17 samples of ovarian cancer, 2 patients in stage I 26.7 + 5.4ng/mL; 6 cases of II patients was 151 + 29ng/mL; 9 cases of III IV patients was 234 + 32ng/mL. The content of free DNA in peripheral blood of patients with epithelial ovarian cancer was significantly higher than that in non cancer patients.2.2.2 fresh ovarian tissue and paraffin embedded sections of p53 gene, D-loop gene mutation and polymorphism site detection efficiency agreement. The detection rate of free DNA in peripheral blood was slightly less than that in the first two groups. There were 4 polymorphic p53 loci, 2 D-loop region and 11 D-loop region. But for the exon 7 p53 in exon 44 codon mutation by C A and D-loop codon 73 mutation by A for both the combined detection of G, can achieve 17 of all positive specimens detected. Show p53 exon exon 7 codon 44 mutation by C A and D-loop codon 73 by A mutation G combination as a potential peripheral blood free DNA detection of ovarian cancer.2.2.3 third generations of cells compared with the original ovarian tissue of the new p53 gene mutation site 2, 11 polymorphic loci, the new D-loop region gene polymorphism of 4 loci, suggesting that the passage of genetic changes. But the detection index of polymorphism gene in epithelial ovarian carcinoma p53 gene, mitochondrial D-loop region detection index potential discovery and drug resistance of recurrence is not changed, can be used as a detection index of subsequent cell level research.ConclusionThe clinical diagnosis of 90 cases of patients with epithelial ovarian cancer and 76 cases of non cancer patients with ovarian tissue samples as the research object, through gene sequencing of the p53 gene, mitochondrial D-loop region, found the detection index of polymorphism of epithelial ovarian cancer p53 gene, mitochondrial D-loop region detection index and potential drug resistance recurrence. In 45 cases of ovarian tissue, disease tissue, peripheral blood and cultured ovarian epithelial cell samples showed further research, found that patients with epithelial ovarian cancer in the peripheral blood of free DNA content than non cancer patients increased significantly, paraffin section and peripheral blood free DNA can replace fresh ovarian tissue as a gene polymorphism detection object. Although the passage will cause changes of gene polymorphism, but the detection index of polymorphism gene in epithelial ovarian carcinoma p53 gene, mitochondrial D-loop region detection index potential discovery and drug resistance of recurrence is not changed, can be used as a detection index of subsequent cell level research.Part two: p53, mitochondrial DNA mutations in combination with metformin in ovarian cancerchemotherapy sensitizer relationsObjectiveThe ovarian cells cultured in vitro, the third generation cells were resistant to multiple drug resistance domestication, simulated phenomenon, and then observe the radiosensitizing effect of metformin in paclitaxel and carboplatin in the treatment of. By detecting p53 and D-loop gene polymorphisms, the relationship between p53, D-loop gene polymorphism and ovarian cancer drug resistance and metformin was detected. In animal models with ovarian tissue of patients with cell passage cells of the third generation model, and using the normal human blood concentration of drug sensitization simulated metformin in the normal medication of chemotherapy, the effects of p53 and D-loop gene polymorphism on the sensitizing effect.Method1 the effect of metformin on the growth of ovarian cancer cellsIn the first part of the second chapter, the third generations of the 17 malignant ovarian tissues were selected as the research subjects, and the SKOV3 cell lines were used as positive control. Each cell in the 5 x 105/mL and 1 x 105/mL two concentrations in the 96 hole cell culture plate on the 48 hole. 48 x105/mL hole cells were resistant to domestication, 1 x 105/mL as the control. In a short period of intermittent resistance domestication domestication, and in accordance with the resistance domestication with paclitaxel(0, 0.6, 1.2 g/mL), carboplatin(0, 10, 25 g/mL), metformin(0, 0.5, 2.5 g/mL) combination of different levels using the method of three factors and three levels orthogonal experiment is divided into 9 a group. In three after domestication, according to the orthogonal experimental group were added by the use of drugs and Domestication Cultivation of 72 h, observe the inhibition of the cells in the experimental group the rate by CCK8 color, of metformin in the sensitization effect of different chemotherapy in ovarian cancer.2 The enhancement effect of metformin on ovarian cancer cells in nude miceThe third generation of cells in 17 cases of canceration of ovarian tissue in the first part of the second chapter of the successful passage 5 times the suspension was injected subcutaneously to construct ovarian cancer xenografts in nude mice, compared with carboplatin and paclitaxel in combination with metformin, carboplatin and paclitaxel combination effect difference.The tumor diameter of not less than 6mm for modeling the standard of success will be successful modeling of each cell strain in nude mice were randomly divided into five groups: the first group with saline as control group; second as low dose group.At the end of the experiment, the mice were dissected, and the ratio of the size of the tumor volume was calculated according to the control group and the experimental group.Resu Lts1 The effect of metformin on the growth of ovarian cancer cells1.1 12 to 17 cells, strains were resistant to the drug. 9 of these 12 cell lines had the tendency of chemotherapy resistance.1.2 The detection rate of 11/12, which was detected by using Exon6-198G、Exon8-67A、D-loop309T、D-loop446 A in 12 cell lines. 8 strains were detected by Exon4-72G、D-loop309 T in 12 cell lines, which were all resistant strains, and the detection rate of resistant strains was 8/9.1.3 Metformin alone, 17 tumor cells and 7 normal cells were in the 72 h appear 2.72-3.56% ranging from inhibition, the inhibition of 3.24-6.83% appear in 120 h. Time dependent inhibition of metformin on cells, but not with the change in the amount of metformin, suggesting that in the two body concentration range, the inhibitory effect of metformin on the same cell, but long-term metformin will be better.2 The enhancement effect of metformin on ovarian cancer cells in nude mice17 cell models were successful in 13 strains, and finally the size and quantity of the tumor were 9 of the experimental requirements. Nude mice experiments showed that the cultured cells in 5 cells containing the drug resistance in the 9 groups(S9, S27, S29, S1, S17), of which 4(S27, S29, S1, S17) were resistant to platinum tendency, 2(S9, S27) with resistant tendency of paclitaxel(S9, 1), paclitaxel combined with platinum resistance), prone to paclitaxel and carboplatin sensitive cells 4. For paclitaxel and carboplatin combined with low dosage comparison, regardless of whether the drug resistance, there were significant positive correlation between the dose. For the control to add metformin in all dose groups of S11, S6, S19, S26 of four strains of paclitaxel and carboplatin in sensitive cells S19 and S26 showed insensitivity to metformin. That other cell lines in nude mice, low doses of paclitaxel and carboplatin in oral metformin on the cell inhibition rate increased significantly. The results were consistent with the experimental results of cell level..ConclusionMetformin is a reversal effect on drug resistance of ovarian cancer cells with Exon4-72 G, D-loop309 T, p53 E7-44 A and D-loop 73 G combined detection of ovarian cancer in the positive cells of polymorphism and easy to relapse, metformin is more sensitive to Exon6-198 G, Exon8-67 A, D-loop309 T, D-loop446 A gene in ovarian cancer cells can be used metformin sensitization. |
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