| Object: To investigate the role of human chorionic gonadotropin ? subunit(?-hCG)in the invasion and metastasis of epithelial ovarian cancer.Method: 1.The lentiviral vector carrying the target gene ?-hCG and the reporter gene GFP and the control lentivirus were infected with epithelial ovarian cancer cell lines SKOV-3 and ES-2,to built ?-hCG stably over-expressing group cells SKOV-3 lv-?-hCG?ES-2 lv-?-hCG and control group SKOV-3 lv-con?ES-2 lv-con.QRT-PCR and western blot were used to detect the expression of target gene.The expression of ?-hCG between over-expressing cell lines SKOV-3 lv-?-hCG?ES-2 lv-?-hCG and control cells SKOV-3 lv-con?ES-2 lv-con was analyzed by single-sample t-test.2.The cells of the experimental group and the control group were transplanted into the abdominal cavity of the 4-6 weeks old female nude mice respectively with the insulin needle to construct the abdominal tumor model.The number of transplanted cells per nude mice was 2×107 cells,and the total volume of cell suspension was 200 uL.There were 6 nude mice in each group.There were no operative causes of death,the normal status of nude mice was observed every 3 days,and the weight change of nude mice was recorded.The tumor formation and metastasis of the tumor cells in the abdominal cavity of the nude mice were observed with the live imaging system once a week.Anatomical nude mice were performed at 4-6 weeks after abdominal transplantation,The abdominal organs and thoracic lung tissues were observed in nude mice,and the ascites and traits were recorded,observe and record the tumor formation,morphology,infiltration and metastasis,calculate the number of abdominal tumor(> 2mm).Part of the tissue was cryopreserved and the other part was fixed with neutral formaldehyde and paraffin-embedded sections.The morphology of the cells was observed by HE staining.western blot detection of expression of ?-hCG two tumors,single-sample t test were used.The two groups of ascites,body weight,tumor formation and the number of metastases compared with Two independent sample t-test.P <0.05 for the difference was statistically significant.3.The cells of the experimental group and the control group were transplanted into one side of the ovarian of the 4-6 weeks old female nude mice respectively by multi-point microscopic injection to construct the ovarian orthotopic transplanted tumor model.The number of transplanted cells per nude mice was5×105 cells,and the total volume of cell suspension was 5uL.There were 6 nude mice in each group.There were no operative causes of death,the normal status of nude mice was observed every 3 days,and the weight change of nude mice was recorded.The tumor formation and metastasis of the tumor cells in the abdominal cavity of the nude mice were observed with the live imaging system once a week.Anatomical nude mice were performed at 4-6 weeks after abdominal transplantation,The abdominal organs and thoracic lung tissues were observed in nude mice,and the ascites and traits were recorded,observe and record the tumor formation,morphology,infiltration and metastasis,Calculate ovarian in situ tumor volume Volume =(long diameter × short diameter 2)/ 2.Part of the tissue was cryopreserved and the other part was fixed with neutral formaldehyde and paraffin-embedded sections.The morphology of the cells was observed by HE staining.western blot detection of expression of ?-h CG two tumors,single-sample t test were used.The two groups of ascites,body weight,ovarian in situ tumor volume and the number of metastases compared with Two independent sample t-test.P <0.05 for the difference was statistically significantResult:1.The SKOV-3 and ES-2 cell lines overexpressing ?-hCG were successfully constructed.The expression efficiency of green fluorescent protein(GFP)in each group was about 90%-95% under inverted fluorescence microscope.The expression rate of ?-hCG was detected by qRT-PCR,and SKOV-3 lv-?-hCG was 21283 times higher than that of SKOV-3 lv-con.ES-2 lv-?-hCG was 72.33 times higher than that of ES-2 lv-con,The statistical results were significantly different(P <0.01).Western blot was used to detect the expression of ?-hCG,SKOV-3 lv-?-hCG was 5.6 times higher than that of SKOV-3 lv-con.ES-2 lv-?-hCG was 4.6 times higher than that of ES-2 lv-con,The statistical results were significantly different(P <0.01).2.The animal models of ovarian cancer in nude mice were successfully constructed,and was confirmed that ?-hCG promoted the invasion and metastasis of ovarian cancer in nude mice intraperitoneal xenograft model.After transplanted tumor in nude mice,the skin was dry and the activity was significantly reduced,the weight loss,the action was slow,the weight gain was slowed down and the cachexia and other cachexia.The fluorescence values of each group were calculated by living imaging system.The experimental group SKOV-3 lv-?-hCG?ES-2 lv-?-hCG was higher than the control group SKOV-3 lv-con?ES-2 lv-con.SKOV-3 lv-con group(46.88 ± 8.23),SKOV-3 lv-?-hCG group(114.6 ± 7.42).The difference of fluorescence between the two groups was statistically significant(P<0.001).ES-2 lv-con group(20.33 ± 5.292),ES-2 lv-?-hCG group(79.81 ± 4.05).The difference of fluorescence between the two groups was statistically significant(P<0.001).Comparison of SKOV-3 lv-con group and SKOV-3 lv-?-hCG group weight after nude mice transplanted tumor model,the weight of them were SKOV-3 lv-con(18.61 ± 0.54)and SKOV-3 lv-?-hCG(18.37 ± 0.27),the difference has no statistically significant(P=0.15).The tumor formation rate of both groups was 100%.Respectively,only two nude mice with a small amount of ascites formation,are 2/6.Comparison of the number of tumor metastases in both groups,the number of them were SKOV-3 lv-con(3.50 ± 0.22)and SKOV-3 lv-?-hCG(6.17 ± 0.79),the difference was statistically significant(P <0.05).And compare the number of tumor formation in both groups,each of them were SKOV-3 lv-con(24.67 ± 1.09)and SKOV-3 lv-?-hCG(48.17 ± 3.75),the difference was statistically significant(P <0.05).Comparison of ES-2 lv-con group and ES-2 lv-?-hCG group weight after nude mice transplanted tumor model,the weight of them were ES-2 lv-con(17.69 ± 0.33)and ES-2 lv-?-hCG(17.83 ± 0.51),the difference has no statistically significant(P=0.37).The tumor formation rate and ascites formation rate of both groups were 100%.It was chylous ascites.Take ascites smear,HE staining morphologically identified.Comparison of two groups of ascites volume,the volume of them were ES-2 lv-con(1.07 ± 0.06)and ES-2 lv-?-hCG(1.09 ± 0.05),there were no statistically significant(P=0.63).Comparison of the number of tumor metastases in both groups,the number of them were ES-2 lv-con(3.67 ± 0.33)and ES-2 lv-?-hCG(6.33 ± 0.92),the difference was statistically significant(P <0.05).And compare the number of tumor formation in both groups,each of them were ES-2lv-con(33.00 ± 0.93)and ES-2 lv-?-hCG(58.33 ± 2.50),the difference was statistically significant(P <0.05).3.The animal model of orthotopic transplanted ovarian cancer in nude mice was successfully constructed,and was confirmed that ?-hCG promoted the invasion and metastasis of ovarian cancer in nude mice orthotopic transplanted tumor model.After transplanted tumor in nude mice,the skin was dry and the activity was significantly reduced,the weight loss,the action was slow,the weight gain was slowed down and the cachexia and other cachexia.The fluorescence values of each group were calculated by living imaging system.The experimental group SKOV-3 lv-?-hCG?ES-2 lv-?-hCG was higher than the control group SKOV-3 lv-con?ES-2 lv-con.SKOV-3 lv-con group(42.18 ± 16.14),SKOV-3 lv-?-hCG group(94.76 ± 15.24).The difference of fluorescence between the two groups was statistically significant(P<0.001).ES-2 lv-con group(23.56 ± 3.60),ES-2 lv-?-hCG group(97.04 ± 8.26).The difference of fluorescence between the two groups was statistically significant(P<0.001)Comparison of SKOV-3 lv-con group and SKOV-3 lv-?-hCG group weight after nude mice were orthotopic transplantation,the weight of them were SKOV-3 lv-con(19.20 ± 0.41)and SKOV-3 lv-?-hCG(18.94 ± 0.26),the difference had no statistically significant(P=0.37).The tumor formation rate of both groups was 100%,but both of them have no ascites formation.Comparison of the number of tumor metastases in both groups,the number of them were SKOV-3 lv-con(2.33 ± 0.21)and SKOV-3 lv-?-hCG(4.33 ± 0.76),the difference was statistically significant(P <0.05).And compare the ovarian in situ tumor volume,each of them were SKOV-3 lv-con(2.57 ± 0.25)and SKOV-3 lv-?-hCG(2.93 ± 0.32),the difference has no statistically significant(P=0.64).Comparison of ES-2 lv-con group and ES-2 lv-?-hCG group weight after nude mice were orthotopic transplantation,the weight of them were SKOV-3 lv-con(18.04 ± 0.24)and SKOV-3 lv-?-hCG(18.53 ± 0.45),the difference had no statistically significant(P=0.20).The tumor formation rate of both groups was 100%,Respectively,only two nude mice with a small amount of ascites formation,are 2/6.Comparison of the number of tumor metastases in both groups,the number of them were ES-2 lv-con(2.50 ± 0.22)and ES-2 lv-?-hCG(4.67 ± 0.80),the difference was statistically significant(P <0.05).And compare the ovarian in situ tumor volume,each of them were ES-2 lv-con(2.56 ± 0.25)and ES-2 lv-?-hCG(2.71 ± 0.29),the difference has no statistically significant(P=0.73).4.Western blot detection In intraperitoneal xenograft model,SKOV-3 lv-?-hCG group was 3.1 times higher than SKOV-3 lv-con group,and ES-2 lv-?-hCG group was 2.4 times higher than that of ES-2 lv-con group;In ovarian orthotopic transplanted tumor model,SKOV-3 lv-?-hCG group was 2.9 times higher than SKOV-3 lv-con group,and ES-2 lv-?-hCG group was 1.4 times higher than that of ES-2 lv-con group.Statistical results were all significantly different(P<0.01)5.Pathological examination Intraperitoneal xenograft model of ovarian and orthotopic xenograft model of ovarian tumor metastasis and various tissues were stained with HE.Microscopic observation showed normal ovarian epithelial cells have been replaced by tumor cells.Situ tumors and metastases of massive diffuse into tablets or slug arranged,Large nuclei and deep staining,cell differentiation is low.SKOV-3 cell model was constructed in ovarian and metastatic tissues,HE staining was observed in line with human ovarian serous adenocarcinoma.ES-2 cell model was constructed in ovarian and metastatic tissues,and HE staining was observed in line with human ovarian clear cell carcinoma.Conclusion: 1.Intraperitoneal injection of 2 × 107 GFP-carrying EOC cell model,can successfully build a dynamic detection of nude mice transplanted tumor model,the model can fully simulate the biological characteristics of intraperitoneal implantation of ovarian cancer,it is suitable as an in vivo model for the study of human ovarian cancer.2.After inhalation of isoflurane anesthesia,middle of the back vertical incision into the abdominal cavity,then micro multipoint injection of GFP-carrying EOC cell model,this method can be successfully constructed dynamic observation of ovarian orthotopic transplantation tumor in nude mice animal models.This animal model is similar to the biological characteristics of human ovarian cancer and is suitable as an in vivo model,to study the development of human ovarian cancer.3.?-hCG can promote the invasion and metastasis of human epithelial ovarian cancer... |
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