| Hydatidiform mole(HM) is a group of benign pregnant trophoblastic cell diseases which related to pregnancy. The average incidence of the disease is about 1/1000 in China. Being the potential malignancy, it may develop into invasive hydatidiform mole and choriocarcinoma. Up to date, the pathological mechanism of the development and neoplasia of HM has been not clear so far. However, It was believed that besides cellular genetic features, the activation of some oncogene and inactivation of tumor suppressor gene might participate the process. To research of function of the known hydatidiform mole-related gene is going up perfection and the novel elated gene and its function may be helpful to the development of the disease.A novel related gene F10 expressed highly in HM was detected and decreased cDNA library between normal villus early pregnant choriocarcinoma and HM tissues achieved by our study with suppression subtractive hybridization technique. A investigation of the features of F10 expression and its essential function was carried out with analysis of the gene silencing technique based on RNA interference (RNAi) in this study.Three topics are discussed in this dissertation:Chapter 1 Detection by In situ hybridization of gene F10 in the trophoblastic Diseases and other tumor tissues1.1 ExperimentalIn situ hybridization was used to study the expression of F10 in 12 cases of early pregnant placenta tissues ,12 cases of HM, 6 cases of invasive mole, 8 cases of choriocarcinoma, 7 cases of uterine intima, 6 cases of cervical carcinoma, 9 cases of ovaria adenocarcinoma, 6 cases of uterine intima cancer, 8 cases of breast cancer, 8 cases of hepatic cancer and 6 cases of gastric cancer those were diagnosed by HE staining pathologic diagnosis. The RNA probe was labeled with Degoxin firstly and then In situ hybridization was used to study the expression of F10.1.2 Results1.2.1 F10 mRNA was negative in 12 cases of early pregnant placenta tissues .F10 mRNA was positive in 12 cases of HM, 10 cases was medium strong positive, 1 cases was weak positive, 1 case was strong positive.1.2.2 F10 mRNA in cytoplasm of chorio-cell was positive in 6 cases of invasive HM and the strong positive rate reached 50%. F10 mRNA in cytoplasm with blue signal was positiv in 8 cases of choriocarcinoma and the strong positive rate reached 75%.1.2.3 The expression of F10 in hydatidiform mole, invasive mole, choriocarcinoma was crescendo trend (P<0.05) which indicated that F10 might participate the development of trophoblastic tumor.1.2.4 F10 mRNA was positive in ovaria adenocarcinoma, uterine intima cancer, breast cancer, hepatic cancer, gastric cancer and most of them were medium strong positive. There was no positive signal of F10 in uterine intima, cervical epithelia and para-tissue of hepatic cancer. The results of our study proved that gene F10 is one of its expression high expression gene which also was highly detected in other tumors such as ovaria adenocarcinoma, uterine intima cancer, breast cancer, hepatic cancer, gastric cancer. It indicated that F10 not only related to the development ofhydatidiform mole but also related to other tumors. It was indicated that there was no expression of FIO in these normal tissues.Chapter 2 Expression of FIO detected with Fluorescence quantitative PCR technique in the gestational trophoblastic tissue and HM as wellas other tumors2.1 ExperimentalFIO was detected and conformed with quantitative PCR technique in the gestational trophoblastic tissue and HM, and the total RNA was abstracted from fresh tissues of normal liver, normal spleen and endometrium of carcinoma, pimeloma and hepatiocarcinoma and its para-tumor tissue.2.2 Results2.2.1 The account of FIO in the HM was over 10-100 fold of copy of FIO in early, medium and advanced pregnant placenta tissues. It indicated that FIO was over expression in the HM.2.2.2 The number of copy of FIO in normal liver, spleen, respectively was 26 and 30 which was as same as that in normal villus, medium and advanced pregnant uterine. The number of FIO copy of uterine intima carcinoma , hepatic cancer and colorectal cancer was 167, 159, 145 respectively that though was lower than that in HM, it still was over 4-6 folds than that in normal liver and spleen.2.2.3 The standardized number of FIO copy was unequally ranged from 3.6 to 27.8 in4cases of hepatic para-tumor tissue, over expression in the another 6 of 8 cases (75%)with hepatiocarcinoma. The all standardized number of FIO copy was 1.5 times than para-tumor tissue, 5.7 and 4.9 fold in another cases. It indicated that the high expression of FIO in original hepatiocarcinoma.Chapter 3 Effection of FIO reticent to proliferation and apoptosisof choriocarcinoma3.1 ExperimentalDouble-stranded RNA (dsRNA) of FIO was created by transcription in vitro technique successfully. After transfection KLE and the FIO reticent conformed by fluorescence quantitative PCR technique, the KLE cellular growth curve was drown in MTT to observe the effection of the FIO to KLE cell growth. To detect the effection of reticent FIO to the changes of the cell growth circle and apoptosis with Flow cytometry.3.2 Results3.2.1 The number of control cells was decreased during 24 hours and then increased soon. It was doubled on the fourth day after inoculation than that on the first day. To transfection FIO dsRNA after 24 hours, the number of KLE was decreased significantly ( about 1/3 of inoculated cells), and this lower level was lasted during 72 hours. It stared to proliferate gradually till the fourth day but the speed was significantly lower that that of control group. So it was indicated that reticent FIO could obviously inhibit proliferation of KLE cell.3.2.2 It was founded that the rate of apoptosis were 15% and 4.1% in the experimental and control groups After 24 hours, the former almost was 3.7 folds than the later(P<0.05). After 48 hours, the rate of apoptosis reached 20.3% in the experimental group 4.14 folds than that (4.9%) in the control(P<0.05). It seemed that there was no significant difference between the two groups during 24 hour and 48 hours(P>0.05). It was indicated that apoptosis was stable after FIO reticent 1-2 days.Conclusions1. It was proved that FIO was an over expression gene in the hydatidiform mole based on the detection of FIO expression using in situ hybridization, fluorescent quantitative PCR technique in the hydatidiform mole.2. FIO mRNA were detected positive in all of hydatidiform mole, invasive mole choriocarcinoma , and the expression intensity was increased in hydatidiform mole, in invasive mole and in choriocarcinoma, The expression of associated new gene FIO of hydatidiform mole may related with expression and invasivesion of trophoblastic diseases.3. It was proved that FIO was an over expression gene in the ovaria adenocarcinoma, uterine intima cancer, breast cancer, gastric cancer and hepatic cancer. It was indicated that FIO was not only related to the development of hydatidiform mole ,but also related to many other tumors.4. It was proved that the proliferation of choriocarcinoma cell was remarkably inhibited after FIO gene silencing in vivo, It was indicated that FIO plays an important role un the proliferation of choriocarcinoma.5. Our data indicated that FIO gene silencing resulte in the increasing of apoptosis of choriocarcinoma cell.
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