| Gestational trophoblastic diseases (GTD) include the abnormal pregnancy, hydatidiform mole (HM), invasive mole, the overtly malignant tumors, choriocarcinoma and placental site trophoblastic tumor. The molecular pathogenesis of GTD remained largely unknown. Normal pregnancy can develop to hydatiditform mole, which can be transformed further to the malignant invasive mole. Choriocarcinoma is consequently evolved from invasive mole. This whole course is highly similar to the cancinogenesis of epithelial tumor. Therefore the developing course of GTD is of the characters of unlimited proliferation, transformation, and multi-step evolvement like most other tumors. It is not one gene or several genes that abnormally express and cause this transformation course.The technology of gene chip is a new technique of gene analysis, going with practicing human genome project. Due to its low cost, high sensitivity and high flux, gene chip is one of the important tools for the study of functional genome, which is obviously better than the previous research model of single gene. The network mechanisms of gene expression regulation are thoroughly studied in the level of whole genome using gene chip.The technique of antisense oligodeoxynucleotides (ASODNs) is one of gene therapies through specifically decreasing the gene expression. ASODNs are used to study the physiological function of the specifically protein and gene as a tool. ASODNs also provide us an important method to down-regulate the gene expression.To identify relevant genes important in the pathogenesis of GTDs, we firstly use microarray (one of gene chips) to identify genes differentially expressed in HM versus normal villi and choriocarcinoma cells versus normal primary culture trophoblasts. Then for validation of microarray results, the traditional methods of RT-PCR, immunoblot, and immunohistochemistry are used. Finally, for the identification of novel therapeutic targets, we investigate whether ASOND-mediated reduction in themRNA level of genes which have the important effect on tumor biological behaviors may resulted the decreased proliferation of choriocarcinoma cells in vitro.Part one Screening of associated genes of hydatidform mole and choriocarcinoma cellsMethods: The differential expressions of 4096 genes were analyzed in two pairs of the tissues of hydatidiform mole versus normal villi, and in two pairs of normal primary culture trophoblasts versus JAR cell line of chariocarcinoma, using cDNA microarray.Results: 89 genes were found differently expressed in all hydatidiform moles, counting for 2.2% of total genes. Among them, 24 genes were up-regulated and 65 genes were down-regulated. Compared with normal primary trophoblasts, 433 genes were up-regulated and 380 genes down-regulated in JAR cells. 46 genes were up-regulated in both hydatidiform mole(HM) and choriocarcinoma, while 13 genes were down-regulated. Genes associated with the inhibition of cell proliferative were significantly down-regulated, whereas genes associated with cell proliferation, malignant transformation and tumor metastasis and drug resistance were up-regulated.Part two Validation of microarray results and mechanism investigation of trophoblastic hyperplasiaMethods: The expressions of the up-regulated genes in microarray analysis, including small subunit ribonucleotide reductase (RRM2), thymidine kinasel (TK1), subunit 2 of replication factor C (RFC2), CyclinBl were examined by immunohistochemistry, immunoblot and RT-PCR in 15 cases of normal villi, 20 cases of fresh tissuses of hydatidiform mole, 98 cases of archived tissuses of GTDs, JAR and JEG-3 cell lines of Choriocarcinoma. Proliferating cell nuclear antigen (PCNA) was detected with immunohistochemistry in 98 cases of archived tissuses of GTDs.Results: Compared with normal villi and primary culture trophoblasts, the levels of mRNA and proteins of TK1 and RRM2 were significantly increased in hydatidiform moles, JAR and JEG-3 cells. The expression of RFC2 protein was also significantly higher. The expre...
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