| Part I : Laser capture microdissection and the disposal of minimalRNAObjective: To find a feasible technique route , acquire pure human normalendometrial glandular epithelial cells and endometrial carcinoma cells by lasercapture microdisection(LCM) and apply in the research of endometrial carcinomarelevant gene's differential expression.Methods: Obtained pure endometrial glandular epithelial cells and endometrialcarcinoma cells from tissue frozen sections by applying LCM, then minimal RNAwas extracted;purified and concentrated, contral experiments were set up Todemonstrate whether the whole processes were capable of preserving the integrity ofRNA. The outcome RNA were measured by spectrophotometer (Nano DropND-1000).Results: 280,000 shottings of endometrial glandular epithelial cells and 196,000shottings of endometrial carcinoma cells was captured by LCM. The RNA integrityis good confirmed by control experiment I and II .The correlation between the LCMshootings and RNA quantity under arranged conditions was preliminary conformed.Conclusion: Laser Capture Microdissection can be apllied to successfully obtainpure objective cells from frozen sections. The integrity of RNA is good after LCMprocess and can be used in downstream experiment.epithelial cell;endometrial carcinoma cell.Part II: Screening, cloning and identificating of cDNA fragments of human endometrial carcinoma relevant genesObjective: To screen > clone and identificate the cDNA fragments of human endometrial carcinoma relevant genes and explore the molecular mechanism of cancer genesis.Methods: Compared LCM captured endometrium glandular cells RNA with endometrial carcinoma cells RNA by using fluoro differential display reverse transcription polymerase chain reaction (FDD-PCR), identified differentially expressed gene fragments that were specially relevant to endometrial carcinoma genesis. The selected fragments were cloned, sequenced and verificated by reverse northern blot analysis, positive fragments were BLAST analysed in Genebank . Results: 38 differential fragments were isolated, 3 of which were expressed more abundantly in normal endometrium and 35 were highly expressed in endometrial carcinoma. 10 fragments were recoverd,cloned and sequenced confirmed by reverse northern blot analysis, 6 fragments were positive. BLAST analysis showed :T1.1 was homologous to cyclin —dependent protein kinase 7( CDK7, 99%);LI .9 was homologous to protein phosphatase 1 regulatory (inhibitor) subunit 12A (PPP1R12A,99%);LI.21 and LI.22 were homologous to cellular repressor of ElA-stimulated genes l(CERQ100%);LI.25 and LI.26 were homologous to solute carrier family 39 (zinc transporter) member 10(SLC39A10, above 98%). Conclusion.- Gene fragments relevant to endometrial carcinoma were obtained by applying LCM and FDD-PCR. It was the first time that the correlation between CDK7, PPP1R12A, CERG ,SLC39A10 and endometrial carcinoma were discovered at mRNA level, their role in cancer genesis molecular mechanism were discussed. CDK7, CERG ,SLC39A10 as new candidate oncogene, PPP1R12A as new candidate anti-oncogene, more deeper layer research about them should be carried out in the future .PartED: The study of CDK7 protein expression and its relationship to the clinical characteristics in human endometrial carcinomaObjective: To investigate the CDK7 protein expression in the endometrialcarcinoma and its relationship to the tumor clinical characteristics.Methods: The expression of CDK7 protein in 47 cases of endometrial carcinomatissues were analysed by applying immunohistochemical staining technique, thecontrol group involved 10 normal endometrium tissues. Statistics analysis wereperformed by SPSS software.Results: The CDK7 protein expression rate in endometrial carcima(74.5%) washigher than that in normal endometrium( %) (P<0.05) .No relation was foundbetween CDK.7 expression and patient's invasive depth and grade.Conclusion: CDK7 is obviously relavant to endometrial carcinoma and can beused as tumor molecular target marker to further research.
|
PDF Full Text Request