【作者】 李斌；

【导师】 丰有吉； 周先荣；

【作者基本信息】 复旦大学 ， 妇产科学， 2008， 博士

【摘要】 同源框基因HOXA10属同源异型盒家族HOXA族。研究显示,HOXA10参与女性苗勒氏管发育并在正常女性子宫、阴道上皮组织中表达。近年有资料显示,HOXA10不仅在孕早期子宫内膜上皮分化和间质蜕膜化中起重要作用,还可能参与调控源于异位子宫内膜的卵巢癌的发生。卵巢透明细胞癌(ovarian clear cell adenocarcinoma,OCCA)的细胞组织结构及免疫表型与子宫内膜及阴道的透明细胞癌极其相似,故目前多数学者认为卵巢透明细胞癌起源于苗勒氏管。由于卵巢透明细胞癌患者合并子宫内膜异位症(endometriosis,EMs)有高达25%—55%,一些学者提出卵巢透明细胞癌很可能来源于异位子宫内膜的恶变,非典型子宫内膜异位症(atypical endometriosis,AEMs)可能是癌前病变,是介于典型子宫内膜异位症与恶变的中间步骤。但是HOXA10在异位子宫内膜恶变和卵巢透明细胞癌形成过程中具有怎样的生物学影响至今尚未见文献报道。本研究分两部分:(1)观察同源框基因HOXA10在卵巢透明细胞癌和异位子宫内膜中的表达及其与临床预后的相关性分析;(2)应用脂质体转染技术观察HOXA10的表达对卵巢透明细胞癌ES-2细胞增殖、运动和侵袭的影响。第一部分HOXA10在卵巢透明细胞癌组织中的表达目的检测HOXA10在卵巢透明细胞癌、正常卵巢、正常在位内膜、卵巢内膜样囊肿及其对应的在位内膜组织中mRNA和蛋白水平的表达,比较它们的表达差异,并分析其与临床预后的相关性。方法应用RT-PCR法检测卵巢透明细胞癌、正常在位内膜、卵巢内膜样囊肿及其相应在位内膜组织中HOXA10基因的表达;应用免疫组织化学法检测卵巢透明细胞癌、正常卵巢、正常在位内膜、卵巢内膜样囊肿及其相应在位内膜石蜡组织中HOXA10蛋白表达。结果(1)卵巢透明细胞癌组织中可以检测到明显的HOXA10表达。卵巢内膜样囊肿上皮中未检测到HOXA10的表达,其相对应的在位内膜,随月经周期的不同有不同程度的表达,其中表达阳性的患者,月经周期多处于围排卵期,病理学检查证实均为分泌期子宫内膜;未合并内膜异位症患者的在位内膜与合并内膜异位症患者的在位内膜的HOXA10表达相似。(2) HOXA10蛋白的阳性表达与卵巢透明细胞癌患者的年龄及临床分期无关,但与患者的术后5年生存率呈负相关(R=-0.442,P=0.043)。结论HOXA10在卵巢透明细胞癌中的异常表达与患者的术后5年生存率密切相关,提示HOXA10基因可能参与卵巢透明细胞癌恶性进展。第二部分HOXA10基因对卵巢透明细胞癌ES-2细胞增殖、运动和侵袭的影响目的研究人同源框基因HOXA10对卵巢透明细胞癌ES-2细胞增殖、迁移和侵袭的影响。方法以脂质体稳定转染建立HOXA10表达的卵巢透明细胞癌ES-2细胞,并筛选高表达阳性克隆,应用Western blot方法检测转染效果。将ES-2细胞分为两组,转染空质粒pcDNA3组和转染质粒HOXA10-pcDNA3组,SRB法分别检测各组细胞HOXA10表达对ES-2细胞增殖的影响;采用细胞划痕试验、Transwell小室检测细胞迁移及侵袭的能力。结果(1)卵巢透明细胞癌ES-2细胞HOXA10转染效率检测,当质粒与脂质体的比例为1:2.5时可以得到较好的转染效率。(2) SRB法检测细胞增殖显示:HOXA10转染组与空载体组A值分别为0.446±0.0024和0.281±0.002(P＜0.01)。(3)细胞划痕5小时后,HOXA10转染组与空载体组的迁移距离分别为0.47±0.07mm和0.25±0.03mm(P＜0.05)。(4)Transwell小室检测HOXA10转染组和空载体转染组24小时跨膜细胞数分别为446±14个和227±12个(P＜0.01)。结论HOXA10N以通过促进细胞增殖,增加细胞的运动、侵袭能力参与卵巢透明细胞癌的恶性进展。以上两部分研究结果显示:(1)同源异型盒转录因子HOXA10在卵巢透明细胞癌组织中的异常表达与预后呈负相关;(2) HOXA10在卵巢透明细胞癌中的表达可以通过促进细胞增殖,增加细胞的运动、侵袭能力参与卵巢透明细胞癌的恶性进展。更多还原

【Abstract】 Homeobox gene HOXA10 belongs to HOXA cluster of homebox family. HOXA10 was reported to participat in the development of female gential tract and was found to express in normal endometrium and vagina muliebris. Recently,it has been found that HOXA10 may be involved in the carcinogenesis of ovarian carcinoma generating from endometriosis(EMs) besides its effect on endometrium epithelial differentiation and stroma decidualization.The cellular structure and immunophenotype of ovarian clear cell adenocarcinoma(OCCA) is very similar with those of endometrium and vagina clear cell adenocarcinoma.Most scholars considered OCCA came from the Muller’s duct.According to the high percentage(25%—55%) of EMs found in the OCCA patients,a part of OCCA was considered to come from the malignant EMs and atypical endometriosis(AEMs) which could be a kind of precancerous lesion.But little is known about the relationship between HOXA10 in malignant EMs and ovarian carcinogenesis.Our experiments are divided into two parts as follows:(1) observeing the expression of homebox gene HOXA10 in ovarian clear cell adenocarcinoma and endometriotic tissues and its correlative analysis of clinical prognosis.(2) Establishing HOXA10 expression ES-2 cell line,an ovarian clear cell adenocarcinoma cell line,and investigating the effects of HOXA10 on proliferation,motility and invasion.SectionⅠExpression of HOXA10 in ovarian clear cell adenocarcinomaObjective To examine the expression of HOXA10 in ovarian clear cell adenocarcinoma,normal ovarian,normal endometrial,ovarian endometriotic and corresponding endometrial tissues in mRNA and protein level.The relationships between expression of HOXA10 and its correlative clinical prognosis were analyzed.Methods RT-PCR was used to detect mRNA expression level of HOXA10 in ovarian clear cell adenocarcinoma,normal endometrial,ovarian endometriotic and corresponding endometrial tissues. Immunohistochemistry staining was used to investigate the expression of HOXA10 protein in ovarian clear cell adenocarcinoma,normal ovarian, normal endometrial,ovarian endometriotic and corresponding endometrial tissues.Results(1) The expression of HOXA10 gene was found in ovarian clear cell adenocarcinoma tissues but not in ovarian endometriosis.Different expression of HOXA10 was found in corresponding normal endometrium in different menstrual cycle.The menstrual cycle of positive expression patients were most in ovulation period and were confirmed to be in secretory phase by pathematology detection.HOXA10 expression of endometrium in patients without endometriosis was the same as that in patients with endometriosis.(2) HOXA10 gene expression level was positively correlated with the stage and grade of ovarian cancers(P＜0.05),but was negatively correlatated with five year survival rate (R=-0.442,P=0.043).Conclusions Overexpression of HOXA10 may be related to the tumorigenesis of epithelial ovarian cancer and canceration of EMs.SectionⅡThe effects of HOXA10 on proliferation,motility and invasion of ovarian clear cell adenocarcinoma ES-2Objective To evaluate the effects of HOXA10 on proliferation,motility and invasion of human ovarian clear-cell carcinoma ES-2.Methods A stable HOXA10-expressed ES-2 cell line was established and the high positive expression clone was screening out.Enhancement of HOXA10 expression was confirmed by Western blot.ES-2 cells were divided into two groups:HOXA10-ES-2 and pcDNA-ES-2.Cell proliferation of each group were evaluated by Sulforhodamine B assay.The motility and invasion of two groups were evaluated by Wound assay and transwell.Results(1) The stable HOXA10 expression clones of ES-2 cells were established by Lipofectamine transfection.The transfection efficiency was satisfactory when ratio between plasmid and Lipofectaminewas 1:2.5.(2) The difference of cell proliferation rates between HOXA10 transfected groups and negative control groups cell was significant(0.446±0.0024 vs 0.281±0.002,P＜0.01) by SRB assay.(3) Using wound healing assays,cell motility for HOXA10 transfection groups were faster than that of empty groups at the fifth hour after scratch.(0.47±0.07mm vs 0.25±0.03 mm,P＜0.05).(4) The invasion activity of HOXA10 transfection cells were significantly higher compared with those of control cells(446±14 vs 227±12,P＜0.01) by transwell.Conclusions HOXA10 could stimulate the carcinogenesis of ovarian clear cell adenocarcinoma by up-regulation proliferation,motility and invasion of cells.The present study has shown that(1) Homebox gene HOXA10 is over-expressed in ovarian clear cell adenocarcinoma.A reverse correlation between prognosis and HOXA10 expression exists in ovarian clear cell carcinoma.(2) HOXA10 could stimulate the carcinogenesis of ovarian clear cell adenocarcinoma by induce the proliferation,motility and invasion of cells.更多还原