【作者】 杨鹰；

【导师】 史常旭；

【作者基本信息】 第三军医大学 ， 妇产科学， 2007， 博士

【摘要】 卵巢癌(ovarian carcinoma)发病率在妇科恶性肿瘤中仅次于宫颈癌,具有起病隐匿、早期不易发现、易转移、预后差等特点,其发生机制及治疗的探索一直是生物医学研究的热点。乙酰肝素酶是一种内糖苷酶,可降解细胞外基质(extracellular matrix,ECM)和基底膜(basement membrane,BM)中的硫酸乙酰肝素(heparin sulfate,HS),在恶性肿瘤细胞中乙酰肝素酶普遍存在,卵巢癌中乙酰肝素酶的表达及与患者临床特征的关系值得进一步探索。实体肿瘤的生长必须依赖持续和广泛的肿瘤血管生成,新生血管为肿瘤组织提供营养物质和氧气,又是肿瘤组织发生转移的重要途径。血管内皮细胞生长因子通过与3种选择性表达在血管内皮细胞上的高亲和力酪氨酸受体VEGFR-1(FLT-1)、VEGFR-2(FLK-1)及VEGFR-3(FLT-4)结合,引起一系列的信号传导,促进血管内皮细胞的增殖与迁移,最终引起新生血管形成,在肿瘤的发生、发展以及转移中发挥重要作用。RNAi技术是一种比较新的生物医学研究方法,将与信使RNA(messenger RNA,mRNA)对应的正义RNA(sense RNA)和反义RNA(anti-senseRNA)组成的双链RNA(double strand RNA,dsRNA)导入细胞,可以使mRNA发生特异性降解,导致相应基因的沉默,这种转录后基因沉默机制(post-transcriptional gene silencing,,PTGS)被称为RNA干扰(RNA interference,RNAi)。RNAi技术将被广泛应用到功能基因组学、基因治疗学、新药开发研究等众多领域。姜黄素有重要的经济价值和广泛的药理作用,如抗氧化、抗炎、抗动脉粥样硬化、降血脂等,是一种具有良好发展前景的抗癌药物,其抗肿瘤作用日益引起人们的重视,成为研究的热点。本研究拟观察乙酰肝素酶在卵巢癌血管生成中的作用及姜黄素对卵巢癌微血管内皮细胞生长的影响。本实验分四部分:第一,选择65例经病理确诊的卵巢癌患者,采用免疫组化方法检测癌组织中乙酰肝素酶、VEGF、VEGFR1和F8因子的表达情况,探寻乙酰肝素酶表达与卵巢癌临床病理特征及肿瘤血管生成的关系。第二,进行卵巢癌微血管内皮细胞原代培养,观测其细胞生物学特性,采用免疫荧光方法及RT-PCR方法检测卵巢癌微血管内皮细胞乙酰肝素酶表达情况。第三,进行卵巢癌微血管内皮细胞乙酰肝素酶表达的RNA干扰实验,检测卵巢癌微血管内皮细胞形态变化、细胞活性、乙酰肝素酶基因mRNA水平及蛋白表达,VEGF蛋白表达及血管内皮细胞增殖和凋亡的变化。第四,在卵巢癌微血管内皮细胞中加入不同剂量的姜黄素共培养,观察卵巢癌微血管内皮细胞存活率、细胞增殖率等变化,采用免疫荧光染色和RT-PCR法检测血管内皮细胞中乙酰肝素酶表达变化,探讨姜黄素对卵巢癌微血管内皮细胞的影响。主要结果及结论如下:第一部分:卵巢癌乙酰肝素酶表达特征及与血管生成关系研究1.12例正常卵巢组织中乙酰肝素酶无阳性表达,阳性率为0%,65例卵巢癌中乙酰肝素酶阳性51例,阳性率为78.5%,两组乙酰肝素酶表达差异非常显著。提示卵巢癌中乙酰肝素酶表达具有肿瘤特异性。2.＜50岁组27例中21例乙酰肝素酶表达阳性,阳性率为77.8%,≥50岁组38例中30例阳性,阳性率为78.9%,无显著性差异。浆液性囊腺癌30例中25例乙酰肝素酶表达阳性,阳性率为83.3%,粘液性囊腺癌21例中16例阳性,阳性率为76.2%,其他类型14例卵巢癌腺癌中10例阳性,阳性率为71.4%,差异不显著。提示卵巢癌乙酰肝素酶表达与卵巢癌组织类型及患者的年龄无相关性。3.早期卵巢癌组织12例中乙酰肝素酶阳性3例,晚期卵巢癌组织53例中乙酰肝素酶阳性48例,组间差异显著。高分化卵巢癌5例中乙酰肝素酶阳性表达1例,中分化卵巢癌15例中乙酰肝素酶阳性表达6例,低分化卵巢癌45例中乙酰肝素酶阳性表达44例,组间差异显著。提示卵巢癌乙酰肝素酶表达与卵巢癌病理分级有显著相关性,分化越低的卵巢癌,乙酰肝素酶表达越强;卵巢癌乙酰肝素酶表达与卵巢癌临床分期有显著相关性,晚期卵巢癌乙酰肝素酶表达强于早期卵巢癌。4.VEGF表达强度在卵巢癌患者不同年龄组中无显著区别,在卵巢癌不同组织学类型中无显著差异,临床晚期卵巢癌中VEGF表达强度高于早期卵巢癌,高中分化卵巢癌和低分化卵巢癌组VEGF表达强度显著不同,以低分化癌组表达为高。卵巢癌乙酰肝素酶表达强度变化与VEGF表达强度呈相同趋势,统计分析表明,卵巢癌VEGF表达强度与乙酰肝素酶表达强度呈正相关,相关系数为0.647。5.卵巢癌不同年龄组及不同组织学类型中VEGFR1表达强度无显著区别,临床晚期卵巢癌VEGFR1表达强度显著高于早期卵巢癌,低分化卵巢癌VEGFR1表达强度显著高于高中分化卵巢癌组。统计分析表明,卵巢癌VEGFR1表达强度与乙酰肝素酶表达强度呈正相关,相关系数为0.612。6.卵巢癌不同年龄组及不同组织学类型中MVD无显著区别,临床晚期卵巢癌MVD显著高于早期卵巢癌,低分化卵巢癌MVD显著高于高中分化卵巢癌。统计分析表明,卵巢癌MVD与VEGF、VEGFR1及乙酰肝素酶表达强度呈正相关,相关系数分别为0.754,0.713和0.669。提示乙酰肝素酶通过释放、激活VEGF来促进血管的生成,乙酰肝素酶和VEGF在卵巢癌新生血管生成过程中发挥协同的作用。第二部分:人卵巢癌微血管内皮细胞分离培养及乙酰肝素酶表达研究1.成功进行人卵巢癌微血管内皮细胞原代培养,细胞形态学观察见人卵巢癌微血管内皮细胞呈短梭形或多边形,细胞排列紧密,边界清晰。细胞活力测定人卵巢癌微血管内皮细胞活力为95%以上。卵巢癌微血管内皮细胞FⅧ因子免疫荧光化学检测,细胞胞浆为绿色荧光,证实为内皮细胞。为研究肿瘤血管新生机制及抗血管生成治疗提供了理想的体外实验模型。2.乙酰肝素酶表达免疫荧光染色见乙酰肝素酶主要表达于血管内皮细胞胞浆,胞核未见染色,OdMEC组乙酰肝素酶表达明显高于HUVEC组。RT-PCR检测结果可见OdMEC组乙酰肝素酶mRNA表达明显高于HUVEC组,提示乙酰肝素酶是OdMEC组内皮细胞区别于HUVEC组内皮细胞的主要生物学标志之一。第三部分:RNA干扰沉默乙酰肝素酶基因表达对卵巢癌微血管内皮细胞影响实验研究1.应用含绿色荧光蛋白的载体作对照转染,发现血管内皮细胞乙酰肝素酶siRNA转染效率可达到95%以上。提示乙酰肝素酶siRNA得到了高效转染。2.实验组以乙酰肝素酶siRNA转染血管内皮细胞,并于2、4、6、8d检测乙酰肝素酶表达,结果发现微血管内皮细胞乙酰肝素酶mRNA水平显著降低、蛋白水平显著降低(免疫荧光法、WB法)。提示乙酰肝素酶siRNA转染可显著抑制卵巢癌微血管内皮细胞乙酰肝素酶的转录及蛋白表达。3.乙酰肝素酶siRNA转染后血管内皮细胞VEGF的WB检测发现,实验组加入不同浓度SiRNA处理后,随着时间增加,VEGF表达降低,对照组无显著改变。提示乙酰肝素酶转录水平及蛋白表达受抑制后降低了VEGF的表达。4.MTT法检测细胞活力发现乙酰肝素酶siRNA转染后血管内皮细胞活力降低,随着时间增加,MTT检测吸光度值变化不大,而正常组和对照组随时间增加呈上升趋势。细胞增殖指数检测发现乙酰肝素酶siRNA转染后血管内皮细胞增殖指数显著下降。流式细胞仪检测发现乙酰肝素酶siRNA转染后血管内皮细胞凋亡指数显著上升。提示RNAi可有效沉默血管内皮细胞乙酰肝素酶表达,降低VEGF表达,抑制血管内皮细胞增殖,并诱导细胞凋亡,RNAi可能为肿瘤治疗提供新的途径。第四部分:姜黄素对卵巢癌微血管内皮细胞生物学特性及乙酰肝素酶表达的影响1.细胞形态观察见姜黄素组培养细胞增殖速度减慢,细胞生长密度减小,细胞出现皱缩,折光性减低,随姜黄素浓度增加可见细胞脱落、脱壁漂浮甚至破碎现象,72h可见细胞成大片脱落,有明显的时间和浓度依赖关系。提示姜黄素(2-250ug/ml)能显著抑制人卵巢癌微血管内皮细胞的活性,具有剂量和时间依赖性。2.细胞生长抑制率检测结果表明,姜黄素对卵巢癌血管内皮细胞抑制作用明显,与阴性对照比较差异显著,有显著的剂量依赖效应。姜黄素组对血管内皮细胞增殖指数影响检查可见姜黄素组细胞增殖指数显著下降,同时观察到G0/G1期前出现明显亚二倍体峰,即凋亡峰。提示姜黄素(2-250ug/ml)能显著抑制人卵巢癌微血管内皮细胞的增殖活性,降低增殖指数,具有剂量和时间依赖性。3.免疫荧光法检测内皮细胞乙酰肝素酶表达结果显示,姜黄素组内皮细胞乙酰肝素酶表达显著下降,有显著的剂量依赖抑制效应。提示姜黄素(2-250ug/ml)能显著降低人卵巢癌微血管内皮细胞乙酰肝素酶基因mRNA转录和蛋白的表达。4.细胞培养上清液MDA及LDH活力检测结果显示,姜黄素组细胞培养液上清LDH活性显著升高,MDA含量显著增加。提示高浓度姜黄素具有显著的细胞损伤效应。更多还原

【Abstract】 Background and AimsThe incidence of ovarian cancer is the second in the malignant tumor of department of gynecology,just next to uterine cervix cancer.It has some characteriscs such as earlier occurrence and metastasis.The development mechanisms and therapeutic study is the topic of biomedicine investigation.Heparanase is a kind of glucosidase,which can degrade heparin sulfate in extracellular matrix and basement membrane.Heparanase exist in malignant tumor widespreadly.The expression of heparanase in ovarian cancer and the relationship with clinical characteriscs of patients need more study.The growth of tumor depended on the persistence and extensive angiogenesis of tumor. New vessels not only provide tumor for nutrient substance and oxygen,but also is the important pathway of tumor metastasis.Vascular endothelial cell growth factor（VEGF） bind with receptorVEGFR-1,VEGFR-2 and VEGFR-3 on the cell membrane.The binding cause a serial signal conduction,which promote the proliferation and migration of vascular endothelial cell and neovascularization.VEGF play an important role in the occurrence, development and metastasis of tumor.RNAi technology is a new biomedical research method.Double strand RNA（dsRNA） consists of sense RNA and anti-senseRNA corresponding to messenger RNA（mRNA）.The mRNA was degraded after double strand RNA imported into cells.The result is the gene silenced.This technology is called RNA interference.RNAi technology will be used in functional gene study,gene therapy and new drug research widespreadly.Curcumine has significant economic value and extensive pharmacological action,such as antioxygen,anti-inflammatory,anti-artherosclerosis and degradation of blood fat. Curcumine is an anticancer drug.More attention was paid to its anti-tumor effect.The aim of this research is to observe the role of heparanase in the angiogenesis of ovarian cancer and the effect of curcumine on the growth of blood vessel endothelium cell from ovarian cancer.The study was divided into four parts.In the 1^st part,65 cases of ovarian cancer were included into the experiment. Immunohistochemistry staining was used to detect the expression of heparanase,VEGF, VEGFR1 and Factor 8 of ovarian cancer.The relationship of heparanase with ovarian cancer clinical pathological characteristics and with the tumor angiogenesis was investigated.In the 2^nd part,the blood vessel endothelium cell from ovarian cancer was primary cultured.The cell bionomics was observed.Immunofluorescence method and RT-PCR method were used to detect the heparanase expression in blood vessel endothelium cell from ovarian cancer.In the 3^rd part,the RNAi experiment of heparanase expression in the blood vessel endothelium cell from ovarian cancer was performed.The cell activity,heparanase mRNA level and protein expression,VEGF expression,cell proliferation and apoptosis were detected.In the 4^th part,curcumine of different dose was added to the the blood vessel endothelium cell from ovarian cancer.The cell survival rate,cell proliferation were observed.Immunofluorescence and RT-PCR were used to detect heparanase expression.The main results and conclusions were summarized as follows:The 1^st part:expression characteristic of heparanase in ovarian cancer and the relationship with angiogenesis1.In the 12 cases of normal ovary,heparanase expression was not observed.The poaitive rate was 0%.In the 65 cases of ovarian cancer,heparanase was positive in 51 cases.The positive rate was 78.5%.The heparanase expression difference was significant.2.In the group of 27 cases whose ages were younger than 50,heparanase expression of 21 cases were positive.The positive rate was 77.8%.In the group of 38 cases whose ages were older than 50,heparanase expression of 30 cases were positive.The positive rate was 78.9%.In the 30 cases of serous cystadenocarcinom,25 cases were heparanase expression positive.The positive rate was 83.3%.In the 21 cases of serous cystadenoma, 16 cases were heparanase expression positive.The positive rate was 76.2%.In the 14 cases of other type groupof ovarian cancer,10 cases were positive.The positive rate was 71.4%. There was not significant difference with them.It shows that there is not dependablity between heparanase expression and ovarian cancer tissue type and patients age.3.In the earlier period group of 12 cases of ovarian cancer,heparanase was positive in 3 cases.In the advanced stage group of 53 cases of ovarian cancer,heparanase was positive in 48 cases.The difference was significant.In the well-differentiated group of 5 cases, heparanase expression was positive in 1 case.In the moderately differentiated group of 15 cases,heparanase expression was positive in 6 cases.In the poorly differentiated group of 45 cases,heparanase expression was positive in 44 cases.The difference was significant.It shows that there was dependablity between heparanase expression and ovarian cancer tissue pathological grade and clinical stage.4.VEGF expression had not significant difference within patients of ovarian cancer of different age.There was not dependablity between VEGF expression and ovarian cancer tissue type.The VEGF expression of ovarian cancer in advanced stage was higher than that of earlier period.The VEGF expression of well-differentiated and moderately differentiated group was lower than that of poorly differentiated group.The VEGF expression of ovarian cancer had the same tendency as heparanase expression.They were positive correlation. The coefficient correlation was 0.647.5.VEGFR1 expression had not significant difference within patients of ovarian cancer of different age.There was not dependablity between VEGFR1 expression and ovarian cancer tissue type.The VEGFR1 expression of ovarian cancer in advanced stage was higher than that of earlier period.The VEGFR1 expression of well-differentiated and moderately differentiated group was lower than that of poorly differentiated group.The VEGFR1 expression of ovarian cancer had the same tendency as heparanase expression.They were positive correlation.The coefficient correlation was 0.612.6.MVD had not significant difference within patients of ovarian cancer of different age.There was not dependablity between MVD and ovarian cancer tissue type.The MVD of ovarian cancer in advanced stage was higher than that of earlier period.The MVD of well-differentiated and moderately differentiated group was lower than that of poorly differentiated group.The MVD ovarian cancer had the same tendency as VEGF,VEGFR1 and heparanase expression.They were positive correlation.The coefficient correlation was 0.754,0.713 and 0.669.It shows that heparanase and VEGF have congenerous effect in the angiogenesis of ovarian cancer.The 2^nd part:blood vessel endothelium cell isolated culture and heparanase expression observation of ovarian cancer1.The blood vessel endothelium cell of ovarian cancer was primary cultured.The cellactivity was above 95%.The 1FⅧimmunofluorescence detection was positive.The blood vessel endothelium cell of ovarian cancer can be used in the study of tumor angiogenesis.2.Heparanase was positive in the cytoplasm of blood vessel endothelium cell in the detection of immunofluorescence.The heparanase expression in the OdMEC group was higher than that in the HUVEC group.In the RT-PCR detection,the heparanase level in the OdMEC group was higher than that in the HUVEC group too.It shows that heparanase maybe the biological marker in the OdMEC.The 3^rd part:The effect of RNA interference on the heparanase expression of blood vessel endothelium cell of ovarian cancer1.The transfection rate of heparanase siRNA in blood vessel endothelium cell of ovarian cancer was above 95%.2.In the experiment group,the blood vessel endothelium cell of ovarian cancer was transfected by heparanase siRNA;the heparanase expression was detected at 2,4,6 and 8 day.The results showed that heparanase mRNA level and protein significantly decreased.It shows that heparanase siRNA transfection can significantly inhibit the heparanase of blood vessel endothelium cell of ovarian cancer.3.In the experiment group of the blood vessel endothelium cell of ovarian cancer transfected by heparanase siRNA,VEGF expression significantly decreased.It shows that heparanase siRNA transfection can significantly inhibit the VEGF expression of blood vessel endothelium cell of ovarian cancer.4.In the MTT detection of the experiment group,cell activity had not significant changes.but in the normal group and the control group,cell activity increased.In the experiment group,the proliferation index significantly decreased and apoptosis index significantly increased.It shows that RNAi interference can inhibit heparanase expression efficiently,and inhibit VEGF expression and cell proliferation too.The cell apoptosis was induced.RNAi maybe is one way for the treatment of tumor. The 4^th part:The effect of curcumine on the bionomics and heparanase expression of blood vessel endothelium cell of ovarian cancer1.In the cfurcumine group,cell proliferation step down and cell growth density decreased.With the dose increase of the curcumine,cells shed,floated or broke into pieces. It shows that curcumine（2-250ug/ml） can significant inhibit the activity of blood vessel endothelium cell of ovarian cancer dose dependently and time dependently.2.In the curcumine group,curcumine can inhibit the cell proliferation significantly.It shows that curcumine（2-250ug/ml） can significant inhibit the proliferation of blood vessel endothelium cell of ovarian cancer dose dependently and time dependently.3.In the curcumine group,the cell proliferation index decreased significantly.The subdiploid was observed.4.According to the immunofluorescence detection,the heparanase expression in the curcumine group decreased significantly.The effect was dose dependent and time dependent.It shows that curcumine（2-250ug/ml） can significant inhibit the heparanase expression of blood vessel endothelium cell of ovarian cancer.5.In the curcumine group,the MDA content and LDH activity of cell culture supernatant significant increased.更多还原