【作者】 王蕾；

【导师】 戴淑真；

【作者基本信息】 青岛大学 ， 妇产科学（专业学位）， 2016， 博士

【摘要】 第一部分Htr A2/Omi在上皮性卵巢癌组织中的表达及临床意义目的检测Htr A2/Omi在上皮性卵巢癌组织及卵巢癌细胞株中的表达,探讨其与临床病理特征之间的关系。方法1.免疫组织化学法检测Htr A2/Omi在60例正常卵巢上皮组织及156例上皮性卵巢癌组织中的表达。2.Real-Time PCR法检测Htr A2/Omi m RNA在正常卵巢上皮组织及上皮性卵巢癌组织中的表达。3.分析Htr A2/Omi的表达与上皮性卵巢癌患者临床病理参数之间的关系。4.Real-Time PCR法和Western-blot法分别检测Htr A2/Omi m RNA及蛋白在正常卵巢上皮细胞株及卵巢癌细胞株中的表达。结果1.Htr A2/Omi在上皮性卵巢癌组织中的表达明显高于正常卵巢上皮组织,两者之间比较差异具有统计学意义(P<0.001)。2.Htr A2/Omi m RNA在在上皮性卵巢癌组织中的表达明显高于正常卵巢上皮组织,两者之间比较差异具有统计学意义(P<0.001)。3.Htr A2/Omi的表达与上皮性卵巢癌患者的FIGO分期、淋巴结转移密切相关(P<0.001),而与患者年龄、肿瘤分化程度无明显相关性(P>0.05)。4.Htr A2/Omi m RNA及蛋白在卵巢癌细胞株OVCAR-3和A2780中的表达均明显高正常卵巢上皮组织,差异具有统计学意义(P<0.001)。结论1.Htr A2/Omi在上皮性卵巢癌组织中呈高表达,在正常卵巢上皮组织中呈低表达。2.Htr A2/Omi的表达与与上皮性卵巢癌患者的FIGO分期、淋巴结转移密切相关,与患者的年龄及肿瘤分化程度无关。3.Htr A2/Omi在卵巢癌细胞株OVCAR-3、A2780中均呈高表达。第二部分Htr A2/Omi基因沉默对卵巢癌细胞OVCAR-3凋亡、侵袭和转移影响的实验研究目的利用RNAi技术沉默Htr A2/Omi基因在卵巢癌细胞株OVCAR-3中的表达,观察干扰前后OVCAR-3细胞凋亡和侵袭能力的变化。方法1.脂质体法转染人卵巢癌OVCAR-3细胞,Real-Time PCR法及Western-blot法检测Htr A2/Omi基因沉默对OVCAR-3细胞中Htr A2/Omi表达的影响。2.MTT法观察Htr A2/Omi基因沉默对OVCAR-3细胞凋亡的影响。3.Transwell侵袭小室法观察Htr A2/Omi基因沉默对OVCAR-3细胞侵袭能力的影响。结果1.RT-PCR结果显示:与si RNA-CN组及对照组相比,OVCAR-3/si RNA-Htr A2/Omi组Htr A2/Omi的m RNA表达下降了91%,较si RNA-CN组及对照组均有统计学差异(P<0.01)。Western-blot结果显示:si RNA-Htr A2/Omi明显抑制了OVCAR-3细胞Htr A2/Omi蛋白的表达,与si RNA-CN组及对照组相比,差异具有统计学意义(P<0.01)。2.MTT结果显示:OVCAR-3/si RNA-Htr A2/Omi细胞凋亡率较si RNA-CN组、对照组降低,从第3天开始其差异具有统计学意义(P<0.05)。3.Transwell侵袭实验结果显示:OVCAR-3/si RNA-Htr A2/Omi组细胞通过人工基底膜的数目与si RNA-CN组、对照组相比明显减少,差异具有统计学意义(P<0.01)。结论1.Htr A2/Omi基因沉默能降低卵巢癌细胞OVCAR-3的凋亡率。2.Htr A2/Omi基因沉默能增强卵巢癌细胞OVCAR-3的侵袭能力。第三部分Htr A2/Omi对OVCAR-3细胞裸鼠移植瘤生长的影响目的建立卵巢癌OVCAR-3细胞裸鼠移植瘤模型,将OVCAR-3/si RNA-Htr A2/Omi、OVCAR-3/si RNA-CN两种卵巢癌细胞分别接种于裸鼠皮下,观察两组裸鼠移植瘤生长情况、组织中细胞凋亡的检测及相关凋亡蛋白的表达情况。方法1.建立卵巢癌细胞裸鼠移植瘤模型:选用健康纯种的BALB/c-nude雌性裸鼠(18~20 g),鼠龄4-5周,在无菌SPF级的动物培养设备中饲养。60只裸鼠随机分为2组,实验组30只皮下注射OVCAR-3/si RNA-Htr A2/Omi细胞,对照组30只皮下注射OVCAR-3/CN细胞,建立裸鼠模型。2.观察裸鼠移植瘤成瘤情况:荷瘤裸鼠接种细胞后,每日观察裸鼠的一般状况,每周测量肿瘤结节的长径(a)和短径(b),并计算肿瘤的体积。连续饲养三周后脱臼处死裸鼠后剥除肿瘤称重。3.采用TUNEL法检测裸鼠移植瘤组织中细胞的凋亡情况。4.Real-Time PCR法和Western-blot法分别检测Htr A2/Omi及凋亡相关蛋白在裸鼠移植瘤中的表达。结果1.实验组裸鼠在接种后1周左右开始出现可触及的瘤体,之后瘤体生长迅速,接种3周以后,裸鼠体重增长缓慢,再之后裸鼠消瘦、体重下降、活动减少。对照组裸鼠于第三周开始出现可触及的瘤体,并且瘤体增长缓慢,裸鼠食欲好,体重增长较快,活动较多。3周以后所有被接种的裸鼠均有瘤体形成,并且全部存活。2.将裸鼠脱臼处死,剥除皮下移植瘤,测量肿瘤的长短径并称重,实验组肿瘤体积分别为1673±105.6mm3,对照组肿瘤体积分别为578±183.4mm3,实验组移植瘤体积均明显大于对照组,差异均具有统计学意义(P<0.001)。实验组瘤体重量分别为2.48±0.24克,对照组瘤体重量分别为0.91±0.25克,实验组瘤体重量明显大于对照组,差异有统计学意义(P<0.001)。3.TUNEL法检测结果表明,实验组移植瘤组织中棕色核细胞的数量相较于对照组而言较多,而且分布比较密集。实验组裸鼠移植瘤组织AI为6.04±0.19%,与对照组(AI=34.63±1.91%)相比,差异具有统计学意义(P<0.01)。4.应用Westem-blot检测裸鼠移植瘤相关凋亡蛋白的表达,结果显示:实验组Htr A2/Omi蛋白表达较对照组降低,XIAP蛋白表达降低,caspase-3、caspase-9活性片段表达降低。结论1.下调Htr A2/Omi的表达对OVCAR-3细胞裸鼠皮下移植瘤生长具有促进作用。2.下调Htr A2/Omi的表达可使裸鼠皮下移植瘤组织细胞的凋亡减少。3.在裸鼠体内下调Htr A2/Omi的表达通过下调XIAP的表达、抑制caspase-3、caspase-9的活性来抑制卵巢癌细胞的凋亡。更多还原

【Abstract】 Part one: the expression and clinical value of Hrt A2/Omi in epithelial ovarian cancerPurpose: Detection of Htr A2/Omi levels both in epithelial ovarian cancer tissue and cell lines and exploration its relationship with clinical pathological features.Methods: 1.Detection the expression of Htr A2/Omi in 60 cases of normal ovarian epithelial tissue and 156 cases of epithelial ovarian cancer tissue using immunohistochemical staining.2.Detection the expression of Htr A2/Omi in 60 cases of normal ovarian epithelial tissue and 156 cases of epithelial ovarian cancer tissue using real-time PCR.3.Analysis of the relationship between the expression of Htr A2/Omi and clinical pathological features in epithelial ovarian cancer patients.4.Detection the expression of Htr A2/Omi both in epithelial ovarian cancer tissue and cell lines using real-time PCR and immunohistochemical staining respectively.Results: 1.The expression of Htr A2/Omi in epithelial ovarian cancer tissue was significantly higher than normal ovarian epithelial tissue(P<0.001).2.The m RNA expression of Htr A2/Omi in epithelial ovarian cancer tissue was significantly higher than normal ovarian epithelial tissue(P<0.001).3.The expression of Htr A2/Omi was closely related with FIGO staging,lymph node metastasis,and had no obvious correlation with patient’s age and tumor differentiation degree(P<0.001).4.The m RNA and protein expression of Htr A2/Omi in ovarian cancer cell lines OVCAR-3 and A2780 were significantly higher than normal ovarian epithelial cells(P<0.001).Conclusions: 1.The expression of Htr A2/Omi in epithelial ovarian cancer tissue was higher than normal ovarian epithelial tissue.2.The expression of Htr A2/Omi was closely related with FIGO staging,lymph nodemetastasis,and had no obvious correlation with patient’s age and tumor differentiation degree.3.Htr A2/Omi was high expressing in both A2780 and OVCAR-3 cell lines.Part two: The experimental research of the influence of Htr A2/Omi gene silencing to apoptosis,invasion and metastasis in OVCAR-3-the ovarian cancer cellsPurpose: We observed its interference in cell apoptosis and invasive ability after silencing the expression of Htr A2/Omi in ovarian cancer cell lines-OVCAR-3 using RNA interference.Methods: 1.Detection the expression of Htr A2/Omi using real-time PCR and Western blot after transfecting OVCAR-3 cells.2.We observed the change of cell apoptosis using MTT method after Htr A2/Omi gene silencing.3.We observed the cell invasive ability using transwell invasive chamber after Htr A2/Omi gene silencing.Results: 1.Compared with si RNA-CN group and control group,the expression of Htr A2/Omi was significantly decreased in OVCAR-3/si RNA-Htr A2/Omi group in m RNA and protein level respectively(P<0.01).2.Compared with si RNA-CN group and control group,the apoptosis rate was significantly decreased starting from the third day in OVCAR-3/si RNA-Htr A2/Omi group(P<0.05).3.Compared with si RNA-CN group and control group,the number of cells passed artificial basement membrane were significantly decreased in OVCAR-3/si RNA-Htr A2/Omi group(P<0.01).Conclusions: 1.Gene silencing of Htr A2/Omi could decrease the apoptosis rates of OVCAR-3 cell lines.2.Gene silencing of Htr A2/Omi could increase the invasive ability of OVCAR-3 cell lines.Part three: the influence of Htr A2/Omi expression on growth of OVCAR-3 nude mouse transplantation tumorPurpose: We constructed subcutaneous transplantation tumor model by inoculated OVCAR-3/si RNA-Htr A2/Omi and si RNA-CN cells in nude mice respectively to observe tumor growth,cell apoptosis and the expression of apoptosis proteins.Methods: 1.Established subcutaneous transplantation tumor model: choosing healthy purebred nude female BALB/C mice(10-20 g,age 4-5 weeks),feeding in a germ-free SPF animal feeding equipment.And 60 nude mice were divided into 2 groups randomly,the experimental group was subcutaneously inoculated OVCAR-3/si RNA-Htr A2/Omi cells and the control group was subcutaneously inoculated si RNA-CN cells.2.Observed the general condition of nude mice daily and measured both of long and short diameters of tumor nodules weekly and calculated the volume of tumor after established the model.All nude mice were killed to weight the tumor after continuous three weeks of feeding.3.Detection of cell apoptosis rates in tumor tissue using TUNEL method.4.Detection the expression of apoptosis protein in subcutaneous transplantation tumor model using real-time PCR and Western blot respectively.Results: 1.Experimental nude mice began to appear palpable tumors and tumor grow rapidly after 1 week inoculation,slow growth in nude mice 3 weeks later and nude mice began to reduce weight and activity.While,control nude mice began to appear palpable tumors and tumor grow slowly after 3 week inoculation,and nude mice had a good appetite,weight growth faster and more activities.All nude mice were survived and administered successful after 3 weeks inoculation.2.All nude mice were killed and removed subcutaneous transplantation tumor to measuring the length and weight of tumor.The tumor volumes were 1673±105.6mm3 and 578±183.4mm3 respectively in experimental group and control group,and the tumor volume was significantly greater than control group(P<0.001).The tumor weights were 2.48±0.24 g and 0.91±0.25 g respectively in experimental group and control group,a nd the tumor volume was significantly greater than control group(P<0.001).3.TUNEL results show that the number of brown nuclear cells in experimental group greater than control group and the distribution densely.Compared with control group(34.63±1.91%),the AI in experimental group was 6.04±0.19% and had statistically significant difference(P<0.01).4.Compared with control group,the expression of Htr A2/Omi protein,XIAP protein,caspase-3 fragment and caspase-9 fragment were decreased in experimental group.Conclusions: 1.Down-regulation of Htr A2/Omi could promote tumor growth of OVCAR-3 nude mice subcutaneous transplantation tumor.2.Down-regulation of Htr A2/Omi could decrease cell apoptosis of OVCAR-3 nude mice subcutaneous transplantation tumor.3.Down-regulation of Htr A2/Omi could inhibit ovarian cell apoptosis by decrease the expression of XIAP,caspase-3 and caspase-9.更多还原