【作者】 王莉；

【导师】 吴小华；

【作者基本信息】 河北医科大学 ， 外科学， 2008， 博士

【摘要】 卵巢上皮性癌(简称卵巢癌)恶性程度高,在妇科恶性肿瘤中死亡率最高,多数患者发现时已是晚期,虽然采取手术和化疗等治疗方法,晚期卵巢癌患者五年生存率仅30%,如能早期发现治疗,五年生存率高达90%。临床迫切需要敏感、特异的诊断和监测卵巢癌病情的指标和有效的治疗手段。间皮素(Mesothelin MSLN)是一种肿瘤分化抗原,通常仅表达在体腔表面的间皮细胞,在恶性胸膜间皮瘤、胰腺癌和卵巢癌等恶性肿瘤中过表达。间皮素基因编码69 kDa的前体蛋白,溶蛋白性裂解导致32 kDa溶解性片段巨核细胞强化因子(Megakaryocyte-potentiating factor MPF)释放入血或退化,剩余的40 kDa片段通过糖基磷脂酰肌醇锚定于细胞表面,即通常所指间皮素。间皮素可能通过与CA125的结合调节细胞间的粘附来促进卵巢癌腹膜转移,这种结合是N聚糖依赖性的相互作用,两者亲和力高且快。间皮素是CA125的配子,间皮素和CA125结合被阻断可能会抑制或延迟卵巢癌腹膜种植转移,可能成为新的治疗靶点来抑制肿瘤的播散。可溶性间皮素相关蛋白(Soluble mesothelin-related protein SMRP)结构与间皮素基本相同,只是羧基端没有锚定于细胞膜而释放入血,可以在肿瘤患者血清中检测到,可用于卵巢癌的诊断、疗效监测和预后。间皮素还是一种新的靶向治疗分子,一种间皮素特异性免疫毒素SS1P在动物实验中证实有效,应用于恶性间皮瘤、卵巢癌、胰腺癌的Ⅰ期临床实验的患者能够耐受其副作用。本课题组前期研究已经发现间皮素基因是卵巢癌组织中表达上调最显著的基因,随后证实间皮素和CA125的蛋白在卵巢上皮性癌组织高表达并与分级和组织学类型密切相关,并成功制备了可溶性间皮素单克隆抗体2H10,具有很高的特异性。但是间皮素的功能还不十分清楚。本课题拟进一步探讨上皮性卵巢癌中间皮素和CA125之间的共定位关系;在体内外水平研究间皮素在卵巢癌细胞生长转移中的作用;初步尝试利用间皮素基因沉默慢病毒对卵巢癌进行基因治疗;自行研发用于SMRP检测的ELISA试剂盒。第一部分间皮素和CA125在卵巢上皮性肿瘤中的表达及其生物学意义目的:研究上皮性卵巢癌细胞系及人体卵巢肿瘤组织中间皮素和CA125的表达及定位,探讨两者之间的共定位关系及生物学意义。方法:间接免疫荧光双标记染色法检测人卵巢上皮性肿瘤组织和卵巢癌细胞系中间皮素和CA125的定位表达。采用Western blotting检测间皮素蛋白在人卵巢癌细胞系中的表达水平。并检测卵巢癌细胞对人工基底膜材料Matrigel的粘附能力。结果:双标记免疫荧光细胞化学染色可见间皮素标记为红色荧光,主要分布在细胞膜,CA125标记为绿色荧光,主要分布在细胞膜,二者完全重叠呈黄色荧光,说明间皮素和CA125共定位表达。卵巢上皮性癌组织间皮素的蛋白表达荧光值(1639.2±181.92)显著高于交界性卵巢上皮性肿瘤(590.8±126.9)、卵巢良性上皮性肿瘤(227.69±26.54)和正常卵巢皮质表面上皮(213.0±24.37) ,差异有统计学意义(F=178.20,p<0.05);病理分级中G2、G3级的蛋白表达(1642.63±44.58;1701.57±54.73)显著高于G1级(1553.99±57.2)( F=10.01, p<0.05);组织学类型中浆液性囊腺癌和宫内膜样癌的蛋白表达(1723.81±33.20; 1673.81±70.27)显著高于粘液性囊腺癌(1466.12±94.93) (F=18.61,p<0.05);间皮素的蛋白表达与年龄、分期及血清CA125的水平均无显著相关性(p>0.05 )。CA125的蛋白表达与间皮素的蛋白表达存在显著一致性(r=0.95, p<0.05)。卵巢癌细胞间皮素蛋白相对表达量依次为OVCAR-3(1.57±0.07)、SKOV3(1.33±0.04)、3AO(1.03±0.08), OVCAR-3、3AO、SKOV3对人工基底膜Matrigel的粘附率分别为(70.4±2.5)%、(45.7±2.1)%、(47.3±3.9)%。OVCAR-3比3AO、SKOV3粘附能力强(p<0.01)。结论:卵巢上皮性癌组织中间皮素和CA125高表达,存在共定位表达关系。间皮素表达与卵巢癌的分级和类型有关,并可能与细胞粘附功能存在正相关关系。第二部分间皮cDNA过表达和RNA干扰重组慢病毒载体及稳转细胞株的构建和鉴定目的:构建间皮素cDNA过表达和RNA干扰重组慢病毒载体,慢病毒介导的基因转移的方法建立间皮素过表达和基因沉默稳转细胞株,用于间皮素的功能研究和基因治疗。方法:根据间皮素基因信息设计合成小干扰序列,插入到慢病毒质粒pRNAT-U6.2/Lenti中,慢病毒包装后感染SKOV3细胞,筛选有效干扰序列,选择干扰效率最高的大量包装。利用反转录的方法获取间皮素cDNA,插入到慢病毒质粒pWPXL-MOD中,测序后慢病毒包装。用慢病毒感染卵巢癌细胞,经筛选获取稳转细胞。用Western blotting、免疫荧光染色法检测间皮素蛋白表达变化。结果:测序结果证明4种小干扰慢病毒质粒、阴性对照质粒和cDNA过表达慢病毒质粒的插入序列完全正确,慢病毒包装成功。慢病毒感染SKOV3细胞后证实重组慢病毒LV-MSLN-shRNA4的干扰效率最高,为90%,LV-MSLN-cDNA可以升调间皮素蛋白表达约50%,慢病毒感染SKOV3细胞后成功筛选出SKOV3-MSLN-cDNA过表达细胞株、SKOV3-MSLN-shRNA基因沉默细胞株和SKOV3-MSLN-neg阴性对照细胞株。荧光免疫组化的共聚焦照片显示间皮素基因沉默细胞SKOV3-MSLN-shRNA定位于细胞膜的间皮素蛋白表达明显弱于阴性对照细胞SKOV3-MSLN-neg、亲本细胞SKOV3和过表达细胞SKOV3-MSLN-cDNA。结论:间皮素cDNA过表达和RNA干扰重组慢病毒载体的构建和慢病毒包装成功,间皮素表达得到了调控;稳转细胞株建株成功。为今后间皮素功能研究和基因治疗奠定了基础。第三部分间皮素对卵巢癌细胞生长及粘附侵袭迁移功能的影响目的:探讨间皮素表达变化对卵巢癌细胞生长及与人工基底膜之间或与间皮细胞之间的体外粘附功能的影响,以及间皮素对卵巢癌细胞体外侵袭迁移功能的影响。方法:利用前面慢病毒介导基因转移方法建立的间皮素过表达和基因沉默卵巢癌稳转细胞株,用细胞增殖实验和克隆形成率测定其生长特性;用人工基底膜材料Matrigel和间皮细胞作为粘附介质进行粘附功能检测;Transwell小室法检测卵巢癌细胞的侵袭迁移能力。结果: SKOV3-MSLN-shRNA、SKOV3、SKOV3-MSLN-neg以及SKOV3-MSLN-cDNA的细胞增殖个数分别为(9.89±2.0)×105 ,(19.81±2.5)×105,(18.9±2.24)×105,(23.68±2.35)×105,结果比较差异有显著性(F=32.905,p<0.01)。SKOV3-MSLN-shRNA的粘附率((12.12±2.21)%)显著低于其余三组((50.74±2.65)% , (49.78±3.2)% , (55.22±3.92)%) (F=213.96,p<0.05)。侵袭实验中,干扰组侵袭细胞数(7.5±1.78)少于其余三组((29.3±2.36),(27.1±3.21)、(32.9±3.67)),差异有统计学意义(F=159.64,p<0.01)。SKOV3-MSLN-shRNA迁移细胞数也少于其余三组。间皮细胞培养成功, SKOV3-MSLN-shRNA粘附于间皮细胞的数目显著下降。结论:间皮素基因沉默后抑制了细胞生长、粘附和侵袭迁移功能,体外实验证明间皮素参与肿瘤细胞的粘附侵袭迁移过程,可能参与肿瘤细胞种植转移到腹腔。第四部分间皮素对卵巢癌裸鼠移植瘤生长的影响及基因治疗研究目的:在体内水平验证间皮素在卵巢癌细胞生长、粘附侵袭转移中的作用,观察慢病毒对实验动物的影响。方法:利用间皮素过表达和基因沉默卵巢癌稳转细胞腹腔注射建立裸鼠移植瘤模型,接种14天后处死荷瘤裸鼠,观察腹腔移植瘤成瘤情况;卵巢癌细胞和慢病毒同时腹腔内注射观察成瘤;慢病毒隔日腹腔内注射观察成瘤;单独慢病毒腹腔内注射,观察慢病毒对全身及腹腔各脏器的毒副作用。结果:SK-MSLN-shRNA腹腔移植瘤个数和体积明显少于SKOV3和SK-MSLN-neg细胞(p<0.01),间皮素基因沉默导致卵巢癌细胞体内生长明显抑制。卵巢癌细胞SK和LV-MSLN-shRNA慢病毒单次同时腹腔内注射后,干扰组腹腔移植瘤个数和体积略少于对照组和过表达组,但没有统计学差异。LV-MSLN-shRNA慢病毒隔日腹腔内注射明显抑制了SKOV3细胞腹腔种植(p<0.01)。三种慢病毒腹腔注射后,裸鼠未见明显的病理变化。结论:间皮素基因沉默时,腹腔种植减少,延缓了卵巢癌的转移。间皮素小干扰慢病毒抑制了卵巢癌细胞的种植转移。慢病毒治疗对实验动物没有毒性,是一种安全的治疗方法。第五部分间皮素血清检测ELISA试剂盒的研制目的:用间皮素原核表达质粒的重组蛋白制备SMRP检测ELISA试剂盒。方法:用真核质粒酶切后将间皮素基因构建到间皮素原核表达质粒,制备间皮素重组蛋白,用纯化后的间皮素重组蛋白免疫小鼠,将小鼠脾细胞和SP2/0骨髓瘤细胞融合,筛选并建立稳定表达SMRP抗体的细胞克隆,抗体效价用ELISA的方法检测,杂交瘤细胞腹腔注射得到高效价的抗体,筛选针对不同抗原表位决定簇的抗体,得到一对最合适的抗体制成ELISA试剂盒。结果:间皮素原核表达质粒pMLSN-cDNAY构建成功,制备的间皮素重组蛋白纯度达到82%,成功筛选出21株稳定表达间皮素抗体的细胞株,得到一对抗体制成ELISA试剂盒,灵敏度(10 ng/ml)、稳定性、重复性等指标均达到标准。结论:检测SMRP的ELISA试剂盒研制获得初步成功。更多还原

【Abstract】 Ovarian cancer is the most common female malignancy and is the leading cause of death from gynecological malignancies. The majority of patients are diagnosed with advanced epithelial ovarian cancer. only 30% of patients with advanced-stage ovarian cancer survive 5 years after initial diagnosis. But the survival rate of patients diagnosed in early stage can reach up to 90%.Additional serum markers will be required to detect all patients in an initial phase by screening, and effective treatment will be required for patients with advanced-stage ovarian cancer.Mesothelin is a differentiation antigen whose expression in normal human tissues is limited to mesothelial cells lining of the pleura, pericardium and peritoneum. However, mesothelin is highly expressed in several human cancers, including mesotheliomas and pancreatic adenocarcinomas, and ovarian cancers. The mesothelin gene encodes a precursor protein of 69 kDa that is processed to a 32 kDa shed protein called megakaryocyte potentiating factor (MPF) and a 40 kDa fragment, mesothelin, that is attached to the cell membrane by a glycosyl-phosphatidylinositol (GPI) anchor. Results of recent studies suggest that the mesothelin may play a role in ovarian cancer metastasis by binding to MLSN/CA125. The binding is a high affinity, N-glycan dependent interaction that facilitates peritoneal metastasis of ovarian tumors. CA125 was reported to be a ligand of mesothelin . Blocking MLSN/CA125-dependent cell attachment may prevent or delay peritoneal metastatic recurrence.A small amount of cell bound mesothelin is shed into the serum and has been shown to be elevated in patients with mesothelioma and ovarian cancer. Studies suggest that serum mesothelin(soluble mesothelin-related protein SMRP) could be useful for diagnosis and follow-up of these patients. Mesothelin is a new target for immunotherapy.SS1P,a recombinant anti-mesothelin immunotoxin, its potent antitumor efficacy in animal models was confirmed, given to patients with mesothelin-expressing mesothelioma, ovarian, and pancreatic cancers is well tolerated.Results of our recent studies suggested that mesothelin was a significantly up-regulated gene in epithelial ovarian carcer by cDNA microarray analysis. The expression of mesothelin and CA125 is high expressed in epithelial ovarian cancer at protein level.There is significant correlation between mesothelin,CA125 expression and grade,histologic type of epithelial ovarian cancer.The anti-SMRP antibody 2H10 were obtained with the compound peptide. The antibody could react with native MSLN and had high specificity.The function of mesothelin is not known clearly. In this study,firstly,the co-located expression of MSLN and CA125 were detected in human ovarian cancer cells and tissue. Secondly, Mesothelin’function was investsigated in the process of epithelial ovarian cancer’s growth and metastasis in vitro and vivo.Thirdly, Lentivirus was given to animal models of ovarian cancer for gene therapy . Finally,a ELISA kit of SMRP was developped.Part I Expressions of mesothelin and CA125 in human epithelial ovarian cancer and its significanceObjective: To investigate the expressions of mesothelin and CA125 and their biological significance in human ovarian cancer cell lines and in human epithelial ovarian neoplasms.Methods: Mesothelin and CA125 protein expressions and locations in human epithelial ovarian cancer tissues and cell lines were detected by indirect immunofluorescence double maker and western blotting. EDTA-induced cell detachment and cell adhesive ability to matrigel were performed.Results: Mesothelin and CA125 specifically bound to cytomembrane of human epithelial ovarian cancer with indirect immunofluorescence double labelling. Mesothelin was red and CA125 was green,they were located together as yellow flourescence. Expressions of mesothelin in epithelial ovarian cancer(1639.2±181.92) was significantly higher than that in borderline adenoma(590.8±126.9) ,benign ovarian tumor(227.69±26.54) and normal ovary(213.0±24.37) (p<0.05).The expression of pathology grade in G2、G3(1642.63±44.58; 1701.57±54.73) was significantly higher than that in G1(1553.99±57.2)( F=10.01, p<0.05).The expression of histologic type in serous、endometrioid(1723.81±33.20; 1673.81±70.27) was significantly higher than that in mucinous(1466.12±94.93) (F=18.61,p<0.05). In age,stage and CA125 in serum,the expression of MSLN have no significant differences(p>0.05). Expressions of mesothelin and CA125 was correlated highly(r=0.95, p<0.05). The expression of mesothelin in the SKOV3(1.33±0.04 ) and 3AO(1.03±0.08) cells were weaker than OVCAR-3(1.57±0.07).Cell adhesion tests showed that the SKOV3 (47.3±3.9)%,and 3AO(45.7±2.1)% adhered less effectively to matrigel than OVCAR-3(70.4±2.5)% (p<0.01).Conclusions: Mesothelin and CA125 is high expressed in human epithelial ovarian cancer tissues and cell lines,and co-located .Mesothelin may be related with cell adhesive ability and gradeing,histologic type of epithelial ovarian cancer.Part II Construction and identification of recombinant lentiviral vectors and cell lines of cDNA and RNA interference of mesothelin geneObjective: To construst recombinant Lentivirus vectors of cDNA and RNA interference of mesothelin gene . To construst cell lines of down- regulated and up-regulated of mesothelin by gene transfer for function study and gene therapy.Methods: According to the Genebank information of mesothelin, 4 interfering sequence and a negative sequence were designed and inserted into plasmid pRNAT-U6.2/Lenti. Mesothelin’cDNA sequence was inserted into plasmid pWPXL-MOD.After packaging,SKOV3 cell was transfected and detected by Western blotting.The plasmid with the highest interfering efficiency was packaged by packaging plasmid mix.LV-MSLN-cDNA , LV-MSLN-shRNA, LV-MSLN-neg were transfered into ovarian cancer cells lines SKOV3,and screened.The expression of MSLN was confirmed by the means of Western blotting and immunofluorescence. Results: DNA sequencing showed that the sequence of recombinant Lentivirus plasmids were correct. The lentivirus with the highest interfering efficiency was LV-MSLN-shRNA4,The interfering efficiency was 90%. MSLN of SKOV3 cells transfered by LV-MSLN-cDNA was up-regulated 50%. It was confirmed that the shRNA,cDNA and negative sequence had been stably integrated into SKOV3 cells lines. Mesothelin specifically bound to cytomembrane of these cells. The expressions of mesothelin in the interfered cell (SKOV3-MSLN-shRNA) was weaker than the control cells (SKOV3-MSLN- neg,SKOV3) and up-regulated cell(SKOV3-MSLN-cDNA).Conclusion: Recombinant lentiviral vectors and cell lines of down- regulated and up-regulated of mesothelin gene were constructed successfully. It can be a useful toll to study the function and gene therapy of mesothelin.PartⅢEffects of mesothelin on cell growth and adhesion,invasion ability in human ovarian cancerObjective: To evalute the effect of mesothelin on the cell growth, adhesion and invasion ability in human ovarian cancer cell line with different expressions of mesothelin.Methods: The cell growth ability was evaluated by proliferation and clone forming experiments. EDTA-induced cell detachment and cell adhesive ability to matrigel were performed. Invasion and migration assay was performed by the Transwell. Mesothelial cells were cultured for adhesive assay.Results: The proliferation of SKOV3-MSLN-shRNA((9.89±2.0)×105) was lower than SKOV3((19.81±2.5)×105)、SKOV3-MSLN-neg((18.9±2.24)×105) and SKOV3-MSLN-cDNA((23.68±2.35)×105) (p<0.01).Cell adhesion tests showed that the SKOV3-MSLN-shRNA adhered less effectively to plastic substance and matrigel than SKOV3,SKOV3-MSLN-neg and SKOV3-MSLN-cDNA(p<0.01).In invasion and migration assay, the invasion cells and migration cells of SKOV3-MSLN-shRNA were lower than that of respective control groups and up-regulated group(p<0.01). Mesothelial cells were cultured successfully. SKOV3-MSLN-shRNA cells adhered less than other groups with mesothelial cells(p<0.01).Conclusions: Down-regulation of mesothelin led to inhibition of epithelial ovarian cancer cell growth and adhesive,invasion and migration ability,and may prevent or delay peritoneal metastatic recurrence.PartⅣEffects of mesothelin on transplanted tumor growth of human epithelium ovarian cancer and gene therapy in nude miceObjective: To study the effects of mesothelin in epithelium ovarian cancer and gene therapy in vivo.Methods: Test one: SKOV3,SKOV3-MSLN-cDNA, SKOV3-MSLN-neg, SKOV3-MSLN-shRNA cells were injected into the peritoneal cavity of nude mice; Test two:tumor cells SKOV3 and LV-MSLN-cDNA,LV-MSLN-neg, LV-MSLN-shRNA,PBS were injected at the same time; Test three:SKOV3 and Lentivirus were injected at the frist day, Lentivirus were injected every other day; Test four: only lentivirus were injected. Weight,distribution and ascites of transplanted tumor were measured after 14 days.Results: Transplanted tumor of SKOV3-MSLN-shRNA cells were suppressed obviously in nude mice compared with the control cells (p<0.01).In test two, the growth of SKOV3 were not suppressed obviously by LV-MSLN-shRNA;But in test three, suppression was obvious by LV-MSLN-shRNA, compared with the control cells; Three kinds of lentivirus have no toxic effec on nude mice.Conclusions: Lentivirus mediated RNA interference of mesothelin suppressed the transplanted tumor growth of human epithelium ovarian cancer.It will be a powerful strategy for cancer gene therapy. PartⅤDevelopment of enzyme-linked immunosorbent assay for SMRPObjective: To develop a indirect ELISA assay with the prokaryotically expressed gene product of SMRP.Methods: The complete length of mesothelin gene was cloned into prokaryotic expression vector pMLSN-cDNAY from eukaryotic expression vector pMLSN-cDNA. The target gene was then expressed in the E.coli cells,the target protein in total bacterial proteins was purified.BALB/c mice were immunized with the recombinant protein.The splenic cells of the mice were fused with SP2/0 mouse myeloma cells.The positive clones were screened.The stable hybridoma cell lines which can excrete antibody were expand cultivation.The titer of antibody were detected with ELISA.BALB/c mice were immunized with some hybridoma cells.Ascites were taken after two weeks. High titer antibodys of anti-MSLN were obtained. Some matched antibodys were selected.Precoating microplate with one anti-MSLN mAb, detecting method includes another anti-MSLN mAb with HRP,and was used in the detection of the recombinant protein at different levels.Results: Recombinant prokaryotic expression vector pMLSN-cDNAY of mesothelin gene was constructed successfully. The concentration of the purified protein was 82%.The stable hybridoma cell lines(21) were screened. Some matched antibodys were selected and the best was detected. This assay had good reproducibility and steadiness,the sensitivity reached to 10 ng/ml.Conclusions: The indirect ELISA assay for the detection of SMRP was established.更多还原