BAMBI基因沉默对人胃癌及裸鼠移植瘤生长影响及调控研究

发布时间：2018-06-20 05:41

本文选题：胃癌 + BAMBI　；参考：《山东大学》2016年博士论文

【摘要】：研究背景：胃癌在全球恶性肿瘤的发病率仅次于肺癌、乳腺癌、大肠癌而排名第四,相关死亡率位居第二位。胃癌的早期临床症状体征无特异性容易被忽视,多数胃癌在确诊时已经属于疾病的进展期,相当部分患者已失去手术根治的机会,总生存时间一直未有明显改善。因此,深入了解胃癌发生、发展的分子机制,寻找与胃癌恶性生物学行为和特点及预后相关的生物学指标,将对胃癌的早期诊断和综合治疗产生极有意义的影响,还可以从分子靶向治疗方面提供新的选择。目前胃癌的病因及发病机制尚未阐明,其发生的机制与细胞信号转导通路的异常密切相关,且信号通路中相关因子也参与了调控胃癌细胞的增殖、分化、侵袭和转移等过程。在肿瘤的分子致病机制研究中发现,TGF-β和Wnt信号通路在各组织器官的肿瘤与疾病的发生及演进中起重要的作用,是受到全世界科研工作者广泛关注的通路之一。骨形成蛋白和激活素的跨膜抑制剂(BAMBI)是TGF-β信号转导通路的伪受体,且BAMBI的表达受Wnt信号的关键组分β-catenin的诱导。目前BAMBI表达异常而引起的各组织器官的疾病或肿瘤细胞的增殖、侵袭和转移机制也逐渐成为研究的热点。目前国内外关于BAMBI在TGF-β和Wnt/β-catenin信号通路中的负调节机制对胃癌的作用研究还很少。本课题通过体内外实验相互结合,研究BAMBI在TGF-β和Wnt/β-catenin信号通路中的调节机制对胃癌的影响,以期为胃癌靶向治疗寻找新的靶点。第一部分BAMBI在人胃癌组织中的表达及意义目的采用免疫组织化学法检测BAMBI在人胃癌组织标本60例和对应癌旁组织标本中的表达情况,探讨其与胃癌发生发展以及不同临床及病理特征的关系,为下一步实验提供理论基础。方法收集2012年10月至2014年10月山东省肿瘤医院病理科胃癌术后蜡块标本60例,每例包括胃癌组织及相应癌旁正常组织。采用免疫组化SP法检测BAMBI在人胃癌组织标本60例和对应癌旁组织标本60例中的表达情况,并分析BAMBI与胃癌及众多临床病理特征之间的相关性。结果BAMBI蛋白定位于腺细胞的细胞浆中呈棕黄色颗粒或团块为阳性表达。胃癌和癌旁正常组织中BAMBI的阳性表达率分别为66.7%和20.0%,胃癌组织中BAMBI的阳性表达率显著高于癌旁正常组织(x2=8.164,P0.05)。在胃癌组织中BAMBI的表达与患者年龄、性别、分化程度以及肿瘤大小均无显著相关性(P0.05)；而与患者TNM分期、淋巴结转移以及浸润浆膜显著相关。有淋巴转移者(91.9%)显著高于无淋巴结转移者的阳性表达率(26.1%)(P0.05)；TNM分期为Ⅲ期患者的BAMBI阳性表达率(88.6%)显著高于Ⅰ期和Ⅱ期患者(36.0%)(P0.05)；有浸润浆膜者(87.9%)显著高于无浸润浆膜者的阳性表达率(40.7%)(P0.05)。结论胃癌组织中BAMBI表达异常增高,推测BAMBI参与了胃癌的发生过程；淋巴结转移,临床分期较晚和浆膜侵犯者BAMBI表达显著增高,表明BAMBI参与了胃癌的发展过程。第二部分慢病毒介导的BAMBI基因沉默对人胃癌细胞生物学行为的影响目的利用慢病毒介导BAMBI基因沉默人胃癌MGC-803细胞,观察沉默BAMBI基因后人胃癌细胞BAMBI蛋白及mRNA的表达变化,检测细胞增殖情况和迁移侵袭能力的变化,有望提供胃癌治疗的新的可能的分子靶点并且为研究该靶点基因的实验奠定基础。方法设计并合成慢病毒shRNA-BAMBI载体,转染入体外培养的人胃癌细胞株MGC-803；采用RT-PCR和Western blot检测转染后MGC-803细胞中BAMBI mRNA和蛋白表达水平；采用MTT法检测转染后MGC-803细胞生长增殖状况；采用Transwe11实验分析慢病毒shRNA-BAMBI载体对MGC-803细胞体外迁移力和侵袭力的影响。结果BAMBI-shRNA慢病毒载体转染细胞后,实验组细胞中BAMBI蛋白及mRNA表达较空白对照组和阴性对照组显著降低(PO.05),阴性对照组与空白对照组相比较两者之间无显著性差异(P0.05)。由此可以证明MGC-803细胞转染BAMBI-shRNA慢病毒后,能特异性沉默BAMBI基因,从而抑制BAMBI基因复制和蛋白表达。BAMBI-shRNA慢病毒载体转染细胞后,对细胞增殖能力和侵袭迁移能力产生显著性影响。实验组细胞抑制率显著高于阴性对照组和空白对照组(P0.05),而侵袭和迁移细胞数显著低于阴性对照组合空白对照组。阴性对照组与空白对照组相比较无显著性差异(P0.05)。提示BAMBI-shRNA慢病毒抑制MGC-803细胞BAMBI基因表达后,可以抑制细胞的增殖能力和侵袭及迁移能力。结论BAMBI-shRNA慢病毒能沉默MGC-803细胞中BAMBI基因,抑制BAMBI mRNA及蛋白的表达,并且能通过该基因沉默抑制细胞增殖,降低细胞的侵袭和迁移能力。第三部分慢病毒介导的BAMBI基因沉默对人胃癌裸鼠原位移植瘤生长、凋亡的影响及机制研究目的培养人胃癌MGC-803细胞,通过接种细胞悬液构建人胃癌裸鼠原位移植瘤模型。使用BAMBI-shRNA慢病毒活体瘤内局部注射,干预活体移植瘤生长,探讨分析BAMBI基因沉默对人胃癌裸鼠原位移植瘤生长增殖影响,检测移植瘤细胞内BAMBI、β-catenin及TGF-β的蛋白及mRNA的变化。分析上述变化的作用及机制。方法体外培养人胃癌MGC-803细胞,裸鼠皮下接种细胞悬液,观察3周记录裸鼠裸鼠成活率及皮下成瘤率。接种成活后切开腹腔将瘤体移植胃体浆膜建立原位移植瘤模型。瘤体内注射慢病毒shRNA-BAMBI溶液(实验组)、生理盐水(空白对照组)和无关序列的慢病毒溶液(阴性对照组),对人胃癌裸鼠原位移植瘤组织进行活体不同干预。按照观察时间点(3天、6天、9天、12天和15天)处死裸鼠,观察BAMBI基因沉默对人胃癌裸鼠原位移植瘤的生长和细胞增殖及凋亡的影响,并应用RT-PCR、Western blot和免疫组化法分别检测移植瘤组织中BAMBI、β-catenin及TGF-β的表达。观察三组间瘤体向周围侵犯程度的不同,比较三组间瘤体大小、瘤体重量、细胞凋亡、细胞增殖、BAMBI、β-catenin及TGF-β的蛋白及mRNA的表达差异。结果瘤体内注射内BAMBI-shRNA慢病毒颗粒的实验组裸鼠,移植瘤体积较小,与周围组织界限清楚,无明显粘连浸润现象。而瘤体内注射生理盐水及无关序列的慢病毒颗粒的裸鼠移植瘤则体积较大,与肝脏、脾脏等周围脏器有不同程度的粘连。三组人胃癌细胞裸鼠原位移植瘤肿瘤体生长曲线比较：实验组与阴性对照组及空白对照组相比生长速度显著减慢(P0.05),空白对照组和阴性对照组相比肿瘤生长速度无显著性差异(P0.05)。实验组裸鼠的瘤重抑瘤率达(47.44±1.64)%。三组原位移植瘤重量进行比较：实验组重量(1.21±0.03 g)与阴性对照组(2.49±0.19g)及空白对照组(2.52±0.17g)相比,实验组瘤体重量显著低于空白对照组、阴性对照组,差异有统计学意义(P0.05),空白对照组和阴性对照组相比无显著性差异(P0.05)。三组原位移植瘤体积进行比较：在活体干预之前实验组体积(0.89±0.35)与阴性对照组(0.92±0.26)及空白对照组(0.87±0.18)相比无显著性差异(P0.05),干预第3天、6天、9天、12天及15天)实验组瘤体体积(1.02±0.29、1.84±0.21、2.34±0.37、2.34±0.37、2.89±0.95和3.14±0.96)显著低于空白对照组(2.12±0.19、3.94±0.28、4.38±0.84、4.96±1.02和5.16±1.18)、阴性对照组(2.18±0.17、3.86±0.27、4.32±0.86、4.95±1.05和5.19±1.12),差异均有统计学意义(P0.05),空白对照组和阴性对照组相比无显著性差异(P0.05)。三组原位移植瘤中细胞抑制率和凋亡率比较：实验组细胞抑制率(24小时、48小时和72小时的百分比,下同)为9.27±2.05,19.24±3.54,28.17±2.94,显著高于空白对照组的0.92±0.09,0.86±0.19,1.42±0.18和阴性对照组的0.85±0.06,0.95±0.13,1.76±0.18。实验组凋亡率为25.18±1.25、30.74±4.29和39.48±5.26显著高于空白对照组的5.02±1.38、7.45±2.35、9.64±2.89和阴性对照组的4.52±1.34、7.19±2.13、8.97±2.74,差异有统计学意义(P0.05)空白对照组和阴性对照组相比无显著性差异(P0.05)。瘤体免疫组化及Western-blot实验结果显示：实验组BAMBI、β-catenin表达的灰度值均明显低于空白对照组和阴性对照组(P0.05),而TGF-β大于阴性对照组及空白对照组(P0.05),空白对照组与阴性对照组之间的差异无统计学意义(P0.05)。RT-PCR检测结果显示实验组的BAMBI、β-catenin的nRNA表达显著低于阴性对照组和空白对照组(P0.05),而TGF-βmRNA表达均显著高于阴性对照组和空白对照组(P0.05),差异有统计学意义,而空白对照组与阴性对照组比较无显著差异(P0.05)。结论1.慢病毒shRNA-BAMBI直接注射人胃癌裸鼠原位移植瘤后可成功转染瘤组织,抑制胃癌移植瘤生长增殖诱导胃癌细胞的凋亡。2.慢病毒shRNA-BAMBI成功转染瘤组织后沉默BAMBI基因,显著促进原位移植瘤组织中TGF-β的mRNA的表达,而抑制BAMBI和β-catenin mRNA的表达。3.慢病毒shRNA-BAMBI成功转染瘤组织,显著促进原位移植瘤组织中TGF-β蛋白的表达,而抑制BAMBI和β-catenin蛋白的表达。
[Abstract]:Background: the incidence of gastric cancer in the world is second only to lung cancer, breast cancer, and large intestine cancer ranks fourth, and the related mortality is the second place. The early clinical symptoms and signs of gastric cancer are easy to be ignored. Most of the gastric cancer has been diagnosed at the time of the disease, and a considerable number of patients have lost a radical operation. There is no significant improvement in the total survival time. Therefore, a thorough understanding of the occurrence of gastric cancer, the molecular mechanism of development, and the search for biological indicators related to the malignant biological behavior, characteristics and prognosis of gastric cancer will have a very significant impact on the early diagnosis and comprehensive treatment of gastric cancer, and can also provide a new selection from molecular targeting therapy. At present, the etiology and pathogenesis of gastric cancer have not been elucidated, and its mechanism is closely related to the abnormal cell signal transduction pathway, and the related factors in the signal pathway also participate in the regulation of the proliferation, differentiation, invasion and metastasis of gastric cancer cells. The TGF- beta and Wnt signaling pathways are found in the study of the molecular pathogenesis of the tumor. The tumor of tissue and organ plays an important role in the occurrence and evolution of the disease. It is one of the pathways widely concerned by researchers all over the world. The transmembrane inhibitor (BAMBI) of bone morphogenetic protein and activin is a pseudo receptor of the TGF- beta signal transduction pathway, and the expression of BAMBI is induced by the key component of Wnt signal, beta -catenin. Currently, BAMBI The pathogenesis of various tissues and organs caused by abnormal expression or the mechanism of proliferation, invasion and metastasis of tumor cells has gradually become a hot spot of research. At present, there are few studies on the role of BAMBI in the TGF- beta and Wnt/ beta -catenin signaling pathway in gastric cancer. This topic combines in vitro and in vivo experiments to study BAMBI The effect of regulation mechanism on gastric cancer in TGF- beta and Wnt/ beta -catenin signaling pathway in order to find new targets for the target therapy of gastric cancer. The expression and significance of BAMBI in human gastric cancer tissue and its purpose were detected by immunohistochemistry in 60 specimens of human gastric carcinoma tissue specimens and the expression of corresponding para cancerous tissue specimens. To discuss the relationship with the development of gastric cancer and the different clinical and pathological features, and to provide a theoretical basis for the next experiment. Methods 60 specimens of paraffin specimens were collected from October 2012 to October 2014 in the pathology department of the Cancer Hospital of Shandong province. Each case included gastric cancer tissue and the corresponding normal tissue adjacent to cancer. BAMBI was detected by immunohistochemical SP method. The expression of 60 cases of gastric carcinoma and 60 cases of paracancerous tissue specimens were analyzed, and the correlation between BAMBI and gastric cancer and many clinicopathological features was analyzed. Results the positive expression of BAMBI in gastric cancer and normal tissues was 6, and the positive expression of BAMBI protein was 6 in the cytoplasm of the gland cells. 6.7% and 20%, the positive expression rate of BAMBI in gastric carcinoma was significantly higher than that of normal tissue adjacent to cancer (x2=8.164, P0.05). There was no significant correlation between the expression of BAMBI and the age, sex, degree of differentiation and tumor size in the gastric cancer tissues (P0.05); there was a significant correlation with TNM staging, lymph node metastasis and infiltrating serosa. The positive expression rate (91.9%) was significantly higher than that of non lymph node metastasis (26.1%) (P0.05), and the positive expression rate of BAMBI in stage III of TNM stage (88.6%) was significantly higher than that in stage I and stage II patients (36%) (P0.05); the positive rate of the infiltrating serosa (87.9%) was significantly higher than that in the non impregnated serosa (40.7%) (P0.05). Conclusion the BAMBI table in the gastric cancer tissue is a conclusion. BAMBI participates in the process of gastric cancer; lymph node metastasis, late stage of clinical stage and BAMBI expression of serous invagger are significantly higher, indicating that BAMBI is involved in the development of gastric cancer. Second the effect of lentivirus mediated BAMBI gene silencing on the biological behavior of human gastric cancer cells is to use the lentivirus to mediate BAMBI Gene silencing human gastric cancer MGC-803 cells, observing the changes in the expression of BAMBI protein and mRNA in human gastric cancer cells after the silence of BAMBI gene, detecting the cell proliferation and the change of migration and invasion ability, may provide a new possible molecular target for the treatment of gastric cancer and provide a basis for the study of the target gene. ShRNA-BAMBI vector was transfected into human gastric cancer cell line MGC-803 in vitro. The expression of BAMBI mRNA and protein in MGC-803 cells after transfection was detected by RT-PCR and Western blot. MTT assay was used to detect the growth and proliferation of MGC-803 cells after transfection, and the Transwe11 experiment was used to analyze the lentivirus shRNA-BAMBI carrier. Results the expression of BAMBI protein and mRNA in the experimental group was significantly lower than that of the blank control group and the negative control group (PO.05) after the BAMBI-shRNA lentivirus vector transfected to the cells (PO.05). There was no significant difference between the negative control group and the blank control group (P0.05). Thus, the transfection of MGC-803 cells to BA was proved to be BA. After the MBI-shRNA lentivirus, it can specifically silence the BAMBI gene and inhibit the replication of BAMBI gene and the transfection of the.BAMBI-shRNA lentivirus vector to the cells. The cell inhibition rate of the experimental group is significantly higher than that of the negative and blank control group and the blank control group (P0.05), and the invasion and migration of the experimental group is significantly higher than that of the negative group and the blank control group. The number of cells in the blank control group was significantly lower than that in the negative control group. There was no significant difference between the negative control group and the blank control group (P0.05). It suggested that the BAMBI-shRNA lentivirus inhibited the BAMBI gene expression in MGC-803 cells and could inhibit the proliferation and invasion and migration ability of the cells. Conclusion BAMBI-shRNA lentivirus can silence the B in MGC-803 cells. AMBI gene, which inhibits the expression of BAMBI mRNA and protein, and can inhibit cell proliferation and reduce cell invasion and migration through the gene silencing. Third the effect of BAMBI gene silencing mediated by Lentivirus on the growth of orthotopic xenografts in human gastric cancer in nude mice, the effect of apoptosis and the mechanism to cultivate human gastric cancer MGC-803 cells by inoculation The model of human gastric carcinoma in situ was constructed by cell suspension. Local injection of BAMBI-shRNA lentivirus was used to interfere with the growth of living xenografts. The effect of BAMBI gene silencing on the growth and proliferation of human gastric carcinoma in situ in nude mice was analyzed. The changes in the protein and mRNA of BAMBI, beta -catenin and TGF- beta in the transplanted tumor cells were analyzed. Methods the human gastric cancer MGC-803 cells were cultured in vitro, and the nude mice were inoculated subcutaneously in the nude mice. The survival rate and subcutaneous tumor formation rate of nude mice were observed for 3 weeks. After inoculation, the tumor body transplanted into the stomach serous membrane was transplanted into the abdominal cavity. The tumor body was injected with lentivirus shRNA-BAMBI solution (experimental group) and physiological salt. The effects of BAMBI gene silencing on the growth, cell proliferation and apoptosis of human gastric cancer in nude mice were observed by different intervention in vivo of the transplanted tumor tissues of human gastric cancer mice (3 days, 6 days, 9 days, 12 days and 15 days) with water (blank control group) and the unrelated sequence of lentivirus (negative control group). The expression of BAMBI, beta -catenin and TGF- beta in the transplanted tumor tissues was detected by RT-PCR, Western blot and immunohistochemistry. The difference of the invasion between the three groups was observed. The size of the tumor body, the weight of the tumor, the cell apoptosis, the cell proliferation, the expression of the protein and mRNA of the BAMBI, beta -catenin and TGF- beta were compared between the three groups. Nude mice in the experimental group with internal injection of BAMBI-shRNA lentivirus particles were small in size, with a clear boundary between the surrounding tissue and no obvious adhesion. The tumor in nude mice injected with normal saline and unrelated lentivirus particles in the tumor was larger, and there were different degrees of adhesion to the surrounding organs such as the liver and the spleen. Three groups of mice. Compared with the negative control group and the blank control group, the growth rate of the tumor cells in the experimental group was significantly slower than that of the negative control group and the blank control group (P0.05). There was no significant difference in the tumor growth rate between the blank control group and the negative control group (P0.05). The tumor suppressor rate of the nude mice in the experimental group was up to (47.44 + 1.64)%. Three groups of orthotopic transplantation The weight of the tumor was compared: the weight of the experimental group (1.21 + 0.03 g) was compared with the negative control group (2.49 + 0.19g) and the blank control group (2.52 + 0.17g). The weight of the tumor body in the experimental group was significantly lower than that in the blank control group, and the negative control group was statistically significant (P0.05). There was no significant difference between the blank control group and the negative control group (P0.05). Three groups of original displacements were found. The volume of the tumor was compared: there was no significant difference between the volume of the experimental group (0.89 + 0.35) and the negative control group (0.92 + 0.26) and the blank control group (0.87 + 0.18) before the living body intervention (P0.05), the intervention for third days, 6 days, 9 days, 12 days and 15 days of the experimental group (1.02 + 0.29,1.84 + 0.21,2.34 + 0.37,2.34 + 0.37,2.89 + 0.87) It was lower than that in the blank control group (2.12 + 0.19,3.94 + 0.28,4.38 + 0.84,4.96 + 1.02 and 5.16 + 1.18), negative control group (2.18 + 0.17,3.86 + 0.27,4.32 + 0.86,4.95 + 1.05 and 5.19 + 1.12), the difference was statistically significant (P0.05). There was no significant difference between the blank control group and the negative control group (P0.05). The cell inhibition rate in the three group in situ xenograft tumor was found. The rate of apoptosis was compared: the rate of cell inhibition (24 hours, 48 hours and 72 hours, the same below) was 9.27 + 2.05,19.24 + 3.54,28.17 + 2.94, significantly higher than that in the blank control group of 0.92 + 0.09,0.86 + 0.19,1.42 + 0.18 and 0.85 + 0.06,0.95 + 0.13,1.76 + 0.18. in the negative control group, and the apoptosis rate was 25.18 + 1.25,30.74 + 4.29 and 39.48 +. 5.26 significantly higher than the blank control group 5.02 + 1.38,7.45 + 2.35,9.64 + 2.89 and negative control group 4.52 + 1.34,7.19 + 2.13,8.97 + 2.74, the difference was statistically significant (P0.05) no significant difference (P0.05) between the blank control group and the negative control group (P0.05). The tumor body immunohistochemistry and the Western-blot test results showed that the experimental group BAMBI, beta -catenin table The gray value of the reach was significantly lower than that of the blank control group and the negative control group (P0.05), but the TGF- beta was greater than the negative control group and the blank control group (P0.05), the difference between the blank control group and the negative control group was not statistically significant (P0.05), the.RT-PCR test results showed the BAMBI in the experimental group, and the nRNA expression of the beta -catenin was significantly lower than that of the negative control group and the empty control group. In the white control group (P0.05), the expression of TGF- beta mRNA was significantly higher than that in the negative control group and the blank control group (P0.05), and the difference was statistically significant, but there was no significant difference between the blank control group and the negative control group (P0.05). Conclusion 1. lentivirus shRNA-BAMBI directly injected into the tumor tissue of human gastric carcinoma in situ and inhibited the gastric cancer migration. The apoptosis of.2. lentivirus shRNA-BAMBI induced by the growth and proliferation of tumor cells successfully transfected the tumor tissue into the BAMBI gene, which significantly promoted the mRNA expression of TGF- beta in the orthotopic xenografts, and the expression of BAMBI and beta -catenin mRNA in the expression of.3. lentivirus shRNA-BAMBI was successfully transfected into the tumor tissue, which significantly promoted TGF- beta in the orthotopic xenografts. The expression of protein inhibited the expression of BAMBI and beta -catenin protein.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.2

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