本文選題：胃癌 + BAMBI��； 參考：《山東大學》2016年博士論文

【摘要】：研究背景：胃癌在全球惡性腫瘤的發(fā)病率僅次于肺癌、乳腺癌、大腸癌而排名第四,相關死亡率位居第二位。胃癌的早期臨床癥狀體征無特異性容易被忽視,多數(shù)胃癌在確診時已經(jīng)屬于疾病的進展期,相當部分患者已失去手術根治的機會,總生存時間一直未有明顯改善。因此,深入了解胃癌發(fā)生、發(fā)展的分子機制,尋找與胃癌惡性生物學行為和特點及預后相關的生物學指標,將對胃癌的早期診斷和綜合治療產(chǎn)生極有意義的影響,還可以從分子靶向治療方面提供新的選擇。目前胃癌的病因及發(fā)病機制尚未闡明,其發(fā)生的機制與細胞信號轉(zhuǎn)導通路的異常密切相關,且信號通路中相關因子也參與了調(diào)控胃癌細胞的增殖、分化、侵襲和轉(zhuǎn)移等過程。在腫瘤的分子致病機制研究中發(fā)現(xiàn),TGF-β和Wnt信號通路在各組織器官的腫瘤與疾病的發(fā)生及演進中起重要的作用,是受到全世界科研工作者廣泛關注的通路之一。骨形成蛋白和激活素的跨膜抑制劑(BAMBI)是TGF-β信號轉(zhuǎn)導通路的偽受體,且BAMBI的表達受Wnt信號的關鍵組分β-catenin的誘導。目前BAMBI表達異常而引起的各組織器官的疾病或腫瘤細胞的增殖、侵襲和轉(zhuǎn)移機制也逐漸成為研究的熱點。目前國內(nèi)外關于BAMBI在TGF-β和Wnt/β-catenin信號通路中的負調(diào)節(jié)機制對胃癌的作用研究還很少。本課題通過體內(nèi)外實驗相互結合,研究BAMBI在TGF-β和Wnt/β-catenin信號通路中的調(diào)節(jié)機制對胃癌的影響,以期為胃癌靶向治療尋找新的靶點。第一部分BAMBI在人胃癌組織中的表達及意義目的采用免疫組織化學法檢測BAMBI在人胃癌組織標本60例和對應癌旁組織標本中的表達情況,探討其與胃癌發(fā)生發(fā)展以及不同臨床及病理特征的關系,為下一步實驗提供理論基礎。方法收集2012年10月至2014年10月山東省腫瘤醫(yī)院病理科胃癌術后蠟塊標本60例,每例包括胃癌組織及相應癌旁正常組織。采用免疫組化SP法檢測BAMBI在人胃癌組織標本60例和對應癌旁組織標本60例中的表達情況,并分析BAMBI與胃癌及眾多臨床病理特征之間的相關性。結果BAMBI蛋白定位于腺細胞的細胞漿中呈棕黃色顆�；驁F塊為陽性表達。胃癌和癌旁正常組織中BAMBI的陽性表達率分別為66.7%和20.0%,胃癌組織中BAMBI的陽性表達率顯著高于癌旁正常組織(x2=8.164,P0.05)。在胃癌組織中BAMBI的表達與患者年齡、性別、分化程度以及腫瘤大小均無顯著相關性(P0.05)；而與患者TNM分期、淋巴結轉(zhuǎn)移以及浸潤漿膜顯著相關。有淋巴轉(zhuǎn)移者(91.9%)顯著高于無淋巴結轉(zhuǎn)移者的陽性表達率(26.1%)(P0.05)；TNM分期為Ⅲ期患者的BAMBI陽性表達率(88.6%)顯著高于Ⅰ期和Ⅱ期患者(36.0%)(P0.05)；有浸潤漿膜者(87.9%)顯著高于無浸潤漿膜者的陽性表達率(40.7%)(P0.05)。結論胃癌組織中BAMBI表達異常增高,推測BAMBI參與了胃癌的發(fā)生過程；淋巴結轉(zhuǎn)移,臨床分期較晚和漿膜侵犯者BAMBI表達顯著增高,表明BAMBI參與了胃癌的發(fā)展過程。第二部分慢病毒介導的BAMBI基因沉默對人胃癌細胞生物學行為的影響目的利用慢病毒介導BAMBI基因沉默人胃癌MGC-803細胞,觀察沉默BAMBI基因后人胃癌細胞BAMBI蛋白及mRNA的表達變化,檢測細胞增殖情況和遷移侵襲能力的變化,有望提供胃癌治療的新的可能的分子靶點并且為研究該靶點基因的實驗奠定基礎。方法設計并合成慢病毒shRNA-BAMBI載體,轉(zhuǎn)染入體外培養(yǎng)的人胃癌細胞株MGC-803；采用RT-PCR和Western blot檢測轉(zhuǎn)染后MGC-803細胞中BAMBI mRNA和蛋白表達水平；采用MTT法檢測轉(zhuǎn)染后MGC-803細胞生長增殖狀況；采用Transwe11實驗分析慢病毒shRNA-BAMBI載體對MGC-803細胞體外遷移力和侵襲力的影響。結果BAMBI-shRNA慢病毒載體轉(zhuǎn)染細胞后,實驗組細胞中BAMBI蛋白及mRNA表達較空白對照組和陰性對照組顯著降低(PO.05),陰性對照組與空白對照組相比較兩者之間無顯著性差異(P0.05)。由此可以證明MGC-803細胞轉(zhuǎn)染BAMBI-shRNA慢病毒后,能特異性沉默BAMBI基因,從而抑制BAMBI基因復制和蛋白表達。BAMBI-shRNA慢病毒載體轉(zhuǎn)染細胞后,對細胞增殖能力和侵襲遷移能力產(chǎn)生顯著性影響。實驗組細胞抑制率顯著高于陰性對照組和空白對照組(P0.05),而侵襲和遷移細胞數(shù)顯著低于陰性對照組合空白對照組。陰性對照組與空白對照組相比較無顯著性差異(P0.05)。提示BAMBI-shRNA慢病毒抑制MGC-803細胞BAMBI基因表達后,可以抑制細胞的增殖能力和侵襲及遷移能力。結論BAMBI-shRNA慢病毒能沉默MGC-803細胞中BAMBI基因,抑制BAMBI mRNA及蛋白的表達,并且能通過該基因沉默抑制細胞增殖,降低細胞的侵襲和遷移能力。第三部分慢病毒介導的BAMBI基因沉默對人胃癌裸鼠原位移植瘤生長、凋亡的影響及機制研究目的培養(yǎng)人胃癌MGC-803細胞,通過接種細胞懸液構建人胃癌裸鼠原位移植瘤模型。使用BAMBI-shRNA慢病毒活體瘤內(nèi)局部注射,干預活體移植瘤生長,探討分析BAMBI基因沉默對人胃癌裸鼠原位移植瘤生長增殖影響,檢測移植瘤細胞內(nèi)BAMBI、β-catenin及TGF-β的蛋白及mRNA的變化。分析上述變化的作用及機制。方法體外培養(yǎng)人胃癌MGC-803細胞,裸鼠皮下接種細胞懸液,觀察3周記錄裸鼠裸鼠成活率及皮下成瘤率。接種成活后切開腹腔將瘤體移植胃體漿膜建立原位移植瘤模型。瘤體內(nèi)注射慢病毒shRNA-BAMBI溶液(實驗組)、生理鹽水(空白對照組)和無關序列的慢病毒溶液(陰性對照組),對人胃癌裸鼠原位移植瘤組織進行活體不同干預。按照觀察時間點(3天、6天、9天、12天和15天)處死裸鼠,觀察BAMBI基因沉默對人胃癌裸鼠原位移植瘤的生長和細胞增殖及凋亡的影響,并應用RT-PCR、Western blot和免疫組化法分別檢測移植瘤組織中BAMBI、β-catenin及TGF-β的表達。觀察三組間瘤體向周圍侵犯程度的不同,比較三組間瘤體大小、瘤體重量、細胞凋亡、細胞增殖、BAMBI、β-catenin及TGF-β的蛋白及mRNA的表達差異。結果瘤體內(nèi)注射內(nèi)BAMBI-shRNA慢病毒顆粒的實驗組裸鼠,移植瘤體積較小,與周圍組織界限清楚,無明顯粘連浸潤現(xiàn)象。而瘤體內(nèi)注射生理鹽水及無關序列的慢病毒顆粒的裸鼠移植瘤則體積較大,與肝臟、脾臟等周圍臟器有不同程度的粘連。三組人胃癌細胞裸鼠原位移植瘤腫瘤體生長曲線比較：實驗組與陰性對照組及空白對照組相比生長速度顯著減慢(P0.05),空白對照組和陰性對照組相比腫瘤生長速度無顯著性差異(P0.05)。實驗組裸鼠的瘤重抑瘤率達(47.44±1.64)%。三組原位移植瘤重量進行比較：實驗組重量(1.21±0.03 g)與陰性對照組(2.49±0.19g)及空白對照組(2.52±0.17g)相比,實驗組瘤體重量顯著低于空白對照組、陰性對照組,差異有統(tǒng)計學意義(P0.05),空白對照組和陰性對照組相比無顯著性差異(P0.05)。三組原位移植瘤體積進行比較：在活體干預之前實驗組體積(0.89±0.35)與陰性對照組(0.92±0.26)及空白對照組(0.87±0.18)相比無顯著性差異(P0.05),干預第3天、6天、9天、12天及15天)實驗組瘤體體積(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)顯著低于空白對照組(2.12±0.19、3.94±0.28、4.38±0.84、4.96±1.02和5.16±1.18)、陰性對照組(2.18±0.17、3.86±0.27、4.32±0.86、4.95±1.05和5.19±1.12),差異均有統(tǒng)計學意義(P0.05),空白對照組和陰性對照組相比無顯著性差異(P0.05)。三組原位移植瘤中細胞抑制率和凋亡率比較：實驗組細胞抑制率(24小時、48小時和72小時的百分比,下同)為9.27±2.05,19.24±3.54,28.17±2.94,顯著高于空白對照組的0.92±0.09,0.86±0.19,1.42±0.18和陰性對照組的0.85±0.06,0.95±0.13,1.76±0.18。實驗組凋亡率為25.18±1.25、30.74±4.29和39.48±5.26顯著高于空白對照組的5.02±1.38、7.45±2.35、9.64±2.89和陰性對照組的4.52±1.34、7.19±2.13、8.97±2.74,差異有統(tǒng)計學意義(P0.05)空白對照組和陰性對照組相比無顯著性差異(P0.05)。瘤體免疫組化及Western-blot實驗結果顯示：實驗組BAMBI、β-catenin表達的灰度值均明顯低于空白對照組和陰性對照組(P0.05),而TGF-β大于陰性對照組及空白對照組(P0.05),空白對照組與陰性對照組之間的差異無統(tǒng)計學意義(P0.05)。RT-PCR檢測結果顯示實驗組的BAMBI、β-catenin的nRNA表達顯著低于陰性對照組和空白對照組(P0.05),而TGF-βmRNA表達均顯著高于陰性對照組和空白對照組(P0.05),差異有統(tǒng)計學意義,而空白對照組與陰性對照組比較無顯著差異(P0.05)。結論1.慢病毒shRNA-BAMBI直接注射人胃癌裸鼠原位移植瘤后可成功轉(zhuǎn)染瘤組織,抑制胃癌移植瘤生長增殖誘導胃癌細胞的凋亡。2.慢病毒shRNA-BAMBI成功轉(zhuǎn)染瘤組織后沉默BAMBI基因,顯著促進原位移植瘤組織中TGF-β的mRNA的表達,而抑制BAMBI和β-catenin mRNA的表達。3.慢病毒shRNA-BAMBI成功轉(zhuǎn)染瘤組織,顯著促進原位移植瘤組織中TGF-β蛋白的表達,而抑制BAMBI和β-catenin蛋白的表達。
[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.
【學位授予單位】：山東大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R735.2

【相似文獻】

相關期刊論文 前1條

1 苗參;高建;趙蕾;王輝;崔澤實;;BAMBI在非小細胞肺癌細胞系中的分布及表達[J];上海醫(yī)學;2009年07期

相關博士學位論文 前1條

1 劉凱;BAMBI基因沉默對人胃癌及裸鼠移植瘤生長影響及調(diào)控研究[D];山東大學;2016年

，

本文編號：2043175