發(fā)布時間：2018-06-04 14:44

  本文選題：轉(zhuǎn)化生長因子-β1 + 膀胱腫瘤��； 參考：《瀘州醫(yī)學(xué)院》2014年碩士論文

【摘要】：膀胱腫瘤是臨床上最常見的一種泌尿系統(tǒng)腫瘤，而且也是一種威脅患者生命的疾病。膀胱癌的生物學(xué)行為特別復(fù)雜，主要變現(xiàn)為：復(fù)發(fā)、多發(fā)、浸潤和轉(zhuǎn)移等。90%的膀胱癌是尿路上皮癌，，且具有高復(fù)發(fā)性率。膀胱癌的發(fā)生、發(fā)展及復(fù)發(fā)機制目前尚不明了，近年來隨著相關(guān)研究的深入，發(fā)現(xiàn)轉(zhuǎn)化生長因子β1（Transforming growth factors β1，TGF-β1）能促進腫瘤血管的生成從而導(dǎo)致癌細胞的擴散轉(zhuǎn)移。目的：探討通過選擇人工設(shè)計合成抑制作用最佳的TGF-β1siRNA轉(zhuǎn)染入人膀胱癌細胞株進行培養(yǎng)，來明確TGF-β1siRNA對膀胱腫瘤血管生成的影響。方法：將膀胱癌細胞株進行培養(yǎng)，取對數(shù)生長期的膀胱癌細胞分為常規(guī)細胞組、脂質(zhì)體組（陰性對照組）和3組（A組、B組和C組）含不同TGF-β1siRNA表達載體組。選擇人工設(shè)計合成好的三種TGF-β1siRNA，通過質(zhì)粒分別轉(zhuǎn)染入A組、B組和C組后，轉(zhuǎn)染細胞培養(yǎng)24小時后使用實時熒光定量聚合酶鏈式反應(yīng)(real-time PCR)與雙抗體一步夾心法酶聯(lián)免疫吸附試驗（Elisa）共同檢測轉(zhuǎn)染TGF-β1siRNA的人膀胱癌細胞株TGF-β1的表達，選出最佳抑制組。取對數(shù)生長期的膀胱癌細胞進行轉(zhuǎn)染，將膀胱癌細胞分為EJ腫瘤細胞組、對照組（TGF-β1組）、重組質(zhì)粒組（TGF-β1siRNA表達載體組）。對照組采用10ng/ml TGF-β1刺激，轉(zhuǎn)染細胞培養(yǎng)24小時后應(yīng)用蛋白印跡法（Western Blot）檢測該組與正常EJ細胞組、TGF-β1組、重組質(zhì)粒組中血管內(nèi)皮生長因子（VEGF）蛋白表達水平。結(jié)果：人膀胱癌細胞株培養(yǎng)良好，TGF-β1siRNA轉(zhuǎn)染成功。RT-PCR檢測發(fā)現(xiàn)：與常規(guī)培養(yǎng)的EJ細胞組比較，TGF-β1mRNA在轉(zhuǎn)染空白質(zhì)粒的EJ細胞組內(nèi)的表達無統(tǒng)計學(xué)意義(P0.05)，表明轉(zhuǎn)染的空白質(zhì)粒對TGF-β1mRNA的表達無明顯干擾；與常規(guī)細胞組和空白質(zhì)粒組比較，TGF-β1mRNA在三個不同TGF-β1SiRNA組EJ細胞內(nèi)的表達均有顯著下降(P0.05)，其中C組最顯著(P0.01)，表明所設(shè)計的三種TGF-β1SiRNA對TGF-β1mRNA的表達均具有抑制作用，其中C組TGF-β1SiRNA序列與其他序列比較具有最佳抑制效果。Elisa檢測發(fā)現(xiàn)：與常規(guī)培養(yǎng)的EJ細胞組比較，TGF-β1蛋白在轉(zhuǎn)染空白質(zhì)粒的EJ細胞內(nèi)的表達無統(tǒng)計學(xué)意義(P0.05)，表明轉(zhuǎn)染的空白質(zhì)粒對TGF-β1蛋白的表達無明顯干擾，與RT-PCR檢測的結(jié)果一致；與常規(guī)細胞組和空白質(zhì)粒組比較，TGF-β1蛋白在三個不同TGF-β1SiRNA組EJ細胞內(nèi)表達均有顯著下降(P0.05)，與RT-PCR檢測結(jié)果一致，TGF-β1蛋白的Elisa檢測也印證了所設(shè)計的三種TGF-β1SiRNA對TGF-β1的表達均具有抑制作用。使用Western Blot檢測各組VEGF蛋白的表達：與正常EJ細胞組比較，VEGF蛋白在轉(zhuǎn)染TGF-β1質(zhì)粒的EJ細胞中的表達量顯著增大(P0.05)，表明VEGF蛋白的表達與TGF-β1呈正相關(guān)；與正常EJ細胞組和TGF-β1組比較，VEGF蛋白在轉(zhuǎn)染TGF-β1siRNA質(zhì)粒的EJ細胞中的表達量顯著減少(P0.05)，表明膀胱癌EJ細胞VEGF蛋白的表達隨著TGF-β1基因沉默而減少。結(jié)論：轉(zhuǎn)染TGF-β1siRNA質(zhì)粒的EJ細胞VEGF蛋白的表達顯著減少，從而肯定了TGF-β1siRNA對膀胱腫瘤血管內(nèi)皮生長因子的合成有明顯的抑制作用。
[Abstract]:Bladder tumor is the most common type of urological tumor in clinic, and it is also a disease that threatens the life of patients. The biological behavior of bladder cancer is particularly complex, which is mainly converted to bladder cancer of.90%, such as recurrence, multiple onset, infiltration and metastasis, and has high recurrence rate. The occurrence, development and recurrence mechanism of bladder cancer It is still unknown at present. In recent years, with the development of related research, it is found that transforming growth factor beta 1 (Transforming growth factors beta 1, TGF- beta 1) can promote the formation of tumor vessels and lead to the proliferation and metastasis of cancer cells. The effect of TGF- beta 1siRNA on the angiogenesis of bladder tumor was carried out. Methods: bladder cancer cell lines were cultured, and bladder cancer cells of logarithmic growth period were divided into conventional cell groups, liposome group (negative control group) and 3 groups (group A, B group and C group) containing different TGF- beta 1siRNA expression vectors. Select three kinds of artificial design. TGF- beta 1siRNA was transfected into A group, B group and C group respectively. After transfection of cell culture for 24 hours, real-time fluorescent quantitative polymerase chain reaction (real-time PCR) was used to detect the expression of TGF- beta 1 transfected with the human bladder cancer cell line of TGF- beta 1siRNA, and the best inhibition group was selected by the simultaneous detection of double antibody and one-step sandwich enzyme linked immunosorbent assay (Elisa). Bladder cancer cells of logarithmic growth period were transfected into EJ tumor cell group, control group (group TGF- beta 1) and recombinant plasmid group (TGF- beta 1siRNA expression vector group). The control group was stimulated by 10ng/ml TGF- beta 1, and after transfecting cell culture for 24 hours, the group and normal EJ cell group, TGF- beta were detected by Western blot method (Western Blot), TGF- beta. The 1 groups, the expression level of vascular endothelial growth factor (VEGF) protein in the recombinant plasmid group. Results: human bladder cancer cell lines were cultured well. The successful.RT-PCR detection of TGF- beta 1siRNA transfection found that the expression of TGF- beta 1mRNA in the EJ cell group transfected with blank plasmids was not statistically significant (P0.05), indicating that the transfection space was empty. The expression of the white plasmid had no obvious interference to the expression of TGF- beta 1mRNA, and the expression of TGF- beta 1mRNA in the EJ cells of three different TGF- beta 1SiRNA groups was significantly decreased (P0.05) compared with the conventional and blank plasmid groups, and the most significant C group (P0.01) was found in the C group (P0.01). Compared with other sequences, the GF- beta 1SiRNA sequence had the best inhibition effect.Elisa detection. Compared with the conventional cultured EJ cell group, the expression of TGF- beta 1 protein in the EJ cells transfected with blank plasmids was not statistically significant (P0.05), indicating that the transfected blank plasmid had no obvious interference to the expression of TGF- beta 1 protein, and the result was one of the results of RT-PCR detection. Compared with the conventional cell group and the blank plasmid group, the expression of TGF- beta 1 protein in the EJ cells of three different TGF- beta 1SiRNA groups was significantly decreased (P0.05), consistent with the results of RT-PCR detection. The Elisa detection of TGF- beta 1 protein also showed that the three TGF- beta 1SiRNA designed to inhibit the expression of TGF- beta 1 was inhibited. The expression of VEGF protein in each group: compared with the normal EJ cell group, the expression of VEGF protein in the EJ cells transfected with TGF- beta 1 plasmids was significantly increased (P0.05), indicating that the expression of VEGF protein was positively correlated with TGF- beta 1, and the expression of VEGF protein in the cells transfected with TGF- beta plasmid was significantly decreased than that of the normal EJ cell group and TGF- beta 1 group. P0.05) showed that the expression of VEGF protein in EJ cell of bladder cancer decreased with the silence of TGF- beta 1 gene. Conclusion: the expression of VEGF protein in EJ cells transfected with TGF- beta 1siRNA plasmid decreased significantly, thus the obvious inhibitory effect of TGF- beta 1siRNA on the synthesis of vascular endothelial growth factor of bladder tumor was affirmed.
【學(xué)位授予單位】：瀘州醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R737.14

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相關(guān)期刊論文 前1條

1 馮傳波;夏春咸;何治軍;邵華;苗永昌;;TGF-β1、TβRⅡ及P38蛋白在胃癌發(fā)生發(fā)展中的意義[J];重慶醫(yī)學(xué);2010年12期

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