TGF-β 1siRNA对膀胱肿瘤血管生成的影响

发布时间：2018-06-04 14:44

本文选题：转化生长因子-β1 + 膀胱肿瘤　；参考：《泸州医学院》2014年硕士论文

【摘要】：膀胱肿瘤是临床上最常见的一种泌尿系统肿瘤，而且也是一种威胁患者生命的疾病。膀胱癌的生物学行为特别复杂，主要变现为：复发、多发、浸润和转移等。90%的膀胱癌是尿路上皮癌，，且具有高复发性率。膀胱癌的发生、发展及复发机制目前尚不明了，近年来随着相关研究的深入，发现转化生长因子β1（Transforming growth factors β1，TGF-β1）能促进肿瘤血管的生成从而导致癌细胞的扩散转移。目的：探讨通过选择人工设计合成抑制作用最佳的TGF-β1siRNA转染入人膀胱癌细胞株进行培养，来明确TGF-β1siRNA对膀胱肿瘤血管生成的影响。方法：将膀胱癌细胞株进行培养，取对数生长期的膀胱癌细胞分为常规细胞组、脂质体组（阴性对照组）和3组（A组、B组和C组）含不同TGF-β1siRNA表达载体组。选择人工设计合成好的三种TGF-β1siRNA，通过质粒分别转染入A组、B组和C组后，转染细胞培养24小时后使用实时荧光定量聚合酶链式反应(real-time PCR)与双抗体一步夹心法酶联免疫吸附试验（Elisa）共同检测转染TGF-β1siRNA的人膀胱癌细胞株TGF-β1的表达，选出最佳抑制组。取对数生长期的膀胱癌细胞进行转染，将膀胱癌细胞分为EJ肿瘤细胞组、对照组（TGF-β1组）、重组质粒组（TGF-β1siRNA表达载体组）。对照组采用10ng/ml TGF-β1刺激，转染细胞培养24小时后应用蛋白印迹法（Western Blot）检测该组与正常EJ细胞组、TGF-β1组、重组质粒组中血管内皮生长因子（VEGF）蛋白表达水平。结果：人膀胱癌细胞株培养良好，TGF-β1siRNA转染成功。RT-PCR检测发现：与常规培养的EJ细胞组比较，TGF-β1mRNA在转染空白质粒的EJ细胞组内的表达无统计学意义(P0.05)，表明转染的空白质粒对TGF-β1mRNA的表达无明显干扰；与常规细胞组和空白质粒组比较，TGF-β1mRNA在三个不同TGF-β1SiRNA组EJ细胞内的表达均有显著下降(P0.05)，其中C组最显著(P0.01)，表明所设计的三种TGF-β1SiRNA对TGF-β1mRNA的表达均具有抑制作用，其中C组TGF-β1SiRNA序列与其他序列比较具有最佳抑制效果。Elisa检测发现：与常规培养的EJ细胞组比较，TGF-β1蛋白在转染空白质粒的EJ细胞内的表达无统计学意义(P0.05)，表明转染的空白质粒对TGF-β1蛋白的表达无明显干扰，与RT-PCR检测的结果一致；与常规细胞组和空白质粒组比较，TGF-β1蛋白在三个不同TGF-β1SiRNA组EJ细胞内表达均有显著下降(P0.05)，与RT-PCR检测结果一致，TGF-β1蛋白的Elisa检测也印证了所设计的三种TGF-β1SiRNA对TGF-β1的表达均具有抑制作用。使用Western Blot检测各组VEGF蛋白的表达：与正常EJ细胞组比较，VEGF蛋白在转染TGF-β1质粒的EJ细胞中的表达量显著增大(P0.05)，表明VEGF蛋白的表达与TGF-β1呈正相关；与正常EJ细胞组和TGF-β1组比较，VEGF蛋白在转染TGF-β1siRNA质粒的EJ细胞中的表达量显著减少(P0.05)，表明膀胱癌EJ细胞VEGF蛋白的表达随着TGF-β1基因沉默而减少。结论：转染TGF-β1siRNA质粒的EJ细胞VEGF蛋白的表达显著减少，从而肯定了TGF-β1siRNA对膀胱肿瘤血管内皮生长因子的合成有明显的抑制作用。
[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.
【学位授予单位】：泸州医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.14

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