下调血管生成素对膀胱癌T24细胞增殖及凋亡的影响及分子机制研究

发布时间：2018-04-22 23:01

本文选题：血管生成素 + 膀胱癌细胞　；参考：《重庆医科大学》2014年硕士论文

【摘要】：目的构建血管生成素（ANG）基因siRNA干扰质粒，并检测其对人膀胱癌T24细胞增殖、凋亡的影响以及初步探讨其分子机制。方法 1.设计并构建针对人ANG基因mRNA的2个特异性siRNA表达载体和1个无同源性的阴性对照载体，通过SalI酶切和DNA测序鉴定载体后，再转染膀胱癌T24细胞，G418筛选稳定表达的细胞株，分别命名为T24-siANG1、T24-siANG2、T24-NC，经Q-RCR，Western Blot检测干扰的有效性。 2.HE染色观察细胞形态；MTT检测细胞增殖能力；流式细胞术检测细胞周期；激光共聚焦检测ANG与RI相互关系；细胞免疫荧光检测蛋白ANG、RI、p-AKT、p-GSK3β、p-mTOR表达水平。 3.流式细胞术检测细胞凋亡率；Tunel和Hochest33342凋亡试剂盒检测细胞凋亡；Western blot检测蛋白ANG、RI，细胞凋亡相关蛋白Bax、Caspase-3、Bcl-2，信号通路蛋白AKT、GSK3β、mTOR、p-AKT、p-GSK3β、p-mTOR表达水平。 4.构建裸鼠移植瘤模型，观察移植瘤生长并计算抑制率；CD31抗体，HE染色观察肿瘤微血管生长以及肺组织自发转移情况；组织免疫荧光检测蛋白RI、ANG、p-AKT、p-GSK3β、p-mTOR表达水平；免疫组织化学检测瘤组织中蛋白ANG、RI、Bax、Caspase-3、Bcl-2、p-AKT、p-GSK3β、p-mTOR、AKT、GSK3β、mTOR表达水平。结果 1.测序结果显示：重组质粒构建成功；Q-PCR，Western Blot分析T24-siANG1组ANG的mRNA，蛋白表达显著抑制，与T24-NC组相比（p0.05）。 2.HE染色结果显示：与对照组相比，实验组T24-siANG1组的细胞恶性程度降低，表现为细胞不重叠生长，细胞核变小，核质比减少，呈弱嗜碱性细胞质；MTT结果显示：T24-siANG1组较T24-NC及T24组细胞增殖能力明显下降；流式细胞分析结果表明：T24-siANG1组较两对照组细胞生长阻滞于G1期，S期和G2期降低；激光共聚焦检测ANG与RI蛋白具有共定位关系；细胞及组织免疫荧光检测结果显示：ANG，信号通路关键蛋白p-AKT，p-GSK3β，p-mTOR表达降低，而RI的表达增高。 3.流式细胞分析结果表明：流式细胞仪检测结果显示：T24-siANG1组细胞有（30.23±15.81）％的凋亡细胞，而T24-NC组和T24细胞组仅有(5.44±3.09)％和(3.96±1.58)％的凋亡细胞，T24-siANG1组较T24-NC组细胞凋亡率明显增高（p0.01）；TUNEL检测结果显示：T24-siANG1组出现大量凋亡细胞；Hochest检测结果显示：T24-siANG1组出现典型凋亡形态特征：染色质凝集，核碎裂和明亮的蓝色荧光等；Western blot结果显示：与T24-NC组相比，T24-siANG1组Bcl-2的表达明显降低(p＜0.05),而Bax和激活的Caspase3的表达明显升高(p＜0.05)；T24-siANG1组较T24-NC组的信号通路蛋白p-AKT，p-GSK3β，p-mTOR的表达显著降低(p＜0.01)，而RI蛋白表达水平明显升高(p＜0.05)。 4.裸鼠在皮下注射各组细胞后，与T24-NC组相比，T24-siANG1组的移植瘤重明显降低；组织免疫荧光CD31及HE染色实验结果均表明：与两对照组相比，T24-siANG1组瘤组织中微血管明显减少。肺组织HE染色结果显示：T24-siANG1组肺组织未见明显转移，而两对照组在大血管附近可见细胞核增大且染色较深的细胞，，表明已发生远处转移。免疫组织化学检测结果显示：T24-siANG1组瘤组织中蛋白RI， Bax，Caspase-3表达明显升高，而Bcl-2，ANG，p-AKT，p-GSK3β，p-mTOR表达明显降低， AKT，GSK3β，mTOR的表达无变化，与Western blot结果一致。结论体内外实验均证实：成功构建的ANG siRNA干扰质粒通过调控ANG信号通路及凋亡蛋白抑制膀胱癌T24细胞的增殖并促进其凋亡。
[Abstract]:Objective To construct siRNA interfering plasmid of angiopoietin ( ANG ) gene , and to examine its effect on proliferation and apoptosis of human bladder cancer cell line 24 , and to explore its molecular mechanism .

method

1 . Two specific siRNA expression vectors and one non - homologous negative control vector were designed and constructed for human ANG gene mRNA .

2 . HE staining was used to observe the morphology of cells .
MTT assay was used to detect cell proliferation .
Flow cytometry was used to detect cell cycle .
laser confocal detection ANG is related to RI ;
The levels of ANG , RI , p - 1 , p - GSK3 & beta ; , p - mtor expression were detected by immunofluorescence assay .

3 . Flow cytometry was used to detect apoptosis rate .
Apoptosis was detected by Tunel and Hooks 33342 apoptosis kit .
Western blot was used to detect the expression level of Bax , Caspase - 3 , Bcl - 2 , signal pathway proteins , GSK3 尾 , mtor , p - 1 , p - GSK3 尾 , p - mtor expression in protein ANG , RI , cell apoptosis related protein Bax , Caspase - 3 , Bcl - 2 , signal pathway protein 3 .

4 . The nude mice transplanted tumor model was constructed , and the growth of transplanted tumor was observed and the inhibition rate was calculated .
CD31 antibody and HE staining were used to observe the growth of tumor microvessels and spontaneous metastasis of lung tissues .
Tissue immunofluorescence assay protein RI , ANG , p - 1 , p - GSK3beta , p - mtor expression level ;
The levels of protein ANG , RI , Bax , Caspase - 3 , Bcl - 2 , p - 1 , p - GSK3 尾 , p - mtor , T , GSK3 尾 and mtor were detected by immunohistochemistry .

Results

1 . Sequencing results showed that the recombinant plasmid was constructed successfully .
The mRNA and protein expressions of ANG mRNA and protein were significantly inhibited by Q - PCR and Western Blot .

2 . The results of HE staining showed that the malignant degree of cells in the experimental group 24 - siANG1 was decreased compared with the control group , which showed that the cells did not overlap and grow , the nucleus became smaller , the nuclear mass ratio decreased , and showed weak basophilic cytoplasm ;
The results showed that the cell proliferation ability was significantly decreased in the 24 - siANG1 group than that in the 24 - NC group .
The results of flow cytometry showed that the growth arrest of the cells was decreased in G1 phase , S phase and G2 phase compared with the control group .
Laser cofocus detection ANG has a co - located relationship with RI protein .
The results of immunofluorescence assay showed that the expression of the key protein of ANG , signaling pathway was decreased , and the expression of p - GSK3 尾 , p - mtor decreased , while RI increased .

3 . The results of flow cytometry showed that there were ( 30.23 卤 15.81 ) % apoptotic cells in 24 - siANG1 group , but only ( 5.44 卤 3.09 ) % and ( 3.96 卤 1 . 58 ) % of apoptotic cells were found in 24 - NC group and 24 - cell group .
TUNEL assay showed that there appeared a significant number of apoptotic cells in 24 - siANG1 group .
The results showed that the typical apoptotic morphological features : chromatin condensation , nuclear fragmentation and bright blue fluorescence appeared in the 24 - siANG1 group .
Western blot showed that the expression of Bcl - 2 was significantly decreased ( p < 0 . 05 ) , but Bax and the activated caspase3 increased significantly ( p < 0 . 05 ) .
The expression of p - gp , p - GSK3 尾 , p - mtor decreased significantly ( p < 0 . 01 ) , while RI protein expression increased significantly ( p < 0 . 05 ) .

4 . After subcutaneous injection of each group of cells in nude mice , the transplanted tumor weight was significantly decreased in the 24 - siANG1 group compared with the 24 - NC group .
The results of HE staining showed that the expression of protein RI , Bax , Caspase - 3 in 24 - siANG1 group was significantly higher than that in the control group , but the expression of Bcl - 2 , ANG , p - 1 , p - GSK3 尾 , p - mtor was significantly decreased .

Conclusion Both in vivo and in vivo experiments confirm that the ANG siRNA interfering plasmid constructed successfully inhibits the proliferation of bladder cancer cell line 24 and promotes its apoptosis by regulating ANG signal pathway and apoptosis protein .

【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.14

【参考文献】