RNA干扰Nodal基因对人胃腺癌癌细胞MNK-45凋亡及血管生成的影响
发布时间：2018-08-24 13:35
【摘要】：目的:构建并鉴定Nodal基因的RNAi慢病毒表达载体,转染人胃腺癌癌细胞MNK-45,沉默人胃腺癌癌细胞MNK-45中Nodal,探讨Nodal基因对人胃腺癌癌细胞MNK-45凋亡及血管生成的影响。方法:1)针对Nodal mRNA设计合成3条siRNA,及设计1条阴性对照siRNA。退火形成双链DNA,与荧光载体(GV248)连接,对构建的3个重组质粒进行DNA测序,如测定序列与目的序列一致,提示插入Nodal基因RNAi序列正确。2)对重组慢病毒载体进行包装及鉴定,对测序正确的菌液进行质粒抽提并与3种质粒载体(重组质粒及pHelper1.0、pHelper2.0)共转染293T细胞,转染293T细胞后96h,荧光显微镜观察细胞,测定病毒滴度。3)将慢病毒感染人胃腺癌细胞MNK-45。感染72h后,进行荧光倒置显微镜观察感染效率。qPCR检测Nodal基因的表达。4)shRNA慢病毒感染MKN-45细胞,培养5天后,使用eBioscience的凋亡检测试剂盒及流氏细胞仪检测细胞凋亡情况。5)采用人脐静脉内皮细胞(HUVECs)三维培养法分析慢病毒感染MKN-45细胞培养上清对血管形成的影响。Cellomics仪观察人脐静脉内皮细胞(HUVECs)血管面积、平均血管长度、平均血管宽度、血管节点数来评估血管生成情况。结果:(1)重组慢病毒载体的测序:对构建的3个重组质粒进行DNA测序,测序结果显示,测定序列与目的序列一致。提示插入Nodal基因RNAi序列正确。(2)重组慢病毒载体的包装及鉴定:将3种质粒载体转染293T细胞后96h,荧光显微镜观察细胞,见细胞生长良好,荧光强烈。测定病毒滴度为5×108TU/ml。(3)qPCR检测RNA干扰后Nodal基因的表达:RNAi慢病毒感染后,MKN-45细胞Nodal基因的表达水平显著下调。LV-NODAL-RNAi(44787-1)感染、LV-NODAL-RNAi(44789-1)感染分别下调了80.3%和84.9%。(4)RNA干扰Nodal基因对MKN-45细胞凋亡的影响:shRNA慢病毒感染MKN-45细胞,培养5天后,干扰组凋亡率与对照组比较明显升高(P0.05)。(5)对体外HUVEC细胞血管生成的影响:96孔板中铺Matrigel,用收集的目的细胞培养上清液重悬HUVEC细胞,铺96孔板。培养后加入Calcein AM,室温孵育。与对照组比较,实验组血管生成主要相关参数中,血管面积、平均血管长度、平均血管宽度、血管节点数无显著性差异(P0.05)。结论:本研究成功构建了人Nodal基因的RNAi慢病毒表达系统。通过RNAi技术有效的抑制了人胃癌细胞MNK-45细胞Nodal基因的表达,并且发现Nodal基因干扰后显著增加胃癌细胞凋亡的数量,但对血管形成影响不明显。
[Abstract]:Aim: to construct and identify the RNAi lentivirus expression vector of Nodal gene, and transfect Nodal, gene into human gastric adenocarcinoma cell line MNK-45, to investigate the effect of Nodal gene on MNK-45 apoptosis and angiogenesis in human gastric adenocarcinoma cell line MNK-45. Methods: we designed and synthesized three siRNA, and one negative control siRNA. for Nodal mRNA. The three recombinant plasmids were sequenced by DNA sequencing. The results showed that the Nodal gene RNAi sequence was correct. 2) the recombinant lentivirus vector was packaged and identified. Recombinant plasmid and pHelper1.0,pHelper2.0 were cotransfected into 293T cells. The cells were observed by fluorescence microscope at 96 h after transfection, and the virus titer was measured. 3) lentivirus was infected into human gastric adenocarcinoma cell line MNK-45.. After 72 hours of infection, the infection efficiency was observed by fluorescence inverted microscope. The expression of Nodal gene was detected by qPCR. The MKN-45 cells were infected by shRNA lentivirus and cultured for 5 days. Using eBioscience apoptosis detection kit and flow cytometer to detect apoptosis.) using (HUVECs) three-dimensional culture method to analyze the effect of MKN-45 cell culture supernatant of lentivirus infection on angiogenesis. Cellomics instrument was used to observe the effect of human umbilical vein endothelial cells on angiogenesis. The vascular area of (HUVECs) in venous endothelial cells, Mean vascular length, mean vascular width, and number of nodes were used to evaluate angiogenesis. Results: (1) sequencing of recombinant lentivirus vector: three recombinant plasmids were sequenced by DNA. The sequencing results showed that the sequencing sequence was consistent with the target sequence. The results showed that the RNAi sequence of Nodal gene was correct. (2) the packaging and identification of recombinant lentivirus vector: 96 h after transfection of three plasmid vectors into 293T cells, fluorescence microscope showed that the cells grew well and the fluorescence was strong. (3) qPCR was used to detect the expression of Nodal gene after RNA interference. (4) the expression level of Nodal gene in MKN-45 cells infected with lentivirus was significantly down-regulated. LV-NODAL-RNAi (44787-1) infection decreased the apoptosis of MKN-45 cells by 80.3% and 84.9%, respectively. (4) RNA interfered with Nodal gene on the apoptosis of MKN-45 cells. The MKN-45 cells are infected with the lentivirus. After 5 days of culture, the apoptotic rate of the interference group was significantly higher than that of the control group (P0.05). (5). The effect on the angiogenesis of HUVEC cells in vitro was observed. The HUVEC cells were heavily suspended in the supernatant of the target cell culture supernatant of Matrigel, and the 96-well plate was covered with the target cell culture supernatant of Matrigel,. After culture, Calcein AM, was added to incubate at room temperature. Compared with the control group, there was no significant difference in the main parameters of angiogenesis between the experimental group and the control group (P0.05). Conclusion: the RNAi lentivirus expression system of human Nodal gene was successfully constructed in this study. The expression of Nodal gene in human gastric cancer MNK-45 cells was effectively inhibited by RNAi technique, and it was found that Nodal gene interference significantly increased the number of apoptosis of gastric cancer cells, but had no obvious effect on angiogenesis.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.2
本文编号：2200975
[Abstract]:Aim: to construct and identify the RNAi lentivirus expression vector of Nodal gene, and transfect Nodal, gene into human gastric adenocarcinoma cell line MNK-45, to investigate the effect of Nodal gene on MNK-45 apoptosis and angiogenesis in human gastric adenocarcinoma cell line MNK-45. Methods: we designed and synthesized three siRNA, and one negative control siRNA. for Nodal mRNA. The three recombinant plasmids were sequenced by DNA sequencing. The results showed that the Nodal gene RNAi sequence was correct. 2) the recombinant lentivirus vector was packaged and identified. Recombinant plasmid and pHelper1.0,pHelper2.0 were cotransfected into 293T cells. The cells were observed by fluorescence microscope at 96 h after transfection, and the virus titer was measured. 3) lentivirus was infected into human gastric adenocarcinoma cell line MNK-45.. After 72 hours of infection, the infection efficiency was observed by fluorescence inverted microscope. The expression of Nodal gene was detected by qPCR. The MKN-45 cells were infected by shRNA lentivirus and cultured for 5 days. Using eBioscience apoptosis detection kit and flow cytometer to detect apoptosis.) using (HUVECs) three-dimensional culture method to analyze the effect of MKN-45 cell culture supernatant of lentivirus infection on angiogenesis. Cellomics instrument was used to observe the effect of human umbilical vein endothelial cells on angiogenesis. The vascular area of (HUVECs) in venous endothelial cells, Mean vascular length, mean vascular width, and number of nodes were used to evaluate angiogenesis. Results: (1) sequencing of recombinant lentivirus vector: three recombinant plasmids were sequenced by DNA. The sequencing results showed that the sequencing sequence was consistent with the target sequence. The results showed that the RNAi sequence of Nodal gene was correct. (2) the packaging and identification of recombinant lentivirus vector: 96 h after transfection of three plasmid vectors into 293T cells, fluorescence microscope showed that the cells grew well and the fluorescence was strong. (3) qPCR was used to detect the expression of Nodal gene after RNA interference. (4) the expression level of Nodal gene in MKN-45 cells infected with lentivirus was significantly down-regulated. LV-NODAL-RNAi (44787-1) infection decreased the apoptosis of MKN-45 cells by 80.3% and 84.9%, respectively. (4) RNA interfered with Nodal gene on the apoptosis of MKN-45 cells. The MKN-45 cells are infected with the lentivirus. After 5 days of culture, the apoptotic rate of the interference group was significantly higher than that of the control group (P0.05). (5). The effect on the angiogenesis of HUVEC cells in vitro was observed. The HUVEC cells were heavily suspended in the supernatant of the target cell culture supernatant of Matrigel, and the 96-well plate was covered with the target cell culture supernatant of Matrigel,. After culture, Calcein AM, was added to incubate at room temperature. Compared with the control group, there was no significant difference in the main parameters of angiogenesis between the experimental group and the control group (P0.05). Conclusion: the RNAi lentivirus expression system of human Nodal gene was successfully constructed in this study. The expression of Nodal gene in human gastric cancer MNK-45 cells was effectively inhibited by RNAi technique, and it was found that Nodal gene interference significantly increased the number of apoptosis of gastric cancer cells, but had no obvious effect on angiogenesis.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.2
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