RNA干擾Nodal基因?qū)θ宋赶侔┌┘毎鸐NK-45凋亡及血管生成的影響
發(fā)布時間：2018-08-24 13:35
【摘要】：目的:構(gòu)建并鑒定Nodal基因的RNAi慢病毒表達載體,轉(zhuǎn)染人胃腺癌癌細胞MNK-45,沉默人胃腺癌癌細胞MNK-45中Nodal,探討Nodal基因?qū)θ宋赶侔┌┘毎鸐NK-45凋亡及血管生成的影響。方法:1)針對Nodal mRNA設(shè)計合成3條siRNA,及設(shè)計1條陰性對照siRNA。退火形成雙鏈DNA,與熒光載體(GV248)連接,對構(gòu)建的3個重組質(zhì)粒進行DNA測序,如測定序列與目的序列一致,提示插入Nodal基因RNAi序列正確。2)對重組慢病毒載體進行包裝及鑒定,對測序正確的菌液進行質(zhì)粒抽提并與3種質(zhì)粒載體(重組質(zhì)粒及pHelper1.0、pHelper2.0)共轉(zhuǎn)染293T細胞,轉(zhuǎn)染293T細胞后96h,熒光顯微鏡觀察細胞,測定病毒滴度。3)將慢病毒感染人胃腺癌細胞MNK-45。感染72h后,進行熒光倒置顯微鏡觀察感染效率。qPCR檢測Nodal基因的表達。4)shRNA慢病毒感染MKN-45細胞,培養(yǎng)5天后,使用eBioscience的凋亡檢測試劑盒及流氏細胞儀檢測細胞凋亡情況。5)采用人臍靜脈內(nèi)皮細胞(HUVECs)三維培養(yǎng)法分析慢病毒感染MKN-45細胞培養(yǎng)上清對血管形成的影響。Cellomics儀觀察人臍靜脈內(nèi)皮細胞(HUVECs)血管面積、平均血管長度、平均血管寬度、血管節(jié)點數(shù)來評估血管生成情況。結(jié)果:(1)重組慢病毒載體的測序:對構(gòu)建的3個重組質(zhì)粒進行DNA測序,測序結(jié)果顯示,測定序列與目的序列一致。提示插入Nodal基因RNAi序列正確。(2)重組慢病毒載體的包裝及鑒定:將3種質(zhì)粒載體轉(zhuǎn)染293T細胞后96h,熒光顯微鏡觀察細胞,見細胞生長良好,熒光強烈。測定病毒滴度為5×108TU/ml。(3)qPCR檢測RNA干擾后Nodal基因的表達:RNAi慢病毒感染后,MKN-45細胞Nodal基因的表達水平顯著下調(diào)。LV-NODAL-RNAi(44787-1)感染、LV-NODAL-RNAi(44789-1)感染分別下調(diào)了80.3%和84.9%。(4)RNA干擾Nodal基因?qū)KN-45細胞凋亡的影響:shRNA慢病毒感染MKN-45細胞,培養(yǎng)5天后,干擾組凋亡率與對照組比較明顯升高(P0.05)。(5)對體外HUVEC細胞血管生成的影響:96孔板中鋪Matrigel,用收集的目的細胞培養(yǎng)上清液重懸HUVEC細胞,鋪96孔板。培養(yǎng)后加入Calcein AM,室溫孵育。與對照組比較,實驗組血管生成主要相關(guān)參數(shù)中,血管面積、平均血管長度、平均血管寬度、血管節(jié)點數(shù)無顯著性差異(P0.05)。結(jié)論:本研究成功構(gòu)建了人Nodal基因的RNAi慢病毒表達系統(tǒng)。通過RNAi技術(shù)有效的抑制了人胃癌細胞MNK-45細胞Nodal基因的表達,并且發(fā)現(xiàn)Nodal基因干擾后顯著增加胃癌細胞凋亡的數(shù)量,但對血管形成影響不明顯。
[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.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R735.2
本文編號：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.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R735.2
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