CELSR1促進血管內(nèi)皮細胞遷移與血管形成作用的研究
發(fā)布時間：2018-09-06 14:43
【摘要】：【背景和目的】 全基因組關(guān)聯(lián)研究發(fā)現(xiàn)CELSR1基因是日本人群缺血性卒中的易感基因，我們課題組前期實驗也發(fā)現(xiàn)CELSR1基因rs6007897和rs4044210與缺血性卒中具有顯著相關(guān)性，與大動脈粥樣硬化具有相關(guān)性；CELSR1是Wnt/PCP通路的核心成員，Wnt/PCP通路在動脈粥樣硬化過程中起作用。本研究觀察過表達和沉默CELSR1基因的人主動脈內(nèi)皮細胞（human aortic endothelial cells，HAECs）血管內(nèi)皮細胞的影響，來闡述CELSR基因在動脈粥樣硬化過程中的作用。 【方法】 首先利用構(gòu)建干擾CELSR1表達的質(zhì)粒，通過慢病毒載體感染HAEC細胞，利用puromycin篩選出表達有CELSR1shRNA的HAEC的細胞株；利用TALEA技術(shù)將攜帶過表達CELSR1質(zhì)粒轉(zhuǎn)染HAEC，利用puromycin篩選出過表達CELSR1的HAEC的細胞株；采用Westernblot法、實時熒光定量PCR法以及MTT法和劃痕實驗、Transwell實驗、成環(huán)實驗檢測過表達和沉默細胞株的CELSR1的表達及HAECs增殖和遷移、成環(huán)能力。 【結(jié)果】 CELSR1shRNA序列可有效抑制HACE細胞CELSR1的表達導(dǎo)入CELSR1序列可明顯提高CELSR1在HAEC細胞中的表達，差異有統(tǒng)計學(xué)意義(P0.05)。MTT實驗結(jié)果示，干擾CELSR1表達的HAEC細胞生長明顯受到抑制,差異有統(tǒng)計學(xué)意義(P0.05)；過表達CELSR1的HAEC細胞與對照組相比，生長速度加快，差異有統(tǒng)計學(xué)意義(P0.05)。劃痕實驗及Transwel實驗結(jié)果示，干擾CELSR1表達的HAEC細胞遷移速度較對照組減慢，差異有統(tǒng)計學(xué)意義(P0.05)，過表達CELSR1的HAEC細胞遷移速度與對照組相比，遷移速度增快，差異有統(tǒng)計學(xué)意義(P0.05)。成環(huán)實驗示，干擾CELSR1表達的HAEC細胞較對照組的成環(huán)數(shù)低于對照組，差異有統(tǒng)計學(xué)意義(P0.05)。過表達CELSR1的HAEC細胞的成環(huán)數(shù)高于對照組，差異有統(tǒng)計學(xué)意義(P0.05)。 【結(jié)論】 CELSR1shRNA能夠下調(diào)HAEC的CELSR1表達，有效地抑制HAEC的增殖和遷移、成環(huán)；轉(zhuǎn)染CELSR1能上調(diào)HAEC內(nèi)源性CELSR1的表達，有效地促進HAEC的增殖和遷移、成環(huán)，提示CELSR1可能在血管生成過程中發(fā)揮重要作用。
[Abstract]:[background and objective] Genome-wide association studies have found that CELSR1 gene is a susceptible gene for ischemic stroke in Japanese population. Our previous study also found that CELSR1 gene rs6007897 and rs4044210 have a significant correlation with ischemic stroke. CELSR1 is a core member of the Wnt/PCP pathway, which plays an important role in atherosclerosis. The purpose of this study was to investigate the effects of human aortic endothelial cells (human aortic endothelial cells,HAECs) expressing and silencing CELSR1 gene on vascular endothelial cells. To explain the role of CELSR gene in atherosclerosis. [methods] first, the plasmid interfering with CELSR1 expression was constructed, and HAEC cells were infected by lentivirus vector. Puromycin was used to screen the cell lines expressing HAEC with CELSR1shRNA; HAEC, was transfected with overexpressed CELSR1 plasmid by TALEA technique; HAEC cell lines expressing CELSR1 were screened by puromycin; Westernblot method, real-time fluorescence quantitative PCR method, MTT method and scratch test were used to screen HAEC cell lines. The expression of CELSR1 and the proliferation and migration of HAECs in overexpression and silencing cell lines were detected by loop forming assay. [results] CELSR1shRNA sequence could effectively inhibit the expression of CELSR1 in HACE cells and induce CELSR1 sequence to increase the expression of CELSR1 in HAEC cells. The difference was statistically significant (P0.05). The growth of HAEC cells which interfered with the expression of CELSR1 was significantly inhibited (P0.05); the growth rate of HAEC cells overexpression of CELSR1 was faster than that of the control group (P0.05). The results of scratch test and Transwel test showed that the migration speed of HAEC cells interfering with CELSR1 expression was slower than that of control group (P0.05). The migration speed of HAEC cells with overexpression of CELSR1 was faster than that of control group (P0.05). The ring-forming experiment showed that the number of HAEC cells interfering with CELSR1 expression was lower than that of the control group (P0.05). The number of HAEC cells with overexpression of CELSR1 was significantly higher than that of control group (P0.05). [conclusion] CELSR1shRNA can down-regulate the expression of HAEC CELSR1 and effectively inhibit the proliferation and migration of HAEC. Transfection of CELSR1 can up-regulate the expression of endogenous CELSR1 in HAEC and effectively promote the proliferation and migration of HAEC, suggesting that CELSR1 may play an important role in angiogenesis.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R743.3
本文編號：2226673
[Abstract]:[background and objective] Genome-wide association studies have found that CELSR1 gene is a susceptible gene for ischemic stroke in Japanese population. Our previous study also found that CELSR1 gene rs6007897 and rs4044210 have a significant correlation with ischemic stroke. CELSR1 is a core member of the Wnt/PCP pathway, which plays an important role in atherosclerosis. The purpose of this study was to investigate the effects of human aortic endothelial cells (human aortic endothelial cells,HAECs) expressing and silencing CELSR1 gene on vascular endothelial cells. To explain the role of CELSR gene in atherosclerosis. [methods] first, the plasmid interfering with CELSR1 expression was constructed, and HAEC cells were infected by lentivirus vector. Puromycin was used to screen the cell lines expressing HAEC with CELSR1shRNA; HAEC, was transfected with overexpressed CELSR1 plasmid by TALEA technique; HAEC cell lines expressing CELSR1 were screened by puromycin; Westernblot method, real-time fluorescence quantitative PCR method, MTT method and scratch test were used to screen HAEC cell lines. The expression of CELSR1 and the proliferation and migration of HAECs in overexpression and silencing cell lines were detected by loop forming assay. [results] CELSR1shRNA sequence could effectively inhibit the expression of CELSR1 in HACE cells and induce CELSR1 sequence to increase the expression of CELSR1 in HAEC cells. The difference was statistically significant (P0.05). The growth of HAEC cells which interfered with the expression of CELSR1 was significantly inhibited (P0.05); the growth rate of HAEC cells overexpression of CELSR1 was faster than that of the control group (P0.05). The results of scratch test and Transwel test showed that the migration speed of HAEC cells interfering with CELSR1 expression was slower than that of control group (P0.05). The migration speed of HAEC cells with overexpression of CELSR1 was faster than that of control group (P0.05). The ring-forming experiment showed that the number of HAEC cells interfering with CELSR1 expression was lower than that of the control group (P0.05). The number of HAEC cells with overexpression of CELSR1 was significantly higher than that of control group (P0.05). [conclusion] CELSR1shRNA can down-regulate the expression of HAEC CELSR1 and effectively inhibit the proliferation and migration of HAEC. Transfection of CELSR1 can up-regulate the expression of endogenous CELSR1 in HAEC and effectively promote the proliferation and migration of HAEC, suggesting that CELSR1 may play an important role in angiogenesis.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R743.3
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