慢病毒介導(dǎo)HO-1基因轉(zhuǎn)染對骨髓間充質(zhì)干細(xì)胞體外缺血性損傷保護(hù)作用的實驗研究
發(fā)布時間：2019-05-17 13:46
【摘要】：目的:通過慢病毒介導(dǎo)HO-1基因轉(zhuǎn)染豬骨髓間充質(zhì)干細(xì)胞(MSCs),并建立體外模擬缺血性損傷模型,探討HO-1基因修飾對MSCs缺血性損傷的保護(hù)作用及其機(jī)制。從而為下一步HO-1基因修飾的MSCs移植治療心肌缺血的研究提供實驗依據(jù)。 方法:采用密度梯度離心結(jié)合貼壁法分離、培養(yǎng)豬MSCs,并用免疫熒光法鑒定;用慢病毒包裝系統(tǒng)介導(dǎo)的HO-1基因轉(zhuǎn)染豬MSCs,通過熒光顯微鏡觀察并繪制轉(zhuǎn)染HO-1基因的MSCs(HO-1-MSCs組)、未轉(zhuǎn)染HO-1基因的MSCs(MSCs組)生長曲線,觀察慢病毒轉(zhuǎn)染對MSCs增殖的影響。PCR檢測HO-1-MSCs組、MSCs組的HO-1mRNA表達(dá);采用血清剝奪和缺氧(SOD)處理建立MSCs體外模擬缺血性損傷模型,通過流式細(xì)胞術(shù)檢測評價HO-1-MSCs組、MSCs組在SOD后3h、6h及9h時的凋亡、死亡情況,并通過RT-PCR、Western blot技術(shù)檢測兩組在SOD各個時間點的HO-1mRNA及HO-1蛋白的表達(dá)情況。 結(jié)果:密度梯度離心結(jié)合貼壁法能有效分離、純化豬MSCs,轉(zhuǎn)染48小時后,可檢測到強(qiáng)熒光出現(xiàn),免疫熒光染色顯示培養(yǎng)的細(xì)胞CD44、CD105表達(dá)陽性;細(xì)胞計數(shù)并繪制生長曲線結(jié)果顯示,轉(zhuǎn)基因的MSCs與未轉(zhuǎn)基因的MSCs生長曲線沒有顯著差別,無統(tǒng)計學(xué)意義;HO-1-MSCs組較MSCs組在常氧培養(yǎng)條件下凋亡率(AR)(P0.05)和死亡率(DR)沒有顯著差別(P0.05),無統(tǒng)計學(xué)意義;兩組MSCs在各SOD時間點的AR和DR較常氧培養(yǎng)條件下均顯著性增高(P0.01),有統(tǒng)計學(xué)意義。在同一SOD時間點,HO-1-MSCs組較MSCs組的AR顯著性降低(P0.01),有統(tǒng)計學(xué)意義。RT-PCR、Western blot結(jié)果顯示:在常氧培養(yǎng)條件下,HO-1-MSCs組較MSCs組的HO-1mRNA和HO-1蛋白表達(dá)均顯著增高(P0.01),有統(tǒng)計學(xué)意義;在各SOD時間點,兩組MSCs的HO-1mRNA和HO-1蛋白表達(dá)較常氧均顯著性增高(P0.01),有統(tǒng)計學(xué)意義;隨著SOD時間延長,兩組MSCs的HO-1mRNA和蛋白表達(dá)均逐步增強(qiáng);在同一SOD時間點,HO-1-MSCs組較MSCs組的HO-1mRNA和HO-1蛋白表達(dá)均顯著性增加(P0.01),有統(tǒng)計學(xué)意義。 結(jié)論: 1.根據(jù)黏附特性可成功分離、培養(yǎng)獲得豬骨髓MSCs。 2.按MOI=20,慢病毒包裝系統(tǒng)可成功將HO-1基因轉(zhuǎn)染MSCs:效率肯定、相對安全,且對MSCs的增殖沒有明顯影響。 3.本實驗結(jié)果顯示:HO-1基因轉(zhuǎn)染及其蛋白的穩(wěn)定表達(dá)可提高缺氧、缺血環(huán)境下MSCs的生存能力,對抗體外模擬缺血性損傷引起的細(xì)胞凋亡,從而具有保護(hù)性作用。
[Abstract]:Aim: to investigate the protective effect and mechanism of HO-1 gene modification on HO-1 ischemic injury induced by lentivirus mediated transfer of (MSCs), from pig bone marrow mesenchymal stem cells (BMSCs) and the establishment of simulated ischemic injury model in vitro. In order to provide experimental basis for the next step of HO-1 gene modified MSCs transplantation in the treatment of myocardial ischemia. Methods: pig MSCs, was isolated by density gradient centrifugation combined with adherent method and identified by immunofluorescence. The HO-1 gene mediated by lentivirus packaging system was transferred into pig MSCs,. The growth curve of MSCs (HO-1-MSCs group) and MSCs (MSCs group without HO-1 gene were observed and drawn by fluorescence microscope. The effect of lentivirus transfer on the proliferation of MSCs was observed. The expression of HO-1mRNA in HO-1-MSCs group and MSCs group was detected by PCR. The model of simulated ischemic injury of MSCs in vitro was established by serum deprivation and hypoxia (SOD) treatment. The apoptosis and death of HO-1-MSCs group were evaluated by flow cytometry at 6 h and 9 h after SOD, and RT-PCR, was used to evaluate the apoptosis and death of MSCs group at 6 h and 9 h after SOD. The expression of HO-1mRNA and HO-1 proteins in the two groups at each time point of SOD was detected by Western blot. Results: density gradient centrifugation combined with adherent method could effectively isolate the purified pig MSCs, for 48 hours, and strong fluorescence could be detected, and the expression of CD44,CD105 in cultured cells was positive by immunofluorescence staining. The results of cell counting and drawing growth curve showed that there was no significant difference in the growth curve between transgenic MSCs and non-transgenic MSCs, and there was no statistical significance. There was no significant difference in apoptosis rate (AR) (P 0.05) and mortality (DR) (P 0.05) between HO-1-MSCs group and MSCs group under normoxic culture (P 0.05), but there was no significant difference in apoptosis rate (P 0.05) and mortality rate (P 0.05). The AR and DR of MSCs in both groups were significantly higher than those in normoxic culture at each time point of SOD (P 0.01). At the same time point of SOD, the AR of HO-1-MSCs group was significantly lower than that of MSCs group (P 0.01). The results of RT-PCR,Western blot showed that under normoxic culture conditions, The expression of HO-1mRNA and HO-1 protein in HO-1-MSCs group was significantly higher than that in MSCs group (P 0.01). At each SOD time point, the expression of HO-1mRNA and HO-1 protein in MSCs was significantly higher than that in normoxic group (P 0.01), and the expression of HO-1mRNA and protein in MSCs increased gradually with the prolongation of SOD time. At the same SOD time point, the expression of HO-1mRNA and HO-1 protein in HO-1-MSCs group was significantly higher than that in MSCs group (P 0.01). Conclusion: 1. According to the adhesion characteristics, pig bone marrow MSCs. could be successfully separated and cultured. 2. According to MOI=20, lentivirus packaging system, HO-1 gene can be successfully transferred into MSCs:, which is relatively safe, and has no significant effect on the proliferation of MSCs. 3. The results showed that the stable expression of HO-1 gene and its protein could improve the survival ability of MSCs under hypoxia and ischemia, and antagonize the apoptosis induced by simulated ischemic injury in vitro, so it had protective effect.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R363
[Abstract]:Aim: to investigate the protective effect and mechanism of HO-1 gene modification on HO-1 ischemic injury induced by lentivirus mediated transfer of (MSCs), from pig bone marrow mesenchymal stem cells (BMSCs) and the establishment of simulated ischemic injury model in vitro. In order to provide experimental basis for the next step of HO-1 gene modified MSCs transplantation in the treatment of myocardial ischemia. Methods: pig MSCs, was isolated by density gradient centrifugation combined with adherent method and identified by immunofluorescence. The HO-1 gene mediated by lentivirus packaging system was transferred into pig MSCs,. The growth curve of MSCs (HO-1-MSCs group) and MSCs (MSCs group without HO-1 gene were observed and drawn by fluorescence microscope. The effect of lentivirus transfer on the proliferation of MSCs was observed. The expression of HO-1mRNA in HO-1-MSCs group and MSCs group was detected by PCR. The model of simulated ischemic injury of MSCs in vitro was established by serum deprivation and hypoxia (SOD) treatment. The apoptosis and death of HO-1-MSCs group were evaluated by flow cytometry at 6 h and 9 h after SOD, and RT-PCR, was used to evaluate the apoptosis and death of MSCs group at 6 h and 9 h after SOD. The expression of HO-1mRNA and HO-1 proteins in the two groups at each time point of SOD was detected by Western blot. Results: density gradient centrifugation combined with adherent method could effectively isolate the purified pig MSCs, for 48 hours, and strong fluorescence could be detected, and the expression of CD44,CD105 in cultured cells was positive by immunofluorescence staining. The results of cell counting and drawing growth curve showed that there was no significant difference in the growth curve between transgenic MSCs and non-transgenic MSCs, and there was no statistical significance. There was no significant difference in apoptosis rate (AR) (P 0.05) and mortality (DR) (P 0.05) between HO-1-MSCs group and MSCs group under normoxic culture (P 0.05), but there was no significant difference in apoptosis rate (P 0.05) and mortality rate (P 0.05). The AR and DR of MSCs in both groups were significantly higher than those in normoxic culture at each time point of SOD (P 0.01). At the same time point of SOD, the AR of HO-1-MSCs group was significantly lower than that of MSCs group (P 0.01). The results of RT-PCR,Western blot showed that under normoxic culture conditions, The expression of HO-1mRNA and HO-1 protein in HO-1-MSCs group was significantly higher than that in MSCs group (P 0.01). At each SOD time point, the expression of HO-1mRNA and HO-1 protein in MSCs was significantly higher than that in normoxic group (P 0.01), and the expression of HO-1mRNA and protein in MSCs increased gradually with the prolongation of SOD time. At the same SOD time point, the expression of HO-1mRNA and HO-1 protein in HO-1-MSCs group was significantly higher than that in MSCs group (P 0.01). Conclusion: 1. According to the adhesion characteristics, pig bone marrow MSCs. could be successfully separated and cultured. 2. According to MOI=20, lentivirus packaging system, HO-1 gene can be successfully transferred into MSCs:, which is relatively safe, and has no significant effect on the proliferation of MSCs. 3. The results showed that the stable expression of HO-1 gene and its protein could improve the survival ability of MSCs under hypoxia and ischemia, and antagonize the apoptosis induced by simulated ischemic injury in vitro, so it had protective effect.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R363
【相似文獻(xiàn)】
相關(guān)期刊論文 前10條
1 李玉明,張敏,劉俊昌,鄭新程,吳立玲,時安云,伍貽經(jīng);LPS作用下的乳鼠心肌細(xì)胞HO-1的表達(dá)與作用[J];中國病理生理雜志;2000年10期
2 李玉明,鄭新程,劉俊昌,張敏,吳立玲,時安云,伍貽經(jīng);LPS對培養(yǎng)的大鼠主動脈平滑肌細(xì)胞HO-1表達(dá)的影響[J];中國病理生理雜志;2000年10期
3 張英,陳容,曾維誠,唐華,李著華;銀杏葉提取物抗大鼠腎缺血/再灌注損傷的實驗研究[J];瀘州醫(yī)學(xué)院學(xué)報;2005年03期
4 顏勝宇,李昌崇,盧荷蓮,葉樂平,李少波;哮喘大鼠的肺組織血紅素氧合酶-1的表達(dá)及意義[J];溫州醫(yī)學(xué)院學(xué)報;2005年02期
5 劉文琪,李雍龍,龍小純;日本血吸蟲成蟲HO-1編碼基因片段的擴(kuò)增及序列分析[J];熱帶醫(yī)學(xué)雜志;2004年02期
6 劉迎;李學(xué)朝;王靜;楊江根;;血紅素加氧酶-1誘導(dǎo)對移植腎的保護(hù)作用[J];中國醫(yī)學(xué)工程;2007年05期
7 袁偉,馮新紅,湯押庚;大鼠急性脊髓損傷后早期血紅素氧合酶-1的表達(dá)[J];中國臨床康復(fù);2004年02期
8 李萍,Jules Sanders,Emma Hawe,Jutta Palmen,David Brull,Sukhbir Dhamrait,Jayshree Acharya,Hugh Montgomery,Steve Humphries;冠脈旁路搭橋術(shù)后的炎癥反應(yīng):HO-1基因多態(tài)的影響[J];介入放射學(xué)雜志;2003年S1期
9 龐東衛(wèi),徐海,劉俊昌,潘桂蘭,韓莉;腎性高血壓大鼠主動脈HO-1表達(dá)的變化及意義[J];中國病理生理雜志;2004年11期
10 張建武;邢志偉;;HO-1的誘導(dǎo)表達(dá)對急性肺損傷小鼠CD_(54)的影響[J];河北醫(yī)藥;2010年14期
相關(guān)會議論文 前10條
1 苗凱;郭敏;安磊;許曉玲;武韓;王棟;吳中紅;田見暉;;低滴度慢病毒生產(chǎn)轉(zhuǎn)基因胚胎和小鼠的新方法[A];中國畜牧獸醫(yī)學(xué)會動物繁殖學(xué)分會第十五屆學(xué)術(shù)研討會論文集（上冊）[C];2010年
2 王婭婷;王季石;李燕;馬丹;楊暢;陳s，
本文編號：2479139
本文鏈接：http://sikaile.net/xiyixuelunwen/2479139.html
最近更新
教材專著
