中华神经医学杂志
中華神經醫學雜誌
중화신경의학잡지
CHINESE JOURNAL OF NEUROMEDICINE
2009年
8期
785-789
,共5页
冯东福%刘勇%陈二涛%汪洋%刘天津
馮東福%劉勇%陳二濤%汪洋%劉天津
풍동복%류용%진이도%왕양%류천진
神经干细胞%脑源性神经营养因子%绿色荧光蛋白%基因转染
神經榦細胞%腦源性神經營養因子%綠色熒光蛋白%基因轉染
신경간세포%뇌원성신경영양인자%록색형광단백%기인전염
Stem cells%Brain derived neurotrophic factor%Green fluorescent protein%Gene transfection
目的 探讨慢病毒载体介导入脑源性神经营养因子(hBDNF)和绿色荧光蛋白(GFP)基因转染大鼠神经干细胞(NSCs)后hBDNF的表达及其生物学特性的变化.方法 构建hBDNF和GFP基因共表达的慢病毒载体并转染NSCs(hBDNF-GFP-NSCs组),同时设GFP转染NSCs组(GFP-NSCs组)和未转染的NSCs组(NSCs组).应用RT-PCR和Western blot法分别检测3组细胞中hBDNFmRNA和蛋白的表达:ELISA检测hBDNF-GFP-NSCs组细胞转染前后培养液中hBDNF含量的变化:使用上述3组细胞的上清液培养背根神经节(DRG)与NSCs,观察DRG的生长情况并应用流式细胞法检测NSCs分化为神经元的比例.结果 RT-PCR、Western blot结果显示转染后7 d hBDNF-GFP-NSCs组hBDNF mRNA和蛋白的表达均明显强于GFp-NSCs组和NSCs组;EUSA检测显示hBDNF-GFP转染NSCs后上清中hBDNF含量增加,第5天分泌达最高峰,与转染前比较差异均有统计学意义(P<0.05);使用hBDNF-GFP-NSCs组上清液培养DRG和NSCs,4 d后DRG很快伸出突起,流式细胞法检测显示NSCs分化为神经元的比例高于其他两组.结论 NSCs可作为基因转染载体,被hBDNF-GFP基因重组慢病毒转染后仍可保持原有生物学特性,并稳定表达和分泌有生物学活性的hBDNF和GFP.
目的 探討慢病毒載體介導入腦源性神經營養因子(hBDNF)和綠色熒光蛋白(GFP)基因轉染大鼠神經榦細胞(NSCs)後hBDNF的錶達及其生物學特性的變化.方法 構建hBDNF和GFP基因共錶達的慢病毒載體併轉染NSCs(hBDNF-GFP-NSCs組),同時設GFP轉染NSCs組(GFP-NSCs組)和未轉染的NSCs組(NSCs組).應用RT-PCR和Western blot法分彆檢測3組細胞中hBDNFmRNA和蛋白的錶達:ELISA檢測hBDNF-GFP-NSCs組細胞轉染前後培養液中hBDNF含量的變化:使用上述3組細胞的上清液培養揹根神經節(DRG)與NSCs,觀察DRG的生長情況併應用流式細胞法檢測NSCs分化為神經元的比例.結果 RT-PCR、Western blot結果顯示轉染後7 d hBDNF-GFP-NSCs組hBDNF mRNA和蛋白的錶達均明顯彊于GFp-NSCs組和NSCs組;EUSA檢測顯示hBDNF-GFP轉染NSCs後上清中hBDNF含量增加,第5天分泌達最高峰,與轉染前比較差異均有統計學意義(P<0.05);使用hBDNF-GFP-NSCs組上清液培養DRG和NSCs,4 d後DRG很快伸齣突起,流式細胞法檢測顯示NSCs分化為神經元的比例高于其他兩組.結論 NSCs可作為基因轉染載體,被hBDNF-GFP基因重組慢病毒轉染後仍可保持原有生物學特性,併穩定錶達和分泌有生物學活性的hBDNF和GFP.
목적 탐토만병독재체개도입뇌원성신경영양인자(hBDNF)화록색형광단백(GFP)기인전염대서신경간세포(NSCs)후hBDNF적표체급기생물학특성적변화.방법 구건hBDNF화GFP기인공표체적만병독재체병전염NSCs(hBDNF-GFP-NSCs조),동시설GFP전염NSCs조(GFP-NSCs조)화미전염적NSCs조(NSCs조).응용RT-PCR화Western blot법분별검측3조세포중hBDNFmRNA화단백적표체:ELISA검측hBDNF-GFP-NSCs조세포전염전후배양액중hBDNF함량적변화:사용상술3조세포적상청액배양배근신경절(DRG)여NSCs,관찰DRG적생장정황병응용류식세포법검측NSCs분화위신경원적비례.결과 RT-PCR、Western blot결과현시전염후7 d hBDNF-GFP-NSCs조hBDNF mRNA화단백적표체균명현강우GFp-NSCs조화NSCs조;EUSA검측현시hBDNF-GFP전염NSCs후상청중hBDNF함량증가,제5천분비체최고봉,여전염전비교차이균유통계학의의(P<0.05);사용hBDNF-GFP-NSCs조상청액배양DRG화NSCs,4 d후DRG흔쾌신출돌기,류식세포법검측현시NSCs분화위신경원적비례고우기타량조.결론 NSCs가작위기인전염재체,피hBDNF-GFP기인중조만병독전염후잉가보지원유생물학특성,병은정표체화분비유생물학활성적hBDNF화GFP.
Objective To explore the expression of human brain-derived neurotrophic factor (hBDNF) and green fluorescent protein (GFP) in hBDNF-GFP gene-transfected rat neural stem cells (NSCs) and the changes in the biological characteristics of the transfected cells. Methods NSCs were transfected with a lentiviral vector carrying hBDNF and GFP genes (hBDNF-GFP-NSCs) or GFP gene only (GFP-NSCs), with normal NSCs as the control. The expression levels of hBDNF mRNA and hBDNF protein in all the 3 groups were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot, respectively. Enzyme-linked immunosorbent assay (ELISA) was performed to detect hBDNF level in the cell culture medium before and after hBDNF-GFP gene transfection. Dorsal root ganglion (DRG) neurons and NSCs were cultured with the supernatants of the transfected NSCs and normal NSCs, and the growth status of the DRG neurons was observed and the proportion of NSCs differentiating into neurons determined. Results Compared with GFP-NSCs and normal NSCs, hBDNF-GFP-NSCs showed obvious hBDNF overexpression at both rnRNA and protein levels 7 days after the transfection, hBDNF content in the supematant of hBDNF-GFP-NSCs culture increased significantly with time and peaked 5 days after the transfecfion (P<0.05). Four days after culture in hBDNF-GFP-NSCs supernatant, the DRG neurons and adherent NSCs extended cells processes, and the ratio of the NSCs differentiating into neurons was higher in cells cultured in hBDNF-GFP-NSCs supematant than in those culture in GFP-NSCs and normal NSCs supematants. Conclusion Lentivitus can be used as the vector for hBDNF and GFP gene transfection into NSCs, and hBDNF-GFP gene-transfected NSCs maintain the basic characteristics of NSCs and are capable of stable expression and secretion of hBDNF and GFP.
