中华创伤杂志
中華創傷雜誌
중화창상잡지
Chinese Journal of Traumatology
2012年
12期
1135-1139
,共5页
钱腾达%代学良%陆晓诚%陶轶%李立新
錢騰達%代學良%陸曉誠%陶軼%李立新
전등체%대학량%륙효성%도질%리립신
脂肪组织%干细胞%神经元%再程序化
脂肪組織%榦細胞%神經元%再程序化
지방조직%간세포%신경원%재정서화
Adipose tissue%Stem cells%Neurons%Reprogrammed
目的 探讨再程序化脂肪干细胞(adipose-derived stem cells,ADSCs)体外定向神经分化的效率和机制. 方法 体外培养大鼠ADSCs并纯化、鉴定,将第3代ADSCs分为三组:未进行慢病毒介导基因转染ADSCs组(空白组)；不含神经元生成素-2(neurogenin-2,Ngn2)基因慢病毒空载体转染ADSCs组(空病毒组)；慢病毒载体介导Ngn2基因转染ADSCs组(Ngn2组)；各组细胞均用含细胞生长因子的诱导培养基诱导15 d.免疫荧光检测各组细胞神经元特异性蛋白(NeuN)阳性表达以观察神经元分化效率；Western blot检测各组细胞Mash1、Hes1、Dll1蛋白表达水平的差异,探索分化机制. 结果 诱导培养基诱导15 d后,Ngn2组、空病毒组、空白组阳性表达NeuN分别为90.12％、45.34％、40.26％,各组比较差异均有统计学意义(P＜0.01).Western blot 结果显示,Ngn2组Dll1蛋白表达水平明显高于空病毒组和空白组(P＜0.01),Mash1、Hes1蛋白表达水平明显低于空病毒组和空白组(P＜0.01)；空病毒组和空白组Dll1、Mash1和Hes1蛋白表达差异均无统计学意义(P＞0.05). 结论 再程序化ADSCs诱导后神经元分化的比例较单纯ADSCs提高近99％,再程序化ADSCs定向分化神经元机制可能是通过上调Dll1蛋白水平,同时下调Mash1、Hes1蛋白水平,抑制notch信号通路,从而促进细胞的定向神经分化.
目的 探討再程序化脂肪榦細胞(adipose-derived stem cells,ADSCs)體外定嚮神經分化的效率和機製. 方法 體外培養大鼠ADSCs併純化、鑒定,將第3代ADSCs分為三組:未進行慢病毒介導基因轉染ADSCs組(空白組)；不含神經元生成素-2(neurogenin-2,Ngn2)基因慢病毒空載體轉染ADSCs組(空病毒組)；慢病毒載體介導Ngn2基因轉染ADSCs組(Ngn2組)；各組細胞均用含細胞生長因子的誘導培養基誘導15 d.免疫熒光檢測各組細胞神經元特異性蛋白(NeuN)暘性錶達以觀察神經元分化效率；Western blot檢測各組細胞Mash1、Hes1、Dll1蛋白錶達水平的差異,探索分化機製. 結果 誘導培養基誘導15 d後,Ngn2組、空病毒組、空白組暘性錶達NeuN分彆為90.12％、45.34％、40.26％,各組比較差異均有統計學意義(P＜0.01).Western blot 結果顯示,Ngn2組Dll1蛋白錶達水平明顯高于空病毒組和空白組(P＜0.01),Mash1、Hes1蛋白錶達水平明顯低于空病毒組和空白組(P＜0.01)；空病毒組和空白組Dll1、Mash1和Hes1蛋白錶達差異均無統計學意義(P＞0.05). 結論 再程序化ADSCs誘導後神經元分化的比例較單純ADSCs提高近99％,再程序化ADSCs定嚮分化神經元機製可能是通過上調Dll1蛋白水平,同時下調Mash1、Hes1蛋白水平,抑製notch信號通路,從而促進細胞的定嚮神經分化.
목적 탐토재정서화지방간세포(adipose-derived stem cells,ADSCs)체외정향신경분화적효솔화궤제. 방법 체외배양대서ADSCs병순화、감정,장제3대ADSCs분위삼조:미진행만병독개도기인전염ADSCs조(공백조)；불함신경원생성소-2(neurogenin-2,Ngn2)기인만병독공재체전염ADSCs조(공병독조)；만병독재체개도Ngn2기인전염ADSCs조(Ngn2조)；각조세포균용함세포생장인자적유도배양기유도15 d.면역형광검측각조세포신경원특이성단백(NeuN)양성표체이관찰신경원분화효솔；Western blot검측각조세포Mash1、Hes1、Dll1단백표체수평적차이,탐색분화궤제. 결과 유도배양기유도15 d후,Ngn2조、공병독조、공백조양성표체NeuN분별위90.12％、45.34％、40.26％,각조비교차이균유통계학의의(P＜0.01).Western blot 결과현시,Ngn2조Dll1단백표체수평명현고우공병독조화공백조(P＜0.01),Mash1、Hes1단백표체수평명현저우공병독조화공백조(P＜0.01)；공병독조화공백조Dll1、Mash1화Hes1단백표체차이균무통계학의의(P＞0.05). 결론 재정서화ADSCs유도후신경원분화적비례교단순ADSCs제고근99％,재정서화ADSCs정향분화신경원궤제가능시통과상조Dll1단백수평,동시하조Mash1、Hes1단백수평,억제notch신호통로,종이촉진세포적정향신경분화.
Objective To investigate the efficiency and mechanism of differentiation of reprogrammed adipose-derived stem cells (ADSCs) to neurons in vitro.Methods ADSCs from rats were cultured in vitro and then purified and identified.ADSCs at the third passage were divided into three groups:ADSCs without lentivirus-mediated gene transfection (blank group),ADSCs transfected with lentivirus carrying no neurogenin2 (Ngn2) (empty virus group) and ADSCs with lentivirus-mediated transfection of Ngn2 (Ngn2 group).All groups were induced in the medium containing cell growth factor for 15 days.The positive expression of neuron-specific nuclear protein (NeuN) in three groups was detected using immunofluorescence method so as to observe the efficiency of neuron differentiation.Expression variances of Mash1,Hes1 and Dll1 in each group were detected by Western blot analysis and the mechanism of differentiation was also discussed.Results After 15 days of induction,positive expression rate of NeuN in Ngn2 group,empty virus group,blank group was 90.12％,45.34％ and 40.26％ respectively,with significant differences among groups (P ＜ 0.01).Western blot analysis showed that Ngn2 group had a significantly higher expression of Dll1 (P ＜0.01) and obvious lower expressions of Hes1 and Mash1 (P ＜0.01),as compared with empty virus group and blank group.However,there were no significant differences of expression levels of Dll1,Mash1 and Hes1 between empty virus group and blank group (P ＞ 0.05).Conclusions After induction,the ratio of neuron differentiation of reprogrammed ADSCs is increased by almost 99％,as compared with simple ADSCs.The increased dfferentiation of reprogrammedADSCs to neurons may be associated with the inhibition of notch signaling through up-regulating Dll1 and down-regulating Mash1 and Hesl.
