CAJ | 학술논문

目的探讨腺病毒(Adv)介导的心肌营养素-1(CT-1)对大鼠骨髓间充质干细胞(rBMSCs)向神经方向分化的影响.方法全骨髓贴壁筛选法分离培养rBMSCs,流式细胞术鉴定细胞表面标记性蛋白.将已纯化细胞分为4组:对照组、重组腺病毒增强型绿色荧光蛋白(Adv-EGFP)+诱导剂组(空病毒组)、Adv-EGFP-CT-1+诱导剂组(CT-1组)及诱导剂组,分别向各转染组中加入对应的腺病毒液.转染后2d,除对照组外,其他各组均予神经分化诱导处理,于诱导后5h、3d和7d观察各组细胞形态,细胞免疫荧光检测各组巢蛋白(Nestin)、神经元特异性核抗原(NeuN)及神经胶质纤维酸性蛋白(GFAP)的表达.结果诱导处理后,除对照组外,其他各组均显示类神经细胞样形态改变,其中CT-1转染组变化最明显.诱导后5 h Nestin检出阳性率最高,其中CT-1组阳性率[(86.31±4.27)％]均高于其他各组,差异均有统计学意义(P均＜0.001),随后逐渐下降,CT-1组较其他各组变化明显,差异均有统计学意义(P均＜0.001);各时间点空病毒组与诱导剂组Nestin的阳性率差异无统计学意义(P均＞0.05).诱导后5h,CT-1组NeuN、GFAP即有少量表达,随后NeuN、GFAP阳性率逐渐升高,至第7天时其阳性率[(64.41±3.65)％、(47.14±4.29)％]最高,其中,CT-1组各时间点NeuN、GFAP阳性率均高于其他各组,差异均有统计学意义(P均＜0.001);各时间点空病毒组与诱导剂组NeuN、GFAP的阳性率差异无统计学意义(P均＞0.05).结论 CT-1可促进rBMSCs向神经方向分化.
목적 탐토선병독(Adv)개도적심기영양소-1(CT-1)대대서골수간충질간세포(rBMSCs)향신경방향분화적영향.방법 전골수첩벽사선법분리배양rBMSCs,류식세포술감정세포표면표기성단백.장이순화세포분위4조:대조조、중조선병독증강형록색형광단백(Adv-EGFP)+유도제조(공병독조)、Adv-EGFP-CT-1+유도제조(CT-1조)급유도제조,분별향각전염조중가입대응적선병독액.전염후2d,제대조조외,기타각조균여신경분화유도처리,우유도후5h、3d화7d관찰각조세포형태,세포면역형광검측각조소단백(Nestin)、신경원특이성핵항원(NeuN)급신경효질섬유산성단백(GFAP)적표체.결과 유도처리후,제대조조외,기타각조균현시류신경세포양형태개변,기중CT-1전염조변화최명현.유도후5 h Nestin검출양성솔최고,기중CT-1조양성솔[(86.31±4.27)％]균고우기타각조,차이균유통계학의의(P균＜0.001),수후축점하강,CT-1조교기타각조변화명현,차이균유통계학의의(P균＜0.001);각시간점공병독조여유도제조Nestin적양성솔차이무통계학의의(P균＞0.05).유도후5h,CT-1조NeuN、GFAP즉유소량표체,수후NeuN、GFAP양성솔축점승고,지제7천시기양성솔[(64.41±3.65)％、(47.14±4.29)％]최고,기중,CT-1조각시간점NeuN、GFAP양성솔균고우기타각조,차이균유통계학의의(P균＜0.001);각시간점공병독조여유도제조NeuN、GFAP적양성솔차이무통계학의의(P균＞0.05).결론 CT-1가촉진rBMSCs향신경방향분화.
Objective To investigate the effect of cardiotrophin-1 (CT-1) mediated by recombinant adenovirus (Adv) on the neural differentiation of bone marrow mesenchymal stem cells (rBMSCs) of rats.Methods The rBMSCs were isolated and cultured by attachment method.The surface marker protein was identified by flow cytometry.The rBMSCs were divided into 4 groups:control group,AdV enhanced green fluorescence protein (Adv-EGFP) + induction group(empty virus group),CT-1 group and induction group.The cells were transfected by Adv in the corresponding multiplicity of infections.Each group was induced by neural induction medium except for control group.The cells morphological changes were observed by microscope and the expressions of Nestin,neuronal nuclei (NeuN) and glial fibrillary acidic protein(GFAP) were detected respectively by cellular immunofluorescence at 5 h,3 d and 7 d after induction.Results After induction treatment,neuron-like cells morphological changes were observed in all the groups except for control group,among which the most obvious change was found in CT-1 group.The positive rate of Nestin was the highest at 5 h after induction.Positive rate of CT-1 group[(86.31 ± 4.27)％] was higher than any other groups,the differences were statistically significant (all P ＜ 0.001);after which its positive rate gradually declined,the positive rate of CT-1 group changed more obviously than other groups,and the differences were statistically significant (all P ＜ 0.001).The difference in Nestin positive rate between the empty virus group and induction group was not statistically significant (all P ＞ 0.05) at various time points.The NeuN and GFAP could be observed in CT-1 group at 5 h after induction.Then the positive rate of NeuN and GFAP increased gradually,and climbed to the highest point [(64.41 ± 3.65)％,(47.14 ± 4.29)％] on 7 d after induction.Positive rate of NeuN and GFAP in CT-1 group at various time points were higher than that of other groups,and the differences were statistically significant (all P ＜0.001).The difference of NeuN and GFAP positive rate between the empty virus group and induction group was not statistically significant (all P ＞ 0.05).Conclusion CT-1 could promote the neural differentiation of rBMSCs.