CAJ | 학술논문

目的探讨甲基磺酸敏感蛋白2(MMS2)在血管紧张素Ⅱ(AⅡ)诱导神经干细胞(NSCs)向多巴胺(DA)能神经元定向分化过程中的作用.方法体外分离培养新生鼠大脑来源的NSCs.通过巢蛋白免疫细胞化学方法对神经前体细胞进行鉴定;在此基础上,将第2代的NSCs按实验设计分成6组:A:对照组;B:AⅡ组;C:AT1受体拮抗剂ZD7155组;D:AT1受体拮抗剂ZD7155+AⅡ组;E:AT2受体拮抗剂PD123319组;F:AT2受体拮抗剂PDl23319+AⅡ组.通过实时荧光定量PCR检测各组细胞MMS2及TH mRNA的表达;转染MMS2基因的小干扰RNA(siRNA)片段到NSCs上沉默MMS2在NSCs的表达,再按以上分组将其诱导分化为DA能神经元,通过实时荧光定量PCR方法检测转染后诱导分化的各组细胞TH mRNA的表达.结果体外分离培养的NSCs,在培养基中呈悬浮生长,神经球表达Nestin.实时荧光定量PCR法检测B组、D组细胞的MMS2及TH mRNA的表达高于对照组,其差异有统计学意义(P<0.05);C、E、F组细胞的MMS2及TH mRNA的表达与对照组比较无统计学意义(P>0.05),转染MMS2-siRNA后诱导分化的各组细胞间TH mRNA的表达无统计学意义(P>0.05 o结论AⅡ通过AT2受体使MMS2在NSCs的表达升高并诱导NSCs向DA能神经元分化,MMS2在AⅡ诱导NSCs向DA能神经元定向分化的过程中可能发挥重要作用.
목적 탐토갑기광산민감단백2(MMS2)재혈관긴장소Ⅱ(AⅡ)유도신경간세포(NSCs)향다파알(DA)능신경원정향분화과정중적작용.방법 체외분리배양신생서대뇌래원적NSCs.통과소단백면역세포화학방법대신경전체세포진행감정;재차기출상,장제2대적NSCs안실험설계분성6조:A:대조조;B:AⅡ조;C:AT1수체길항제ZD7155조;D:AT1수체길항제ZD7155+AⅡ조;E:AT2수체길항제PD123319조;F:AT2수체길항제PDl23319+AⅡ조.통과실시형광정량PCR검측각조세포MMS2급TH mRNA적표체;전염MMS2기인적소간우RNA(siRNA)편단도NSCs상침묵MMS2재NSCs적표체,재안이상분조장기유도분화위DA능신경원,통과실시형광정량PCR방법검측전염후유도분화적각조세포TH mRNA적표체.결과 체외분리배양적NSCs,재배양기중정현부생장,신경구표체Nestin.실시형광정량PCR법검측B조、D조세포적MMS2급TH mRNA적표체고우대조조,기차이유통계학의의(P<0.05);C、E、F조세포적MMS2급TH mRNA적표체여대조조비교무통계학의의(P>0.05),전염MMS2-siRNA후유도분화적각조세포간TH mRNA적표체무통계학의의(P>0.05 o결론AⅡ통과AT2수체사MMS2재NSCs적표체승고병유도NSCs향DA능신경원분화,MMS2재AⅡ유도NSCs향DA능신경원정향분화적과정중가능발휘중요작용.
Objective To explore the possible effects of methyl methanesulfonate sensitive 2(MMS2)in the process of angiotensin Ⅱ inducing differentiation of neural stem cells (NSCs) into dopaminegic phenotype neurons. Methods NSCs were isolated from the brain of newborn rats and were cultured in the serum-free medium.Identification of neural precursor cells was done by Nestin immunocyt ochemical staining. Then the second generation of NSCs was divided into the following six groups: A, control; B, AⅡ; C, AT1 antagonist ZD7155; D, ZD7155+AⅡ; E, AT2 antagonist PD123319; F, PD123319+AⅡ. The detection of expression of MMS2 and TH mRNA level was done by real-time PCR. The silence of the expression of MMS2 in NSCs was brought about via the transfection of MMS2-siRNA, and then the NSCs were induced to differentiate into dopaminegic neurons. The expression of TH mRNA level in the cells of the groups after transfection was detected by real-time PCR. Results Nestin-positive cells were observed in suspended growth in the medium.Real-Time PCR revealed that the MMS2 and TH mRNA expression of group B and D were significantly higher than that of the control group(P<0.05), There was no significant difference in MMS2 and TH mRNA expression between group C, E, F and the control, respectively. Conclusion AⅡ increased the expression of MMS2 mRNA in NSCs and induced the differentiation of NSCs into DA neurons via AT2 recepter. MMS2 may play important roles in the process of angiotensin Ⅱ inducing NSCs to differentiate into dopaminergic neurons.