目的 研究牛磺酸对宫内生长受限(intrauterine growth restriction,IUGR)胎鼠神经干细胞(NSCs)增殖及Ras同源基因A(RhoA)、Rho相关螺旋卷曲蛋白激酶(ROCK)Ⅱ表达的影响.方法 采用低蛋白饮食法建立IUGR胎鼠模型.取胎鼠室管膜下生发基质区(SVZ)脑组织进行NSCs培养.将所得NSCs分为5组:正常对照组(Ⅰ组)、IUGR组(Ⅱ组)、IUGR+牛磺酸组(Ⅲ组)、IUGR+ ROCK阻断剂组(Ⅳ组)、IUGR+牛磺酸+ROCK阻断剂组(Ⅴ组).采用免疫荧光法检测各组脂肪酸结合蛋白7(FABP7)阳性细胞数;实时荧光定量PCR法检测各组细胞RhoA、ROCKⅡmRNA水平;采用Western blot法检测各组细胞RhoA、ROCKⅡ蛋白水平.结果 1.以Ⅰ组为标准计数,Ⅰ组、Ⅱ组、Ⅲ组、Ⅳ组、Ⅴ组FABP7阳性细胞计数比例分别为(100.0±1.6)％、(63.8±5.2)％、(82.8±4.3)％、(78.6±2.7)％、(87.6±3.2)％,Ⅱ组较Ⅰ组减少约36％,差异有统计学意义(t=14.98,P＜0.01);Ⅱ组、Ⅳ组、Ⅲ组、Ⅴ组FABP7阳性细胞数呈逐渐增加趋势,组间比较差异有统计学意义(F=66.53,P＜0.01).2.Ⅰ组、Ⅱ组、Ⅲ组、Ⅳ组、Ⅴ组RhoA mRNA表达水平分别为1、3.05±0.36、2.15±0.19、1.89±0.11、1.66±0.11,ROCKⅡmRNA表达水平分别为1、2.78±0.27、2.12±0.21、1.74±0.12、1.53±0.12,Ⅱ组RhoA及ROCKⅡmRNA水平均较Ⅰ组显著增加(t=-12.61、-14.67,均P＜0.01),Ⅱ组、Ⅲ组、Ⅳ组、Ⅴ组中RhoA及ROCKⅡmRNA的表达呈逐渐下降趋势,组间比较差异均有统计学意义(F=73.20、76.38,均P＜0.01).3.Ⅰ组、Ⅱ组、Ⅲ组、Ⅳ组、Ⅴ组RhoA蛋白相对表达分别为0.37±0.03、0.87±0.02、0.73±0.04、0.72±0.04、0.55±0.04,ROCKⅡ蛋白相对表达分别为0.46±0.03、0.96±0.02、0.74±0.01、0.63±0.02、0.55±0.03,Ⅱ组RhoA及ROCKⅡ蛋白水平较Ⅰ组均显著增加(t=-27.60、-35.77,均P＜0.01);Ⅱ组、Ⅲ组、Ⅳ组、Ⅴ组中ROCKⅡ蛋白的表达呈逐渐下降趋势,组间比较差异有统计学意义(F=390.16,P＜O.01);RhoA蛋白在Ⅱ组、Ⅲ组、Ⅴ组中的表达呈逐渐下降趋势,组间比较差异有统计学意义(F=130.51,P＜0.01),Ⅲ组及Ⅳ组间比较,差异无统计学意义(=0.46,P＞0.05).结论 牛磺酸可能通过抑制Rho/ROCK信号通路促进IUGR胎鼠NSCs增殖,从而改善其脑发育.%Objective To investigate the effects of Taurine on the proliferation of neural stem cells(NSCs) with intrauterine growth restriction(IUGR) in fetal rats and the expressions of Ras homolog gene family member A(RhoA) and Rho-associated coiled-coil containing protein kinase (ROCK) Ⅱ in the process of proliferation.Methods IUGR fetal rats models were established with low protein diet.NSCs from subventricular zone were isolated and cultured in vitro.The NSCs were divided into 5 groups:normal control group(group Ⅰ),IUGR group(group Ⅱ),IUGR + Taurine group (group Ⅲ),IUGR + ROCK blockers group(group Ⅳ),IUGR + Taurine + ROCK blockers group(group Ⅴ).The cells were examined by immunofluorescence for counting fatty acid binding protein 7 (FABP7)-positive cells.mRNA and protein levels of RhoA and ROCK were detected by quantitative real time polymerase chain reaction (PCR) and Western blot,respectively.Results (1) Data of group Ⅰ were presented as standard,the number of FABP7 positive cells in group Ⅰ,group]Ⅱ,group Ⅲ,group Ⅳ,group Ⅴ was (100.0 ±1.6)％,(63.8 ±5.2)％,(82.8 ±4.3)％,(78.6 ± 2.7) ％,and (87.6 ± 3.2) ％,respectively.Compared with group Ⅰ,FABP7 positive cells in group Ⅱ decreased significantly by approximately 36％ (t =14.98,P ＜0.01).The number of FABP7 positive cells in group Ⅱ,group Ⅳ,group Ⅲ and group Ⅴ increased gradually and there were significant differences among the groups(F =66.53,P ＜0.01).(2) The levels of RhoA mRNA in group Ⅰ,group Ⅱ,group Ⅲ,group Ⅳ,group Ⅴ were 1,3.05 ± 0.36,2.15 ± 0.19,1.89 ± 0.11,and 1.66 ± 0.11 respectively;the levels of ROCK Ⅱ mRNA in group Ⅰ,group Ⅱ,group Ⅲ,group Ⅳ,group Ⅴ were 1,2.78 ± 0.27,2.12 ± 0.21,1.74 ± 0.12,and 1.65 ± 0.05,respectively.Cells of group Ⅱ showed a statistically higher level of RhoA and ROCK Ⅱ mRNA than those of control cells(t =-12.61,-14.67,all P ＜ 0.01).The levels of RhoA and ROCK Ⅱ mRNA in group Ⅱ,group Ⅲ,group Ⅳ,group Ⅴ decreased gradually,and there were significant differences among the groups(F =73.20,76.38,all P ＜ 0.01).(3)The levels of RhoA protein in group Ⅰ,group Ⅱ,group Ⅲ,group ⅢⅣ,group Ⅴ were 0.37 ± 0.03,0.87 ± 0.02,0.73 ± 0.04,0.72 ± 0.04,and 0.55 ± 0.04 respectively;the levels of ROCK Ⅱ protein in group Ⅰ,group Ⅱ,group Ⅲ,group Ⅳ,group Ⅴ were 0.46 ± 0.03,0.96 ± 0.02,0.74 ± 0.01,0.63 ± 0.02,and 0.55 ± 0.03,respectively.The levels of RhoA and ROCK Ⅱ protein were significantly higher in group Ⅱ than those in group Ⅰ (t =-27.60,-35.77,all P ＜ 0.01).The levels of ROCK Ⅱ protein in group Ⅱ,group Ⅲ,group Ⅳ,group Ⅴ reduced gradually,and there were significant differences among the groups(F =390.16,P ＜ 0.01).The levels of RhoA protein in group Ⅱ,group Ⅲ,group Ⅴ reduced gradually,and there were significant differences among the groups (F =130.51,P ＜ 0.01),but there was no significant difference between group Ⅲ and group Ⅳ (t =0.46,P ＞ 0.05).Conclusions Taurine supplementation can promote the proliferation of NSCs in IUGR fetal rats by inhibiting Rho/ROCK signaling pathway,thus to improve IUGR fetal brain development.
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