脐血源神经干细胞在SDF-1/CXCR4趋化下对脑出血大鼠神经损伤恢复的作用

Effects of umbilical cord blood neural stem cells via stromal cell-derived factor-1/CXC chemokine receptor 4 signaling on neural recovery in rat models of intracerebral hemorrhage

目的探讨移植的脐血源神经干细胞(UCBNSCs)在基质细胞衍生因子-1(SDF-1)/CXC趋化因子受体4(CXCR4)作用下对脑出血大鼠神经功能修复的影响。方法(1)体外迁移实验中,将UCBNSCs置入Transwell小室的上室,在下室中分别加入0、30、60和120ng/mL浓度的SDF-1。(2)体内实验中,将60只脑出血模型大鼠按照随机数字表法分为UCBNSCs移植组和对照组,每组30只。造模后2d分别向2组大鼠颅内移植UCBNSCs悬液10μL和等量的磷酸盐缓冲液(PBS),同时腹腔注射5-溴脱氧尿嘧啶核苷(BrdU)标记内源性神经干细胞。分别于移植后1d、1周、2周对2组大鼠进行改良神经功能缺损评分(mNSS)。移植后2周处死全部大鼠,进行SDF-1、BrdU、血管内皮生长因子(VEGF)、神经胶质纤维酸性蛋白(GFAP)及双肾上腺皮质激素(DCX)免疫荧光染色;同时采用转移酶介导的三磷酸脱氧鸟苷-生物素刻痕末端标记(TUNEL)检测试剂盒检验细胞凋亡情况。结果(1)体外实验中,UCBNSCs中能够检测到CXCR4的表达;60ng/mLSDF-1对UCBNSCs迁移作用最大,与其他浓度SDF-1比较,差异有统计学意义(P<0.05)。(2)体外实验中,移植2周后,UCBNSCs组大鼠室管膜下区BrdU标记的细胞数量增多,BrdU/DCX、BrdU/GFAP细胞数量明显比对照组增多,差异有统计学意义(P<0.05);UCBNSCs移植组脑损伤区神经营养因子VEGF阳性细胞数[(88.30±7.21)个/视野]明显高于对照组[(53.20±4.45)个/视野],差异有统计学意义(t=4.144,P=0.000);UCBNSCs移植组脑损伤区凋亡细胞的数量[(34.30±2.44)个/视野]明显低于对照组[(47.70±1.98)个/视野],差异有统计学意义(t=4.266,P=0.001)。移植后2周时UCBNSCs组mNSS评分[(6.40±0.163)分]明显低于对照组[(7.50±0.17)分],差异有统计学意义(t=4.714,P=0.002)。结论SDF-1/CXCR4可以趋化移植的UCBNSCs到达脑出血损伤区,促进大鼠神经功能恢复,其作用机制可能与促进神经发生、VEGF的分泌和抑制细胞凋亡有关。

Objective To investigate the effects of umbilical cord blood neural stem cells(UCBNSCs)via stromal cell-derived factor-1(SDF-1)/CXC chemokine receptor 4(CXCR4)signaling on neural recovery in rat models of intracerebral hemorrhage.Methods(1)In migration assay in vitro,UCBNSCs were distributed in the upper wells of Transwell plates,and SDF-1 at concentrations of 30,60 and 120 ng/mL was placed in the lower wells.(2)Sixty rat models of intracerebral hemorrhage were randomly divided into UCBNSCs-transplanted group and phosphate buffer(PBS)-transplanted group(n=30);two d after modeling,10 L UCBNSCs suspension and same amount of PBS were,respectively,transplanted into the two groups,and intraperitoneal injection of deoxyuridine(BrdU)labeled endogenous neural stem cells was performed;neurological functions were assessed with modified Neurological Severity Scale(mNSS)one d,and one and two weeks after cell transplantation;the expressions of SDF-1,vascular endothelial growth factor(VEGF),glial fibrillar acidic protein(GFAP),and doublecortin(DCX)were detected by immunofluorescence when the rats were sacrificed two weeks after cell transplantation;cell apoptosis was detected by TUNEL.Results(1)In the in vitro experiment,CXCR4 expression could be detected in UCBNSCs;60 ng/mL SDF-1 had the greatest migration effect on UCBNSCs,and this effect showed statistically significant difference as compared with that at other concentrations(P<0.05).(2)In the in vivo experiment,two weeks after transplantation,the UCBNSCs-transplanted group had significantly increased number of BrdU-labeled cells in the subventricular zone,and significantly larger number of BrdU/DCX and BrdU/GFAP cells than the PBS-transplanted group(P<0.05);the VEGF expression in the brain injury area of the UCBNSCs-transplanted group([88.30±7.21]/field)was significantly higher than that of PBS-transplanted group([53.20±4.45]/field,t=4.144,P=0.000);the number of apoptotic cells in the brain injury area of the UCBNSCs-transplanted group([34.30±2.44]/field)was significantly smaller than that of PBS-transplanted group([47.70±1.98]/field,t=4.266,P=0.001);two weeks after transplantation,the mNSS scores of UCBNSCs-transplanted group(6.40±0.163)were significantly lower than those of the PBS-transplanted group(7.50±0.17,t=4.714,P=0.002).Conclusion SDF-1/CXCR4 can reach the injured area of cerebral hemorrhage after chemotactic transplantation of UCBNSCs and promote the recovery of nerve function in rats,whose mechanism may be that it can promote neurogenesis and VEGF secretion and inhibit apoptosis.