间充质干细胞条件培养基对新生大鼠缺氧缺血脑损伤移植神经干细胞凋亡的影响  

Conditioned media of mesenchymal stem cells alleviate apoptosis of transplantated neural stem cell for hypoxic-ischemic brain injury in a neonatal rat model

作　　者：张艳莎[1] 杨印祥[1] 栾佐[1] 何志旭[2] 汪兆艳[1] 章波[1] 

机构地区：[1]海军总医院儿科,北京100048 [2]贵阳医学院儿科

出　　处：《中华细胞与干细胞杂志（电子版）》2014年第3期25-31,共7页Chinese Journal of Cell and Stem Cell（Electronic Edition）

基　　金：国家科技部国际合作研究项目(2012DFA30880)

摘　　要：目的研究来源于人脂肪间充质干细胞（MSCs）的条件培养基对新生大鼠缺氧缺血脑损伤移植NSCs凋亡的影响。方法孕14.5天SD大鼠取乳鼠海马NSCs培养至第2-3代,DAPI标记后用于移植。7d龄SD大鼠根据Rice法进行改进,制作缺氧缺血脑损伤模型。造模成功后分为3组,每组20只：模型组（Ⅰ）、脂肪MSCs条件培养基（ASCCM）联合神经干细胞（NSCs）移植组（Ⅱ）及NSCs移植组（Ⅲ）。建模24h后,Ⅱ行右侧颈总静脉注射50μl ASC-CM,Ⅲ注射等体积生理盐水。注射完成后止血,Ⅱ、Ⅲ于左侧海马进行NSCs移植,Ⅰ不采取任何注射和移植。分别于移植后24、48h,3组脑组织冰冻切片行TUNEL染色观察细胞凋亡情况;提取左侧损伤区脑组织蛋白质,测定活化caspase-3凋亡蛋白的表达。采用t检验和方差分析进行统计学分析。结果移植后48h,组Ⅱ、Ⅲ的脑组织外源性NSCs凋亡数分别为230.00±8.94、305.00±20.70,两两比较,差异有统计学意义（P〈0.05）;但移植后24h两组之间的脑组织外源性NSCs凋亡数分别为481.25±39.99、512.22±47.11,差异无统计学意义。三组左侧损伤区脑组织的活化caspase-3凋亡蛋白表达,于移植后24h,其灰度分别为234.08±1.78、234.37±4.59、235.10±2.74,三组间差异无统计学意义;移植后48h,三组灰度分别为229.85±1.63、211.35±2.83、232.20±1.22,三组间差异有统计学意义（P〈0.05）;这表明与其它两组比较,ASC-CM联合NSCs移植可明显降低外源性NSCs的凋亡率,降低活化的Caspase-3表达。结论 MSCs条件培养基可以改善新生大鼠缺氧缺血脑损伤模型移植的NSCs存活,减少其凋亡,降低活化caspase-3凋亡蛋白的表达。Objective The study investigated the effects of conditioned media of mesenchymal stem cells from human adipose-derived stem cells on the apoptosis of neural stem cell transplanted for hypoxic-ischemic brain injury in a neonatal rat model. Methods Pregnant Sprague-Dawley rats were sacrificed and hippocampuses of the fetus were removed at gestational day 14.5. The neural stem cells were isolated, cultured to 2-3 passage and labeled by DAPI before transplantation. A total of 60 pups were randomly divided into three groups after hypoxic-ischemia was induced： （ Ⅰ ） control group, （Ⅱ） ASC-CM plus NSC group and （Ⅲ） NSC group. 24 hours after hypoxic-ischemia was induced, pups in group Ⅱ received injections of 50 μl of ASC-CM plus NSCs via the left jugular vein, and pups in group Ⅲ received 50 μl of NS plus NSCs. Pups in the blank control group received no injection and transplantation. Pups were killed 24 hours and 48 hours after transplantation, respectively. Brain tissues were subject to TUNEL staining for observing cell apoptosis. The expression of the cleavage caspase-3 in the left injured brain tissue was evaluated. T test and ANOVA were used for statistical analysis. Results 24 hours after transplantation, exogenous neural stem cell apoptosis of the brain was similar between groups Ⅱ （481.25 ± 39.99） and Ⅲ（512.22 ± 47.11）. 48 hours after transplantation, there was significant differences between group Ⅱ （230.00 ± 8.94） and group Ⅲ（305.00 ± 20.70）. 24 hours after transplantation, there was no significant differences in expression of cleavage caspase-3 in the three groups （234.08 ±1.78,234.37 ± 4.59,235.10 ± 2.74, P = 0.88 ）. 48 hours after transplantation, there was significant differences in the three groups （229.85 ± 1.63,211.35 ± 2.83,232.20 ± 1.22, P = 0.00 ）.Expression of cleavage caspase-3 was significantly lower in group Ⅱthan the other two groups. Conclusions Our data suggest that treatment with ASC-CM and neural stem cells transplantation

关 键 词：间充质干细胞 细胞培养技术 神经干细胞 细胞移植 细胞凋亡 

分 类 号：R722.1[医药卫生—儿科]