摘要
目的观察应用维生素C(Vc)和碱性成纤维因子(bFGF)对骨髓间充质干细胞(BMSCs)体外增殖培养,移植到脑梗死大鼠体内后,在脑内的存活情况。方法①采用密度梯度离心及贴壁法分离大鼠BMSCs。按培养液中加入的不同因子分为4组:对照组、Vc组(50μg/ml)、bFGF组(1μg/L)以及Vc+bFGF组(50μg/ml Vc和1μg/L bFGF),在体外对BMSCs进行培养并观察各组细胞的增殖情况。采用MTT比色法分别于培养第1、2、3、5和7天,测定细胞在450nm波长处的吸光度(A)值;采用流式细胞仪检测培养96 h后的细胞周期。②采用线栓法制备20只大鼠右侧大脑中动脉闭塞2 h模型,并随机分为2组:联合Vc++bFGF培养的BMSCs移植组(10只)和对照BMSCs移植组(10只)。将相应组别的BMSCs细胞移植入脑梗死24 h后的大鼠体内。分别于移植后第1、2和3周,制备大鼠脑组织切片并行BrdU免疫组化染色。结果①形态学观察显示,Vc+bFGF组的BMSCs增殖最快,对照组则相对缓慢。②各组细胞的A值于培养第2天开始增加,Vc组和bFGF组在第3天达到高峰(F=728.52和F=197.18,P<0.05),Vc+bFGF组在第5天达到高峰(F=1771.32,P<0.05),第7天均有所下降。第2天起,bFGF组、Vc组与bFGF+Vc组的,A值均高于对照组;第3天起,Vc+bFGF组高于Vc组与bFGF组,差异均有统计学意义(P<0.05)。③Vc组、bFGF组与 Vc+bFGF组细胞处于增殖期(S+G2+M期)的开分比均较对照组高,差异有统计学意义(P<0.05)。④BMSCs移植后第2周,联合Vc+bFGFt培养的BMSCs移植组大鼠脑组织切片中BrdU阳性细胞计数较对照组高;第3周两组阳性细胞计数均有下降,但前者仍较后旨高。差异均有统计学意义(P<0.01);Brdu阳性细胞主要分布在梗死灶周围。结论 Vc与bFGF均可促进大鼠BMSCs的增殖,二者联用较单用其一的效果更好;脑梗死大鼠移植增殖培养后的BMSCs,其在脑组织中的成活能力明显增强。
Objective To study the effects of vitamin C and basic fibroblast growth factor （bFGF） on proliferation in vitro and transplantation in vivo of bone marrow mesenchymal stem cells （ BMSCs ） in rats with cerebral infarction. Methods （1）BMSCs of rats were isolated by using density gradient centrifugation and adherent method. The rats were divided into four groups according to the different growth factors added in the medium： control group, Vc group （50 μg/mL）, bFGF group （1μg/L） , and Vc ＋ bFGF group （50 μg/mL Vc and 1 μg/L bFGF）. The cellular morphology in each group was observed. MTF colorimetric method was used to detect the absorbance （ A value） at the wavelength of 450 nm at day 1, 2, 3, 5, and 7 after the culture ; flow cytometer was used to detect the cell cycle cultured after 96 hours. （2）A model of right middle cerebral artery occlusion （MCAO） for 2 hours was induced by the suture method in 20 rats, and they were randomly divided into 2 groups： the BMSC transplantation group cultured by the combined Vc ＋ bFGF and the control BMSC transplantation group. BMSCs in the corresponding groups were transplanted in rats 24 hours after cerebral infarction. At 1,2, and 3 weeks after transplantation, the brain tissue slices of the rats were prepared, and they were revealed by BrdU immunohistochemistry staining. Results （1）Morphological observation showed that the proliferation of BMSCs was most fast in the Vc ＋ bFGF group, and it was relatively slow in the control group. （2）The A value of cells in each group began to increase at day 2 after the culture. It reached the peak at day 3 in the Vc group and bFGF groups （ F = 728.52 and F = 197. 18, P 〈 0. 05） ; it reached the peak at day 5 in the Vc ＋ bFGF group （F = 1771.32, P 〈 0. 05）, and then both deceased at day 7. From the second day, the A value in the bFG, Vc and Vc ＋ bFGF groups was significantly higher than that in the control group ; from the third day, Vc ＋ bFGF group was higher than the Vc and bFGF group, and there was significant difference （P 〈 0. 05 ）. （3）The percen- tage of ceils in the proliferative phase （ S ＋ G2 ＋ M） in the Vc, bFG and Vc ＋ bFGF groups was higher than that in the control group, and there was significant difference （ P 〈 0. 05 ）. （4）Two weeks after the BMSC transplantation, BrdU-positive cell counts in the brain tissue slices in the BMSC transplantation group cultured by the combined Vc ＋ bFGF was higher than that in the control BMSC transplantation group ; the positive cell counts in both groups decreased at week 3, however, the former was still higher than the latter, and there was significant difference （P 〈 0. 01 ）. Most BrdU labeled BMSCs appeared in the centre or around the infarct brain. Conclusion Both Vc and bFGF may promote proliferation of BMSCs in rats. The efficacy of the combination of both is better than any one alone. Survival ability of the BMSCs are obviously strengthened when they are transplanted into rats infarcted brain.
出处
《中国脑血管病杂志》
CAS
2009年第9期475-480,共6页
Chinese Journal of Cerebrovascular Diseases
基金
广东省建设中医药强省科研课题(2009319)
