骨髓基质细胞与pLXSN-bcl-2基因联合治疗大鼠脑缺血

Effect of bone marrow stromal cells combined with pLXSN-bcl-2 gene on rat cerebral ischemia

目的:用质粒pLXSN携带抗凋亡基因bcl-2,观察分析骨髓基质细胞与其联合应用对局灶性脑缺血大鼠的治疗效果。方法:实验于2005-05/2006-10在中国医科大学完成。①实验材料:雌性Wistar大鼠40只,随机数字表法分为模型对照组、细胞移植组、基因质粒组、联合组,10只/组。另选取雄性Wistar大鼠10只用于骨髓基质细胞的提取。质粒pLXSN-bcl-2由钱其军博士惠赠。②实验方法:无菌条件下取大鼠肱骨、胫骨和股骨,去除干骺端,冲出骨髓,离心后重悬为单细胞悬液进行原代培养。待细胞生长到80%铺满瓶底时胰蛋白酶消化,接种密度1×104/cm2,传至3代后用于细胞移植,移植前24h行BrdU标记。扩增、提取、鉴定并纯化pLXSN-bcl-2质粒,-20℃备用。各组均采用改良线栓法建立大鼠大脑中动脉闭塞再灌注模型。质粒于造模后3h经鼠颈动脉注入,总量为10μg,总体积200μL。骨髓基质细胞于造模后24h经鼠尾静脉注入,总量为3×106个,总体积1mL。模型对照组给予等量生理盐水。③实验评估:各组大鼠分别于移植术后3,14d清醒状态下进行神经运动功能评分。麻醉后处死,免疫组织化学法观察脑源性神经营养因子及Bcl-2蛋白的表达、BrdU标记的骨髓基质细胞在脑内的分布及数量,TUNEL法检测脑内细胞凋亡情况。结果:①神经功能评分:移植术后3d联合组神经功能评分明显小于模型对照组,至14d明显低于其余各组(P<0.05)。②移植入脑的Brdu阳性骨髓基质细胞数:联合组和细胞移植组在梗死半球可见大量BrdU阳性的骨髓基质细胞,主要存在于梗死灶周边,其次为海马、纹状体区,梗死对侧也有少量细胞存在。移植术后3,14d,联合组Brdu阳性细胞数均明显多于细胞移植组(P<0.05)。③脑源性神经营养因子及Bcl-2蛋白的表达:移植术后3,14d,联合组脑源性神经营养因子、Bcl-2蛋白的表达均明显高于其余3组(P<0.05)。④细胞凋亡情况:移植术后3,14d,联合组细胞凋亡数量均低于其余各组(P<0.05)。结论:骨髓基质细胞与pLXSN-bcl-2两种治疗因素叠加应用可明显改善大鼠缺血性脑损伤,且效果优于其各自的单独作用。其机制可能是bcl-2基因在抗脑内细胞凋亡的同时,也抑制移植入脑的骨髓基质细胞凋亡,从而更大限度发挥骨髓基质细胞的治疗作用。

AIM： To discuss the therapeutic effect of grafting bone marrow stromal cell （MSC） and plasmid pLXSN2-mediated transfer of bcl-2 gene to treat local cerebral ischemia in rats. METHODS： The experiment had been completed at the China Medical University from May 2005 to October 2006. （1）Forty Wistar female rats were randomly divided into control group, cell transplantation group, gene plasmid group and combination group, each contained 10 rats. Ten wistar male rats were adopted to extract the MSC. Plasmid pLXSN-bcl-2 gene was offered by Doctor Qian Qi-jun. （2）bone marrows were isolated from rat humerus, tibia and femur sterilely, followed by removing metaphysis. And the cell suspension after centrifugation was used for primary culture. When reaching 80% confluence, the cells covering the bottom of culture bottle were digested using trypsin and passaged to the third generation at the density of 1 ×10^4/cm^2 for transplantation, 24 hours before which the cells were labeled with BrdU. Then plasmid pLXSN-bcl-2 was amplificated, extracted, identified and purified for preservation at -20 ℃. The ischemia-reperfusion rat models were established by the transient occlusion of left middle cerebral artery using modified thread, 3 hours later the plasmid was injected into rat carotid artery at the total dose of 10 μg and total volume of 200 μL. Moreover 1 mL suspension containing 3×10^6 MSC was injected into the tail vein of rats. While equal volume of physiological saline was given in the model control group.（3） Neurological Deficits Score （NDS） was observed in all the conscious rats at days 3 and 14 after transplantation. Then the cerebral tissues were obtained after the rats were anesthetized to death. Double-staining immunohistochemistry of brain sections was used to identify brain-derived neurotrophic factor （BDNF）, Bcl-2 protein expression, the distribution and quantity in the brain of the BrdU-labeled MSC. TUNEL method was employed to detect the cell apoptosis. RESULTS： （1）NDS in the combination group of 3 days postoperation was significantly lower than that in the model control group, and also significantly lower than other groups （P 〈 0.05）.（2）BrdU-positive MSC was observed around the ischemic brain tissues, then hippocampus and corpora striatum, additionally the contralateral area of infarction appeared a few cells. BrdU, BDNF and bcl-2 protein expressions in brain tissues of the combination group were higher than those in other 3 groups （P 〈 0.05）. CONCLUSION： MSC and pLXSN-bcl-2 gene combined transplantation has the obvious therapeutic effects in rats with cerebral ischemia, and the effect is superior to the simple administration. Its mechanism possibly is that bcl-2 gene can reduce both the apoptosis of grafted MSC and the cellular apoptosis in the brain.