绿色荧光蛋白转基因小鼠骨髓间充质干细胞脑内移植后的成活及迁移

Survival and migration of mesenchymal stem cells from green fluorescent protein transgenic mice after transplantation into rats with Parkinson's disease

目的建立绿色荧光蛋白(GFP)转基因小鼠骨髓间充质干细胞(MSCs)细胞系,探讨MSCs移植对帕金森病(PD)模型大鼠行为学的影响及MSCs在脑组织中的成活及迁移分布情况。方法通过全骨髓贴壁法体外分离、培养、纯化GFP转基因小鼠的MSCs,将PD模型大鼠按随机数字法分为MSCs移植组(MSCs组)、DMEM/F12培养液对照组(DMEM/F12组)和PD空白对照组,MSCs组进行纹状体MSCs细胞移植。于MSCs细胞移植术前及术后第2、4和6周,计数阿扑吗啡(APO)诱发下30min内的平均旋转圈数,动态观察PD模型大鼠行为学变化,并观察移植部位脑组织形态学及MSCs成活和迁移情况。结果 MSCs传代培养后形成形态均一的梭形细胞,呈GFP强阳性表达;MSCs纯度得到提高,与原代培养3d的MSCs比较,传代培养5代后MSCs的CD44、CD54阳性率增高〔(95.8±0.8)%vs.(64.4±2.2)%,(91.4±1.3)%vs.(63.2±0.5)%,〕,CD44阳性率减低〔(33.6±4.1)%vs.(2.1%±1.8)%〕(均P<0.05)。移植术后第2周,MSCs组在APO诱发下30min内的平均旋转圈数为(9.2±1.8)次/min,较移植术前〔(10.8±1.8)次/min〕明显减少(P<0.05),亦低于DMEM/F12组〔(10.1±2.1)次/min〕和PD空白对照组〔(10.8±2.2)次/min〕(P<0.05);在移植术后第4周〔(5.7±2.1)次/min〕和第6周〔(6.2±1.9)次/min〕平均旋转圈数减少最为明显(均P<0.05),亦均低于DMEM/F12组〔(10.6±1.8)、(10.2±1.4)次/min〕和PD模型组〔(11.1±2.1)、(10.4±1.7)次/min〕(均P<0.05);DMEM/F12组和PD空白对照组平均旋转圈数移植术前后比较无明显变化(P>0.05)。荧光染色结果显示,移植细胞在PD模型大鼠脑组织内均能存活,并与大鼠组织产生整合;大鼠脑组织结构无破坏,无胶质瘢痕形成。结论成功建立了GFP转基因小鼠的MSCs细胞系;MSCs脑内移植能有效改善PD模型大鼠的行为学表现,与宿主组织相容性良好。

Objective To establish a basis for the tracer study of the transplantaton into rats with Parkinson＇ s disease （PD） by isolating, incubating and identifying the mesenchymai stem cells （MSCs） from green fluorescent protein （GFP） mouse. Methods MSCs obtained from the GFP transgenic mouse were isolated, cultured and purified by the whole bone marrow adherence method, for morphology observations; MSCs were identified by immunochemistry; and the GFP protein was observed with fluorescence. The successful rat models of PD were divided into 3 groups： MSCs group （transplanted were 5 ）〈10s MSCs）, DMEM/F12 group and blank control group. MSCs group DMEM/F12 group, respectively, 5 μL of cell suspension and an equal volume of DMEM/F12 medium were injected into the right striatum. The rotation scores were assessed 2 weeks, 4 weeks and 6 weeks a{ter transplantation. At different time after transplantation the brains of PD model rats were examined with fluorescent microscope. Results MSCs have been harvested from GFP transgenic mouse in vitro,and formed uniform spindle cell morphology after subculture with the ability of self-renewals and stable expression of GFP protein. CD44, CD54-positive rate was significantly higher [ （95.8±0.8）% vs. （64.4± 2.2）%, （ 91.4±1.3）% vs. （63.2±0.5）%], CD44-positive rate to reduce [ （33.6±4.1 ）% vs. （2.1%±1.8） % ] after subculture 5 generations （P〈0.05）. MSCs transplantation could effectively improve the behavioral performance in rats. At the 2nd week after cell transplantation, after an injection of apomorphine （APO）, rotation frequency decreased in MSCs group （9.2±1.8） circles/min versus （10.8± 1.8） and （10.8±2.2） circles/min before cell transplantation and PD groups （P〈0.05）. At the 4 th and 6 th week, rotation frequency decreased in MSCs group （5.7±2.1） and （6.2±1.9） circles/minversus （10.6±1.8）, （10.2±1.4） and （11. 1 ±2.1）, （10.4±1.7） circles/min in DMEM/F12 and PD groups （P〈0.05）. MSCs survived in the brains of PD model rats, and showed no signs of destroying the host and the glial cicatrisation. Conclusions MSCs have been harvested from GFP transgenic mouse by the whole bone marrow adherence method; The behavior of rat with PD was improved significantly while transplanting MSCs injected in right striatum, MSCs survived in the brains of PD model rats and showed good compatibility with the host cells.