神经干细胞移植对脑损伤大鼠整合素表达的影响

Effects of Tansplantation of Neural Stem Cells on Integrin Expression in a Rat Model of Traumatic Brain Injury

目的探讨神经干细胞(NSCs)移植对大鼠创伤性脑损伤(TBI)整合素(integrin)表达的影响。方法从E14大鼠胚胎分离NSCs,进行原代培养及传代培养;对NSCs进行诱导分化;采用免疫细胞化学技术对NSCs和其分化为神经元的表型进行鉴定。采用改良的Feeney法制备创伤性脑损伤模型。利用脑立体定位仪和微量注射泵进行NSCs脑内移植。采用免疫组织化学技术、免疫印迹技术和RT-PCR技术检测在移植后不同时间脑组织损伤区整合素的表达。结果在培养基中,NSCs呈球团状悬浮生长,Nestin表达阳性。用含10%胎牛血清的培养基对NSCs进行体外诱导分化后第2d,多数细胞伸出突起,以后突起逐渐延长,分支增加。分化后第5d,部分细胞呈βIII-微管蛋白阳性。整合素阳性产物主要表达于细胞膜,呈棕黄色。在对照组及移植组均可见阳性细胞表达。在不同时间点,NSCs移植组移植点及其周围脑组织中整合素的mRNA表达均显著高于对照组(P<0.01)。整合素的蛋白表达结果和mRNA表达结果相一致。结论移植NSCs至TBI大鼠损伤脑组织,在移植点周围脑组织中整合素的表达显著增加。

Objective To explore the effects of tansplantation of neural stem cells （NSCs） on integrin expression in a rat model of traumatic brain injury （TBI）. Methods Primary NSCs were isolated from the embryonic 14-day （El4） Wistar rats and cultured. To induce differentiation of NSCs, a single cell suspen- sion of NSCs was switched into the basic neurosphere medium with 10% FBS. Immunocytechemical stai- ning was conducted to identify anti-nestin for NSCs and anti-βⅢ-tubulin for neurons. The model of TBI was built by modified Feeney method. The model rats of TBI received delivery of the NSC suspension with the stoelting quintessential injector. The expressions of integrin in the injuried brain tissue were detected by irnmunohistochemistry, immunoblotting and RT-PCR during different times. Results Primary NSCs were isolated and cultureed as suspension, neurospheres were formed. All the clones were nestin （＋）. Clonally derived spheres were allowed to differentiate on the adhesive substrate in the medium with the presence of 10% FBS for 5 days followed by βⅢ-tubulin for neurons. Integrin positive products were brown-yellow, mainly expressed in the cell membrane. There were positive cells in both control and trans- plant groups. The expressions of integrin positive cells were significantly higher in the NSC-transplanted group than in the control group at different time points. Comparing the two groups at the same time point, the difference was significant （P〈0.05）. Significantly higher mRNA expression of integrin was found in the NSC-transplanted group than in the control group （P〈0.01）. The expression of protein was consistent with that of mRNA. Conclusion Significantly higher expression of integrin was found in the transplant core and border of the NSC-transplanted rats after TBI.