叶酸联合成体神经干细胞治疗创伤性脑损伤大鼠的实验

Study on Folic Acid Combined with Adult Neural Stem Cells in the Treatment of Traumatic Brain Injury in Rats

目的观察叶酸联合成体神经干细胞对创伤性脑损伤大鼠的治疗作用,探讨其可能作用机制。方法 120只Wistar大鼠随机分为6组,正常组,模型组,假手术组,叶酸注射组,成体神经干细胞移植组,成体神经干细胞移植+叶酸注射组。倒置显微镜下观察神经干细胞形态学变化;流式细胞仪检测神经干细胞表面标记物CD105、CD45、CD44、CD29的表达;免疫荧光法检测神经元特异性烯醇酶(NSE成熟神经元的特异性标志)、胶质纤维酸性蛋白(GFAP胶质细胞的标记物)的表达;平衡木实验检测大鼠运动协调与整和能力;Morris水迷宫实验测试各组大鼠的学习记忆能力;HE染色及Brdu免疫组化实验观察脑组织形态学变化;酶联免疫吸附试验检测大鼠脑组中脑源性神经生长因子(BDNF)、神经生长因子(NGF)的表达;蛋白质印迹法检测脑组织中凋亡相关蛋白BCL-2、Bax、Caspase-3的表达。结果分离所得细胞能在体外传代培养,流式细胞仪检测发现细胞阳性表达CD44、CD29,阴性表达CD105、CD45,细胞经胎牛血清诱导分化后能形成NSE或GFAP阳性细胞。实验表明,叶酸与成体神经干细胞干预创伤性脑损伤大鼠模型后能显著改善其行为学变化,减轻脑组织的炎症反应,恢复受损神经细胞,增加脑组织内BDNF、NGF的含量,上调BCL-2的表达,下调Bax、Caspase-3的表达。结论叶酸联合成体神经干细胞干预创伤性脑损伤大鼠能显著改善中枢神经功能,对维持神经元微环境稳态具有重要的作用。

Objective To explore the folic acid and adult neural stem cells’ joint influencing mechanism on the rat with Traumatic Brain Injury,and to find out its possible mechanism.Methods Divide 120 rats to 6 groups randomly—normal group,mode group,sham operation group,folacin injection group,adult stem cells transplant group,and folacin injection plus adult stem cells transplant group.Observe the morphological change under the microscope,then,do flow cytometry test and detect the expression of the neural stem cells’ facial notation—CD105、CD45、CD44、CD29.Examine the expression of neuron special enolase（NSE） and the expression of gelatinous fibre acidic protein（GFAP） with immunofluorescence.Examine the rats’ ability to motor-coordinate and conform with balance beam.Test each group’s learning and memorizing ability by conducting the Morris water maze experiment.Then,conduct the HE chromosome and Brdu immunohistochemistry experiment to detect morphological change of the brain tissue.After this,do the enzyme-linked immunosorbent assay（ELISA） to detect the expression of brain-derived neurotrophic factor（BDNF） and the expression of nerve growth factor.The last but not least,use western blotting to examine the expression of related dead protein in the brain tissue：BCL-2、Bax、Caspase-3.Results The separated cells can be vitro subcultured,by doing flow cytometry test,we find that positive cells express CD44 and CD29 while negative express CD105,CD45.Cells induced by fetal bovine serum can produce NSE or GFAP positive cells.Experiments suggest that traumatic brain injured rats can significantly improve their behavior after jointly influenced by the folic acid and adult neural stem cells.Besides,they can also reduce brain tissue inflammation,restore damaged nerve cells,increase brain tissue of BDNF and NGF’s content,increase BCL 2 expression,and lower the expression of Bax,caspase-3.Conclusion The folic acid combined with adult neural stem cell intervention in traumatic brain injury in rats can significantly improve the central nervous system dysfunction,plays an important role to maintain the steady microenvironment in the neuron.