【作者】 张磊；

【导师】 汤永红；

【作者基本信息】 南华大学 ， 神经病学， 2011， 硕士

【摘要】 目的通过尾静脉、颈内动脉和大脑立体定位不同途径,联合移植大鼠间充质干细胞(MSCs)和血管内皮祖细胞(EPCs)到MCAO模型大鼠体内,观察不同移植途径对大鼠缺血性脑损伤后神经功能修复的影响及作用机制的探讨。方法取体重约100g雄性SPrague一Dawley(SD)大鼠长骨的骨髓细胞,经过不同的培养方式,分别获得MSCs及EPCs。利用改良线栓法制作SD大鼠大脑中动脉缺血再灌注模型(MCAO)。造模成功的MCAO40只,完全随机均分为4组,尾静脉PBS对照组(A组),尾静脉途径组(B组),颈内动脉途径组(C组),大脑立体定位移植组(D组)。缺血2h后,再灌注24小时后,B组、C组及D组将2×10~6个MSCs和2×10~6个EPCs分别通过尾静脉、颈内动脉和大脑立体定位途径移植入MCAO大鼠体内。A组做好MCAO后尾静脉注入PBS液。第14天后,分别观察神经行为学评分,脑梗死体积,HE染色,检测脑源性神经营养因子(BDNF)和血管内皮生长因子(VEGF)的表达。结果1脑梗死体积:A组的梗死体积为216.3+4.86mm3,B组为206.0+5.33mm3,C组为198.2+4.20mm3,D组191.2+4.96mm3,各组间梗死体积两两比较差异均有统计学意义(P <0.05),D组脑梗死体积最小。2神经行为学评分:移植前模型神经功能评分:A组(3.4+0.67)分、B组(3.1+0.77)分、C组(3.4+0.52)分、D组(3.3+0.67)分。A组B、C、D各组之间比较(P>0.05)差异均无统计学意义,移植前各组模型纳入标准无差异(神经功能缺损严重程度类似)。移植后二周神经功能评分:A组(3.0+0.82)分、B组(2.4+0.84)分、C组(2.0+0.67)分、D组(1.2+0.42)分。A与B比较(P=0.066),两者比较差异无统计学意义;A与C、D比较P值分别为0.003和0.000,差异有统计学意义;B与C比较(P=0.214),两组比较差异无统计学意义;但D组与各组比较(P<0.05),两组比较差异有统计学意义;D组神经功能改善最好。3:病理形态学(1)BDNF的表达:移植14天后, A组鼠脑组织中BDNF阳性细胞数表达较少,B组、C组与D组BDNF表达较多,阳性细胞胞浆呈棕褐色,而细胞核不着色,多为海马和皮层的锥体细胞或颗粒细胞,而D组BDNF表达比C组、B组增多,C组比B组增多。各组间BDNF表达两两比较差异均有统计学意义(P<0.05),D组BDNF表达最高。(2)VEGF的表达:A组鼠脑组织中VEGF阳性细胞数表达较少,阳性细胞主要分布在皮质、海马和皮质下结构;B组、C组与D组VEGF表达较多,阳性细胞为血管内皮细胞、胶质细胞和神经干细胞,A组与B、C、D组比较(P<0.05),差异有统计学意义;B与C、D比较(P<0.05),差异有统计学意义;D组VEGF表达量最高,A组最低,C组表达高于B组。(3)脑组织切片HE染色显示:低倍镜下,D组皮层神经细胞数量多,胞浆丰富,镜下可见散在的小软化灶,其余组织结构相对较好。细胞受损较A组、B组、C组明显减轻;A组组织明显水肿,神经细胞体积增大,空泡变性,细胞间隙增大;而B组较其C组又有所差别,组织结构较差,细胞水肿较C组重。结论1大鼠间充质干细胞和血管内皮祖细胞联合移植对缺血性脑损伤有修复作用,其机制可能是通过促进脑内BDNF,VEGF等因子的分泌,促进神经细胞的新生和新生血管形成。2大鼠间充质干细胞和血管内皮祖细胞联合移植对缺血性脑损伤的修复,大脑立体定位移植途径优于动脉移植和尾静脉移植。更多还原

【Abstract】 OBJECTIVETo observe the rat neurological function recovery and explore the protective mechanism and correct transplanting path which were respectively through stereotaxis, vena caudalis and carotid artery path treated middle cerebral artery occlusion(MCAO) model by mesenchymal stem cells(MSCs) and endothelial progenitor cells(EPCs).METHODSMSCs and EPCs were respectively isolated, proliferated, purified and passaged from the long bone of 100g SD rats.MACO model was established by improved method.Forty MCAO rats were divided into BPS control group (A), vena caudalis group (B), carotid artery group(C) and stereotaxis group(D) at random. 2×10~6MSCs and 2×10~6EPCs were injected into MACO model rats in group B, C and D through vena caudalis, carotid artery and tereotaxis paths respectively and 10μl BPS fluid transplanted into MACO model rats in A group at 24-hour after made modes. Bederson neurological score, cerebral infarction volume, HE dyeing, brain-derived neurotrophic factor(BDNF), Vascular endothelial growth factor (VEGF) were investigated after 14 days.RESULTS1 The volume of cerebral infarction: The infarction volumes of group A, B, C and D were 216.3+4.86mm3, 206.0+5.33mm3, 198.2+4.19mm3 and 191.2+4.96mm3 respectively. The infarction volumes between each two groups were statistically significant(P<0.05) and they in group D was the smallest.2 The scores of Nerve behavior: The Bederson neurological score of group A, B, C and D were 3.4+0.67, 3.1+0.77, 3.4+0.52 and 3.3+0.67 respectively and not statistically differences(P>0.05),While They were 3.0+0.82, 2.4+0.84,2.0+0.67 and 1.2+0.42 respectively in the 14-day after transplanted. The scores at each two groups were statistically significant except between group A and group B, group B and group C, (P<0.05). Neurological function was recovered bestly in group D. 3 Pathological morphology(1) BDNF expressions: The positive expressions of BDNF in group A were less than those in group B, group C and group D. The cells with positive expressions of BDNF almost were pyramidal cells or granulosa cells at hippocampus and cortex, and their cytoplasm were tan and nuclei was not stained. The positive expressions of BDNF between each two groups were statistically significant(P<0.05) and they in group D was the highest.(2) VEGF expressions: The numbers of VEGF positive cells in cerebral penumbra of group A were 10.50+1.96 which is the lowest and less than those in group B, group C and group D. The numbers of VEGF positive cells between each two groups were statistically significant(P<0.05). They were 26.4+1.90 in group D and the highest.(3) HE staining: The cortex nervous cells in group D which were rich,abundant cytoplasm and damaged slighter than those in group A, B and C were relatively well except some small softening lesions at low power microscope. The cells in group A damaged most seriously than those in other groups. The cells in group B which structure were bad and edema damaged more seriously than those in group C.CONCLUSION1 The mechanisms of transplantation MSCs with EPCs treating ischemic brain injury were upgrading secretion of BDNF、VEGF in brain, promoting new nerve cells neogeneses and angiogenesis and increasing blood flow to ischemic areas to improve nerve functions.2 The stereotaxis injection was the best method which was more effective than vena caudalis and carotid artery path. The carotid artery was better than vena caudalis path.更多还原