人脐带间充质干细胞移植在颅脑损伤的作用研究

发布时间：2018-06-15 13:27

  本文选题：脐带间充质干细胞 + 细胞移植　； 参考：《河北医科大学》2013年硕士论文

【摘要】：目的：建立大鼠液压冲击颅脑损伤模型,探讨人脐带间充质干细胞(human umbilical cord mesenchymal stem cells, hUC-MSCs)移植对颅脑损伤大鼠神经功能恢复的影响以及移植细胞的存活、迁移和神经分化情况，为其在颅脑损伤治疗的应用提供理论依据。 方法：取足月妊娠剖宫产健康新生儿的脐带，利用酶消化法和贴壁法获得原代细胞，传3~5代备用。收集消化后的细胞，流式细胞学检测CD45PE、CD90PE、 CD105PE、CD73PE、CD11b PE和CD34FITC、CD19FITC， HLA-DR PE，并以小鼠IgG1-PE和小鼠IgG1-FITC作为同型对照。 选取成年健康雄性Sprague-Dawley (SD)大鼠，体重约260g～320g，制作液压冲击颅脑损伤模型。模型制备成功24小时后，采取改良神经功能评分（modified Neurological Severity Score, mNSS）对其进行神经功能评价（Table1），选取评分为7～12分（中度）的50只大鼠进入实验。将其随机分为三组：⑴hUC-MSCs移植组：20只，细胞移植前用噻唑蓝[3-(4,5)-dimethylthiahiazol-2-y1-2,5-diphenytetrazolium bromide，MTT]检测细胞活力。将第5代hUC-MSCs移植到大鼠颅脑损伤部位。⑵磷酸盐缓冲液（phosphate buffered saline, PBS）组：20只，用PBS替代hUC-MSCs注射到颅脑损伤部位。⑶单纯颅脑损伤：10只，行开骨窗和颅脑损伤处理，但不移植hUC-MSCs。另外，再随机选取10只大鼠为⑷假手术组，只开骨窗，不行颅脑损伤处理，亦不行细胞移植。 四组大鼠分别采用改良的神经功能评分(modified neurologicalseverity score，mNSS)在hUC-MSCs移植或PBS注射后的第１天、7天、14天、21天、28天进行神经功能评分，观察其神经功能改善情况。评分结束后采用RT-PCR技术检测移植细胞胶质纤维酸性蛋白(glial fibrillaryacidic protein,GFAP)、神经元特异性烯醇化酶(neuron specific enolase,NSE)、巢蛋白(Nestin)以及髓鞘碱性蛋白(myelin basic protein，MBP)的表达情况，并进行比较。 结果： 细胞接种4～6h后观察到少量细胞贴壁，，12h以后大部分细胞贴壁。接种7d时，细胞汇合达到80％～90％，消化后按1:2或1:3的比例传代。细胞传代后，形态均一，生长迅速，3-4d可传一代。流式细胞学检测结果显示：hUC-MSCs表达间充质干细胞表面抗原CD105、CD73、CD90，不表达B型淋巴细胞表面抗原CD19、造血前体细胞表面抗原CD34、白细胞表面抗原CD45、中性粒细胞表面抗原CD11b和HLA-DR，表示该细胞是hUC-MSCs且具备异体移植的可行性。细胞移植前采用MTT法检测hUC-MSCs细胞活力好，台盼蓝染色显示细胞存活率达95%以上。 hUC-MSCs移植后28d内，hUC-MSCs移植组死亡2只，死亡率为10.0%；PBS组死亡6只，死亡率为30.0%；单纯颅脑损伤组死亡4只，死亡率40.0%。hUC-MSCs移植组死亡率低于单纯颅脑损伤组和PBS组，差异有统计学意义（P0.05）。 神经功能评分结果（Table3）显示，hUC-MSCs移植组随着时间的变化神经功能恢复优于单纯颅脑损伤组，差异有统计学意义（P0.05）。 hUC-MSCs移植28天，神经功能评分结束后，取新鲜脑组织提取总RNA,应用RT-PCR从分子生物学证实了移植的hUC-MSCs在大鼠颅脑损伤部位存活并表达GFAP、NSE、Nestin，未表达MBP。 结论： 1人脐带间充质干细胞移植促进大鼠颅脑损伤后神经功能的恢复。 2移植的hUC-MSCs在大鼠颅脑损伤部位存活并表达GFAP、NSE、Nestin，未表达MBP。
[Abstract]:Objective: to establish a model of hydraulic impact craniocerebral injury in rats and to explore the effect of human umbilical cord mesenchymal stem cells (hUC-MSCs) transplantation on the recovery of neural function in rats with craniocerebral injury and the survival, migration and differentiation of the transplanted cells in order to provide a theory for the application of the brain injury in the treatment of brain injury. Basis.
Methods: the umbilical cord of the healthy newborn in the term of cesarean section was taken. The original cells were obtained by enzyme digestion and adherence method. The cells were collected for 3~5 generation. The cells after digestion were collected, and the flow cytometry was used to detect CD45PE, CD90PE, CD105PE, CD73PE, CD11b PE and CD34FITC, CD19FITC, HLA-DR PE, and the mice IgG1-PE and mice were used as the same control.
The adult healthy male Sprague-Dawley (SD) rats were selected and weighed about 260g to 320G to make a model of hydraulic shock craniocerebral injury. After 24 hours of successful model preparation, the modified neural function score (modified Neurological Severity Score, mNSS) was used to evaluate the nerve function of the brain injury (Table1), and 50 rats with a score of 7~12 (moderate) were selected. The experiment was randomly divided into three groups: (1) hUC-MSCs transplantation group: 20, cell viability was detected by thiazolium [3- (4,5) -dimethylthiahiazol-2-y1-2,5-diphenytetrazolium bromide and MTT] before transplantation. Fifth generation hUC-MSCs was transplanted into the brain injury site of rats. (2) phosphate buffer solution (phosphate buffered saline, PBS) group: 20 PBS instead of hUC-MSCs was injected into the site of craniocerebral injury. (3) simple craniocerebral injury: 10 rats were treated with open bone window and craniocerebral injury, but no transplantation of hUC-MSCs., 10 rats were randomly selected as a sham operation group, only open bone window, no craniocerebral injury treatment, and no cell transplantation.
The four groups of rats were treated with improved neural function score (modified neurologicalseverity score, mNSS) on the first day after hUC-MSCs transplantation or PBS injection, 7 days, 14 days, 21 days and 28 days to evaluate the neural function improvement. After the score, the RT-PCR technique was used to detect the glial fibrillary acidic protein (glia) of the transplanted cells (glia). L fibrillaryacidic protein, GFAP), the expression of neuron specific enolase (neuron specific enolase, NSE), nestin (Nestin) and myelin basic protein (myelin basic protein), and compared.
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本文编号：2022156