人雪旺细胞促进人脐带间充质干细胞向神经方向分化的研究

发布时间：2018-06-01 08:28

  本文选题：脊髓损伤 + 细胞移植　； 参考：《天津医科大学》2012年硕士论文

【摘要】：[目的] 观察人雪旺细胞(human Schwann cells, hSCs)对人脐带间充质干细胞(human umbilical cord-mesenchymal stem cells, hUC-MSCs)在脊髓损伤(spinal cord injury, SCI)大鼠体内存活、分化的影响；描述胶质纤维酸性蛋白(glial fibrillary acidic protein, GFAP)、髓磷脂碱性蛋白(myelin basic protein, MBP)与高分子量神经丝蛋白(high molecular weight neurofilament, NF-H)表达的时间特征,并探讨hSCs促进hUC-MSCs向神经方向分化的可能机制。 [方法] 无菌条件下取出健康、因外伤行截肢治疗患者的坐骨神经,应用双酶消化组织块结合机械分离法分离培养hSCs、采用低浓度胰蛋白酶消化和差速贴壁法对细胞进行纯化,传至第4代(P4)的hSCs用S-100抗体对其进行鉴定,本实验植入的hSCs为P5；实验所用的hUC-MSCs由天津协和干细胞基因工程有限公司提供,细胞经过流式细胞仪鉴定符合干细胞标准,植入的hUC-MSCs亦为P5。取8周龄雌性Wistar大鼠80只,以Impactor Model-Ⅱ型打击器制作脊髓胸10(T10)损伤模型。实验随机均分为4组：DMEM对照组(A), hSCs移植组(B),hUC-MSCs移植组(C), hUC-MSCs与hSCs联合移植组(D)。移植后2w,4w,各组实验动物随机取出2只经心脏灌注后取材固定(脊髓长度包括损伤中心头尾端长约1.5cm),制成石蜡切片后行GFAP, MBP与NF-H免疫荧光染色,植入的细胞行MAB1281免疫荧光染色进行体内示踪。各组分别于移植后1W,2w,3w,4w以同样方法取材,分别行Western-Blot, Real-Time PCR检测。上述数据用SPSS16.0软件方差分析进行统计分析。 [结果] hSCs在原代培养第3天即可从组织块边缘爬出,至第9天时即可观察到有大量的细胞长出,2w后进行细胞传代,该细胞可以在体外稳定传至5代以上；P5代的hUC-MSCs在镜下观察可见其形态规则、大小均一、呈漩涡状生长。 免疫荧光染色显示：A组未见到MAB1281阳性染色细胞,而其它各组为阳性染色；D组可观察到MBP与NF-H阳性染色细胞,其它各组为阴性染色。 Western-Blot, Real-Time PCR检测结果显示：A组3种神经标志物在蛋白与mRNA水平的表达量随时间延长均逐渐增高,且GFAP的表达量在移植后第2w明显增高(Western-blot所测第2w与第1w的GFAP条带灰度值比值为1.34而PCR所测mRNA比值为2.86±0.33),差异有统计学意义(P0.05)；而MBP与NF-H的表达量在移植后第3w明显增高(Western-blot所测第3w与第1w的MBP条带灰度值比值为3.43而PCR所测mRNA比值为6.47±0.62,NF-H所测结果的比值则分别为4.12,6.78±0.77),差异有统计学意义(P0.05)。与A组相对应时间点比较,细胞移植组MBP,NF-H的表达量较高而GFAP的表达量较低,其中D组的MBP, NF-H的表达量最高(D组与A组MBP在第3w时条带灰度值比值为2.11而NF-H在第3w时条带灰度值比值为2.11),差异有统计学意义(P0.05)；虽然D组GFAP的表达在第2w较第1w增高,但其在第2,3与4w时间点之间的表达量差异(各周所测条带灰度比值依次为1：1.12：1.13：1.14)无统计学意义(P0.05)。 [结论] 在hSCs影响下,hUC-MSCs至少可在SCI动物体内存活4周；hSCs能促进hUC-MSCs向神经元与少突胶质细胞方向分化；hSCs与‘(?)UC-MSCs在SCI大鼠体内共同作用后,能够抑制胶质瘢痕的形成：GFAP, MBP,与NF-H等3种神经标志物的表达有一定的时间特征。
[Abstract]:Purpose of the project

To investigate the effects of human Schwann cells ( hSCs ) on survival and differentiation of human umbilical cord - mesenchymal stem cells ( hUC - MSCs ) in spinal cord injury ( SCI ) rats .
This paper describes the time characteristics of glial fiber acidic protein ( GFAP ) , myelin basic protein ( MBP ) and high molecular weight neurofilament ( NF - H ) expression , and discusses the possible mechanism of hSCs to promote the differentiation of hUC - MSCs into nerve .

Methodology

The hSCs were isolated and cultured under the condition of aseptic condition . The hSCs were isolated by double enzyme digestion and mechanical separation , and the cells were purified by low concentration trypsin digestion and differential adherent method . The hSCs transferred to 4th generation ( P4 ) were identified by S - 100 antibody , and the hSCs implanted in this experiment were P5 ;
The experimental animals were divided into four groups : DMEM control group ( A ) , hSCs transplantation group ( B ) , hUC - MSCs transplantation group ( C ) , hUC - MSCs and hSCs combined transplantation group ( D ) . The experimental animals were randomly divided into 4 groups : DMEM control group ( A ) , hSCs transplantation group ( B ) , hUC - MSCs transplantation group ( C ) , hUC - MSCs and hSCs .

The result is not valid .

The hSCs can be isolated from the edge of the tissue mass on the third day of primary culture , and a large number of cells can be observed at the 9th day , and cell passages can be carried out after 2w , and the cells can be stably transferred to more than 5 generations in vitro ;
The expression of hUC - MSCs in P5 was observed under the microscope , and its size was uniform and vortex - like growth was observed .

Immunofluorescence staining showed that MAB1281 positive staining cells were not seen in group A , while other groups were positive staining .
In group D , MBP and NF - H positive staining cells were observed , and the other groups were negative staining .

The results of Western - Blot and Real - Time PCR showed that the expression level of three kinds of nerve markers in group A was gradually increased with time , and the expression of GFAP increased significantly after transplantation ( 2 . 86 卤 0.33 ) .
Compared with group A , the expression of MBP and NF - H was 4.12 , 6.78 卤 0.77 respectively . The expression of MBP and NF - H in group D was higher than that in group A . The ratio of MBP and NF - H was 2.11 in group D and 2.11 in group A , and the difference was significant ( P0.05 ) .
Although the expression of GFAP in group D was higher at 2w compared to 1w , there was no significant difference in the expression of GFAP ( 1 : 1.12 : 1.13 : 1.14 ) between the 2nd , 3rd and 4w time points ( P0.05 ) .

Conclusion

Under the influence of hSCs , hUC - MSCs could survive at least 4 weeks in SCI animals .
hSCs can promote the differentiation of hUC - MSCs into neurons and less glial cells ;
hSCs and ' ( ? ) UC - MSCs can inhibit glial scar formation in SCI rats . The expression of GFAP , MBP and NF - H has a certain time characteristic .
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

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