神经母细胞瘤细胞提取液诱导脐带间充质干细胞对坐骨神经损伤修复的实验研究

发布时间：2018-05-10 19:34

本文选题：神经修复 + 神经营养因子　；参考：《新乡医学院》2014年硕士论文

【摘要】：背景和意义神经母细胞瘤细胞(SHSY5Y)是神经系统疾病研究中重要的细胞系,该细胞表达神经营养因子。本实验制备的SHSY5Y提取液是此细胞内的蛋白,对中枢和外周神经系统有营养作用。干细胞疗法是研究组织修复的重要方法。脐带间充质干细胞(UC-MSCs)是一类具有多向分化潜能和自我更新能力的细胞群。UC-MSCs可分泌细胞因子,如神经生长因子(NGF)、脑源性神经营养因子(BDNF)等,在神经损伤后的修复过程中发挥重要作用。研究表明增加损伤部位神经营养因子可促进神经修复。本实验用SHSY5Y提取液处理UC-MSCs,检测处理后细胞分泌NGF和BDNF的变化,观察处理后的干细胞对坐骨神经损伤的促修复作用,为周围神经损伤修复提供一种有益的思路。材料与方法应用超声破碎法得到SHSY5Y提取液,BCA法进行蛋白定量。复苏UC-MSCs,采用无血清培养基接种至6孔板,105/孔。在实验组的UC-MSCs中加入SHSY5Y细胞提取液,浓度为50ul/ml诱导7天。7天后分别检测两组细胞的表面标志物；分别检测目的基因BDNF和NGF在两组细胞中的表达情况。采用大鼠坐骨神经挤压损伤动物模型,36只SD雌性大鼠,随机分成6组(A-F),分别是生理盐水组,SHSY5Y提取液组,MSC组,SHSY5Y提取液和MSC组,诱导后的MSC组和正常对照组。术后观察各组大鼠肢体状况：步态变化,足趾挛缩、肌肉萎缩及恢复情况。参照沈江宁的足迹分析法,采用Brain公式计算坐骨神经功能指数(SFI)。术后3周检测大鼠小腿三头肌萎缩恢复情况,测定肌湿重恢复率(术侧小腿三头肌湿重/正常对照侧×100%),以评价再生神经对靶组织的支配程度。分析靶肌肉和神经病理学HE染色组织切片。结果 1.应用胶原酶消化法分离脐带间充质干细胞,应用超声破碎法得到SHSY5Y提取液； 2.培养对照组和实验组两组细胞,对两组细胞分别进行细胞表型鉴定,实验组MSC的CD29, CD44, CD105和HLA-ABC结果显示仍为阳性； 3.分别提取两组细胞总RNA进行RT-PCR,实验组NGF和BDNF均有一定增加； 4.制作坐骨神经挤压损伤模型,结果显示大鼠肌肉痉挛缓解程度均优于生理盐水对照组； 5.坐骨神功能指数测定结果显示各实验治疗组与生理盐水组之间均有显著差异(P0.05)； 6.小腿三头肌湿重恢复率测定结果显示各治疗组与生理盐水组间对比有显著差异(P0.05); 7.术后3w取术侧小腿三头肌及损伤处坐骨神经做横切片HE染色,显示诱导后MSC组肌肉只有极少量的中性粒细胞,相对其他各组其神经髓鞘恢复较好。结论1. SHSY5Y提取液可使UC-MSCs分泌的NGF和BDNF核酸表达量有所增加；2. SHSY5Y提取液诱导的UC-MSCs能够更好地促进坐骨神经损伤后的修复。
[Abstract]:Background and significance Neuroblastoma cell line SHSY 5Y is an important cell line in the study of nervous system diseases, which expresses neurotrophic factor. The SHSY5Y extract prepared in this experiment is the protein in this cell and has nutritional effects on the central and peripheral nervous system. Stem cell therapy is an important method to study tissue repair. Umbilical cord mesenchymal stem cells (UC-MSCs) are a group of cells with multidirectional differentiation potential and self-renewal ability. UC-MSCs secrete cytokines, such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and so on. It plays an important role in the repair of nerve injury. It has been shown that increasing neurotrophic factor in injured site can promote nerve repair. In this experiment, UC-MSCs were treated with SHSY5Y extract, the changes of NGF and BDNF secreted by the treated cells were detected, and the effects of the treated stem cells on the repair of sciatic nerve injury were observed, which provided a useful way of thinking for the repair of peripheral nerve injury. Materials and methods The SHSY5Y extract was obtained by ultrasonic crushing method for protein quantification. UC-MSCs were resuscitated and inoculated with serum-free medium to 6 hole plate (10 5 / well). After 7 days of 50ul/ml induction, the surface markers of the two groups of cells were detected, and the expression of the target gene BDNF and NGF in the two groups were detected respectively. Thirty-six SD female rats were randomly divided into 6 groups: normal saline group, SHSY5Y extract group, MSC group and normal control group. The changes of gait, toe contracture, muscle atrophy and recovery were observed after operation. According to Shen Jiangning's footprint analysis, the sciatic nerve function index (SFI) was calculated by Brain formula. Three weeks after operation, the recovery of triceps muscle atrophy and the recovery rate of muscle wet weight (wet weight of triceps muscle / normal control side 脳 100) were measured to evaluate the degree of regenerative nerve innervation to target tissue. The HE staining sections of target muscle and neuropathology were analyzed. Result 1. Umbilical cord mesenchymal stem cells were isolated by collagenase digestion and SHSY5Y extract was obtained by ultrasonic fragmentation. 2. The cell phenotypes of the control group and the experimental group were identified respectively. The results of CD29, CD44, CD105 and HLA-ABC of MSC in the experimental group were still positive. 3. The total RNA of the two groups were extracted for RT-PCR.The NGF and BDNF of the experimental group were increased to a certain extent. 4. The model of sciatic nerve crush injury was made. The results showed that the remission degree of muscle spasm in rats was better than that in normal saline control group. 5. The results of ischium function index test showed that there was significant difference between the experimental treatment group and normal saline group (P 0.05). 6. The results showed that there was significant difference between the treatment group and the normal saline group (P 0.05). 7. Three weeks after operation, the triceps muscle of the leg and the sciatic nerve were stained with HE. The results showed that there were only a few neutrophils in the muscles of the MSC group after induction, which was better than that of the other groups. Conclusion 1. SHSY5Y extract could increase the expression of NGF and BDNF nucleic acids secreted by UC-MSCs. UC-MSCs induced by SHSY5Y extract can promote the repair of sciatic nerve injury.
【学位授予单位】：新乡医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R739.4

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