静脉注射人脐血间充质干细胞治疗大鼠创伤性脑损伤的实验研究

发布时间：2018-04-21 15:28

本文选题：人脐血间充质干细胞 + 创伤性脑损伤　；参考：《华北煤炭医学院》2009年硕士论文

【摘要】：目的:静脉注射人脐血间充质干细胞(Human umbilical cord blood mesenchymal stem cell,HUCBMSCs)治疗大鼠创伤性脑损伤(Traumatic brain injury,TBI),观察治疗前后大鼠神经损伤严重程度变化、神经生长因子(Never growth factor,NGF)、脑源性神经营养因子(Brain-derived neurotrophic factor,BDNF)表达以及凋亡细胞数量的改变,旨在说明HUCBMSCs通过影响损伤局部NGF、BDNF表达,从而改善损伤局部微环境,减少细胞凋亡,促进神经功能恢复。方法:取第4代HUCBMSCs,于注射前72小时,用5-溴-2-脱氧尿嘧啶核苷(5-bromo-2-deoxyuridine,Brdu)标记,移植备用。标记后的细胞用免疫细胞化学检测细胞标记率,用台盼蓝排斥试验检测标记后细胞的活性。取清洁级健康雄性SD大鼠90只随机分为假伤组(30只)、损伤对照组(以下简称“对照组”)(30只)和注射人脐血间充质干细胞治疗组(以下简称“治疗组”)(30只)。然后每组再随机分为注射后3天、7天、14天、21天和28天组,每个亚组6只大鼠。采用改进的Feeney自由落体方法制作大鼠TBI模型。造模成功后24小时,治疗组所有老鼠经鼠尾静脉注入3×106个HUCBMSCs,以1ml的PBS液溶解;假伤组及对照组所有老鼠经鼠尾静脉注入等体积的PBS液。3组大鼠均未用免疫抑制剂。于相应时间点处死大鼠之前行神经损伤严重程度评分(Neurological Severity Score Points,NSS)。取出标本后,采用HE染色、免疫组化法、原位杂交法对TBI大鼠注射后不同时间点的脑组织在光镜下行形态学变化、Brdu、BDNF和NGF因子、以及细胞凋亡的观测。结果:刚传代的细胞呈圆形,很快贴壁,伸展为三角形、多角形,并逐渐变成梭形,传代24h内完全贴壁,贴壁细胞均匀分布,恢复原来形态,3～5天迅速增殖,约6～7天长满培养瓶底。传至第4代细胞形态较先更均匀,排列更有序。取传至第4代的HUCBMSCs掺入Brdu,72h后免疫组化染色显示80～90%细胞Brdu表达阳性。表明HUCBMSCs的标记率达到80～90%。移植后剩余细胞行台盼蓝染色,结果显示90%以上细胞存活。大鼠NSS评分显示,自注射HUCBMSCs后第7天,各时间点治疗组的神经功能改善均显著优于对照组(P0.05)。光镜下假伤组大鼠脑组织细胞结构基本正常;对照组大鼠受损皮层可见组织结构破坏、脱落,神经元变性,胞体皱缩。治疗组光镜下病理改变较损伤对照组轻微。治疗组可见Brdu标记细胞在损伤周边区明显聚集并存活。随着时间延长,Brdu标记细胞逐渐减少。假伤组仅有极少数神经细胞呈TUNEL阳性。对照组脑损伤周边区出现大量TUNEL染色阳性细胞,和假伤组比较有显著差异(P0.05),相同时间点治疗组脑损伤周边区TUNEL阳性细胞明显减少,但仍显著高于假伤组(P0.05)。随着时间推移,对照组和治疗组TUNEL阳性细胞数量逐渐减少。假伤组仅有少量的BDNF、NGF阳性表达细胞。对照组BDNF及NGF表达显著增加,以损伤周边区最为明显,约注射后14天达到高峰,之后逐渐下降,但仍高于假伤组。治疗组BDNF、NGF阳性细胞的表达明显增加,与相同时间点的对照组比较有显著差异(P0.05),注射后14天达高峰,21d、28d阳性表达明显下降,但仍高于相同时间点的对照组。结论: 1.经尾静脉注射HUCBMSCs可以迁移至脑创伤灶的周边区。 2.未用免疫抑制剂下,HUCBMSCs在免疫功能健全的异种(大鼠)脑内至少能存活4周,且大鼠未发生移植相关的死亡。 3.注入HUCBMSCs能促进脑创伤灶周围区神经元功能恢复,并且NSS评分下降,BDNF、NGF基因蛋白增多,BDNFmRNA表达增强,神经元凋亡减少。
[Abstract]:Objective: intravenous injection of human umbilical cord blood mesenchymal stem cells (Human umbilical cord blood mesenchymal stem cell, HUCBMSCs) in the treatment of traumatic brain injury in rats (Traumatic brain injury, TBI), the changes in the severity of nerve injury in the rats before and after treatment, the neurotrophic factor, and brain derived neurotrophic factor (brain-derived neurotrophic factor) The expression of rived neurotrophic factor, BDNF) and the changes in the number of apoptotic cells are designed to indicate that HUCBMSCs can improve the local microenvironment, decrease the apoptosis and promote the recovery of nerve function by affecting the local NGF and BDNF expression of damage.
Methods: fourth generations of HUCBMSCs, 72 hours before the injection, were labeled with 5- bromine -2- deoxy uridine (5-bromo-2-deoxyuridine, Brdu). The labeled cells were labeled with immunocytochemistry, and the activity of the labeled cells was detected by trypan blue rejection test.
90 healthy male SD rats were randomly divided into a false injury group (30 rats), the injury control group (the following "control group") (30) and the injection of human umbilical cord blood mesenchymal stem cells (the following "treatment group") (30). Then each group was randomly divided into 3 days after the injection, 7 days, 14 days, 21 days and 28 days, and 6 rats in each subgroup. The improved Feeney free fall method was used to produce rat TBI model.
24 hours after the success of the model, all rats in the treatment group were injected with 3 x 106 HUCBMSCs into the tail vein of the rat and dissolved in the PBS solution of 1ml. All rats in the false injury group and the control group were injected with the same volume of the.3 group of the PBS solution in the tail vein of the rat. The severity score of the nerve injury (Neurological Se) before the rats were executed at the corresponding time point. Verity Score Points, NSS). After taking out the specimens, using HE staining, immunohistochemistry and in situ hybridization, the morphological changes of the brain tissues at different time points after injection of TBI rats were observed under light microscopy, Brdu, BDNF and NGF, and the observation of cell apoptosis.
Results: the cells in the new generation were round, quickly adhered to the wall, stretched into triangular, polygonal, and gradually turned into spindle shape. The cells in 24h were completely adhered to the wall, the adherent cells were evenly distributed, restored to the original form, proliferated rapidly in 3~5 days, and about 6~7 days were filled with the bottom of the culture bottle. The form of the fourth generation of fine cells was more uniform and ordered more orderly. Then to the fourth generation of HUCB MSCs was added into Brdu, and the expression of Brdu in 80 ~ 90% cells was positive after 72h. It showed that the labeling rate of HUCBMSCs reached 80 ~ 90%. after transplantation of trypan blue, and the results showed that more than 90% cells survived.
The NSS score of rats showed that the neurological function improvement of the treatment group at each time point was significantly better than that of the control group on the seventh day after injection of HUCBMSCs (P0.05).
The structure of brain tissue in the rats with false injury under light microscope was basically normal, and the damaged cortex of the control group could see the destruction of tissue structure, the degeneration of the neurons, the degeneration of the neurons and the contraction of the cells. The pathological changes under the light microscope in the treatment group were slightly more than that of the control group.
In the treatment group, Brdu labeled cells obviously clustered and survived in the injured peripheral area, and the Brdu labeled cells decreased gradually as time went on.
Only a few nerve cells in the false injury group were TUNEL positive. There were a large number of TUNEL positive cells in the peripheral area of the control group, and there was a significant difference between the false group and the false group (P0.05). The TUNEL positive cells in the peripheral area of the brain injury at the same time point were significantly reduced, but still significantly higher than those in the false injury group (P0.05). The number of TUNEL positive cells in the group decreased gradually.
There were only a small amount of BDNF and NGF positive cells in the false injury group. The expression of BDNF and NGF in the control group increased significantly, which was the most obvious in the peripheral area, and reached the peak at 14 days after the injection, and then decreased gradually, but it was still higher than that in the false group. The expression of BDNF, NGF positive cells in the treatment group was significantly increased, and there was a significant difference compared with the control group at the same time point (P0.05 After injection, the positive expression of 21d and 28d decreased significantly in 14 Tianda peak, but still higher than that in the control group at the same time point.
Conclusion:
1. HUCBMSCs injected into caudal vein can migrate to the peripheral area of the brain injury foci.
2. without immunosuppressive agents, HUCBMSCs could survive at least 4 weeks in the brain of a rat with immunological function, and there was no death related to transplantation.
3. injection of HUCBMSCs could promote the recovery of neuron function around the brain wound area, and the NSS score decreased, BDNF, NGF gene protein increased, the expression of BDNFmRNA was enhanced, and the neuron apoptosis decreased.

【学位授予单位】：华北煤炭医学院
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329.2

【参考文献】