| 付小洋,张丰识,李启程,等.过表达肝细胞生长因子的内皮祖细胞移植促进坐骨神经挤压伤修复的研究[J].中国临床保健杂志,2024,27(2):241-249. |
| 过表达肝细胞生长因子的内皮祖细胞移植促进坐骨神经挤压伤修复的研究 |
| Endothelial progenitor cell transplantation overexpressing hepatocyte growth factor for sciatic nerve crush injury repair |
| 投稿时间：2023-11-10 |
| DOI：10.3969/J.issn.1672-6790.2024.02.021 |
| 中文关键词: 坐骨神经 周围神经损伤 肝细胞生长因子 内皮祖细胞 模型,动物 |
| 英文关键词: Sciatic nerve Peripheral nerve injuries Hepatocyte growth factor Endothelial progenitor cells Models,animal 〖FL |
| 基金项目:国家重点研发计划项目(2023YFC2509901)；北京市自然科学基金项目(7222198) |
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| 中文摘要: |
| 目的 探讨过表达肝细胞生长因子(HGF)慢病毒转染的内皮祖细胞对大鼠坐骨神经损伤的修复作用。方法 体外实验方面,提取大鼠骨髓的内皮祖细胞(EPC)并利用DiI-acLDL和FITC-UEA-1双染法进行鉴定。EPC转染慢病毒后采用qRT-PCR实验检测HGF基因表达水平。采用CCK-8实验、Transwell实验以及血管形成实验对转基因EPC的体外功能进行评估。体内实验方面,随机将25只SD大鼠平均分为5组,即对照组(Con组)、正常EPC组(N-EPC组)、空载病毒转染EPC组(LV-NC组)、过表达HGF病毒转染EPC组(LV-HGF组)和假手术组(Sham组)。各组大鼠暴露出右侧坐骨神经后,在坐骨神经分叉处上方10 mm处使用止血钳形成2 mm宽的坐骨神经挤压伤,假手术组在暴露神经后不做处理;分别将转染空载慢病毒EPC、转染过表达HGF慢病毒EPC或正常EPC与基质胶按1∶1体积比混合后注入伤处神经外膜下,而对照组不注入EPC。术后4周,运用Catwalk检测大鼠运动功能的恢复情况,电生理实验检测神经传导功能的恢复情况,透射电镜观察髓鞘厚度,甲苯胺蓝染色观察神经纤维密度,腓肠肌湿重称重检测肌肉萎缩程度,马松染色检测各组手术侧的腓肠肌肌纤维面积。结果 体外实验结果显示,与正常EPC组相比,LV-HGF组EPC在CCK8实验中细胞增殖速度更快(P<0.05),Transwell实验中细胞迁移速率更高(P<0.01),在血管形成实验中形成更多血管状结构(P<0.01)。动物实验结果显示,LV-HGF组的坐骨神经功能指数值显著高于其他3组(Con组,P<0.01；N-EPC组,P<0.01；LV-NC组,P<0.01)；复合肌肉动作电位波幅更高(Con组,P<0.01；N-EPC组,P<0.05；LV-NC组,P<0.05)；再生坐骨神经的髓鞘较厚(Con组,P<0.01；N-EPC组,P<0.01；LV-NC组,P<0.01)；有髓神经纤维密度更高 (Con组,P<0.01；N-EPC组,P<0.05；LV-NC组,P<0.05)；腓肠肌湿重比值更高 (Con组,P<0.01；N-EPC组,P<0.01；LV-NC组,P<0.01)；肌纤维平均面积较大(Con组,P<0.01；N-EPC组,P<0.05；LV-NC组,P<0.05);LV-HGF组以上实验结果与Sham组相比,差异有统计学意义(P<0.01)。结论 过表达HGF基因可以增强EPC的增殖、迁移及分化功能。在坐骨神经挤压伤后,HGF基因转染的EPC相较于正常EPC,能更明显地促进了受损周围神经结构和功能的恢复,其机制与促进受损周围神经的轴突再生及髓鞘形成有关。 |
| 英文摘要: |
| Objective To investigate the role of endothelial progenitor cells transfected with lentivirus overexpressing hepatocyte growth factor in repairing sciatic nerve injury in rats.Methods In vitro experiments,rat bone marrow endothelial progenitor cells (EPC) were extracted and identified using DiI-acLDL and FITC-UEA-1 double staining.qRT-PCR detected the gene expression level after EPC were transfected with lentivirus.CCK-8 assay,Transwell assay,and tube formation assay were used to evaluate the function of EPC in vitro.In vivo experiments,25 SD rats were randomly divided equally into 5 groups,i.e.,the blank group (Con group),the normal EPC group (N-EPC group),the empty virus-transfected EPC group (LV-NC group),the overexpression of HGF virus-transfected EPC group (LV-HGF group),and the sham operation group.After exposing the right sciatic nerve in each group of rats,a 2-mm-wide sciatic nerve crush injury was formed by using hemostatic forceps 10 mm above the bifurcation of the sciatic nerve;the sham-operated group was left untreated after exposing the nerve.After surgery,EPC transfected with empty lentivirus,EPC transfected with lentivirus overexpressing HGF,or normal EPC mixed with matrigel at a 1:1 volume ratio were respectively injected into the neuroepithelium of the nerves,whereas no EPC were injected into the blank group.Four weeks after surgery,Catwalk was used to detect the recovery of motor function in rats,electrophysiological tests to detect the recovery of nerve conduction function,transmission electron microscopy to observe the thickness of myelin sheaths,toluidine blue staining to observe the density of nerve fibers,Matson staining to detect the area of gastrocnemius muscle fibers,and gastrocnemius muscle wet weight weighing to detect the degree of muscle atrophy.Results Compared with normal EPC,EPC in the LV-HGF group had faster cell proliferation in the CCK8 assay (P<0.05),higher cell migration rate in the Transwell assay (P<0.01),and formed more vessel-like structures in the tube formation assay (P<0.01);the sciatic nerve function index values of the LV-HGF group were significantly higher than those of the other three groups (Con group,P<0.01;N-EPC group,P<0.01;LV-NC group,P<0.01);the compound muscle action potential amplitude was higher (Con group,P<0.01;N-EPC group,P<0.05;LV-NC group,P<0.05);thicker myelin sheaths in regenerating sciatic nerves (Con group,P<0.01;N-EPC group,P<0.01;LV-NC group,P<0.01);higher densities of myelinated nerve fibers (Con group,P<0.01;N-EPC group,P<0.05;LV-NC group,P<0.05);and higher wet-to-weight ratios of gastrocnemius muscles (Con group,P<0.01;N-EPC group,P<0.01;LV-NC group,P<0.01);and a larger mean area of muscle fibERs (Con group,P<0.01;N-EPC group,P<0.05;LV-NC group,P<0.05);however,the above experimental results were still significantly different from the sham-operated group (P<0.01).Conclusions Overexpression of the HGF gene could enhance the proliferation,migration,and differentiation functions of EPC.After sciatic nerve crush injury,EPC transfected with the HGF gene promotes the restoration of damaged peripheral nerve structure and function more significantly compared with normal EPC.The mechanism is related to the promotion of axon regeneration and myelin sheath formation in damaged peripheral nerves. |
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