NT-3转染的SC对坐骨神经缺损大鼠腓肠肌细胞的影响

Affect of gastrocnemius muscle cell of neurotrophic factor-3 modified Schwann cells on sciatic nerve defect in rats

目的观察神经营养因子-3(NT-3)基因修饰的施旺细胞(SC)对大鼠坐骨神经缺损模型腓肠肌细胞的影响。方法取3 d龄Wistar大鼠2只,分离、培养SC。将重组人真核细胞质粒(pcDNA3)-NT-3通过阳离子脂质体转染SC,用免疫组化染色观察转染前后SC的生长速度,测定SC含量。Wistar成年大鼠80只制成坐骨神经缺损模型,向坐骨神经断端分别注入ECM凝胶(A组)、SC-PLGA ECM凝胶(B组)、NT-3基因-PLGA ECM凝胶(C组)、NT-3基因修饰SC-PLGA ECM凝胶(D组)各20μL,每组20只。分别于术后1、4、8、12周每组各取5只大鼠,制作腓肠肌标本,常规HE染色,测量肌纤维横截面积。采用Tunel法对肌细胞凋亡率进行检测。结果 NT-3经阳离子脂质体转染SC后3、5、7、9、14、21 d SC细胞数、纯度与转染前相比,P均<0.05。术后12周腓肠肌纤维横截面积B、C、D组与A组相比,B、C组与D组相比,P均<0.05。转染后12周A、B、C、D组腓肠肌细胞凋亡率分别为32.09%±1.02%、15.84%±0.63%、11.67%±0.48%、6.83%±0.25%,A组>B组>C组>D组,P均<0.05。结论 NT-3基因经阳离子脂质体转染SC能促进SC的分泌,修复损伤神经,防止失神经肌细胞凋亡。

Objective To observe the affect of gastrocnemius myofibercross sectional area and muscle cell apoptosis of cationic liposome transfer neurotrophic factor-3（NT-3） modified Schwann cells（SC） on sciatic nerve defect.Methods The 2 Wistar three-day age rats were isolated and cultured SC,pcDNA3-NT-3 genes were transfected into SC with cationic liposome,the speed of growth and content of SC were measured by S-100 immunocytochemical stain after modified.The 80 adult rats were made sciatic nerve transected model and divided into four groups.To the stump of the sciatic nerve were injected into the ECM gel（group A）,SC-of PLGA ECM gel（group B）,NT-3 gene-of PLGA the ECM gel（group C） of NT-3 gene modification of the SC-of PLGA the ECM gel（group D） each 20 μL（n=20）.5 rats in each group were sacrificed respectively on 1,4,8,12 weeks after surgery,gastrocnemius muscle specimens of the rats were taked and treated by HE staining,muscle fiber cross-sectional area were detected.Muscle cell apoptosis rate were detected by the Tunel method.Results The cell numbers and content of SC were singnificantly difference after modified with NT-3（all P0.05）.Compared with group A and group B,C,there was higher rates in group B,C in myofibercross sectional area and cell apoptosis on different times potoperatively.Compared with group D and group B,C,the results have statistical significance（all P0.01）.There was no singnificant difference in group B,C of sectional area（P0.05）.But which in group B were more than group C for the number and rate of muscle cell apoptosis detection.Conclusion NT-3 genes modified by cationic liposomes can efficienly promote the secretion of SC,repair the damage nerve and prevent muscle cell apoptosis of denervation amyotrophy.