神经干细胞植入大鼠缺损皮肤对创面神经修复的影响

Effects of neural stem cell transplantation on neural regeneration in rats’ skin wounds

摘要:

目的：探讨酸性成纤维细胞生长因子（acidic fibroblast growth factor，aFGF）体外诱导大鼠骨髓间质干细胞（bone marrow mesenchymal stem cells，BMSCs）分化为神经干细胞（neural stem cells，NSCs）后，植入缺损皮肤对创面神经修复的作用及方式。方法：分离培养SD大鼠BMSCs，利用aFGF诱导其向NSCs分化，并用DAPI荧光标记物进行标记。将 81只SD大鼠背部作皮肤全层缺损创面，每组9只。随机分为局部创面注射NSCs（A）组、尾静脉注射NSCs （B）组、NSCs+血浆支架（局部外敷料组）（C）组、局部创面注射BMSCs（D）组、尾静脉注射BMSCs（E）组、BMSCs+血浆支架组（局部外敷料组）（F）组、血浆支架（局部外敷料）（G）组、局部创面注射等量生理盐水（H）组、尾静脉注射等量生理盐水（I组），创面模型在各种移植处理后的第4、5、7周取材，免疫荧光及RT-PCR检测皮肤表皮下及真皮层蛋白基因产物（protein gene product，PGP）9.5的表达。结果：PGP 9.5在创面愈合后第4、5、7周的荧光密度分布，在时间上采用重复资料分析，差异有统计学意义（F=427.03，P=0.000），干细胞移植对神经修复特异性表达有差异；在分组上采用重复资料分析，差异有统计学意义（F=130.44，P=0.000），干细胞移植在时间及分组上的相互作用结果差异有统计学意义（F=3.543，P=0.001）。NSCs局部注射移植后神经修复作用较NSCs尾静脉注射移植明显（P=0.001），NSCs尾静脉注射移植后神经修复较NSCs外敷料移植明显（P=0.000），而NSCs局部注射移植后神经修复作用较BMSCs局部注射移植后明显（P=0.000），BMSCs局部注射移植后神经修复作用较生理盐水局部注射移植后明显（P=0.000），血浆局部外辅料移植与生理盐水的局部注射移植之间差异有统计学意义（P=0.014），生理盐水的局部注射移植与尾静脉注射移植之间差异无统计学意义（P=0.446），RT-PCR检测PGP 9.5基因表达的测定更进一步说明了以上结果。结论：aFGF诱导BMSCs分化为NSCs后，植入皮肤缺损促进皮肤创面神经修复的效果较单纯植入BMSCs强，且以局部注射效果最为显著。

Abstract:

Objective：To observe the effects of neural stem cells （NSCs） in vivo on neural regeneration in skin wounds after acidic fi－broblast growth factor（aFGF） being used to induce rat bone marrow mesenchymal stem cells（BMSCs） into neural stem cells（NSCs） in vitro. Methods：SD rat BMSCs were isolated and cultured by the whole bone marrow adherent culture method，induced by aFGF to differentiate into NSCs，and was marked with DAPI. Eighty-one SD rats were made back full-thickness defect skin wounds and each group had 9 SD rats. NSCs were randomly divided into the local wound injection（group A），the tail vein injection（group B），the NSCs with plasma graft implantation（group C）. BMSCs were divided into the local wound injection（group D），the tail vein injection of（group E），BMSCs with plasma graft implantation（group F），the plasma graft transplantation（group G）. Tail veins were injected with normal saline（group H） and local wounds were injected with normal saline（group I）. The expression of protein gene product（PGP） 9.5 in wound skin after transplantation at the 4th，5th，7th week was detected by immunofluorescence and RT-PCR in order to reflect the skin nerve regeneration. Results：There were statistically significant differences in fluorescence density value of PGP 9.5 at the 4th，5th，7th week after the wound healing based on the results of repeated data analysis of design information（F=27.03，P=0.000）. There were differences in specific expression of nerve restoration after stem cell transplantation based on the results of repeated data analysis of design information（F=130.44，P=0.000）. The inter－action of stem cell transplantation in terms of time and group was statistically significant（F=3.543，P=0.001）. Local injection of NSCs transplantation significantly improved nerve restoration than tail vein injection of NSCs transplantation（P=0.001）. Tail vein injection of NSCs transplantation significantly improved nerve restoration than NSCs plasma graft transplantation（P=0.000）. Local in－jection of NSCs transplantation significantly improved nerve restoration than local injection of BMSCs transplantation（P=0.000）. Local injection of BMSCs transplantation significantly improved nerve restoration than local injection of saline transplantation（P=0.000）. There were statistically significant differences between local plasma graft transplantation and local injection of saline transplantation（P=0.014） an there was no difference between local injection of saline transplantation and tail vein injection transplantation（P=0.446）. Detection of PGP 9.5 with RT-PCR further illustrated the above results. Conclusion：NSCs transplanted into skin wounds in the local wound injection can promote the neural regeneration of wound and it was the most effective pattern.