脂肪间充质干细胞鞘内移植对脊髓栓系综合征大鼠松解术后神经损伤的修复作用及机制研究

Repairing effect of intrathecal transplantation of adipose mesenchymal stem cells on nerve injury in rats with tethered cord syndrome and its mechanism

目的探讨脂肪间充质干细胞(AMSCs)鞘内移植对脊髓栓系综合征(TCS)大鼠松解术后神经损伤的修复作用及其机制。方法将80只大鼠随机分为4组,即假手术组(双侧椎板切除术+鞘内注射PBS)、假手术移植组(双侧椎板切除术+鞘内注射AMSCs)、TCS松解术组(TCS松解术+鞘内注射PBS)、TCS松解术移植组(TCS松解术+鞘内注射AMSCs),每组各20只;采用改良Tarlov分级法评估移植前、移植14、28 d后双后肢运动能力;移植28 d后采用体感诱发电位(SEP)进行神经电生理检测;采用苏木精-伊红(HE)染色和尼氏体染色观察脊髓组织病理学改变;采用Western blot法检测脊髓组织中脑源性神经营养因子(BNDF)、酪氨酸蛋白激酶B(TrkB)蛋白表达水平。结果与假手术组和假手术移植组比较,移植前、移植14、28 d后TCS松解术组和TCS松解术移植组改良Tarlov评分均较低,移植28 d后SEP潜伏期延长、波幅减小(P<0.05);与TCS松解术组比较,移植14、28 d后TCS松解术移植组改良Tarlov评分均较高,移植28 d后SEP潜伏期缩短、波幅增大(P<0.05);随时间的延长,TCS松解术移植组改良Tarlov评分逐渐升高(P<0.05),而其余各组无明显变化(P>0.05)。HE染色和尼氏体染色显示,假手术组和假手术移植组脊髓组织结构完整,灰、白质结构致密、交界清楚,尼氏体含量丰富;TCS松解术组脊髓组织结构紊乱,灰、白质界限模糊,灰质区域内呈现大小不一空洞,尼氏体明显减少;AMSCs移植后TCS松解术移植组脊髓组织损伤减轻,白质区域内空洞形成减少,尼氏体含量明显增多。与假手术组和假手术移植组比较,TCS松解术组和TCS松解术移植组脊髓组织中BNDF、TrkB蛋白相对表达水平均降低(P<0.05);与TCS松解术组比较,TCS松解术移植组脊髓组织中BNDF、TrkB蛋白相对表达水平均升高(P<0.05);假手术组与假手术移植组比较,脊髓组织中BNDF、TrkB蛋白相对表达水平均无明显差异(P>0.05)。结论 AMSCs鞘内移植可有效促进TCS松解术后神经损伤的修复能力,改善后肢运动功能,其作用机制可能与促进脊髓组织中BNDF、TrkB蛋白表达有关。

Objective To explore the repairing effect of intrathecal transplantation of adipose mesenchymal stem cells(AMSCs) on nerve injury in rats with tethered cord syndrome(TCS) rats and its mechanism.Methods 80 rats were randomly divided into 4 groups, sham operation group(bilateral laminectomy + intrathecal injection of PBS), sham operation transplantation group(bilateral laminectomy + intrathecal injection of AMSCs), TCS release group(TCS release + intrathecal injection of PBS), TCS release transplantation group(TCS release + intrathecal injection of AMSCs), 20 rats each group.The sham operation group and the sham operation transplantation group underwent bilateral laminectomy, and 14 days later underwent intrathecal catheterization, and rats with successful surgery were injected intrathecally with 100 μL of PBS buffer. The TCS release group and the TCS release transplantation group were first prepared TCS rat model, 14 days later the release operation and intrathecal catheterization were performed, and the successful rats were injected intrathecally with 106/100 μL AMSCs cells 100 μL. The modified Tarlov classification method was used to evaluate the motor ability of both hind limbs before transplantation and after 14 and 28 days of transplantation. after 28 days of transplantation, neurophysiological examination was performed using somatosensory evoked potential(SEP). Hematoxylin-eosin(HE) staining and Nissl body staining were used to observe the histopathological changes of spinal cord. Western blot was used to detect the expression levels of brain-derived neurotrophic factor(BNDF) and tyrosine protein kinase B(TrkB) protein in spinal cord tissue.Results Compared with the sham operation group and the sham operation transplantation group, the modified Tarlov score was lower in the TCS release group and the TCS release transplantation group before transplantation and after 14 and 28 days of transplantation, and the SEP latency and the amplitude decreased after 28 days of transplantation(P<0.05). Compared with the TCS release group, the modified Tarlov score was higher in the TCS release transplantation group after 14 and 28 days of transplantation, and the SEP latency was shortened and the amplitude increased at 28 days after transplantation(P<0.05). With the prolongation of time, the modified Tarlov score of the TCS release transplantation group gradually increased(P<0.05), while there was no significant change in the other groups(P>0.05). HE staining and Nissl body staining showed that there were complete spinal cord tissue structure, dense gray and white matter structures, clear junctions, and abundant Nissl body content in sham operation group and sham operation transplantation group. In the TCS release group the spinal cord tissue was disorganized, the gray and white matter boundaries were blurred, the gray matter area showed different sizes, and the Nissl body was significantly reduced. After the transplantation of AMSCs, the spinal cord injury of the TCS release transplantation group was reduced, the formation of voids in the white matter area was reduced, and the content of Nissl body was significantly increased. Compared with the sham operation group and the sham operation transplantation group, the relative expression levels of BNDF and TrkB protein in the spinal cord tissue of the TCS release group and the TCS release transplantation group were both reduced(P<0.05). Compared with the TCS release group, the relative expression levels of BNDF and TrkB protein in the spinal cord tissue of the TCS release transplantation group were increased(P<0.05). There were no significant differences in the relative expression levels of BNDF and TrkB protein in spinal cord tissue between sham operation group and sham operation transplantation group(P>0.05).Conclusion Intrathecal transplantation of AMSCs could effectively promote the repair of nerve injury after TCS release and improve the motor function of hind limbs, which might be related to the promotion of BNDF and TrkB protein expressions in spinal cord tissue.