中国神经再生研究(英文版)

中国神经再生研究(英文版) ›› 2019, Vol. 14 ›› Issue (10): 1765-1771.doi: 10.4103/1673-5374.257540

• 原著:脑损伤修复保护与再生 • 上一篇    下一篇

TrkA可调节骨髓基质干细胞在神经移植物中的再生能力

  

  • 出版日期:2019-10-15 发布日期:2019-10-15
  • 基金资助:

    国家自然科学基金(81372041，81801220)

TrkA regulates the regenerative capacity of bone marrow stromal stem cells in nerve grafts

Mei-Ge Zheng 1, 2, Wen-Yuan Sui 1, Zhen-Dan He 3, Yan Liu 4, Yu-Lin Huang 1, Shu-Hua Mu 5, Xin-Zhong Xu 2, Ji-Sen Zhang 2, Jun-Le Qu 6, Jian Zhang 3, Dong Wang 1   

  1. 1 Department of Orthopedics, The Seventh Hospital of Sun Yat-sen University, Shenzhen, Guangdong Province, China
    2 Department of Orthopedics, The Second Hospital of Anhui Medical University, Hefei, Anhui Province, China
    3 School of Medicine, Shenzhen University, Shenzhen, Guangdong Province, China
    4 Department of Scientific Research, The Seventh Hospital of Sun Yat-sen University, Shenzhen, Guangdong Province, China
    5 Psychology & Social College of Shenzhen University, Shenzhen, Guangdong Province, China
    6 Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province, College of Optoelectronic Engineering, Shenzhen University, Shenzhen, Guangdong Province, China
  • Online:2019-10-15 Published:2019-10-15
  • Contact: Dong Wang, MD, david74429@126.com; Jian Zhang, PhD, jzhanghappy@163.com
  • Supported by:

    This work was supported by the National Natural Science Foundation of China, No. 81372041 (to DW), and No. 81801220 (to MGZ)

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摘要：

课题组前期研究已证实了过表达原肌球蛋白相关激酶A(TrkA)能显著促进骨髓基质干细胞(BMSCs)在神经移植体内的存活和向类许旺细胞分化，并能有效促进周围神经再生与功能恢复。实验将进一步探讨TrkA在BMSCs神经移植体内发挥功能效应的可能分子机制。实验利用分别携带大鼠TrkA cDNA及其空载体Vector，大鼠TrkA-shRNA及其空载体Control的慢病毒载体感染SD大鼠BMSCs，构建过表达TrkA的Over-TrkA BMSCs及其对照Vector BMSCs，TrkA表达抑制的TrkA-shRNA BMSCs及其对照 Control BMSCs；分别接种于同种异体大鼠去细胞神经支架后，用于桥接大鼠右侧坐骨神经1 cm缺损处。术后8周，(1)利用苏木精伊红染色对各组神经移植体进行组织学检测显示：与Vector和Control移植组相比，Over-TrkA移植组中的细胞和纤维更密集并且均匀排列，而在TrkA-shRNA移植组中它们的分布则更稀疏和无序；(2)利用western blot技术检测各组神经移植体内神经再生相关蛋白，以及TrkA信号通路相关分子表达情况的结果显示：与Vector和Control移植组相比，Over-TrkA移植组的髓鞘标记物髓鞘碱性蛋白(MBP)和轴突标记物神经丝蛋白200(NF200)的表达量显著增高，TrkA信号通路相关分子TrkA、pTrkA、Erk1/2、pErk1/2蛋白水平明显增高；抗凋亡蛋白Bcl-2和Bcl-xL的表达明显增多。而TrkA-shRNA移植组则正相反，MBP、NF200、Erk1/2、pErk1/2、Bcl-2和Bcl-xL等表达明显减少，促凋亡蛋白Bax和Bad表达显著增高。TrkA下游信号分子Akt和pAkt的表达水平在各组之间无明显差异；(3)上述结果表明，TrkA可能是通过Erk1/2途径激活靶分子Bcl-2等的表达从而发挥其促进BMSCs在移植体内的存活和分化，并显著影响周围神经的修复与再生等功能效应。上述实验经中国深圳大学动物伦理委员会于2014年12月批准(批准号：AEWC-2014-001219)。

orcid: 0000-0002-2345-4457 (Dong Wang)
         0000-0003-4126-0223 (Jian Zhang)

关键词: 骨髓基质干细胞, TrkA受体, 慢病毒载体, shRNA, Erk1/2, Bcl-2, 神经移植体, 周围神经再生, 存活, 神经再生

Abstract:

We previously demonstrated that overexpression of tropomyosin receptor kinase A (TrkA) promotes the survival and Schwann cell-like differentiation of bone marrow stromal stem cells in nerve grafts, thereby enhancing the regeneration and functional recovery of the peripheral nerve. In the present study, we investigated the molecular mechanisms underlying the neuroprotective effects of TrkA in bone marrow stromal stem cells seeded into nerve grafts. Bone marrow stromal stem cells from Sprague-Dawley rats were infected with recombinant lentivirus vector expressing rat TrkA, TrkA-shRNA or the respective control. The cells were then seeded into allogeneic rat acellular nerve allografts for bridging a 1-cm right sciatic nerve defect. Then, 8 weeks after surgery, hematoxylin and eosin staining showed that compared with the control groups, the cells and fibers in the TrkA overexpressing group were more densely and uniformly arranged, whereas they were relatively sparse and arranged in a disordered manner in the TrkA-shRNA group. Western blot assay showed that compared with the control groups, the TrkA overexpressing group had higher expression of the myelin marker, myelin basic protein and the axonal marker neurofilament 200. The TrkA overexpressing group also had higher levels of various signaling molecules, including TrkA, pTrkA (Tyr490), extracellular signal-regulated kinases 1/2 (Erk1/2), pErk1/2 (Thr202/Tyr204), and the anti-apoptotic proteins Bcl-2 and Bcl-xL. In contrast, these proteins were downregulated, while the pro-apoptotic factors Bax and Bad were upregulated, in the TrkA-shRNA group. The levels of the TrkA effectors Akt and pAkt (Ser473) were not different among the groups. These results suggest that TrkA enhances the survival and regenerative capacity of bone marrow stromal stem cells through upregulation of the Erk/Bcl-2 pathway. All procedures were approved by the Animal Ethical and Welfare Committee of Shenzhen University, China in December 2014 (approval No. AEWC- 2014-001219).

Key words: nerve regeneration, bone marrow stromal stem cells, tropomyosin receptor kinase A receptor, lentiviral vector, shRNA, extracellular signal-regulated protein kinases 1/2, Bcl-2, nerve grafts, peripheral nerve regeneration, survival, neural regeneration