犬同种异体椎间盘复合髓核细胞移植初探

Tissue engineered allograft total disc transplantation using exogenous nucleus pulposus cells: an experimental study in a beagle model

中文关键词:  椎间盘退变  椎间盘移植  同种异体  人端粒酶逆转录酶  基因转染  髓核细胞  犬

英文关键词:Intervertebral disc degeneration  Intervertebral disc transplantation  Human telomerase reverse transcriptase  Viral transfection  Nucleus Pulposus cells  Beagle dogs

中文摘要:

  【摘要】目的:探讨同种异体椎间盘复合髓核细胞移植的转归，观察转人端粒酶逆转录酶（human telomerase reverse transcriptase，hTERT）基因髓核细胞介入椎间盘移植的生物学效应。方法：常规分离8周龄比格犬髓核细胞，将PKH-26标记的传2代髓核细胞（NPc）和转hTERT基因髓核细胞（hTERT-NPc）分别注射入冷冻保存的同种异体椎间盘内构建组织工程化同种异体椎间盘。选择12月龄同种犬18只随机分为3组，分别植入hTERT-NPc组织工程化的同种异体椎间盘（A组）、NPc组织工程化的同种异体椎间盘（B组）以及未组织工程化的同种异体椎间盘（C组）。每只犬于术后4、8、12周行正侧位X线片及MRI检查，观察移植椎间盘同宿主椎体愈合情况、移植椎间盘高度和信号变化情况；手术后12周麻醉状态下处死实验动物取L1~L7段脊柱标本进行生物力学测试，取移植椎间盘进行组织学观察。结果：X线及MRI检查结果显示3组移植椎间盘与宿主椎体均实现了良好的骨性融合，其中C组椎间盘术后出现了呈进展趋势的退行性改变，至移植后12周，其椎间盘高度及髓核信号比灰度值明显低于A、B两组（P<0.05）。生物力学显示术后12周时，C组移植椎间盘在屈伸和旋转方向的活动度显著性大于A、B两组（P<0.05），A组与B组无显著性差异（P＞0.05）。激光共聚焦显微镜观察到A、B组移植椎间盘髓核区域内有红色荧光细胞；光学显微镜下观察A、B两组移植椎间盘结构保持较好，外形类似软骨细胞的髓核细胞数量较多，排列较为规则；C组髓核形态保持欠佳，结构较为紊乱，髓核细胞数量明显减少，细胞形态欠饱满，退行性改变明显。RT-PCR分析显示术后12周时A组髓核内可检测到hTERT mRNA的阳性表达。结论：犬同种异体椎间盘移植后可在宿主体内存活，通过复合NPc可延缓椎间盘移植后的退行性改变；hTERT-NPc介入具有更好的保持异体椎间盘功能的潜力，有望保证同种异体椎间盘移植的远期疗效。

英文摘要:

  【Abstract】 Objectives: To investigate in vivo rehepitation of the transplanted tissue engineered allograft total disc and to explore the biological effect of nucleus pulposus(NP) cells or hTERT gene transfected NP cells on allograft total disc transplantation. Methods: Eighteen canine lumbar intervertebral discs were obtained from 5 canines and cryopreserved in liquid nitrogen. Canine nucleus pulposus cells were isolated and transduced with rAAV-hTERT. The cells were injected into the discs to construct a "tissue-engineered" allograft disc(group A). NP cells and DMEM/F12 were used for positive control(group B) and blank control(group C) respectively. 18 beagle dogs received the 3 groups of allograft IVD composites implantation respectively. Radiographic examination was performed at 4, 8 and 12 weeks after implantation. At 12 weeks after operation, all dogs were sacrificed and the lumbar spines were harvested for the biomechanical test and histological analysis, ectogenic NP cell tracing and hTERT mRNA analysis. Results: Bony fusion between intervertebral disc allograft and adjacent host intervertebral body was observed in all animals. The disc height and T2 signal intensity preservation in group A and B were better than group C. MRI showed typical degenerative changes in group C. In group A, the normalized grayscale of the transplanted disc in MRI image was significant higher than that of the controls at 12 weeks. Biomechanical test showed a poor stability preservation in group C compared with group A and B. PKH-26 positive cells were identified within the allograft discs in group A at 12 weeks, which provided matrix for cell survival. Histological analysis showed the NP cell morphology, cell number and distribution of the allograft discs were better preserved in group A and B than group C at 12 weeks of follow-up. Conclusions: NP cells or hTERT loaded NP cells intervention can effectively resist the degeneration of the allogenic transplanted intervertebral discs in beagle model. The hTERT loaded NP cells show better anti-degeneration effect than NP cells. This modified disc regeneration technique through NP cell injection may preserve the disc function better, which can ensure the long-term outcome of allograft disc transplantation.