脐带华通胶间充质干细胞移植对退变椎间盘影响的实验研究

The effect of Wharton′s jelly-derived mesenchymal stem cells in a xenograft canine model for intervertebral disc degeneration

中文关键词:  华通胶间充质干细胞  细胞移植  椎间盘退变  动物实验

英文关键词:Wharton′s jelly-derived mesenchymal stem cells  Cell transplantation  Intervertebral disc degeneration  Animal experiment

中文摘要:

  【摘要】 目的：探讨人华通胶间充质干细胞（Wharton′s jelly-derived mesenchymal stem cells，WJMSCs）移植对犬退变椎间盘的影响。方法：从新生儿脐带中提取WJMSCs，取增殖良好的第3代细胞，用含有绿色荧光蛋白的腺相关病毒（rAAV2-EGFP）感染标记细胞。选择20只健康成年比格犬作为实验动物，使用穿刺抽吸髓核组织法建立椎间盘退变模型（L4/5、L5/6、L6/7）。4周后将犬各节段椎间盘进行分组：L3/4为对照组（A组）；L4/5为退变组（B组）；L5/6为注射组（C组），注射生理盐水；L6/7为移植组（D组），移植绿色荧光蛋白标记的WJMSCs细胞悬液。造模术前、术后4、8、12、24周行腰椎X线及MRI检查。24周后处死动物取材进行冰冻切片荧光、HE染色及番红O染色等组织学检测，提取髓核组织总RNA，反转录后行Real Time PCR检测，观察蛋白多糖、Ⅱ型胶原、SOX-9及Ⅰ型胶原基因表达变化。结果：分离培养的WJMSCs贴壁生长，呈梭形形态，rAAV2-EGFP病毒感染后第3天表达绿色荧光。影像学检查结果显示各组椎间盘高度指数及相对灰度指数在造模术前、术后第4周无统计学差异，术后8、12、24周，D组椎间盘相对高度指数及相对灰度指数较B、C组高（P<0.05），比A组低（P<0.05）。术后24周，D组髓核组织冰冻切片内能够检测到GFP阳性的WJMSC细胞，HE染色显示D组髓核组织退变比B组和C组轻，番红O染色结果显示D组染色较B组和C组深，基因表达检测结果显示D组Ⅱ型胶原、蛋白多糖及SOX-9基因表达比B、C组高（P<0.05），但比A组低（P<0.05）。结论：人WJMSCs移植入犬退变椎间盘内能够存活，促进椎间盘细胞外基质Ⅱ型胶原及蛋白多糖合成，维持椎间盘高度及髓核含水量，能够有效延缓椎间盘退变进展。

英文摘要:

  【Abstract】 Objectives: To assess the effect of human umbilical cord Wharton′s jelly-derived mesenchymal stem cells(WJMSCs) in a xenograft canine model for intervertebral disc degeneration. Methods: WJMSCs were isolated from human umbilical cords and labeled with rAAV2-EGFP. Twenty healthy adult Beagle dogs were used for animal experiment. Degenerative models were established in L4/5, L5/6, L6/7 intervertebral discs by aspiration of the nucleus pulposus. For each canine, L4/5 was used as degenerative segments(group B), L5/6 was injected with 0.9% saline 4 weeks after first operation(group C), L6/7 was injected with EGFP-labbled WJMSCs 4 weeks after first operation(group D), and the intact disc L3/4 served as an un-injured control (group A). The animals were followed up for 24 weeks after the initial operation. Spinal image was evaluated at 0, 4, 8, 12 and 24 weeks after operation respectively. Histologic analyses were performed at 24 weeks. Gene expression analysis for aggrecan, type Ⅱ collagen, SOX-9 and type Ⅰ collagen was performed. Results: The WJMSCs in vitro were adherent growth and exhibited fibroblast-like morphology. GFP expression was confirmed using fluorescence microscope 3 days after rAAV2-EGFP infection. Disc height and T2-weighted signal intensity were higher in group D than group B and C at 8, 12 and 24 weeks(P<0.05), and didn′t show significantly changes in all groups at 0 and 4 weeks. GFP-positive WJMSCs were detected in the nucleus pulposus of the canine in group D 24 weeks after first operation. Histological assay showed better preserved in group D as compared to the operated degenerated control discs(group B and C). Safranin O staining showed higher aggrecan content in group D. Disc regeneration was also confirmed at the gene expression level by using real-time polymerase chain reaction. The relative gene expressions of matrix-related genes(aggrecan and type Ⅱ collagens) were significantly increased in group D when compared to those of group B and group C(P<0.05). Conclusions: The transplanted WJMSCs can survive in the disc of canine. The increased amount of aggrecan and type Ⅱ collagen in the nucleus pulposus suggests that WJMSCs may serve as a new valuable resource in cell transplantation therapy for degenerative disc disease.