hTGF-β1基因增强组织工程化髓核修复退变腰椎间盘的实验研究

Tissue engineered nucleus pulposus composite by hTGF-β1 gene transferred bone mesenchymal stem cells for intervertebral disc degeneration

目的:探讨人转化生长因子-β1(hTGF-β1)基因修饰山羊骨髓基质干细胞(BMSCs)为种子细胞构建的组织工程化髓核对山羊退变腰椎间盘的修复作用。方法:用粗针穿刺山羊L1/2～L5/6纤维环抽取髓核制作椎间盘退变模型,同时体外培养山羊自体BMSCs并分为3组:实验组以携hTGF-β1基因重组腺病毒(Ad/hTGF-β1)转染;阴性对照组以携lacZ基因腺病毒(Ad/LacZ)转染,空白对照组为未转染细胞。抽取髓核后15d,收集3组细胞与透明质酸(HA)复合为组织工程化髓核,以细针从椎间盘原穿刺孔回植入髓核中央。L1/2至L3/4依次植入实验组、阴性对照组及空白对照组细胞;L4/5注入不含细胞的HA;L5/6不注入任何物质。术后观察24周,定期行放射学、组织学和生化检测。结果:X线片椎间隙测量显示,24周时L1/2～L4/5的椎间隙狭窄均较L5/6明显延迟(P<0.05);4周以内L2/3间隙的X-gal染色保持阳性;组织学评估示L1/2～L4/5退变均较L5/6为轻;免疫组化示L1/2在4周内高表达hTGF-β1、CollagenⅡ和Aggrecan,但8周后L1/2至L4/5未见显著性差异;GAG定量显示整个观察期内L1/2～L4/5均高于L5/6,其含量依次为L1/2>L2/3=L3/4>L4/5;对4周时L1/2至L3/4髓核组织的RT-PCR显示,L1/2的hTGF-β1、CollagenⅡ及Aggrecan的mRNA表达显著增强(P<0.05)。结论:体外构建的组织工程化髓核回植可延缓椎间盘早期退变的进程,经hTGF-β1基因增强的组织工程化髓核对退变延缓作用尤为显著,但均无法完全逆转整个椎间盘退变进程。

Objective：To explore the possibility of human transforming growth factor-β1（hTGF-β1） gene induction for BMSCs（bone mesenchymal stem cells） as nucleus pulposus（NP） tissue-engineering seed cells in repair of intervertebral disc degeneration（IDD）. Method：The NP from L1/2～5/6 discs of goats were aspirated with a thicker needle to induce IDD.At the same time,the autologous BMSCs were cultured in vitro,and then divided into 3 groups（5×10^6 cells/group）.Experimental group was infected with adenoviral hTGF-β1 ,adenoviral β-gal infected cells as control group and no infected as blank control.15 days after the aspiration,the BMSCs （107 cells） were harvested and mixed with hyaluronan（HA）,and then injected into the disc＇s centre through the previous puncturation opening by a thinner needle,L1/2 with experimental group cells,L2/3 with control group,L3/4 with blank control,L4/L5 with HA/medium composite only and L5/6 without injection as sham operation.Every 4 goats were sacrificed at 2,4,8,12,24 weeks respectively and discs radiography,tissue glycosaminoglycan（GAG） quantification,histological evaluation,and immunohistochemistry（IH） were examined.RT- PCR were also performed to verify mRNA expression of hTGF-β1,collagen Ⅱ and aggrecan in nucleus pul- posus tissue 4 weeks after operation.Result：The radiography showed postponed disc space narrowing in L1/2～L4/5 24 weeks later.The X-gal staining was positive before 4 weeks in 12/3.Histological evaluation agreed with less degenerated presentation in L1/2～4/5 than in L5/6,and IH showed strong positive expression of hTGF-β1 ,collagen Ⅱ and aggrecan in L1/2 level before 4 weeks.GAG quantification showed the most in L1/2, least in L4/5,and no difference between 12/3 and L3/4（P〈0.05）.The RT-PCR also showed stronger expression of hTGF-β1,collagen Ⅱ and aggrecan mRNA in experimental group （P〈0.05）.Conclusion： Reinsertion of tissue engineered nucleus pulposus could decelerate the early IDD,and this effect will be more remarkable by the hTGF-β1 gene transferred BMSCs as seed cells.However,the disc degeneration process could not be reversed by current methods used in this study.