富含血小板血浆凝胶复合脂肪间充质干细胞构建可注射组织工程髓核

Construction of injectable tissue engineered nucleus pulposus by using complex of platelet-rich plasma gel scaffold and adipose derived stromal cells in vitro

目的:探讨以自体富含血小板血浆(PRP)凝胶复合脂肪间充质干细胞(ADSCs)构建可注射组织工程髓核的可行性。方法:将体外扩增的第3代兔ADSCs经流式细胞仪鉴定后接种至自体PRP凝胶中,体外立体培养2周、4周、8周时分别行大体观察、组织学检查、5-溴-2-脱氧尿嘧啶(BrdU)免疫荧光法观察细胞在PRP凝胶中的分布及存活情况;分光光度法检测PRP凝胶-细胞复合体中糖胺聚糖(GAG)含量,实时荧光定量PCR法检测低氧诱导因子(HIF-1α)、蛋白多糖(Aggrecan)、Ⅱ型胶原(CollagenⅡ)基因表达情况。结果:流式细胞仪检测显示体外扩增的第3代细胞CD90阳性率为95.2%,CD45阳性率为0.9%。培养2、4、8周时PRP凝胶细胞复合体均为表面光滑的凝胶状,弹性较好;番红O染色2周时细胞外基质几乎不着色,4周时可见细胞周围呈粉色的弱阳性染色,8周时多数细胞周围呈红色阳性染色。HE染色和扫描电镜各时间点均可见细胞均匀分布于网络状支架内;BrdU免疫荧光法显示细胞在支架中生存状态良好,培养4周时阳性细胞数较培养2周时明显增多(P<0.05),8周时较4周时明显增多(P<0.05)。培养4周时GAG含量较培养2周时明显增高(P<0.05),8周时较4周时明显增高(P<0.05);培养4周时与培养2周时比较3个目的基因mRNA表达量均增高,差异有显著性(P<0.05),8周时与4周时比较亦明显增高(P<0.05)。结论:以兔自体PRP凝胶与ADSCs构建的复合体在体外培养时细胞可以向类髓核样细胞分化,用此方法构建可注射组织工程髓核具有可行性。

Objective：To explore the feasibility of complex of platelet-rich plasma（PRP） gel scaffold and adipose derived stromal cells（ADSCs） as seed cells to construct injectable tissue engineered nucleus pulposus in vitro.Methed：The third massaging rabbit ADSCs were identified by flow ctytometry in vitro,the ADSCs were seeded into PRP gel scaffolds and continued culturing in vitro.At the 2nd,4th and 8th week,the PRP gel-cell complexes were examined macroscopically and histologically.The vitality of cells in the PRP gel scaffold was measured by BrdU immunofluorescence method.The glycosaminoglycans（GAG） in the PRP gel-cell complexes were measured by spectrophotometry.Messenger ribonucleic acids（mRNA） of hypoxia-inducible factor-1α（HIF-1α）,Aggrecan,type Ⅱ collagen were determined by real-time PCR.Result：Flow cytometry showed that ADSCs were positive for CD90 and negative for CD45.Macroscopically,the PRP gel-cell complexes presented with smooth surface and well elasticity at each time point.The safranin O staining showed no positive dyeing cells at the 2nd week,weak positive dyeing cells at surrounding region at the 4th weeks,and enhanced obviously at 8th week,which was visible to surrounding cells with red positive dyeing.The results of HE staining and scanning electron micrograp（SEM） demonstrated that the cells were well-distributed in the reticulate scaffold.BrdU immumofluorescence showed that most ADSCs can survive and proliferate in the PRP gel scaffold.Positive cells at the 4th week were much more than those at the 2nd week（P0.05）,and significant differences were also noted between the 4th and 8th week（P0.05）.The content of glycosaminoglycan at the 4th week was higher than those at the 2nd week（P0.05）,and significant differences were also noted between the 4th and 8th week（P0.05）.Messenger ribonucleic acids（mRNA） of 3 genes at the 4th week were much more than those at the 2nd week（P0.05）,and significant differences were also noted between the 4th and 8th week（P0.05）.Conclusion：The PRP gel scaffold allows for rabbit ADSCs differentiated into nucleus pulposus-like cells after compound culture in vitro,the complexes can be used as injectable tissue engineered nucleus pulposus.