TGF-β3对PLGA支架3-D培养兔髓核细胞影响的体外研究

Effect of TGF-β3 on rabbit nucleus pulposus(NP) cells cultured in three-dimensional polylactic-co-glycolic acid scaffold in vitro

目的评价转化生长因子β3(transforming growth factor-β,TGF-β3)对兔髓核细胞在聚乳酸-羟基乙酸共聚物(polylacticco-glycolic acid,PLGA)支架3-D培养的影响。方法应用粒子沥滤法制备多孔PLGA支架培养兔髓核细胞(1×106/PLGA),分为100ng/m L TGF-β3/PLGA,500 ng/m L TGF-β3/PLGA,1μg/m L TGF-β3/PLGA,PLGA对照组。通过MTT分析细胞增殖分化,1,9-二甲基亚甲蓝(DMMB)法检测糖胺多糖(GAG),q PCR检测Ⅱ型胶原(COLⅡ)、Aggrean差异,组织学观察TGF-β3对兔髓核细胞在PLGA支架微孔环境生长形态学改变。结果培养7、14、21 d后TGF-β3/PLGA组的髓核细胞活性、GAG产量、COL II及Aggrecan mRNA表达均高于PLGA对照组(P<0.05)。随着TGF-β3浓度递增,细胞活性及基质表达有显著增加(P<0.05)。21 d组织学HE染色显示TGF-β3/PLGA组较PLGA对照组中微孔支架吸附、积聚更多髓核细胞生长。结论 TGF-β3对PLGA三维培养髓核细胞更好提高分化增殖能力,促进细胞基质分泌。TGF-β3/PLGA支架可作为生物学治疗退变椎间盘病变潜在选择方法。

Objective To evaluate the effect of TGF-β3 on rabbit nucleus pulposus（NP） cells cultured in three-dimensional polylactic-co-glycolic acid（PLGA） scaffold in vitro. Methods PLGA scaffolds were fabricated by particulate leaching method and soaked in rabbit NP cells suspension（1 ×106/scaffold）. PLGA-seeded NP cells were devided into 4 groups： 100 ng/m L TGF-β3/PLGA,500 ng/m L TGF-β3/PLGA,1 μg/m L TGF-β3/PLGA,PLGA control group. Cell proliferation activity was measured using MTT assay. The glycosaminoglycan（GAG） analysis were performed by 1,9-dimethylmethylene blue（DMMB） assay. mRNA expression was measured by quantitive PCR at each time point. Histological observation was performed to elucidate the morphological changes of NP cells in PLGA effected by TGF-β3. Results Higher cellular proliferation activity,GAG production,Collagen type Ⅱ,Aggrean expression were observed in TGF-β3/PLGA-seeded NP cells compared with PLGA control group on day-7,day-14,day-21（P〈0. 05）.Higher dose of TGF-β3 exhibited intense cellular proliferation activity and peri-cellular matrix by increasing trend（P〈0. 05）. Histological observation showed TGF-β3/PLGA developed more significant disc cells cluster than PLGA groups on day-21. Conclusion The 3D porous PLGA scaffold-seeded cells using TGF-β3 can promotes cell proliferation,and prompt extracellular matrix（ECM） production. It is a potential biotherapy for the treatment of disc degeneration.