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（polylactic-co-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100 ng／mL TGF-β3／PLGA，500 ng／mL TGF-β3／PLGA，1μg／mL 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），qPCR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/mL TGF-β3/PLGA,500 ng/mL TGF-β3/PLGA,1 μg/mL 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.
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