Sox9基因转染的人脐带血干细胞复合骨基质明胶/生物蛋白胶构建组织工程软骨的实验研究

Construction of tissue engineered cartilage with Sox9 gene transfection human umbilical cord blood-derived mesenchymal stem cells and BMG/fibrin

目的探讨Sox9基因转染的人脐带血干细胞(hUCMSC)复合骨基质明胶/生物蛋白胶体内外构建组织工程软骨的效果。方法体外分离培养hUCMSC;采用脂质体转染Sox9载体质粒,将转染的细胞在骨基质明胶/生物蛋白胶材料上体外培养8周,行扫描电子显微镜和组织学观察。选择雄性SD大鼠55只,体质量(150±20)g。将体外形成的软骨样组织移植入SD大鼠背部皮下,8周时行组织学检查,观察软骨形成能力,并进行宿主免疫反应监测。结果体外培养8周后hUCMSC在支架材料上生长良好,分泌大量蛋白多糖和Ⅱ型胶原。移植入大鼠体内8周后,形成软骨样组织,形态学和免疫组织化学染色显示蛋白多糖和Ⅱ型胶原阳性。移植后IgG、IgA、IgM、C3和C4水平与对照组差异无统计学意义[IgG(1.105 6±0.019 2)μg/mL vs(1.145 0±0.023 1)μg/mL(术后28 d),IgM(0.219 1±0.034 3)μg/mL vs(0.348 7±0.030 3)μg/mL(术后28 d),IgA(0.350 7±0.058 5)μg/mL vs(0.367 7±0.042 4)μg/mL(术后28 d),C3(122.65±4.40)μg/mL vs(130.63±2.98)μg/mL(术后28 d),C4(49.89±2.11)μg/mL vs(51.45±3.74)μg/mL(术后28 d)]。Sox9基因转染的hUCMSC复合骨基质明胶/生物蛋白胶构建的组织工程软骨移植入体内后未引起明显的宿主免疫反应。结论Sox9基因转染的hUCMSC可作为组织工程软骨的一种良好的种子细胞来源。

Objective To evaluate the chondrogenesis of Sox9 gene transfection human umbilical cord mesenchymal stem cells（h UCMSC） seeded onto bone matrix gelatin（BMG）/fibrin hybrid scaffolds in vitro and in vivo. Methods The hUCMSC were isolated and stably transfected with Sox9 gene plasmid and grown on the three-dimensional BMG/fibrin hybrid scaffold for 8 weeks. Scanning electron microscopy and histochemistry were performed. The h UCMSC-loaded scaffolds were implanted into the subcutaneous layer of immunocompetent rats and chondrogenesis and host immune responses were monitored at 8-week.Results After cultured 8-week in intro, hUCMSC spread well and produced abundant glycosaminoglycans and collagen Ⅱ.At 8-week after implantation into rats, which formed cartilage-like tissue and displayed positive staining in toluidine blue,safranin O, Masson＇s trichrome and collagen Ⅱ. There were no significant differences in serum levels between implantation and control of IgG[（1.105 6 ± 0.019 2） μg/mL vs（1.145 0 ± 0.023 1） μg/mL（postoperative 28-day）], IgA [（0.350 7 ± 0.058 5） μg/mL vs（0.367 7 ± 0.042 4） μg/mL（postoperative 28-day）, IgM[（0.219 1 ± 0.034 3） μg/mL vs（0.348 7 ± 0.030 3） μg/mL（postoperative 28-day）], C3[（122.65 ± 4.40） μg/mL vs（130.63 ± 2.98） μg/mL（postoperative 28-day）] and C4[（49.89 ± 2.11） μg/mL vs（51.45 ± 3.74） μg/mL（postoperative 28-day）]. The xenogeneic implantation of Sox9 gene transfection hUCMSC embedded in BMG/fibrin scaffolds promoted the formation of cartilage-like tissue without evident host immune response. Conclusion It is demonstrated that Sox9 gene transfection hUCMSC represent apromising cell candidate for cartilage tissue engineering.