转染转化生长因子-β3对前软骨干细胞增殖及向成软骨方向分化的影响

Proliferation and differentiation of precartilaginous stem cells in response to stable expression of hTGF-β3

目的探讨稳定表达重组转化生长因子-β3（hTGF-β3）对前软骨干细胞（precartilaginous stem cells，PSCs）增殖及向成软骨方向定向分化的诱导作用。方法免疫磁珠法分离纯化得到新生大鼠PSCs后，用线性化的聚乙烯亚胺转染pcDNA3．1（＋）-hTGF-β3到体外单层培养的PSCs中，抗生素筛选使其稳定表达。采用四甲基偶氮唑盐比色法（MTT法）、流式细胞术（FCM）测定转染对PSCs增殖和DNA合成的影响，实时定量RT-PCR、免疫组化及Western blot方法比较转染后目的基因和软骨特异性标志物表达的情况。结果hTGF-β3在分离纯化的PSCs中稳定表达。与未转染组细胞相比，转染后细胞DNA合成增多，增殖速度加快。PCR和免疫组化学结果表明，转染后软骨标志物基因上调明显，分泌软骨多糖基质及Ⅱ型胶原蛋白也明显增加。结论通过hTGF-β3基因强化的PSCs可以稳定表达高效的hTGF-β3蛋白，从而促进PSCs的增殖并向成软骨方向分化，为软骨缺损的高效修复提供了新的思路。

Objective To study the effect of stable expression of reconstructed human transforming growth factor-β3 （ hTGF-β3 ） on proliferation of precartilaginous stem cells （PSCs） and their differentiation into cartilage chondrecytes. Methods After isolated and purified by immunological microbeads, PSCs of rats were transfected with peDNA3. 1 （ ＋ ） -hTGF-β3. MTr reduction assay （MTT） and flow cytometry were performed to investigate the effect of transduction on proliferation and DNA synthesis. Biosynthesis of hTGF-β3 and expressions of cartilage associated genes and proteins were examined by qRT-PCR, immunohistology and Western blot. Results hTGF-β3 was expressed in PSCs stably. Compared with non-transfection group, PSCs＇ DNA synthesis level and proliferation rate were significantly increased after transfection. Quantitative real-time PCR and immunological investigation suggested up-regulated expression of specific genes and proteins of chondroeyte and increase of deposition of chondrocyte typical extracellular matrices proteoglycan and collagen type Ⅱ. Conclusions Gene enhanced PSCs can stably express hTGF-β3 protein to promote proliferation of PSCs and induce differentiation of PSCs into chondrocytes, which provides a fresh approach to cartilage tissue engineering.