课题背景：课题受国家自然科学基金和卫生部部属临床重点学科项目资助，在之前的研究中已顺利构建转化生长因子β2的真核表达载体，并在转染人关节软骨细胞和维持其表型的研究中获得成功。

偏倚或不足：①虽然在转染后48 h和4周均检测到目的基因的表达，但基因整合和表达的长期稳定性尚需进一步确定，采用病毒载体进行转化实验或许可以提高基因整合率和表达稳定性。②骨髓间质前体细胞在体外单层培养中可诱导表达软骨相关基因和蛋白，但尚不能证实其能分化为成熟软骨细胞，尤其不能确定其具有关节软骨的生物学特性，下一步可利用生物反应器或者进行体内实验深入分析。

术语解析：基因转染是将具生物功能的核酸（RNA或DNA）转移或运送到细胞内，并使核酸在细胞内维持其生物功能的核酸转移技术。在组织工程研究中，靶细胞不仅是作为目的基因的携带者，而且还是组织修复的细胞成分。基因转染的方法包括磷酸钙共沉淀法、电穿孔法、机械法以及脂质体法等，其中脂质体转染的转染效率相对较高，操作简便，是实验常用的转染方法。

摘要
目的：骨髓中包含的间质前体细胞在适宜的条件下可以被许多细胞因子诱导分化为软骨。采用基因转染技术观察转化生长因子β2对体外培养的骨髓间质前体细胞向软骨分化的影响。
方法：实验于2005-12/2007-01在中日友好医院临床研究所骨科实验室完成。①骨髓标本来源于6名志愿者，对治疗及实验均签署知情同意书，实验经医院医学伦理委员会批准。②实验方法：志愿者局麻下行骨髓穿刺，抽取7.0~10.0 mL骨髓抗凝，以密度梯度离心法分离人骨髓间质前体细胞，通过脂质体介导转染技术，将pcDNA3.1(+)TGF-β2载体导入骨髓间质前体细胞中，体外单层培养。③实验评估：利用RT-PCR、Western Blotting和免疫组化法检测转化生长因子β2和软骨相关基因、蛋白的表达情况。
结果：①转染后48 h，可检测到明显的转化生长因子β2 mRNA表达，Ⅱ型胶原A1和Aggrecan mRNA均有轻微表达；转染后4周，转化生长因子β2 mRNA表达稍有降低，Ⅱ型胶原A1和Aggrecan mRNA表达均明显上调。②转染后48 h和4周，间质前体细胞培养上清液中均有转化生长因子β2蛋白的表达。③转染后48 h部分细胞显示Ⅱ型胶原蛋白免疫组化阳性信号，转染后4周Ⅱ型胶原蛋白阳性细胞数增加。
结论：pcDNA3.1(+)/TGF-β2真核表达载体在骨髓间质前体细胞内可获得短暂和长期表达。在单层培养的情况下，骨髓间质前体细胞可被转化生长因子β2诱导向软骨方向分化。
关键词：组织工程；软骨；TGF-β2；基因转染；间质前体细胞

王卫国，孙伟，鞠晓东，娄思权.基因转染促骨髓间质前体细胞体外向软骨的分化[J].中国组织工程研究与临床康复，2008，12(12):2211-2215 [www.zglckf.com/zglckf/ejournal/upfiles/08-12/12k-2211(ps).pdf]

Gene transfection to induce in vitro chondrogenesis of human bone marrow-derived mesenchymal progenitor cells

Abstract

AIM：Bone marrow-derived mesenchymal progenitor cells could be induced by various cytokines to differentiate into chondrocytes under specific condition. This study investigated the effect of human transforming growth factorα2 (TGF-α2) on mesenchymal progenitor cell chondrogenesis in monolayer culture.

METHODS: The experiment was performed at Clinical Institute Orthopaedic Laboratory of China-Japan Friendship Hospital from December 2005 to January 2007. ①Bone marrow was harvested from 6 volunteers. Informed consents of treatment and experiment were signed previously, and the experiment was approved by medical ethics committee of the hospital.②Bone marrow aspiration was performed under local anesthesia and 7.0-10.0 mL of marrow was harvested from each volunteer. Mesenchymal progenitor cells were isolated by density gradient centrifugation. Recombinant pcDNA3.1(+)/TGF-β2 was transfected into mesenchymal progenitor cells by liposome technique, and cells were cultured in monolayer in vitro. ③RT-PCR, Western blotting and immunohistochemistry analyses were performed to identify the expression of TGF-β2 and cartilage associated genes and proteins.

RESULTS: ①RT-PCR results showed that marked expression of TGF-β2 and slight expression of both type Ⅱ collagen A1 (COL2A1) and Aggrecan mRNAs were detected in transfected cells after 48 hours. At the culture interval of 4 weeks, the expression of TGF-β2 was slightly decreased, and the expression of COL2A1 and Aggrecan was significantly up-regulated. ②Western blotting suggested that TGF-β2 protein was detected in the culture medium of transfected cells at both 48 hours and 4 weeks after transfection. ③Immunohistochemistry suggested that type II collagen protein was detected in some of the transfected cells 48 hours after transfection. More cells with positive signal were detected 4 weeks after transfection.

CONCLUSION: Transfection of pcDNA3.1(+)/TGF-β2 into mesenchymal progenitor cells is able to provide transient and persistent expression. The differentiation of human marrow-derived mesenchymal progenitor cells into chondrocyte in monolayer culture is feasible and may be induced by TGF-β2.

Wang WG, Sun W, Ju XD, Lou SQ.Gene transfection to induce in vitro chondrogenesis of human bone marrow-derived mesenchymal progenitor cells.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(12):2211-2215
[www.zglckf.com/zglckf/ejournal/upfiles/08-12/12k-2211 (ps).pdf]