巢式PCR扩增性别决定基因序列检测兔骨髓间充质干细胞移植治疗关节软骨缺损的修复组织来源  

Cell sources for cartilage defect repair with rabbit bone marrow mesenchymal stem cells:PCR amplification for sex-determining gene sequence

作　　者：杨亚冬[1] 张文元[1] 房国坚[1] 沃恩康[1] 何卓晶[1] 洪艳[1] 陈勇[1] 

机构地区：[1]浙江省医学科学院生物工程所,浙江省杭州市310013

出　　处：《中国组织工程研究与临床康复》2007年第37期7341-7344,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：浙江省科技计划重大项目(2004F11018)~~

摘　　要：目的:明确骨髓间充质干细胞移植到关节软骨损伤处后,该处修复组织的细胞来源,以了解组织修复的过程。方法:实验于2006-01/2007-01在浙江省医学科学院生物工程所完成。①实验材料:3月龄新西兰大白兔,清洁级,体质量约2kg,由浙江省实验动物中心提供。②实验方法:利用兔性别决定基因序列设计两对PCR引物。取雄性新西兰大白兔进行骨髓间充质干细胞的分离培养与扩增。取雌性新西兰大白兔,暴露其膝关节软骨,用电钻钻孔造成膝关节软骨内侧直径4.5mm深3.5mm的全层关节软骨缺损,达软骨下骨。一侧植入骨髓间充质干细胞/胶原海绵,另一侧仅植入胶原海绵。③实验评估:于术后1,2,3,4,5个月取兔关节软骨缺损修复处组织提取总DNA,进行PCR扩增,鉴定移植物中是否存在性别决定基因,以明确细胞来源。结果:移植后1,2,3,4,5个月移植雄兔骨髓间充质干细胞及胶原海绵的关节修复处组织提取的DNA,用设计的引物通过PCR扩增出一条约200bp大小的条带,大小与阳性对照一致;而另一侧仅移植胶原海绵的关节软骨修复处组织提取的DNA,用设计的引物通过PCR扩增后未发现有条带。PCR扩增阳性样本测序分析显示,每个样本碱基序列一致,与GenBank上性别决定区Y基因组gb|AY785433.1|比较,扩增的样本序列与标准序列完全一致,序列同源性达100%。结论:雌兔关节骨髓间充质干细胞/胶原海绵复合物移植处的新生组织中含有雄性组织,提示新生软骨中有移植的雄性骨髓间充质干细胞分化来的软骨细胞。AIM： To confirm the call source of cartilage defect after bone marrow mesenchymal stem calls （MSCs） transplantation, so as to know the recovery process. METHODS： The experiment was conducted in the Institute of Bioengineering, Zhejiang Academy of Medical Sciences from January 2006 to January 2007. ①Three-month-old New Zealand Rabbit of clean grade and about 2 kg was selected （experimental animal center of Zhejiang Province）. Two pairs of PCR primers were designed by using the sex-determining gene sequence. MSCs were isolated from the male New Zealand Rabbit and cultured. The cartilage of knee joint of female rabbits was exposed to establish the full-thickness cartilage defect of 4.5 mm in diameter and 3.5 mm in depth by electrodrill. One side was implanted with MSCs/collagen sponge, and the other with collagen sponge only. At postoperatively 1, 2, 3, 4, and 5 months, the total DNA was extracted from the cartilage defects for PCR amplification to identify whether there was sex-determining gene in the implants, so as to confirm the call source. RESULTS： At 1, 2, 3, 4, and 5 months after transplantation, there was a band （about 200 bp） of DNA extracted from the tissues implanted with MSCs and collagen sponge by PCR; it was of the same size as the positive control, but no band was found in the other side. The sequencing of PCR amplified positive sample showed that the base sequence of every sample was uniform, and accorded with the standard sequence of Y-genome gb ｜AY785433.1｜ from GenBank. The sequencing homology was 100%. CONCLUSION： Male tissues are found in the defects implanted with female MSCs and collagen sponge, indicating that there are chondrocytes that are differentiated from the implanted male MSCs in new formation cartilage.

关 键 词：性别决定基因 骨髓间充质干细胞 关节软骨缺损 

分 类 号：R394.2[医药卫生—医学遗传学]