RNA干扰技术构建组织工程软骨的实验研究  

TISSUE ENGINEERED CARTILAGE USING CHITOSAN/GELATIN AND NORMAL OR POST-RNA INTERFERENCE-CHONDROCYTES IN VITRO

作　　者：王正辉[1] 吴宝俊[1] Kamal Mustafa 

机构地区：[1]西安交通大学医学院第二附属医院耳鼻咽喉-头颈外科,西安710004 [2]挪威卑尔根大学牙科学院

出　　处：《中国修复重建外科杂志》2012年第1期106-111,共6页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家自然科学基金资助项目(81000416);西安交通大学医学创新基金(GH-0203202);中央高校基本科研业务费专项资金(西安交通大学国际合作类;交叉学科类)~~

摘　　要：目的种子细胞来源是软骨组织工程的研究热点。探讨以RNA干扰(RNA interference,RNAi)技术干扰多聚蛋白聚糖酶(Aggrecanase)后的软骨细胞作为组织工程种子细胞的可行性。方法取成年SD大鼠肋软骨采用消化法分离培养软骨细胞。取第1代细胞分为空白阴性对照组(对照组)及慢病毒转染组(实验组)两组。将两组细胞分别与壳聚糖/明胶材料进行体外复合培养制备组织工程软骨,于培养后不同时间点通过HE、Masson染色,扫描电镜观察以及RT-PCR方法检测两组软骨中蛋白聚糖(Aggrecan)以及Aggrecanase-1、2的变化情况。结果组织学观察示培养2周时,对照组与实验组相比细胞数量及细胞外基质未见明显差别;随着培养时间延长,实验组较对照组细胞外基质分泌增多,细胞数目更多。RT-PCR结果显示培养4、8周,实验组细胞Aggrecan mRNA表达量明显高于对照组,Aggrecanase-1和Aggrecanase-2 mRNA表达量明显低于对照组,比较差异均有统计学意义(P<0.05)。扫描电镜显示实验组细胞数量明显多于对照组,且细胞间连接紧密。结论采用RNAi技术干扰Aggrecanase后的软骨细胞可以作为组织工程软骨种子细胞,其在材料上分泌Aggrecan的含量明显高于正常软骨细胞,其生物学活性需进一步研究。Objective The seed cells source is a research focus in tissue engineered cartilage.To observe whether the post-RNA interference（RNAi） chondrocytes could be used as the seed cells of tissue engineered cartilage.Methods Chondrocytes were separated from Sprague Dawley rats.The first passage chondrocytes were used and divided into 2 groups： normal chondrocytes（control group） and post-RNAi（experimental group）.Normal and post-RNAi chondrocytes were seeded into chitosan/gelatin material and cultured in vitro to prepare tissue engineered cartilage.The contents of Aggrecan and Aggrecanase-1,2 were measured by HE and Masson staining,scanning electron microscope（SEM）,and RT-PCR.Results The histological results： no obvious difference was observed in cell number and extracellular matrix（ECM） between 2 groups at 2 weeks;when compared with control group,the secretion of ECM and the cell number increased in experimental group with time.The RT-PCR results： the expression of Aggrecan mRNA in experimental group was significantly higher than that in control group（P 0.05）;but the expressions of Aggrecanase-1,2 mRNA in experimental group were significantly lower than those in control group（P 0.05）.The SEM results： the cell number in experimental group was obviously more than that in control group,and the cells in experimental group were conjugated closely.Conclusion The post-RNAi chondrocytes can be used as the seed cells for tissue engineered cartilage,which can secrete more Aggrecan than normal chondrocytes.But their biological activities need studying further.

关 键 词：组织工程软骨 RNA干扰 软骨细胞 蛋白聚糖 多聚蛋白聚糖酶 大鼠 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]