增强型绿色荧光蛋白标记技术示踪组织工程化骨形成

Enhanced green fluorescent protein labeling technology in monitoring the formation of tissue-engineered bone

目的:观察以脂质体介导法转染的增强型绿色荧光蛋白质粒作为骨组织工程种子细胞示踪剂的可行性。方法:实验于2005-07/2006-11在南昌大学第二附属医院分子医学实验室完成。选取12个月龄中国青山羊2只作为骨髓基质干细胞供体。另取BACB/un裸鼠9只作为骨髓基质干细胞-珊瑚复合物受体。①将已转化pEGFP-N2大肠杆菌109菌种,进行质粒提取并经AvaⅡ酶切鉴定。②脂质体lipofectamine2000介导转染羊骨髓基质干细胞。分别于转染24h、6个月后计算荧光的转染效率。标记与未标记细胞经成骨诱导分化后,检测碱性磷酸酶活性和四环素钙结节染色,以未标记的同期细胞作对照。③将标记细胞接种于珊瑚支架,形成骨髓基质干细胞-珊瑚复合物,分别于体外培养4,7,14d后进行电镜扫描观察。④将体外培养7d的骨髓基质干细胞-珊瑚复合物移植于裸鼠皮下,8周后取出,荧光显微镜下进行示踪观察,苏木精-伊红染色观察组织结构。以未标记的骨髓基质干细胞-珊瑚复合物、单纯珊瑚作对照。结果:共纳入中国青山羊2只,BACB/un裸鼠9只,作为受体的9只裸鼠进入结果分析。①质粒经酶切后电泳,可见3条带,分别为445bp,1938bp,2354bp,确定所提质粒为pEGFP-N2。②转染24h后绿色荧光表达率35%;经G418筛选,转染6个月后绿色荧光表达率75%;与对照组相比,标记细胞碱性磷酸酶活性差异无统计学意义(P>0.05),均具有钙结节形成能力。③标记细胞与材料贴附生长良好,并分泌胶原纤维及钙盐结晶样基质。④组织学示新生骨样组织围绕材料孔隙生成;新生组织细胞内有绿色荧光表达,同时对照组未观察到绿色荧光。结论:脂质体介导法转染的pEGFP-N2标记骨髓基质干细胞,可用于裸鼠体内示踪,是一种理想的组织工程化骨组织的示踪方法。

AIM： To explore the feasibility of liposome-mediated enhanced green fluorescent protein （EGFP） plasmid as a tracer of seed cells for bone tissue engineering. METHODS： The experiment was conducted in the Molecular Medical Laboratory of Second Affiliated Hospital of Nanchang University from July 2005 to November 2006. Two Chinese green goats of 12 months old were selected as donors of bone marrow stromal cells （BMSC）; meanwhile, nine BACBlun nude mice were selected as recipients of BMSCs-coral composite. ①Bacillus coli 109 strain, which was transfected with pEGFP-N2, was plasmid extracted and identified by restriction enzyme Ava Ⅱ digestion. ②Lipofectamine 2000 was used to transfect the goat BMSCs, ＇and the transfection efficiency was counted respectively after 24 hours and 6 months transfection. Both labeled and unlabeled BMSCs were induced to osteoblastic differentiation to detect the alkaline phosphatase （ALP） activity and tetracin calcium nodule staining, and the unlabeled BMSCs served as control. ③The labeled BMSCs were seeded into coral scaffold to form BMSCs-coral complex, which was observed under scanning electron microscope after 4, 7, and 14 days culture in vitro. ④The BMSCs-coral complex that had been cultured for 7 days in. vitro was implanted into the nude mice subcutaneously, and taken off 8 weeks later, then was evaluated by HE staining and traced under fluorescence microscope. The unlabeled BMSCs-coral complex, and coral scaffold acted as the control. RESULTS： Two Chinese green goats and nine mice were involved in the result analysis. ①The plasmid was digested into three bands of 445 bp, 1 938 bp, and 2 354 bp, respectively in gel electrophoresis, and was identified as pEGFP-N2. ②The green fluorescence expression efficiency was 35% after transfection for 24 hours; After being screened by G418, the efficiency reached to 75% till the sixth month. Compared with the control group, there was no statistical significance in ALP expression activity （P 〉 0.05）. Both the labeled and unlabeled BMSCs displayed the capability of forming calcium nodules. ③The labeled-cells were able to grow into the coral scaffold and secret collagen fiber bundles and mineralized nods. ④The histological observation suggested that there were newly formed trabeculae around the pores of coral. Fluorescence microscope detected that labeled cells existed in many newborn tissues, and coral was partly degraded.There was no green fluorescence expression in the control group. CONCLUSION： The labeled BMSCs, which are transfected with liposome-mediated pEGFP-N2, could be used to trace the formation of tissue-engineered bone. in nude mice. It may be an optimal labeling technique to monitor tissueengineered bone formation.