Aggrecan Northern探针的构建及其在组织工程研究中的应用

目的：构建猪aggrecan探针，用于对组织工程化软骨及软骨细胞中aggrecan mRNA表达水平的Northern检测。方法：取猪软骨细胞，RT—PCR扩增aggrecan球间区域(IGD)片段。将纯化的RT—PCR产物克隆入pUCm—T载体，酶切、测序鉴定。32P标记aggrecan探针，Northern杂交法检测组织工程化软骨和软骨细胞中aggrecan mRNA的表达状况。结果：构建产物经鉴定，证实其包含了猪aggrecan IGD的基因序列。Northern结果显示，该探针可用于对组织和细胞中aggrecan mRNA表达水平的柃测。结论：在组织工程化软骨的研究中，利用Northern杂交法对样品中aggrecan mRNA的水平进行检测，具有良好的灵敏度、可信度和特异性。

Construction of Plasmid Containing Porcine Aggrecan IGD Gene Utilized as Probe for Northern Hybridization

Objective To quantify the aggrecan tnRNA expression level in tissue engineered cartilage and in vitro cultured chondrocytes by the constructed Northern hybridization probe. Methods Aggrecan IGD cDNA was amplified by RT - PCR and inserted into the pUCm - T vector. The products were analyzed by restriction mapping and sequencing. The probe was labeled by P and hybridized with total RNA from tissue engineered cartilage and cultured chondrocytes. Results The constructed plasmid contained porcine aggrecan IGD cDNA sequences, and the probe could be utilized to evaluate aggrecan mRNA expression level. Conclusion Aggrecan mRNA in tissue engineered cartilage and chondrocytes can be quantified by Northern hybridization with the constructed probe,which is sensitive,reliable and specific.