人软骨蛋白聚糖Northern杂交探针的构建及其应用

目的　构建人软骨蛋白聚糖 (aggrecan)探针 ,应用于整复外科研究中Northern法对aggrecanmRNA表达水平的检测。 方法　取人软骨细胞 ,逆转录 多聚酶链反应 (RT PCR )扩增ag grecan球间区域 (IGD)片段。将纯化的RT PCR产物构建入 pUCm T载体 ,酶切及测序鉴定。32 P标记aggrecan探针 ,Northern杂交法检测人软骨组织和培养软骨细胞 (各 3组 )以及人正常和瘢痕疙瘩成纤维细胞 (各 6组 )中aggrecanmRNA的表达。 结果　构建产物经鉴定 ,显示其包含了人ag grecanIGD的基因序列 ,与GeneBank已收录序列 (M 5 5 172 ) 10 0 %相同。Northern结果显示 ,该探针可用于aggrecanmRNA表达水平的定性和定量检测。对正常人软骨组织和体外培养人软骨细胞的aggrecan β actin的吸光度比值进行比较 ,发现其间 ( 0 .5 75 6± 0 .0 15 2 1.36 90± 0 .0 2 0 6 )差异具有显著性 (P <0 .0 1)。结论　Northern杂交法可以对整复外科研究中常见的软骨和瘢痕疙瘩成纤维细胞中aggrecanmRNA的表达水平进行检测 ,且具有较好的灵敏度和特异性。

Construction of northern hybridization probe to human aggrecan

Objective To construct Northern hybridization probe for quantitative analysis of aggrecan mRNA expression level in plastic surgery research.Methods Aggrecan IGD cDNA was amplified by RT-PCR and inserted into pUCm-T vector.The products were identified by restriction mapping and sequencing.The probe was labeled by 32 P and hybridized with RNA samples.Results The constructed plasmid contained human aggrecan IGD gene sequences,which was 100 % identical to published human IGD sequence (GeneBank,M55172).Additionally,the probe was demonstrated to be able to hybridize IGD mRNA from chondrocytes and keloid fibroblasts.Conclusion Aggrecan mRNA in different types of cells can be quantified by Northern hybridization,which is sensitive and specific.