PCR标记前胶原基因探针及其检测肝星状细胞前胶原基因表达

THE LABELING OF PROCOLLAGEN GENE PROBE BY PCR AND THE DETECTION OF PROCOLLAGEN GENE EXPRESSION IN HEPATIC STELLATE CELLS

目的 :建立一种新的前胶原基因探针制备方法 ,并用其检测HSC的前胶原mRNA表达。方法 :从NCBIGeneBank查询Ⅰ、Ⅲ、Ⅳ型前胶原基因的序列 ,根据基因序列用OLIGO软件设计其引物 ;RT PCR扩增基因 ,并用不对称PCR方法和DIG dUTP标记前胶原基因探针 ,用其原位杂交检测HSC前胶原基因表达。结果 :用所设计的引物和RT PCR扩增得到目的基因 ,制备了DIG标记的前胶原基因探针 ,并用其检测到HSC的前胶原基因表达。结论 :建立了一种新的、较简易的前胶原基因探针标记方法 。

Aim:To establish a new method for labeling of procollagen gene probe and the detection of procollagen mRNA expression in HSC. Methods: According to gene sequences from NCBI Gene Bank,the primers for the amplification of type Ⅰ?Ⅲ and Ⅳ procollagen genes were designed by OLIGO software, the procollagen genes were amplified by RT PCR and were labeled by PCR and DIG dUTP, and the probes were applied to detect procollagen mRNA expression in cultured hepatic stellate cells by in situ hybridization.Results: The procollagen genes were successfully amplified by the Primers and RT PCR and were labeled by DIG dUTP and PCR,the procollagen mRNA expression in cultured hepatic stellate cells was detected by the probes. Conclusions:A new simplified method for labeling of procollagen genes is successfully found and it can be used for other gene amplification and labeling.