Immortal Mouse Hepatic Stellate Cells Generated By HTERT Gene Transfer

Objective to establish a long-term passage immortalized mouse hepatic stellate cells with stable morphological biological characteristics.Methods purfusing the liver of mice with the 0.1%collagenase IV through the inferior vena cava below liver, isolate and purificate the hepatic stellate cells of the mice through density gradient centrifugation method with percoll, then transfected the primary HSCs by retrovirus carrying hTERT gene. Filtering by G418and select single cell to culture and passage(named it hTERT-mHSCs). Detecting Telomerase activity byTRAP-ELISA method; The hTERT-mHSCs was Identified with desmin ,vmentin andα-SMA immunostaining. The mRNA Expression level of Associated genes(MMP-2 ,MMP-14, TIMP-1 ,TIMP-2, OB-RL, PDGF-α,PDGF-β, PDGFR-α,PDGFR-β, DDR-2 ,TGF-β1) were detected by Real-time quantitative PCR(qRT-PCR). Expression of cell surface molecules was determined via ? ow cytometric analysis. TGF-β1 stimulating expression of a(1)I procollagen mRNA was also deticted by qRT-PCR .Results The hTERT-mHSCs could be cultured and long-term passaged. The 50 Generation of hTERT-mHSCs had telomerase activity , expressing desmin ,vmentin andα-SMA.There were not obvious difference between hTERT-mHSCs and Primary-mHSCs in expression of associated genes(MMP-2,MMP-14,TIMP-1,TIMP-2,OB-RL,PDGF-α,PDGF-β, PDGFR-α,PDGFR-β, DDR-2 ,TGF-β1),and also the cell surface molecules(p>0.05).The expression level of a(1)I procollagen mRNA was Increased significantly after stimulated with TGF-β1.Conclusion Successfully established immortalized mouse hepatic stellate cell line,which was similar to the activation of primary mouse hepatic stellate cells in basic biological characteristics.