The Expression Of CXCL12-CXCR4and Its Function In Liver Fibrosis Disease

ObjectiveStudies have suggested that CXCL12(Chemokine C-X-C motif ligand12) andits receptor CXCR4(Chemokine C-X-C motif receptor4) were associated with somechronic liver disease recently. Our study is designed to study the function ofCXCR4-CXCL12in liver fibrosis by detecting the expression of CXCR4-CXCL12axis in the fibrotic liver tissues, and cell experiments in vitro.MethodsEight normal and20fibrotic liver tissue specimens by HBV were collected andpreserved in-80℃.The cDNA specimens were obtained by RNA extraction and reversetranscription in liver tissues. The primers were designed. Specific gene segments ofβ-actin, CXCL12α, CXCL12β and CXCR4were amplified by PCR and linked toT-vector. Reconstructed plasmids were transformed into competent cells, and thenamplified and obtained by plasmid extraction. After quantification，the4kinds ofplasmids were used to detect β-actin, CXCL12α, CXCL12β and CXCR4asstandards by Q-PCR.Frozen section of the liver tissues was prepared. CXCL12α, CXCL12β andCXCR4were localized by immunohistochemical method, and analyzed by seniorpathologist. CXCR4and Collagen type1were detected by immunofluorescencestaining. LX-2cell was cultured and intervened by PDGF, total protein of the cells wasextracted and quantified. The expression of CXCR4and DAPDH were detected byWB method.The chemotaxis of HSC by CXCL12α, CXCL12β was detected by cellmigration experiments. After counting，LX-2cells were cultured in transwel inserts,PDGF(positive control), PBS(negative control), CXCL12α and CXCL12β wereadded into the lower chambers. The cells which had migrated across thepolycarbonate membrane were fixed，stained and counted.ResultsThe R-squared values of the standard curve in Q-PCR were all higher than0.995,and the results could be ensured. At the transcriptional level, CXCL12α, CXCL12βand CXCR4were all expressed in normal and fibrotic liver tissues, the level ofCXCL12α transcript was higher than CXCL12β. The expression of CXCR4infibrosis group is much higher than normal group statistically (P=0.019).At the translational level, CXCL12α was expressed in normal and hyperplasticbiliary epithelial cells of both normal and fibrotic liver tissues. CXCL12β was notfound positive in all liver tissues. CXCR4was found positive in both normal andfibrotic liver tissues, and the expression was lower in normal liver tissues, onlypositive in biliary epithelial cells. In fibrotic tissues, CXCR4was expressed inhyperplastic biliary epithelial cells and fibrous septum. MFB was also positive forCXCR4.In vitro, LX-2cells express CXCR4, and the expression of CXCR4was higherafter PHGF intervention. In cell migration experiments, we found LX-2cells could beinduced by CXCL12α and CXCL12β efficiently (P<0.05). Conclusions1. The expression of CXCL12-CXCR4in fibrotic liver tissues is higher thannormal tissues. Compared to CXCL12β, CXCL12α transcript was more expressed.CXCR4was highly expressed in fibrotic liver tissues at both transcriptional andtranslational level, indicating CXCR4is associated with the progression of liverfibrosis.2. CXCR4can be expressed in HSCs, and was positive in MFB and fibrousseptum by immunohistochemistry. The results could be evidence suggesting CXCR4can play a part in the HSC activation and fibrous septum formation.3. CXCR4expression of LX-2cells was increased after cell activation in vitro,indicating CXCR4can participate in the HSC activation.4．CXCL12could induce LX-2cells in vitro, indicating CXCL12is associatedwith HSC migration and fibrous septum formation in vivo.