Effect Of IL-10 On Activating Of Rat Primary Stellate Cells By Ultraviolet Light-induced Apoptotic Body

Backgrounds Liver fibrosis is a necessary stage in the development of various chronic liver diseases. The present study suggests that liver fibrosis can be reversed to normal state. As a indispensible stage in the development of liver cirrhosis,researching the pathogenesis and treatment of liver fibrosis is of great importance to control the occurrence and development of liver cirrhosis. The activation of hepatic stellate cells plays a key role in the course of hepatic fibrosis. In recent years, it has been found that the apoptosis of hepatocytes is closely related to the activation of hepatic stellate cells. IL-10 was confirmed to play a protective role,which is resistant to hepatic fibrosis in liver injury. So,exploring the effect and mechanism of IL-10 on the activation of stellate cells stimulated by apoptotic cells may provide theoretical basis for the intervention and treatment of liver fibrosis.Objectives 1.To observe the Change of morphology and function of the stellate cells after the induction of apoptosis caused by ultraviolate in the liver cells and co-cultured with the primary stellate cells. 2. To observe the effects of IL-10 on the autom-activation of HSC and its activation stimulated by apoptotic bodies so as to explore the role of IL-10 in the activation of hepatic stellate cells.Methods Hepatic stellate cells were isolated in situ separation and in vitro circulation perfusion, then 5 ×107 cells were obtained to detect its viability and purity.Oil red 0 staining was used to observe the storage capacity of the stellate cells. The primary HSC was inoculated in 6-well plates in the density of 2.0×105 /well,adding the DMEM supplemented with 20%FBS 2ml for 48 hours, the cells were divided into 2 groups.1.Cultured with high-glucose DMEM, IL-10(2?10?20ng/ml)for 24?48?72?96 hours each, use QRT-PCR to detect the effect of IL-10 on the gene expression of ?-SMA?TGF-?1 and Col I m RNA at different concentration and different time, make sure the best concentration and the best time to inhibit the activation of HSCs was 20ng/ml at 96 hours. 2.Co-culturing the apoptotic bodies and HSCs for 24?48?72?96 hours each, treated with IL-10 20ng/ml, use RT-PCR to detect the gene expression of ?-SMA?TGF-?1 and Col I m RNA in HSCs,observing the effect of apoptotic bodies on primary HSCc and the intervention of IL-10 on the function of HSCs stimulated by apoptotic bodies.Results 1. The number of HSCs reaches to 5.0×107, its purity 91% and its vitality 98%. 2. Co-culturing IL-10 at different concentrations, high-glucose DMEM for 24?48?72?96 hours each, use QRT-PCR to detect the gene expression of ?-SMA?TGF-?1 and col I m RNA in HSCs,significant difference can be found between each group(p<0.05),the HSCs in IL-10 group(20ng/ml)was totally down-regulated in comparison with the blank control group(p<0.05). 3. Co-culturing apoptotic bodies and HSCs for 24?48?72?96 hours each, using QRT-PCR to detect the gene expression of ?-SMA ? TGF-?1 and Col I m RNA in HSCs,it was totally up-regulated in comparison with blank control group(p<0.05), especially the longer, the stronger. 4. Co-culturing IL-10(20ng/ml)?apoptotic bodies and HSCs for 24?48?72?96 hours each, using QRT-PCR to detect the gene expression of ?-SMA?TGF-?1 and col I m RNA in HSCs,it was totally down-regulated in comparison with apoptotic body group(p<0.05).Conclusion: 1. IL-10 can inhibit the spontaneous activation of primary HSCs. 2.The ultraviolate-induced apoptotic bodies could stimulate the activation and proliferation of HSCs, especially prolong the effection with time lasting. 3. IL-10 can inhibit the activation of HSCs stimulated by apoptotic body.