The Function Of Connexin In The Activation Of Hepatic Stellate Cells

[background]Cirrhosis is the terminal stage of progressive liver fibrosis.If it is not prevented,it may develop into liver cirrhosis,even liver cancer and liver failure.The activation of hepatic stellate cell(HSC)plays a core role in the progress of liver fibrosis.The current research is mainly focused on the role of cytokines in promoting activation.We have elucidated the molecular mechanism of many factors,but the intervention of cytokine activation pathway can not achieve the desired effect on antifibrosis.The intercellular contact and connection is an important manifestation of activation of HSC,the research group also found that before the cell contact and non-contact co culture,HSC will exhibit different activation state,therefore,treatment breakthrough to explore new mechanisms for hepatic cirrhosis will contribute to hepatic fibrosis.[Objective]:the quiescent hepatic stellate cells and activation of hepatic stellate cells were cultured in contact and non-contact co culture,toobserve the intercellular junction protein CX-43 expression and activation of hepatic stellate cells after blocking effect,so as to reveal its effect on HSC activation.[method]1.perfused the liver of male S-D rats by sequential infusion of protease,collagenase and post-dispersion,and dispersed the digestive cells.Then the hepatic stellate cells were obtained by density gradient centrifugation,andmarksed by quantum dot.Inoculate the isolated primary HSC and HSC-T6 cellsinto a transwellsystem whichcoated with Matrigel.(1)Non-blockers:Group A(non-contact and non-co-culture group):the upper chamber was inoculated with 1.3×10~5 cells/ml,2ml HSC-T6 cells,the lower chamber was inoculated 1.3×10~5 cells/ml,primary HSCs.Group B(contact co-culture group):Each inoculated 2ml of the suspension with the primary HSC 1.3×10~5 cells/ml and HSC-T6 cell lines 1.3×10~5个/ml,according to the rate of 1:1,into each upper chamber and lower chamber.Group C(negative control):The upper chamber and the lower chamber were added 1.3×10~5/ml,2ml.Blocker group(Bi group):Each inoculated 2ml of the suspension with the primary HSC 1.3×10~5cells/ml and HSC-T6 cell lines 1.3×10~5个/ml,according to the rate of 1:1,into each upper chamber and lower chamber,and add the gap26 into each chamber,which is the blocker of CX-43.2、Observations:Obeservingthe expression ofα-smooth muscle actin(α-SMA)by immunofluorescence technique at 24h and 48h,to influence the situation of cell activation.And observe the expression level of connexin-43(CX-43)to examine the relationship with activity of HSCs.[Results]:Non-blocker groupα-SMA:Group A:The primary HSCs were not activated at 24h and 48h,and did not expressα-SMA.In group B,primary HSCs started to activate at 24h,expressed a small amount ofα-SMA,activated obviously at 48h,changed cell morphology,increased cell body,extended cell processes,extended pseudopodia,and expressed a large amount ofα-SMA.Group C:The primary HSCs were not activated at 24h and 48h,and did not expressα-SMA.Blocker group:Bi group,the primary HSC 24h has not activated,does not expressα-SMA.Group A:Primary HSCs were not activated at 24h and 48h and did not express CX-43.Group B:primary HSCs started to activate and expressed a small amount of CX-43 at 24h.And it started to changes in cell morphology,increased cell body,cell protrusion,extending pseudopodia,the expression of a large number of CX-43 and activate significant at 48h.Group C:The primary HSCs were not activated at 24h and 48h and did not express CX-43.Blocker group:(Bi group):the primary HSC has not activated at 24h,does not express CX-43.[Conclusion]:1.After the co-culture of quiescence primary HSCs with activated HSCs,the activation was accelerated,suggesting that the intercellular junctions could promote the activation of HSC.2.Activated HSC expression the cell connexin CX-43 and block the CX43can inhibit the process of activation of HSC,indicating that CX-43 play a important role in the activation of primary HSC.