The Study Is To Discovery And Identify Autophagy Of Hepatic Stellate Cell And Understand Its Biological Significance

Aims: On the base of affirmance the autophagy of hepatic stellate cell, Toobserve the effect of autophagy on the proliferation and procollagen byregulating the level of HSC autophagy in vitro, in order to investigate thefeasibility of treatment in liver fibrosis when macrophage signaling pathway isto be a target.Method：1. To separate the hepatic stellate cell from liver by gradientdensity method, and culture the primary HSC; to observe the cell growth bymicroscopy.2. We observed the morphology of autophagy by transmissionelectron microscopy.3. To observe the morphology of autophagolysome byMDC staining and fluorescence microscopy.4. The expression of the ATGfamily proteins were detected by western-blot.5. The expression of procollagenmRNA was detected by real-time PCR.6. The proliferation of HSC wasanalyzed by CellTiter96Non-Radioactive Cell Proliferation Assay (MTT) kit.7. The apoptosis rate of HSC was detected through flow cytometer.8. Toobserve the response to changes of autophagy level in HSC by using accelerant and inhibitor of macrophage signaling pathway.Result: In this study，primary HSC was successfully isolated by densitygradient method and cultured successfully, on this basis:1.The morphologicalcharacteristics of autophagy were observed by fluorescence microscopy andtransmission electron microscopy; The expression of autophagy pathway-relatedmolecules was detected in the hepatic stellate cell.2. After HSC was culturedwith EBSS for6hours, the quantity of autophagosome was significantlyincreasing; Rapamycin which was the autophagy enhancers stimulated theexpression of ATG5, beclin-1, LC3B in the HSC, on the contrary,3MA whichwas the autophagy inhibitors reduced those protein.3.Real-time PCR analysisshowed that3MA reduced the expression of Procollagenα2（1）mRNA, on thecontrary, rapamycin increased the expression of Procollagenα2（1）mRNA. MTTanalysis showed that3MA inhibited the proliferation of HSC in dose-dependentmanner.4. TGFβ1significantly stimulated the expression of Procollagenα2（1）mRNA in the HSC; after HSC was incubated with rapamycin, the level ofProcollagenα2（1）mRNA was further increasing, but3MA inhibited theexpression of Procollagenα2（1）mRNA which was induced by TGFβ1.5. HCQreduced the expression of procollagenα2(1) mRNA and inhibit the proliferationof the hepatic stellate cell in dose-dependent manner. The level ofprocollagenα2(1) mRNA and the inhibitor rate of cell proliferation is negativelycorrelated, p=0.02,correlation coefficient is-0.882.6. The expression of beclin-1and Atg5increased with the extension of the Ccl4induction time in the liverfibrosis model by western-blot analysis.7. after HCQ and3MA were incubatedfor24hours, combination therapy increased apoptosis rate of hepatic stellatecell than single agent, P＜0.05. Conclusion:1.this study confirmed that Hepatic stellate cell had thephenomenon of autophagy.2. autophagy took part in the liver fibrosis byaffecting HSC proliferation and collagen generation, inhibiting the level ofautophagy in the hepatic stellate cell would inhibitor the proliferation and theexpression of procollagen mRNA，which prompted that autophagy signalingpathway is a new target to cure the liver fibrosis.3. HCQ significantly inhibitedthe signal transmission of autophagy, as the same time, it significantly reducedthe expression of procollagenα2(1) mRNA, prompting the tantalizing prospectof anti-liver fibrosis treatment.