A Study Of Serum Jaundice Regulate The Proliferation And Phenotypic Conversion In Human PASMCs

Hepatopulmonary syndrome (HPS) is a kind of serious liver-derived pulmonarydiseases, which takes the main feature is that "chronic liver diseases-pulmonarymicrovascular dilation and angiogenesis-hypoxemia ". In the pathological process of thosechronic liver diseases, a lot of stimulating factors (such as cytokines, growth factors,chemokines, etc.) are released, to reach the lung through the blood circulation and effect onpulmonary microvascular endothelial cells (PMVECs), to cause pulmonary vasodilationand angiogenesis, thus resulting in pulmonary hypoxemia.Currently, the pathogenesis of HPS remains unclear.The thickening pulmonary arterycaused obstructive pulmonary vascular disease was found in common bile duct ligation(CBDL) rats, and our previous study found that CBDL serum could promote PASMCsphenotypic conversion and proliferation. In addition, a large number of studies also showthat phenotypic conversion and proliferation of PASMCs play the core role in hypoxicpulmonary vascular remodeling, those results suggest that hypoxia may contribute to thechanges of rat PASMCs phenotype and proliferation in HPS, leading to the pulmonaryartery wall thickening, which aggravate pulmonary hypoxemia. Jaundice is more commonin patients with chronic liver dieases. In view of this, we hypothesized that: A largeamounts of cytokines, chemokines and growth factors produced by chronic liver diseasesreleased into the blood, caused by the pathological pulmonary vascular remodeling inPASMCs through the blood circulation to lung, resulting in the the pathogenesis of HPS,and this is also the purpose of this study. This study primary cultured human PASMCs,observed the changes of jaundice serum human PASMCs proliferation and phenotypicconversion, while the use of luminex-XMAP microarray technology to detect the changesof serum cytokines in patients with cirrhosis, in order to provide a theoretical basis onprevention and treatment in liver-derived pulmonary diseases.Methods:1. Establishment of primary culture human PASMCs. An improved method of primary cultured human PASMCs with the right amount of PDGF in high glucose medium, whichwere identified by light microscopy, immunofluorescence staining.2. Jaundice serum of cirrhosis patients and normal serum of volunteers was collected,and then normal and jaundice serum stimulated human PASMCs, respectively, theproliferation of human PASMCs was detected by CCK-8, the changes of human PASMCscalponin and α-SM-actin protein were detected by western boltting.3.61cytokines in serum with liver cirrhosis patients were detected by luminex-XMAPmicroarray technology.Results:1. Improved method for primary culture of human PASMCs were spindle shaped,protruding contact each other, showing a typical "peak valley" signs under light microscope,the calponin and α-SM-actin immunofluorescence staining identified successfully culturedhuman PASMCs.2. Jaundice serum promoted the proliferation of human PASMCs. Compared to normalserum, the proliferation of human PASMCs of jaundice serum in vitro significantlyenhanced. In different time, the strongest proliferation human PASMCs of jaundice serumwithin48h, the proliferation ability during form48h to72h is weaker, but still strongproliferation ability than24h.This showed that jaundice serum of cirrhosis patients couldpromote their proliferation PASMCs in vitro.3. Compared to normal serum jaundice, jaundice serum stimulated human PASMCswhich expressed α-SM-actin not obviously，while calpnion expression were significantlyincreased after24h; however, jaundice serum stimulated human PASMCs which expressedα-SM-actin and calpnion was significantly reduced from48h to72h.4. Luminex-XMAP test result shows that61kinds of cytokines were detected, theexpression these cytokines(such as IL-6, IL-15, IL-23, TNF-α, IL-28A, LIF, I-309, IP10,MCP-4,6Ckine, BCA-1, MIP-1δ, INF-γ) were significantly increased, while the othercytokines (such as IL-3, IL-7, TRAC, TRAIL, GRO, MCP-1, MCP-2, ENA-78and sCD40L)were significantly decreased.Conclusion:1. An improved methods of human PASMCs overcome these problems which aredifficult to culture of human PASMCs in vitro, appling it to the study more accorded withthe features of human cells in morphology and Biology.2. Serum jaundice promote human PASMCs phenotypic conversion and proliferation. 3. Cytokines which are more obvious changes in patients with cirrhosis were screenedby luminex-XMAP microarray technology, providing a theoretical basis for the treatmentof liver-derived lung disease.