Aquaporins (AQPs)Expression In Human Pleural Mesothelial Cell And It's MRNA Regulation In Cell Culture

PrefaceTight control of the volume and composition of the pleural liquid is necessary toensure an efficient mechanical coupling between lung and chest wall. Liquid enters thepleural space through the parietal pleura down a net filtering pressure gradient. Liquidremoval is provided by an absorptive pressure gradient through the visceral pleura, bylymphatic drainage through the stomas of the parietal pleura, and by cellularmechanisms. Indeed, contrary to what was believed in the past, pleural mesothelial cellsare metabolically active, and possess the cellular features for active transport of solutes,including vesicular transport of protein. Furthermore, the mesothelium was shown, onthe basis of recent experimental evidence,both in vivo and in vitro, to be a lesspermeable barrier than previously believed, being provided with permeabilitycharacteristics similar to those of the microvascular endothelium.Some researchs foundthat mesothelial cell was not only constituent part of complete serous cavity also beareda part in fluid transport and had the function of stem cell, and participated in damagerepair of pleural cavity.Water and the transport of water through biological membrane is very important.To adapt to transport of water, there are water selective transporter proteins on cellmembrane: aquaporins. Thirteen subtypes of aquaporins have been found: AQP_0～12. AQPsparticipated in transport of water and small nonpolar molecule through cellmembrane. AQP maybe have vital role in water transport inside and outside the pleural mesothelial cell. Past research found that AQP1 expressed abundantly in the mousevisceral pleura. The expression of AQP1～AQP10 in human pleura did't clear now.To date, in the HMPC and human pleura tissue, AQP1 protein and AQP1～AQPmRNA expression are unknown. The molecular mechanisms for regulatingAQP1mRNA expression in HMPC are unknown. We cultured primary HMPC in vitroand detected the expression of AQP1～10mRNA in human pleural mesothelial cell invitro. The cell cultures were treated with dexamethasone (DEX) and study whetherAQP1 mRNA expression upregulated by DEX. We studied the expression of AQP1protein in the human pleura, We hope providing some theory basement in the treatmentof pleural effusion and it have important value in investigating the mechanism of fluidtransport in the pleural cavity.Meterials and Methods1 Isolation and Culture of Human Pieural Mesothelial CellUse two methods to establish human pleural mesothelial cell culture reproduciblemodel in vitro and compare the advantage and disadvantage of them. Mesothelial cellswere isolated from human pleura by trypsin EDTA disaggregation and pleural effusionfluid; Human pleural mesothelial cells(HPMCs) were identified by morphology andImmunocytochemistry(ICC).2 The Expression of AQP_1～10 mRNA in the Human MesothelialCellsMesothelial cells were isolated from pleural effusion fluid of nonmalignant pleuraleffusion patients and cultured in vitro. RT-PCR was used to detect the expression ofAQP_1～10 mRNA in human pleural mesothelial cells.3 Effect of dexamethasone to the expression of AQP₁ mRNA in theHuman mesothelial cells Culture reproducible model was established of human pleural mesothelial cells invitro; RT-PCR was Used to detect the expression of AQP₁ mRNA in human pleuralmesothelial cells. Experiment was divided to three group:â… , dexamethasonegroup;â…¡, dexamethasone and RU486 group;â…¢, control group. AQP1 protein expressionin the human pleura was studied by Immunohistochemistry(IHC).Results1 Isolation and Culture of Human Pleural Mesothelial CellConfluent HPMCs appeared multipolar and like cabblestone; Numerous surfacemicrovilli and abundant endoplasmic reticulum were observed under electronmicroscopy. HPMCs expressed cytokeratin and vimentin;â…§factor associated antigenand CD₄₅ were negtive. Two methods achived success in establishment of reproduciblemodel for culture of human puaral mesothelial cells; Those methods were feasibility inmethodology.2 Expression of AQP_1～10 mRNART-PCR studies showed that all of the AQP_1～10 mRNA expressed inHPMCs; AQP₁,AQP₉,AQP₁₀ mRNA have abundant expression in mesothelial cells andsignificant deviation was found between AQP₁,AQP₉,AQP₁₀ and AQP₂,AQP₃,AQP₇,AQP₈.3 Effect of dexamethasone to the expression of AQP₁ mRNAUsing IHC, we indentified AQP1 protein expression in human pleura. Showed thatAQP1 protein expressed in mesothelial cell and red blood cell (RBC) and vascularendothelial cell. RT-PCR studies showed that AQP₁ mRNA expressed in mesothelialcells; Significant deviation of AQP₁ mRNA expression was found between the group ofdexamethasone and other two group. Dexamethasone up-regulated the expression ofAQP₁ mRNA in the human mesothelial cells, The expression of AQP₁ mRNA havepositive correlation with the dexamethasone dose. Conclusions1 Two methods achived success in establishment of reproducible model for cultureof human puaral mesothelial cells; Those methods were feasibility in methodology. Thecells isolated from human pleura had higher purity quotient. Pleural effusion fluid waseasyer to obtain.2 Human pleural mesothelial cells expressed AQP_1～10 mRNA, All of them haverelation with the function of human pleural mesothelial cells;. AQPs are involved inrapid and active gating of water across biological membranes. Confirmed thatmesothelial cells have important contribution in pleural fluid dynamics.3 Human pleural mesothelial cell expressed AQP1mRNA and AQP1 protein,Dexamethasone up-regulated the expression of AQP₁ mRNA. It give us some theorybasement in the treatment of pleural effusion.