A Study On The Construction Of SEXPAR-SPR And The Role Of Annexin A1 In HPS-related Inflammatory Phenotype And Proliferation

Background and objective:This subject is a study on the molecular mechanism of the clinical disease by combine medical laboratory diagnosis,critical care medicine,surgery and other surgical disciplines.The main research direction of our team is the basic and clinical research of lung injury caused by distal organs,which is about the critical care medicine.In clinical practice,we study on the molecular mechanism of pathophysiology of hepatopulmonary syndrome(HPS).At the same time,combine medical laboratory diagnosis which is use of solid phase exponential amplification reaction(sEXPAR)and surface plasmon resonance(SPR)biosensor as a screening tool for the post-control targets of key regulatory proteins associated with HPS.The advantage of this technology is high efficiency and high throughput.Screening results can reveal the mechanism of pathophysiology o f HPS directive and operability by combining with literature and previous experiments.HPS is a clinical syndrome occurs mainly in patients with end-stage liver disease.The incidence of HPS is already greater than 15% in patients with advanced liver disease.Gradually,the mortality rate of HPS shows an increasing trend.The study on the pathophysiological molecular mechanism of HPS become a hot topic.Liver-derived cytokines leading to the muscle differentiation and abnormal proliferation of pulmonary microvascular endothelial cells(PMVECs),which is the main reason of intrapulmonary vasodilatation(IPVD).Hypoxemia and hypoxic environment in lung further promoted the phenotype modulation and abnormal proliferation of pulmonary artery smooth muscle cells(PASMCs),which aggravated the pulmonary vascular remodeling(PVR)and finally promoted the development of HPS.Therefore,the major pathological changes in HPS-related PVR are the cell differentiation,phenotypic transformation and abnormal proliferation of two major cells which are PMVECs and PASMCs.This study was aimed at the molecular mechanism of pathophysiological changes about PASMCs.Previous studies have reported that in the pathogenesis of HPS,a variety of cytokines is secreted into the cycle,leading to the activation of a great many signaling pathways,resulting in phenotype modulation and abnormal proliferation of PASMCs.These signal pathways are intertwined with each other,which lead to difficulty to completely block.So the research needs to be further explored.In recent years,the study on the mechanism of inflammatory phenotype of PASMCs found that hypoxia,a variety of growth factors and other stimulating factors can mediate PASMCs inflammatory phenotype in hypoxic pulmonary hypertension and other pathological conditions.A variety of cell inflammatory mediators release by the development of HPS,vasoconstriction and oxygenation disorder also further aggravate the release of IL-1,IL-6,prostaglandin E2(PGE2)and other inflammatory mediators.PASMCs develop inflammatory phenotypes and enter the cell cycle from G0/G1 phase,which lead to the proliferation of PASMCs.Therefore,changes in the inflammatory phenotype of PASMCs may be a prerequisite for other pathophysiological changes.of the cells.Based on the above reasons,this study is aimed at the regulatory mechanism of inflammatory phenotype and proliferation of PASMCs in HPS-related PVR,and find some new gene therapy targets for HPS.SEXPAR-SPR biosensor detection technology,which is a new detection technology combined s EXPAR and SPR.SPR biosensors is detection technology based on the optics physics principle,which through the chip detection pool or bond biological macromolecules on its surface to work.And make use of the changes of its refractive index by the interaction of these biological macromolecule too.Eventually achieve the fast,easy,accurate and sensitive detection of biological macromolecules.SEXPAR is a method for the expansion of DNA by constant temperature index,which through the formation of polymerization-replacement-cut-polymerization cycle.In order to achieve the amplification by constant temperature index.By combining the two techniques,primers and probes were designed for the detection of target DNA,which achieve the sEXPAR of target DNA.The same chip of SPR have different detection pool which can be fixed to different probes,in order to achieve a high-throughput,fast,accurate and sensitive detection of target DNA.MicroRNA(mi RNA)is a very conserved single-stranded,non-coding small RNA which about 20 to 24 nucleotides in length.More and more literature suggests that mi RNAs can serve as new targets for early diagnosis or treatment of disease.In this study,we intend to use the technique of sEXPAR-SPR to do a high-throughput,high-sensitivity screening of mi RNA expression in HPS-related PVR.The positive results of miRNA detection,which corresponding some target proteins.And these proteins may be play important regulatory role of regulation of inflammatory phenotype and proliferation of PASMCs in HPS.Annexin A1(ANNA1)is one of the members of the annexin family and is a calcium-dependent and phospholipid binding protein.ANXA1 plays a variety of biological functions in different tissues and parts,including anti-inflammatory effects,mediated cell signal transduction.The expression of mi R-30a-5p in the HPS group was significantly up-regulated.And the results screening by sEXPAR-SPR biosensor technology have statistical significance.It was predicted that mi R-30a-5p could regulate the expression of ANXA1 by post-transcriptional regulatory mechanism.Combined with previous studies and previous experimental results,it is speculated that ANXA1 may be involved in regulation of inflammatory phenotype and proliferation of PASMCs in HPS.At the same time,due to the pathological changes of lung tissue in HPS rats and the results of previous studies,Our study group also selected endothelin-1(ET-1)to detect.ET-1 was closely related to vascular remodeling and pulmonary hypertension.Further research on ET-1 may help to improve the molecular mechanism of ANXA1 in HPS-related PVR.Methods:1.The establishment of EXPAR system and the optimization of the conditions.To explore the appropriate conditions for EXPAR by using fluorescence quantitative PCR and the temperature gradient of 55.0?~65.0?.SPR-related probes were prepared,prepared the SPR biosensor matrix membrane chip,made fixation of needle,detected the SPR angle which through the sensor by real-time PCR.At last completed the s EXPAR-SPR biosensor technology.2.In vivo HPS animal model was established by CBDL,and Judged the success of the model based on common standard.The expression of mi RNAs in the lung tissue of HPS rats was detected by s EXPAR-SPR.Predicted the target gene which is regulated by miRNA through the http://www.mirbase.org/ website.3.Combined with previous studies and previous experimental results,ANXA1 was selected as the target protein.The expression of ANXA1 in HPS-related model was detected by immunohistochemistry and Western-blot.In order to improve the study of ANXA1,ET-1 was selected and detect the expression of ET-1 in HPS model.4.The expression of ANXA1 protein in PASMCs was up-regulated by adenovirus transfection,and the transfection efficiency was detected by RT-PCR,Western-blot and immunofluorescence.5.Up-regulated the expression of ANXA1.The expression of IL-1?,TNF-? and IL-6 in each group was detected by ELISA.The proliferation of PASMCs was detected by CCK-8 and 3H-Td R.The expression of p-ERK1/2 and cyclin D1 were detected by Western-blot.6.The transcription and expression of ANXA1 in PASMCs which was pretreated with ET-1 by RT-PCR and Western-blot.The effect of ET-1 on ROS release in PASMCs was detected by ROS assay.7.The expression of ANXA1 and carbonylated ANXA1 was detected by immunoprecipitation.The protein expression of ANXA1 in each group which combined with MG-132 by Western-blot.Result:1.The establishment of the EXPAR reaction system and the appropriate conditions for the EXPAR is 50?.Target gene concentration was linear correlated with the take-off time of EXPAR curve and T50 value.The high concentration was accompanied by early the curve jump and low T50 value.The liquid EXPAR detection limit the concentration of 100 fM.The pool curve of the target gene was exponentially increased.And the SPR angle was about 500 milligrams.SEXPAR-SPR biosensor was measured in real time.When the concentration of DNA was 10-14 M to 10-8M,the T50 value was linearly related to the logarithm(lgC)of the target concentration.At last the s EXPAR-SPR biosensor was constructed successfully.2.The expression of mi RNA-30a-5p in HPS group was significantly shorter than that in control group(P <0.05),suggesting that it was up-regulated in HPS group.Predicted by using the http://www.mirbase.org/ website,found that it can regulate ANXA1 as post-transcriptional.3.The expression of ANXA1 in lung tissue of HPS rats was significantly down-regulated and the expression of ET-1 was up-regulated detected by immunohistochemistry and Western-blot.The expression of ANXA1 in PASMCs which was pretreated with ET-1 decreased in a dose-dependent manner.4.AD-ANXA1 transfected into PASMCs efficiently(transfection rate> 90%)detected by immunofluorescence,RT-PCR and Western-blot.And the levels of mRNA and protein of ANXA1 in AD-ANXA1 group was significantly increased.5.AD-ANXA1 transfection could inhibit the release of inflammatory factors such as IL-1?,TNF-? and IL-6 significantly in PASMCs detected by ELISA.The release of inflammatory cytokines in PASMCs which was pretreated with ET-1 increase obviously.6.AD-ANXA1 transfection could inhibit the proliferation of PASMCs significantly detected by CCK-8 and 3H-TdR.ET-1 pretreatment could promote the proliferation of PASMCs.7.AD-ANXA1 transfection could inhibit the production of p-ERK1/2 and expression of cyclin D1 significantly detected by Western-blot.After adding ET-1,p-ERK1/2 and cyclin D1 expression was upregulated significantly.8.The transcription level of ANXA1 in ET-1 pretreated PASMCs was not changed detected by RT-PCR and Western-blot.The reduced levels of ANXA1 by ET-1 were very similar in cells treated with or without Act-D or CHX.9.The expression of ROS in PASMCs pretreated with ET-1 was significantly increased detected by ROS assay.The expression of carbonylated ANXA1 in PASMCs was increased by ET-1 pretreatment significantly.10.The down-regulated expression of the expression of ANXA1 induced by ET-1were reversed by using the proteasome inhibitor MG-132.Conclusion:1.sEXPAR-SPR biosensor is an efficient,high-throughput detection technology.The concentration of the gene related the curve take-off time and T50 value.This technology can be used as a screening tool at research related HPS.2.The expression of mi R-30a-5p was up-regulated in HPS lung tissue.It was predicted that mi R-30a-5p could regulate the expression of ANXA1.It was presumed that ANXA1 may played an important role in HPS-related PVR.3.The expression of ANXA1 and ET-1 were changed in HPS-related models.The expression of ANXA1 in PASMCs which was pretreated with ET-1 decreased in a dose-dependent manner.It is speculated that ET-1 plays an important role in HPS by regulating the expression of ANXA1.4.The release of inflammatory cytokines in PASMCs was significantly inhibited by AD-ANXA1 transfection.ET-1 may stimulate the release of inflammatory factors,which may attribute to the down-regulated expression of ANXA1.5.The proliferation of PASMCs was inhibited by AD-ANXA1 transfection significantly.The formation of p-ERK1/2 and transfer from the cytoplasm to the nucleus were inhibited too.And the expression of cyclin D1 also was inhibited.After treatment with ET-1,lead to the proliferation of PASMCs and the up-regulation of proliferating-associated proteins.And this function may attribute to the down-regulated expression of ANXA1.6.ET-1 does not affect the transcription level of ANXA1.It may promote the carbonylation of ANXA1 by release of ROS,which lead to the de-gradation of ANXA1 at last.It was predicted that,PASMCs developed inflammatory phenotypes and proliferated in HPS-related PVR.The expression of ANXA1 was down-regulated in lung tissue of HPS rats and the expression of ET-1 was up-regulated.ET-1 could mediate the release of ROS,result in the carbonylation and degradation of ANXA1.Thus reverse the inhibition of inflammatory phenotype and proliferation of PASMCs by ANXA1,and promote the development of HPS-related PVR ultimately.