Ⅰ.Expression Of TIPE2 In PBMCs From CHB And Its Implications Ⅱ.Functions Of CPI-17 On Atherosclerosis By Regulating The Phenotype Of VSMCs

ObjectivesTIPE2, the tumor necrosis factor (TNF)-alpha-induced protein 8-like 2 (TNFAIP8L2), plays an essential role in the maintenance of immune homeostasis by negatively regulating T cell receptor and Toll-like receptor (TLR) signaling. TIPE2 was identified as a highly expressed gene in inflamed tissues. It can prevent hyper-responsiveness. Hepatitis B Virus (HBV) infection affects more than 350 million people world wide. The development of molecular biology enabled researchers to understand more about the hepatitis B virus (HBV), but a lot is still unknown about the immune mechanism causing chronic hepatitis B (CHB). Recent studies suggest that the host immune response plays a critical role in the outcome of HBV infection. The hepatic inflammation caused by immune response mediated by T cells is the major form of cell death in the pathological process. So the aim of this research is to detect the TIPE2 mRNA in peripheral blood mononuclear cells from chronic Hepatitis B patients, and to study whether TIPE2 takes part in the pathogenesis caused by hepatitis B virus and what role it may play in HBV related inflammation.Methods1. Case CollectionFresh heparin-treated blood of chronic Hepatitis B patients (51 cases), from Jinan city hospital for infectious diseases and department of infectious diseases, Shandong provincial hospital without Hepatitis C and other infectious diseases were collected. Diagnosis of viral hepatitis was in accordance with the prevention and treatment programs in the year 2000. Meanwhile we collected 21 cases healthy controls.2. Detection of TIPE2 expression on peripheral blood mononuclear cells from chronic Hepatitis B patients2.1 Collection of peripheral blood of chronic Hepatitis B patients and Isolation of peripheral blood mononuclear cells from chronic hepatitis B patients.Fresh heparin-treated bloods from chronic Hepatitis B patients, healthy controls were collected and peripheral blood mononuclear cells were separated by the method of Ficoll-Hypaque density gradient centrifugation.2.2 Extraction of total RNA of peripheral blood mononuclear cells and the preparation of cDNATotal RNA was extracted from peripheral blood mononuclear cells using Tizol reagent. RNase free DNase was used to remove genomic DNA contamination. The purity and concentration of RNA was detected by ultraviolet spectrophotometer, if the absorbance (A) on wavelength 260/280 nm is between 1.8～2.0, it is available. cDNA was synthesized using Reverse Transcription System (RNA 2.0μg).2.3 Detection of TIPE2 mRNA level by real-time fluorescence quantitative PCRAccording to the sequence of human TIPE2 gene in PubMed, specific primers for the TIPE2 fragments were designed using Primer Premier5.0 software. The levels of TIPE2 mRNA was detected by real-time fluorescence quantitative PCR.2.4 Detection of TIPE2 expression of the chronic Hepatitis B patients before and after treatmentsFor the same case, we analysized the TIPE2 mRNA level before and after treatments.3. Correlation between TIPE2 expression on peripheral blood mononuclear cells and clinical parameters of hepatitis B virusesCorrelation analysis was performed between TIPE2 expression on peripheral blood mononuclear cells and serum levels of alanine aminotransferase (ALT) activities, glutamic oxaloacetic transaminase (AST) activities, total bilirubin (Tbil) activities, HBV-DNA copies in chronic Hepatitis B patients with Prism Graphpad software 5.1.Results1. TIPE2 expression was decreased on peripheral blood mononuclear cells from chronic Hepatitis B patientsReal-time fluorescence quantitative PCR analysis showed that TIPE2 expression was much lower on peripheral blood mononuclear cells from chronic Hepatitis B patients than that of the healthy controls (3.91±0.57 vs 6.9±1.2, P=0.0121). In some HBV patients, the result showed that the expression of TIPE2 mRNA on peripheral blood mononuclear cells from chronic Hepatitis B patients was up-regulated dramatically after clinical treatments compared to that before treatments.2. Correlation between TIPE2 expression and clinical parameters of hepatitis B virusesAccording to serum ALT levels, we divided chronic hepatitis B patients into two groups:high-level group (ALT> 47IU/mL) and low-level group (ALT< 47IU/mL). Detection result showed that TIPE2 mRNA level on peripheral blood mononuclear cells of low-level group was significantly higher than that in high-level group (6.551±2.15 vs 3.409±0.477, P=0.043). Statistical analysis showed that TIPE2 mRNA level on peripheral blood mononuclear cells was negatively correlated with serum levels of ALT activities (r=-0.3269, P=0.0395)Similarly, according to serum AST,Tbil,HBV-DNA levels, we divided chronic hepatitis B patients into AST high-level group (AST> 47IU/mL) and low-level group (AST< 47IU/mL),Tbil high-level group (Tbil> 20.5μmol/L) and low-level group (Tbil< 20.5μmol/L),HBV-DNA high-level group (HBV-DNA> 1000 copies/mL) and low-level group (HBV-DNA< 1000 copies/mL), TIPE2 mRNA levels of high-level group were significantly higher than the low-level group (AST group, 5.086±1.413 vs 2.595±0.3035,P=0.0276; Tbil group,4.497±0.7694 vs 2.474±0.3468, P= 0.0219). Furthermore, the level of TIPE2 mRNA was higher in low HBV-DNA patients than that in high ones. A significant difference existed between the two groups (5.435±1.458 vs 3.132±0.3842, P=0.0398). But statistic analysis showed that there was no direct correlation between TIPE2 mRNA expression on peripheral blood mononuclear cells and levels of AST activities, Tbil activities, HBV-DNA copies.Conclusion1. TIPE2 expression was much lower on peripheral blood mononuclear cells from chronic Hepatitis B patients than that of the healthy controls and in some HBV patient, TIPE2 expression was significantly increased after treatments.2. TIPE2 expression level may reflect the degree of liver cell damage:TIPE2 mRNA expression level was negatively correlated with levels of ALT activities which can be a clinical parameters response to liver cell injury. But there was no direct correlation between TIPE2 expression and levels of AST activities, Tbil activities, and HBV-DNA copies.3. TIPE2 may involve in the progression of chronic hepatitis B by regulating the cellular immunity.Innovation1. We first studied the relationship between anti-inflammatory protein TIPE2 and hepatitis B virus infection.2. The results indicate that TIPE2 may involve in the process of chronic hepatitis B through regulating the cellular immunity. ObjectiveCPI-17 (PKC-potentiated myosin phosphatase inhibitor of 17 kDa) is a phosphorylation-dependent myosin phosphatase inhibitory protein (17 kDa) that is primarily expressed in smooth muscle tissue, in internal organs, especially in arteries. It can inhibit the main enzyme activity regulatory subunit of MLCP-38 kDa catalytic subunit of PP1c, to enhance smooth muscle contraction. When vascular smooth muscle cells were stimulated with ox-LDL,hypoxia and other stimulitions, the phenotype changed from contractile phenotype into synthetic type. This is the crital step during the development of atherosclerosis. The main objective of the resaech is to investigate the role of CPI-17 in atherosclerosis by inducing the phenotype changing in vascular smooth muscle cells.Methods1. Established the atherosclerotic plaque modelWe used ApoE-knockout mice of 8～10 weeks, they were fed a high-fat diet throughout the experiment, and carotid atherosclerotic lesions were induced by placement of a constrictive perivascular collar. After 8 weeks, took blood from their tails and detected the serum lipoprotein levels. Took the carotid artery in the side of implementing operation, used pathological section, HE staining, Oil red O staining to confirm the formation of plaque. 2. Detected the mRNA level of CPI-17 and smooth muscle phenotype marker proteins by quantitative fluorescence real-time PCR2.1 Extraction of the carotid total RNA of the mice and preparation of cDNAAnesthetized the ApoE-knockout plaque mice and used saline infusion for 10 min, took the carotid artery in the side of implementing operation.The carotid of ApoE-knockout mice atherosclerotic plaque were grinded in liquid nitrogen. Total RNA was extracted from carotid using Tizol and treated with RNase-free DNase to remove genomic DNA contamination. RNA (2 fig) was reverse transcribed to cDNA using reverse transcription system kit. We also extracted the carotid total RNA of C57BL/6 mice as controls.2.2 Detected CPI-17 mRNA level by quantitative fluorescence real-time PCRThe expression of CPI-17 gene of ApoE-knockout mice atherosclerotic plaque carotid was evaluated by quantitative fluorescence real-time PCR, using cDNA obtained by reverse transcriptase as a template and adding CPI-17 specific primers. Analysised the expression changes of CPI-7 bertween C57BL/6 mice carotid and ApoE-knockout mice atherosclerotic plaque carotid. SMCs differentiated markers sm-MHC, a-actin was also quantified by quantitative fluorescence real-time PCR.2.3 Detected phenotypic markers mRNA level by quantitative fluorescence real-time PCRDetected SMCs phenotypic markers——sm-MHC, a-actin mRNA level of ApoE-knockout mice atherosclerotic plaque carotid, using cDNA as a template and adding sm-MHC, a-actin specific primers. We used carotid artery of C57BL/6 mice as control. Analysised the phenotype conversion of vascular smooth muscle cells during the formation of atherosclerosis and analysised correlation between CPI-17 and sm-MHC, a-actin.3. Studied the effect of ox-LDL stimulation,hypoxia stimulation on the expression of CPI-17 of VSMCs in vitro3.1 The effect of ox-LDL stimulation on the expression of CPI-17 of MOVAS cellsMOVAS cells were stimulated with ox-LDL at the concentration of 50μg/ml. Cells were harvested at 0 h,12 h,24 h and 48 h after stimulated with ox-LDL. Total RNA was extracted from these cells using Tizol and treated with RNase-free DNase to remove genomic DNA contamination. The expression of CPI-17 gene was evaluated by quantitative real-time PCR.3.2 The effect of hypoxia stimulation on the expression of CPI-17 of MOVAS cellsMOVAS cells were cultured under hypoxia for 0 h,12 h and 48 h respectively. Cells were harvested and washed to extract total RNA using Tizol method. The RNA was dissolved in RNase-free DNase water to remove genomic DNA contamination. RT-PCR was performed to produce cDNA. The expression of CPI-17 gene was evaluated by quantitative real-time PCR.Results1. Successfully established the atherosclerotic plaque modelThe leves of lipoprotein of ApoE-knockout mice with placement of a constrictive perivascular collar and fed a high-fat diet in 8 weeks, the HE staining result and the Oil red O staining result all showed that the models were successfully established.2. The mRNA level of CPI-17 and phenotypic markers proteins in atherosclerotic plaque organizationsQuantitative real-time PCR results showed, CPI-17 mRNA level was significantly decreased in ApoE-knockout mice atherosclerotic plaque carotid campared to C57BL/6 mice carotid (90.09±27.40 vs 650.2±193.9, P=0.0004) Meanwhile, the mRNA level of sm-MHC, a-actin was also significantly decreased in ApoE-knockout mice atherosclerotic plaque carotid campared to C57BL/6 mice carotid (sm-MHC:24.82±14.19 vs 124.9±35.48, P= 0.0074; a-actin:1065±442.6 vs 16740±8467, P= 0.0092)3. The expression of CPI-17 was decreased in MOVAS cells after stimulation with ox-LDL,hypoxia in vitro.Quantitative real-time PCR results showed, CPI-17 mRNA level was significantly decreased in the cells treated with ox-LDL and hypoxia compared to blank control. ox-LDL stimulation,12 h group vs 0 h group:0.7869±0.04393 vs 3.964±0.3973, P= 0.0014; 24 h group vs 0 h group:1.694±0.1290 vs 3.666±0.2073, P= 0.0013; 48 h group vs 0 h group:1.590±0.09089 vs 3.501±0.2341, P= 0.0016.Conclusion:1. The level of CPI-17 mRNA in ApoE-knockout mice atherosclerotic plaque carotid was down-regulated campared to the wild type C57BL/6 mice;2. The level of sm-MHC, a-actin mRNA in ApoE-knockout mice atherosclerotic plaque carotid was down-regulated compared to the nomarl carotid arteries; But there was no apparent correlation between CPI-17 and sm-MHC, a-actin;3. The level of CPI-17 mRNA was down-regulated in MOVAS cells stimulated with ox-LDL and cultured under hypoxia;4. CPI-17 may affect atherosclerosis by regulating the phenotype of vascular smooth muscle cells.Inovation:1. The first to study the CPI-17 expression changes in plaque tissue of ApoE-knockout induced atherosclerotic plaque model;2. Proposed the new argument that CPI-17 could regulate the phenotype conversion of vascular smooth muscle cells affect the occurrence and development of atherosclerosis.