Downregulation of cellular prion protein inhibited the proliferation and invasion and induced apoptosis of Marek's disease virus-transformed avian T cells

Cellular prion protein (PrP(C)) is ubiquitously expressed in the cytomembrane of a considerable number of eukaryotic cells. Although several studies have investigated the functions of PrP(C) in cell proliferation, cell apoptosis, and tumorigenesis of mammals, the correlated functions of chicken PrP(C) (chPrP(C)) remain unknown. In this study, stable chPrP(C)-downregulated Marek's disease (MD) virus-transformed avian T cells (MSB1-SiRNA-3) were established by introducing short interfering RNA (SiRNA) targeting chicken prion protein genes. We found that downregulation of chPrP(C) inhibits proliferation, invasion, and migration, and induces G1 cell cycle phase arrest and apoptosis of MSB1-SiRNA-3 cells compared with Marek's disease virus-transformed avian T cells (MSB1) and negative control cells. To the best of our knowledge, the present study provides the first evidence supporting the positive correlation between the expression level of chPrP(C) and the proliferation, migration, and invasion ability of MSB1 cells, but appears to protect MSB1 cells from apoptosis, which suggests it functions in the formation and development of MD tumors. This evidence may contribute to future research into the specific molecular mechanisms of chPrP(C) in the formation and development of MD tumors.

Ca~(2+) signaling mediated salidrosides promotes directional differentiation of mouse bone marrow mesenchymal stem cells into nerve cells / 中国组织工程研究

BACKGROUND:Tranditional Chinese medicine,which possesses anti-oxidation properties,can promote directional differentiation of mouse bone marrow mesenchymal stem cells(BMMSCs)into nerve cells.Salidrosides,as the effective constituent of Rhodiola,have strong anti-oxidation function.OBJECTIVE:To investigate the molecule mechanism of salidrosides induced differentiation of mouse BMMSCs into nerve cells.METHODS:When in vitro cultured BMMSCs reached 80% confluency,the cells were assigned into 3 groups.Cells in the control group were cultured by complete culture medium;those in the induction and positive control groups were cultured by complete culture medium adding 20 mg/L salidrosides or 0.1 mg/L nerve growth factors(NGF).The related gene and protein of nerve cells were detected using RT-PCR and Western blot method at 12 hours after culture.After that,the cells in the induction group were divided into 3 groups,the blocking agents EGTA(Ca~(2+) chelator),Nifedipine(L-type Ca~(2+) channel blocker)and LY294002(IP3 receptor blocking pharmacon)were applied to block the cellular Ca2* signal pathway respectively for 12 hours.RT-PCR and Western blot methods were used to study the signal transduction of the salidrosides.RESULTS AND CONCLUSION:①The expression of neuron specific enolase(NSE),B-Tubulin III,Nurri mRNA could be found ir the induction and positive control groups,instead of the control group;The expression abundance of the positive control group was smaller than that of the induction group.The expression abundance of GFAP mRNA was very low in each group,but the c-fos mRNA was expressed abundantly in the induction group.②Compared with the positive control group,the induction group could promote the NSE expression obviously,which was no expressed in the blank control group.?The expression of NSE and Nurri were conspicuously down-regulated when the Ecto Ca~(2+) and L-type Ca~(2+) channel and IP3 receptor were blocked respectively.Salidrosides can induce the differentiation BMMSCs into nerve cells. Ca~(2+) signaling and IP3 dependent Ca~(2+) signaling pathway play an important role in transduction salidrosides signal in BMMSCs differentiation.

Cloning the Structure Genes and Expression the N Gene of Porcine Epidemic Diarrhea Virus DX / 中国病毒学·英文版

The structure genes spike (S), nucleocapsid (N), membrane (M), small membrane (sM) of a porcine epidemic diarrhea virus (PEDV) strain DX isolated in Gansu province, North-west of China, were cloned, sequenced and compared with published sequences of PEDV strains. The nucleotide sequences encoding the entire S, sM, M and N genes open reading frame (ORF) of DX were 4 152, 231, 681 and 1 326 bases long respectively. There were transcription regulatory sequences (TRSs) upstream of the initiator ATG of the S, N and M genes. The amino acids sequences of S, M and N contained 30, 3 and 7 potential asparagine (N)-linked glycosylation sites. Homologous analysis and phylogenetic trees showed that DX had the closest relationship with strains LJB/06, JS-2004-2Z and CH/HLJH/06 that were also isolated from China and indicated the prevalence of some PEDV isolates in China were widespread since the JS-2004-2Z strain originated from the south of the China, and LJB/06 and CH/HLJH/06 were isolated from northeast China. The N gene was cloned using two primers which contained Nco I and BamH I restriction enzyme sites and subcloned into expression vector pET30a. The recombinant plasmid was then transformed into E.coli Rossta. SDS-PAGE showed there was a protein of about 55kDa as expected and Western blot indicated the N protein had biological activity.

The effect of urokinase on hepatic fibrogenesis in rats / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the effect of urokinase on hepatic fibrogenesis in rats.</p><p><b>METHODS</b>Hepatic fibrosis was induced in rats by complex pathogenic factors including subcutaneous injections of carbon tetrachloride, alcohol and cholesterol feeding. Animals were randomly divided into 3 groups: normal control group, hepatic fibrosis group (complex pathogenic factors for 6 weeks), UK prevention group (complex pathogenic factors+UK for 6 weeks). The animals were sacrificed at the end of week 6. The expression of alpha-SMA, uPA, PAI-1, TGFb1, TIMP-1, collagen type I and type III proteins in hepatic fibrosis tissue was detected by immunohistochemistry, the expression of PAI-1 and TGFb1 mRNA in the hepatic fibrosis tissue was quantified by real time RT-PCR. The serum levels of hyaluronicacid (HA), alanine aminotransferase (ALT), aspartate aminotransferase (AST), bilirubin (TBil) and the content of liver hydroxyproline (Hyp) were detected using ELISA kits.</p><p><b>RESULTS</b>The serum ALT, AST, TBil, HA and the content of liver Hyp were (46.66+/-6.30) U/L, (126.26+/-31.65) U/L, (31.11+/-4.20) micromol/L, (109.70+/-18.81) microg/L and (0.98+/-0.09) mg/(g liver), respectively, in UK prevention group, which were significantly lower than those [(101.57+/-11.97) U/L, (205.89+/-56.26) U/L, (67.75+/-2.75) micromol/L, (184.43+/-32.36) microg/L and (1.65+/-0.16) mg/(g liver), respectively] in hepatic fibrosis group (q = 3.3801-20.0061, P < 0.01). The levels of a-SMA, collagen type I, type III, TIMP-1, PAI-1, TGFb1 proteins were (299.27+/-37.36), (210.05+/-27.17), (192.94+/-24.48), (213.70+/-32.21), (204.25+/-17.92), (205.97+/-23.81), respectively, in UK prevention group, which were significantly lower than those [(418.83+/-30.21), (323.77+/-21.53), (302.37+/-31.43), (376.63+/-25.19), (313.53+/-26.67) and (327.42+/-36.75), respectively] in hepatic fibrosis group. The level of uPA protein was increased, and the expression of PAI-1, TGFb1 mRNA in hepatic fibrosis tissue was decreased in UK prevention group.</p><p><b>CONCLUSION</b>In the early stage of hepatic fibrogenesis, urokinase can attenuate the progression of rat hepatic fibrosis via upregulation of uPA, downregulation of TGFb1, and inhibition of HSC activation.</p>

The plasma levels of urokinase plasminogen activator and urokinase plasminogen activator receptor and plasminogen activator inhibitor-1 in patients with different stages of liver cirrhosis following chronic hepatitis B / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To measure the plasma levels of urokinase plasminogen activator (uPA), urokinase plasminogen activator receptor (uPAR) and plasminogen activator inhibitor-1 (PAI-1), and study the relationship between the plasma levels of uPA, PAI-1 and the serum albumin (Alb), collagen type IV (CIV), the serum hyaluronic acid (HA), prothrombin time (PT) and prothrombin activity (PTA) in patients with different stages of liver cirrhosis following chronic hepatitis B.</p><p><b>METHODS</b>72 cases with liver cirrhosis of different stages were classified according to child-pugh's categories A, B, C, in which there were 23 cases in child A, 29 cases in child B, and 20 cases in child C. The plasma levels of uPA, uPAR, PAI-1 and the serum levels of HA, CIV were detected by ELISA. The serum PCIII concentration was determined by radioimmunoassay.</p><p><b>RESULTS</b>With the progression of hepatic fibrosis, the plasma levels of uPA, uPAR and PAI-1 were (1.36+/-0.43) microg/L, (3.03+/-1.48) microg/L and (24.09+/-7.14) microg/L respectively in group A, (1.79+/-0.62) microg/L, (4.80+/-2.22) microg/L and (41.40+/-17.52) microg/L respectively in group B. The highest levels were in child C, whose levels were (1.88+/-0.64) microg/L, (4.82+/-2.02) microg/L and (52.60+/-16.87) microg/L respectively, compared with group A and group B, t value were from 2.81 to 7.38, all of P value were less than 0.01. There was negative correlation between the plasma levels of uPA and the serum PCIII (r=-0.4785, P<0.05) in child A, but, positive correlation between the plasma PAI-1 and the serum HA (r=0.5447, P<0.01) in child C. The value of PAI-1/uPA was significantly decreased in child A, but increased in child B and child C.</p><p><b>CONCLUSION</b>In the late of liver cirrhosis, increased PAI-1 together with uPA, uPAR are associated with overall inhibition of matrix degradation. The plasma levels of uPA and PAI-1 were correlation to the progression of liver cirrhosis.</p>

The plasma levels of transforming growth factor beta1 and the protein expressions of alpha-SMA, urokinase plasminogen activator and plasminogen activator inhibitor-1 in liver of patients with different grades of hepatic fibrosis / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To measure the plasma levels of transforming growth factor beta1 (TGFbeta1), the protein expression of alpha-SMA in hepatic stellate cells and urokinase plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1), and study on the relationships between the plasma levels of TGFbeta1, the protein expression and the serum hyaluronic acid (HA) in patients with different grades of hepatic fibrosis.</p><p><b>METHODS</b>Thirty seven cases with hepatic fibrosis of different grades were classified according to HE and VG staining categories from 0 to 4, in which there were 8 cases in grade 1, 9 cases in grade 2, 7 cases in grade 3, 13 cases in grade 4. The plasma levels of TGFbeta1 and the serum levels of HA were detected by ELISA. The protein expressions of a-SMA, uPA and PAI-1 in fibrotic liver tissue were observed by immunohistochemistry.</p><p><b>RESULTS</b>With the progression of hepatic fibrosis, the plasma levels of TGFbeta1 and the protein expression of a-SMA, uPA and PAI-1 in fibrotic liver tissue were increased. In grade 3 and 4, the plasma levels of TGFbeta and the protein expression of a-SMA and PAI-1 in fibrotic liver tissue were significantly increased, but the protein expression of uPA in cirrhosis liver tissue did not increased.</p><p><b>CONCLUSION</b>TGFbeta1, a-SMA, uPA and PAI-1 play an important role in the progression of hepatic fibrosis. Inhibiting the early activation of latent TGFbeta1 or increasing uPA and inhibiting PAI-1 over express may contribute to matrix degradation and retard the progression of hepatic fibrosis.</p>