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Objective To evaluate the effect of over-expression of phosphatase and tensin homolog does on chromosome ten (PTEN) in podocytes on kidney under high fat diet (HFD) in vivo and clarify the mechanism how PTEN regulates scavenger receptor A (SR-A) expression exposed to oxidized low density lipoprotein (ox-LDL) in podocytes in vitro.Methods The podocyte-specific PTEN knockin (PPKI) mice were fed with HFD to establish mouse model of lipid-induced renal injury.Mice were divided into four groups:ND+Ctrl group,ND+PPKI group,HFD+Ctrl group and HFD+PPKI group.After 24 weeks of dietary intervention,all mice were tested for clinical and biochemical parameters,including serum creatinine (Scr) as well as urine albumin excretion rate (UAER);renal lipid content was measured by oil red O staining and cholesterol quantitative analysis;the pathological changes of glomeruli were observed by PAS staining and electron microscope.Podocyte injury was induced by ox-LDL in vitro.Western blotting was used to detect the changes of SR-A expression induced by ox-LDL after YAP-siRNA interfering (si-YAP),as well as YAP phosphorylation induced by ox-LDL after interfering by PTEN-siRNA (si-PTEN) and PTEN phosphatase inhibitor (Bpv-PTEN),and overexpressing by recombinant adenovirus (ad-PTEN).Results Compared with ND+Ctrl group,HFD+ Ctrl group significantly aggravated the levels of Scr and UAER,the expression of SR-A in podocytes,renal lipid content,mesangial matrix expansion,effacement of podocyte foot processes,and incrassation of glomerular basement membrane (all P ＜ 0.05).Conversely,compared with HFD+Ctrl group,HFD+ PPKI group obviously alleviated the above lipid-induced renal damage (all P ＜ 0.05).In vitro,the expression of SR-A in podocytes was up-regulated when stimulated with ox-LDL (P ＜ 0.05),and the knockout of YAP significantly down-regulated the expression of SR-A induced by ox-LDL (P ＜ 0.05).Exposed to ox-LDL,the expression of p-YAP increased in podocytes (P ＜ 0.05);over-expression of PTEN inhibited p-YAP up-regulation induced by ox-LDL (P ＜ 0.05),while either knockdown of PTEN or inhibition of PTEN phosphatase activity displayed opposite effect (all P ＜ 0.05).Conclusions Over-expression of PTEN in podocytes protected the kidney against damage from HFD in vivo and PTEN might suppress SR-A mediated lipid uptake via dephosphorylating p-YAP to prevent podocyte injury from ox-LDL.
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Objective The expression of agr and sigB regulation system in Staphylococcus aureus with different infection types were assessed by analyzing the characteristics of m /z value generated by MALDI-TOF-MS.Methods A total of 50 isolates with specific genotypes were collected from Tianjin First Center Hospital during Jun 2013 to Feb 2014 for retrospective study .The pattern profiles of these isolates were obtained by MALDI-TOF-MS with RUO model, and the m/z value was also analysed to evaluate the expression the agr and sigB regulation system .The phylogenetic tree based on mass spectrum peak feature was constructed using SARAMIS software .Results A total of 50 strains of Staphylococcus aureus were divided into two groups: acute infection and chronic persistent and recurrent infection .The expression of delta toxin in acute infection and in chronic infection was 99.2 ±4.1 and 60.5 ±10.1 ( t =16.83, P<0.05), respectively.The regulation of stress proteins of sigB system was enhanced in chronic persistent and recurrent infections , and the expression intensities of SAS 030, SAS049 and SA0772 were 27.1 ±14.7, 54.8 ±21.5 and 51.6 ±19.2, respectively; while in acute infections , those were 4.9 ±1.9, 12.4 ±2.8 and 15.7 ±6.9, respectively.The t values between the two groups were -6.88 (P<0.05),-8.98 (P<0.05) and -1.87 (P<0.05), respecitively.The expression of phenol-soluble modulins (PSMs) was inconsistent , and the relative strength of PSMα3 was 100%in the colony variants small strains .Conclusions Different types of the Staphylococcus aureus infections could be evaluated through the assessment of the agr and sigB regulation system .The m/z value obtained by MALDI-TOF mass spectrometry is a marker for the expression of agr and sigB regulation system .The application of this technology needs further development .
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AIM: To construct the prokaryotic expression system containing protein transduction domain (PTD) with heat shock protein 27 (HSP27) in order to prepare and purify the recombinant protein , and to verify whether the recombinant protein PTD-HSP27 has the ability to penetrate the human lens epithelial cell ( HLEC) membrane and the rabbit cornea.METHODS:The plasmid pKYB-PTD-HSPB1-6His was constructed by the technique of overlap extension PCR.The plasmid was transformed and PTD-HSP27 was purified through nickel affinity chromatography column and identi-fied by Western blotting.PTD-HSP27-6His was labeled with the fluorescein isothiocyanate (FITC).The penetrating ability of PTD-HSP27 into HLECs and rabbit cornea was tested .RESULTS:The recombinant PTD-HSP27 plasmid was success-fully cloned and effectively expressed .The correctness of the recombinant protein PTD-HSP27 was demonstrated .Fluores-cence microscopic examination showed that PTD-HSP27-FITC was internalized by HLECs .Fluorescent labeled PTD-HSP27 was then observed in the rabbit aqueous humor .CONCLUSION:The recombined gene PTD-HSPB1 was constructed by o-verlap extension PCR technique and the PTD-HSP27 fusion protein was prepared and purified by nickel affinity chromatog-raphy column.Using the technique of PTD-fusion protein, HSP27 was transduced into HLECs and passed through the cor-nea .
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Objective To evaluate the clinical value of the immunochemistry staining of thyroid transcription factor-1 (TTF-1), cytokeratin7 (CK7), P63, and cytokeratin5/6 (CK5/6) in the bronchoscop-ic forceps biopsy specimen in the differential diagnosis of non-small cell lung cancers.Methods Totally 143 cases of non-small cell lung cancers from Department of Respiratory and Department of Oncology from Nov 2012 to Jan 2014 were diagnosed with pathological examinations of the bronchoscopic forceps biospy.The sen-sitivity and specificity of TTF-1, CK7, P63, and CK5/6 of forceps biopsy at the diagnosis of lung adenocarci-noma and squamous cell carcinoma were calculated.Results The expressions of TTF-1 and CK7 in the pul-monary adenocarcinomas were higher than the pulmonary squamous cell cancer ( P <0.01).The diagnosis sensitivity and specificity of TTF-1 in the pulmonary adenocarcinoma were 85.1%(63/74) and 98.6%(68/69), respectively.The diagnosis sensitivity and specificity of CK7 in the pulmonary adenocarcinoma were 82.4%(61/74) and 91.3%(63/69), respectively.The diagnosis sensitivity and specificity of P63 in the pulmonary squamous cell cancer were 97.1% ( 67/69 ) and 89.2% ( 66/74 ) , respectively.The diagnosis sensitivity and specificity of CK5/6 in the pulmonary squamous cell cancer were 79.7%(55/69) and 89.2%(66/74), respectively.The combined expression of CK7(+)/TTF-1(+) in the pulmonary adenocarcinoma was higher than the pulmonary squamous cell cancer ( P <0.01).The combined expression of P63(+)/CK5/6(+) in the pulmonary squamous cell cancer was higher than the pulmonary adenocarcinoma ( P <0.01).Conclusions The combination use of the immunochemistry staining of TTF-1, CK7, P63, and CK5/6 helps to differentiate lung adenocarcinoma and squamous cell cancer.
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Objective To investigate the relationship between the expression of β-catenin and drug-resistance mechanism of choriocarcinoma according to the expression of β-catenin in JEG-3 cells (human choriocarcinoma cell line) and drug resistant JEG-3/VP16 cells.Methods The mRNA and protein expressions of β-catenin were analyzed with reverse transcription-polymerase chain reaction(RT-PCR) and Western blotting.Flow cytometry was used to determine the percentages of β-catenin-positive cells in the two choriocarcinoma cell lines.Results Both drug resistant choriocarcinoma cells and drag sensitive cells were found to express β-catenin; but the expression of β-catenin mRNA (1.43 ±0.24) and protein(1.49 ±0.17)in drug resistant choriocarcinoma cells was found much higher than that in drug sensitive cells(0.65 ±0.14,0.66 ±0.16,P ＜0.01).And according to detect by flow cytometry,we found the number of β-catenin-positive cells in JEG-3/VP16 cells [(40.13 ±5.17) ％] was much more than that in JEG-3 cells [(13.15 ± 1.48) ％,P ＜ 0.01].Conclusions β-catenin was highly expressed in the drug resistant choriocarcinoma cell line (JEG-3/VP16).It indicates β-catenin might be involved in the drug resistance mechanism of choriocareinoma.
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Objective To investigate the expressions of signal transducer and activator of transcription factor 3 (STAT3),Bcl-2,and matrix metalloproteinase 2 (MMP2) in colorectal adenomas and adenocarcinomas,and the relationship of those factors with the colorectal clinicopathological features and prognosis of intestinal adenocarcinomas,and explore their roles in invasion,metastasis,and prognosis of a colorectal cancer.Methods The samples were selected from Dongyang City People's Hospital from January 2011 to January 2012,including 50 cases of paraffin-coded colorectal mucosas with chronic inflammation,50 cases of paraffincoded colorectal adenomas,and 100 cases of paraffin-coded colorectal adenocarcinomas (35 cases with metastasis and 65 cases without distant metastasis).Envison two method was used to detect the expressions of STAT3,Bcl-2,and MMP2 in each sample.Results The expressions of STAT3,Bcl-2,and MMP2 in colorectal adenomas and adenocarcinomas were significantly higher than those in colorectal mucosas with chronic inflammation (P ＜ 0.05).The expressions of STAT3 and MMP2 in colorectal adenocarcinomas with distant metastasis were significantly higher than that those without distant metastasis (P ＜ 0.05).The expression of BCL-2 had no significant difference between colorectal adenocarcinomas with and without distant metastasis (P ＞ 0.05).Conclusions STAT3,Bcl-2 and MMP2 were associated with occurrence and development of colorectal carcinoma.STAT3 was associated with distant metastasis of colorectal carcinoma and might indicate a poor prognosis.STAT3 might be used as a candidate clinical sign for recurrence,metastasis,and poor survival prognosis of colorectal adenocarcinoma.
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MicroRNAs (miRNAs) are evolutionarily conserved small noncoding RNAs of-22 nucleotides that exist in a wide variety of organisms,including animals,plants and virus.Mature miRNAs are able to control gene expression at a post-transcriptional level,either by blocking mRNA translation or inducing their degradation.Hepatitis B virus X protein (HBX) is a 17000 protein that is implicated to play a crucial role in hepatocarcinogenesis.Recently,many studies have shown that HBX is associated with miRNA regulation,and is involved in regulating fundamental biological processes of tumor in cell proliferation,differentiation and apoptosis.
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Objective To explore influence of JAK/STAT3 signaling pathway on the apoptosis of HCC cells.Methods DNA-vector-based RNAi approach silence was used to down-regulate STAT3 expression in Bel-7402 cells.According to the STAT3 cDNA sequence in the GeneBank database,the plasmid pGCsi.U6/neoRFP-STAT3 that was designed for expression of STAT3 siRNA was constructed and synthesized,and then transfected into the Bel-7402 cells with iipofectamine 2000.The apoptotic rate was measured with flow cytometry (FCM) and annexinV/PI apoptosis detection kit staining.The mitochondrial membrane potential (BΨm) was visualized by the JC-1 fluorescence staining and the inverted fluorescence microscope.Moreover,the expression of caspase-3 protein was analyzed by Western blotting.Results The apoptotic ratio of STAT3-siRNA group was (38.82 ± 0.88) ％,which was significantly higher than that in other group [control group (9.22 ± 0.38) ％,scramble-siRNA group (16.47 ± 1.04) ％,P ＜ 0.05].The mitochondrial membrane potential of STAT3-siRNA group observed by the JC-1 fluorescence staining was decreased significantly[(91.33 ± 1.78) ％] and [(89.90 ± 1.92) ％ vs (59.06 ± 1.89) ％,P ＜ 0.05].The Western blot results showed that the protein expression of active caspase-3 in STAT3-siRNA group was significantly higher than other groups (0.48 ± 0.05 vs 0.22 ± 0.04 and 0.26 ± 0.06,P ＜ 0.05).Conclusions STAT3 gene silencing significantly improves the apoptotic effect in the Bel-7402 ceils.
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Objective To further localize 5' distal enhancer-like region of the Toll-like receptor 4 (TLR4) gene, and identify the trans-acting factors involved in its transcriptional regulation. Methods The molecular cloning technique and reporter gene analysis were used to analyze the effects of different regions of TLR4 gene 5' flank on its transcriptional activity. The pGL-3 vectors with TLR4 gene 5' flank -1337 to -683bp from transcriptional start site and their deletants were constructed and transferred into K562 cell line by liposomal transfection method. The instant luciferase expression of different clones was detected and the effects of the deletants on the transcriptional activity of TLR4 gene were evaluated. The electrophoretic mobility shift analysis and transcription factor database search were used to locate and identify the trans-acting factors. Results A 245 bp enhancer-like region was further identified in the range of -923 to -679bp from the TLR4 gene transcriptional start site, and -923 to -742 and -721 to -679 regions were refined as two functional units. The activator protein 1 (AP1) was confirmed as a trans-acting factor in the -721 to -679 functional units, and lymphoid enhancer-binding factor (LEF-1) may be another one. Conclusion AP1 acts as a trans-acting factor interacting with -923 to -679 region of TLR4 5' flank, and plays an enhancer-like role together with LEF-1.
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Objective To evaluate the feasibility of transfection of Sonic hedgehog gene (SHH)into bone marrow mesenchymal stem cells(BMMSC).Methods After the SHH gene was transfected into BMMSC by electroporation apparatus,the transfection rate was evaluated by fluorescence inverted microscope.The growth curves of untransfected and transfected BMMSC were drawn,respectively,to observe the influence of transfection on cells.The expression of SHH gene in the BMMSC was detected by PCR,RT-PCR,Western-blot analyses.Results Through fluorescence inverted microscope,the observed transfection rate was appropriately 30％,PCR showed a obvious increase of SHH expression in transfected cells than that in untransfected cells,and it is quantified by qPCR for appropriately 7 times.Western-blot further demonstrated that the SHH protein expression in transfected cells had a distinct increase.However,it was observed that the exponential phase of BMMSCSHH growth curve delayed.The growth curves of both overlap 12 days after transfection.Conclusions This electroporation method can transfect exogenous SHH gene into BMMSC sufficiently with the effective protein expression in BMMSCSHH.It is the foundation of further research of genetic therapy for ischemic heart disease.
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Objective To explore the effects of pretreatment with different doses of curcumm on the expression of p-CREB and PGC-1α in hippocampus after global cerebral ischemia-reperfusion (IR) injury in rats.Methods Three hundred male SD rats weighing 200-250 g were randomly divided 5 groups ( n = 60 each): sham operation group (group S), IR group, low, median and high dose curcumin group (group LC, MC, HC). Global cerebral ischemia was produced by occlusion of 4 vessels (cauterization of bilateral vertebral arteries and 15 min occlusion of bilateral common carotid arteries) according to the method described by Finkbeiner. Bilateral common carotid arteries were only exposed but not ligated in group S. Intraperitoneal curcumin 30, 100 and 300 mg/kg were injected at 1 h before ischemia in group LC, MC and HC respectively. Equal volume of dimethylsulfoxide (DMSO) was injected intraperitoneally in group S and IR. The rats were killed at 2, 6, 24 and 72 h and 7 d after reperfusion (12 at each time point). Brains were immediately removed and hippocampus was isolated. The number of apoptosis neurons was counted using TUNEL. The expression of p-CREB and PG C-1α protein in hippocampus was detected by Western blot. Results The number of apoptosis neurons, p-CREB and PG C-1α protein expression were significantly higher at each time point in the other 4 groups than in group S ( P ＜ 0.05). The number of apoptosis neurons was significantly lower at T2-4 in group LC and MC, while p-CREB and PG C-Ⅰα protein expression wes significantly higher at T1-4 in group LC, MC and HC than in group IR (P ＜ 0.05). The number of apoptosis neurons was significantly higher, while p-CREB and PGC-1α protein expression was significantly lower at T2-4 in group LC and HC than in group MC ( P ＜ 0.05). Conclusion Curcumin can inhibit neuronal apoptosis in hippocampus and reduce global cerebral IR injury by up-regulating p-CREB and PG C-1α expression in rats and the effect was dose-related.
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Objective To study the expression and significance of CD10, SMA, P63 on human breast myoepithelial cells. Methods SP immunohistochemical method was used to examine the expression of CD10, SMA, P63 on human breast myoepithelial cells from 68 breast diseases samples, including breast fibroadenoma, breast fibroadenesis, breast atypical ductal hyperplasias, ductal carcinomas in situ and breast invasive carcinomas. Results 2 +/3 + circumferential CD10, SMA and P63 staining of MEC was seen in 10 patients with breast fibroadenoma, breast fibroadenoma and 13 patients with atypical ductal hyperplasias. In an analysis of total ducts by 15 patients with breast ductal carcinomas in situ, 3 + circumfenrential staining was seen in 92 of 384 ducts(23.96%) stained for CD10 versus 185 of 384 ducts (48. 18%) stained for SMA and 80 of 384 ducts(20. 83%) stained for P63. All differences among percentage positively stained of these three antibodies was significant, p-values was lower 0. 05. MECs were not found with immunohistochemistry in 170 of 384 duets (44. 27%)with anti-CD10,74 of 384 ducts(19.27%) with anti-SMA and 127 of 384 ducts(33.07%) with anti-P63. MECs were not found with CD10,SMA and P63 in 30 patients with breast invasive carcinomas. Conclusion We concluded that CDIO could be another useful marker of breast myoepithelial cells after SMA and P63. It provided more sophisticated information about presence or absence of breast myoepithelial cells in confusing breast lesions.
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Objective To express nonstructural protein 2 transactivated protein (NS2TP) of hepatitis C virus (HCV) in the prokaryotic expression system and prepare polyclonal antibody,and to study the expressions in different liver tissues.Methods NS2TP gene was amplified by polymerase chain reaction (PCR) technique and cloned into the prokaryotic expression vector pET-32a(+),which was transformed into E.coli BL21.The protein was induced by isopropyl thiogalactose (IPTG) and analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and confirmed by Western blotting assay.The recombinant protein were expressed and purified in a large amount.The rabbit was immunized with the purified protein to prepare polyelonal antibody.The liver tissues of patients with chronic HCV infection and healthy controls were detected by immunohistochemistry method.Results The recombinant NS2TP protein (relative molecular mass:33×103 ) and polyclonal antibody with high titer and specificity were successfully prepared.NS2TP expressions in the liver of patients with chronic HCV infection were higher than those of healthy controls,and were mainly distributed in the nucleus of hepatocytes.Conclusions The NS2TP expression level and intracellular location in liver tissue of patients with chronic HCV infection are understanded,which could bring new clues for further study of the biological function of NS2TP and the pathogenesis of HCV infection.
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0bjective To observe the cell membrane penetration of protein transduction domain (PTD)-HBeAg fusion protein in vitro.Methods The sequence of trans-activator of transcription (Tat)-PTD was synthesized and the whole HBcAg gene was amplified by polymerase chain reaction (PCR).Overlap extension PCR was employed to fuse Tat-PTD and whole HBcAg gene.Then the fusion gene was cloned into prokaryotic expression vector pMAL-c2X.The correct vector was transformed into E.coli Rosetta-gamiTM 2(DE3),and the protein was induced by isopropyl β-D-1-thiogalactopyranoside(IPTG).Western blot was used tO identify the protein. Furthermore,the fusion protein PTD-HBcAg was purified by affinity chromatography.HBcAg protein expressed using the same methods was employed as eontr0l.The purified protein was added tO HuH-7 cell culture,then the transduction of PTD-HBcAg and HBcAg in cells were detected by indirect immunofluorescence assay (IFA).Results The fusion protein was effectively expressed in E. Coli and purified by affinity chromatography.Both purified PTD-HBcAg and HBcAg could be recognized by HBeAg monoclonal antibody in Western blot analysis.IFA visualization showed that PTD-HBeAg could be introduced into HUH-7 ceils while HBcAg only could not be detected in cells.Conclusions PTD-HBcAg fusion protein can be expressed effectively and purified in prokaryotic expression system.PTD could mediate HBcAg penetrating eell membrane into the cells.
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Replication of the hepatitis B virus is suppressed by deficiency of the X protein. Although several molecules that block cellular targets of X protein reduce the production of hepatitis B virus progeny, the effect of a specific inhibitor of X protein on viral replication has not been investigated. To block X protein specifically, we adopted an intracellular expression approach using H7 single chain variable fragment (H7scFv), an antibody fragment against X protein. We previously demonstrated that cytoplasmic expression of H7scFv inhibits X protein-induced tumorigenicity and transactivation. In this study, intracellular H7scFv expression inhibits reporter gene transactivation but not viral replication determined by endogenous hepatitis B virus polymerase activity assay and real-time PCR. Our findings imply that intracellular expression of antibody fragment against X protein may not be an alternative therapeutic modality for inhibition of hepatitis B virus replication.
Sujets)
Réplication virale/effets des médicaments et des substances chimiques , Transactivateurs/antagonistes et inhibiteurs , Région variable d'immunoglobuline/génétique , Virus de l'hépatite B/effets des médicaments et des substances chimiques , Antigènes e du virus de l'hépatite virale B/métabolisme , Lignée cellulaireRésumé
Objective:To explore the expression levels of MHCⅡ molecules and its regulator genes CⅡTA on bleomycin-induced pulmonary fibrosis in rats,and to investigate the underlying immunologic mechanisms of pulmonary fibrosis.Methods:The rats were treated with either a single intratracheal bleomycin injection(fibrosis group)or a normal saline injection(control group).The pathologic changes of lung tissues stained with HE and Masson were observed,and the contents of hydroxyproline were detected on the 7th and 28th day respectively after bleomycin administration.The expression of MHCⅡ molecules in the lung tissues was evaluated with immunohistochemistry techniques,and the percentage of MHCⅡ+ cells was measured.The amounts of total CⅡTA and typeⅠ,Ⅲ and Ⅳ CⅡTA mRNA of lung tissues were measured by real-time PCR using Taqman probe.Results:(1)The percentage of MHCⅡ+ cells in lung tissues increased significantly in fibrosis group compared with that of control group on the 7th day and the 28th day[(0.10?0.03)vs(0.06?0.02),P0.05];In fibrosis group,type Ⅳ CⅡTA mRNA was 667.3% [(2.98?0.92)vs(0.39?0.15),P
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Objective To investigate the roles of transcription factor GATA-3 and T-bet at the fetal- maternal interface in the pathogenesis of unexplained recurrent spontaneous abortion(URSA).Methods The expression of GATA-3 and T-bet mRNA was examined by in situ hybridization.Decidua was obtained from 20 women with URSA and 20 normal pregnant(NP)women.Results(1)The number of GATA-3 positive cells per high power field in women with URSA(25?16)was significantly lower than those in NP women(38?16)(P