RESUMO
Aim To explore the protective effect of Yishen Huashi Granule (YSHS) on streptozotocin (STZ) - indueed diabetes nephropathy (DN) in mice and its possible mechanism. Methods The STZ induced DN mice model was established, which was randomly divided into model group, YSHS group and YAP inhibitor Vertepofin group, and the eontrol group was also established. The intervention was started eight weeks after the successful modeling with the course of four weeks. Urine protein concentration before and after intervention in each group as well as serum creatinine (Scr) and blood urea nitrogen (Bun) levels after intervention were measured. After the treatment, the mice were sacrificed, and the pathological changes of glomeruli were observed by light microscope HE staining. The protein expression of YAP, p-YAP S127 and CTGF were detected by Western blot, and the mRNA expressions of YAP, CTGF and podocyte functional proteins nephrin, podophyxin and WT1 were detected by RT-PCR. Results The biochemical indexes of YSHS group were better than those of model group, and HE staining showed that the pathological injury of glomeruli was improved. In the model group the protein expression of YAP, p-YAP (S127) and CTGF as well as the mRNA expression of YAP and CTGF increased, while the mRNA expression of nephrin, podocalyxin and WT1 decreased. After YSHS treatment, the protein expression of YAP, p-YAP S127, CTGF and the mRNA expression of YAP and CTGF decreased, while the mRNA expression of nephrin, podocalyxin and WT1 increased. Conclusions YSHS can reduce urinary protein, protect renal function and alleviate glomerular pathological injury in DN mice. Its possible mechanism may be related to the down-regulation of YAP in renal tissue, the reduction of CTGF expression level and the up-regulation of podocyte protein mRNA expression.
RESUMO
Objective To preliminarily discuss the feasibility of geolocation inference of forensic individual origin by soil metagenomic analysis. Methods The 33 soil samples from Heilongjiang, Qinghai and Tibet were collected, total bacterial DNA in the samples were extracted, and universal primers were used to amplify the V3 and V4 hypervariable region of bacterial 16S rDNA. The region was sequenced by high-throughput sequencing (HTS) with the MiSeq sequencer. Bioinformatics analysis such as species composition and sample comparison was performed on sequencing data. The richness index and diversity index were calculated based on operational taxonomic unit (OTU) results. Results A total of 2 720 149 sequences were generated by sequencing. Those sequences were clustered into 114 848 OTUs. The Chao1 indexes of soil microorganisms in Heilongjiang, Qinghai, and Tibet were 797.45, 745.11 and 535.98, respectively, and Shannon indexes were 6.46, 6.36 and 6.25, respectively. The number of bacterial species and the community diversity in the soil from high to low were Heilongjiang > Qinghai > Tibet. The composition of soil bacteria in three provinces at various classification levels were obtained, the dominant genuses in Heilongjiang were Chthoniobacteraceae DA101 and an unannotated genus of Thermogemmatisporaceae; the dominant genuses in Qinghai were an unannotated genus of Cytophagaceae and an unannotated genus of Nocardioidaceae; the dominant genuses in Tibet were an unannotated genus of Comamonadaceae and Verrucomicrobiaceae Luteolibacter. The results of principal co-ordinates analysis demonstrated that, according to the weighted UniFrac analysis, the three principle components represented 56.36% of the total variable, and according to the unweighted UniFrac analysis, the three principle components represented 34.81% of the total variable. The samples from the same province could be clustered together, and the species and content of soil microorganisms from different provinces were significantly different. Conclusion Based on the metagenomic analysis method, soil samples from different regions can be effectively distinguished, which has potential application value in geolocation inference of forensic individual origin in the future.
Assuntos
Bactérias/genética , Sequenciamento de Nucleotídeos em Larga Escala , RNA Ribossômico 16S/genética , Solo , Microbiologia do SoloRESUMO
@#【Objective】To investigate the effect of Imatinib(Ima)on the inflammatory phenotype of RAW264.7 mac⁃ rophages induced by lipopolysaccharide(LPS).【Methods】RAW264.7 cells were treated by LPS(0.1 μg/mL)and/or Ima(1 μmol/L,5 μmol/L). The mRNA levels of IL-1β,IL-10,CCL2,iNOS,TNF-α and Arg1 in RAW264.7 cells were tested by Q-PCR. The iNOS protein level and the activation of NF-κB and MAPK signal pathways were detected by Western Blot. The protein levels of IL-1β,CCL2,IL-10 and TNF-α in cell supernatant were detected by ELISA. 【Results】Compared with the normal control group,the mRNA levels of IL-1β,CCL2,iNOS,TNF-α and IL-10 were significantly increased in RAW264.7 cells treated with LPS for 8 h(P < 0.001). In addition,the cell supernatant protein levels of IL-1β,IL-10,CCL2 as well as TNFα and the iNOS protein level were significantly increased in RAW264.7 cells stimulated by LPS for 24 h(P < 0.001). Moreover,the phosphorylation levels of p65,p38,ERK and AKT were enhanced in RAW264.7 cells after treatment with LPS for 24 h. Compared with LPS group,pretreatment with Ima decreased the mRNA and protein levels of IL-1β,CCL2,iNOS and TNF-α(P < 0.01,P < 0.001),but increased the mRNA and protein level of IL-10(P < 0.001). And this effect of Ima was dose-dependent. The phosphorylation levels of p65,p38, ERK and AKT were decreased in LPS+Ima group compared with that in LPS group. The functional status of RAW264.7 cells did not change significantly after treatment with Ima alone.【Conclusions】The effect of Imatinib to inhibit the inflammato⁃ ry phenotype of macrophage is related to retarding the overactivation of NF-κB and MAPK signaling pathways.
RESUMO
Single-cell sequencing is a technique that analyzes DNA and RNA sequences on the cellular level with next generation sequencing. The ultra high resolution of single-cell sequencing provides new perspectives and opens new frontiers for our understanding of many areas of life sciences, including forensic genome. This paper summarizes the recent advancements in single-cell sequencing and the prospect of its forensic application.
Assuntos
Humanos , DNA , Genética Forense , Sequenciamento de Nucleotídeos em Larga Escala , Análise de Sequência de DNA/métodosRESUMO
Objective To detect the acetylation of decorin (DCN) and its influence on DCN ubiquitination in mesangial cells in rats.Methods Mesangial cells of rats were cultured in vitro.The immunoprecipitation,Western blot assay and RT-PCR were used to determine the acetylation of DCN.Results DCN was acetylated in renal mesangial cells in rats.The acetylated DCN promoted its stability via inhibiting of its degradation through polyubiquitination.Moreover,transforming growth factor-β1 (TGF-β1) and type Ⅳ collagen expression of mesangial cells decreased,and cell growth was inhibited when acetylation of DCN was enhanced in mesangial cells.Conclusions Acetylation of DCN inhibited DCN ubiquitination degradation,which enhances DCN's antagonistic effect against nephritis.These results may provide a potential target for further study of prevention and treatment of mesangial cell proliferative glomerulonephritis.
RESUMO
<p><b>BACKGROUND</b>Clarifying the mechanisms underlying vascular smooth muscle cell (VSMC) proliferation is important for the prevention and treatment of vascular remodeling and the reverse of hyperplastic lesions. Previous research has shown that the gaseous signaling molecule sulfur dioxide (SO2) inhibits VSMC proliferation, but the mechanism for the inhibition of the angiotensin II (AngII)-induced VSMC proliferation by SO2has not been fully elucidated. This study was designed to investigate if SO2inhibited VSMC proliferation in mice with hypertension induced by AngII.</p><p><b>METHODS</b>Thirty-six male C57 mice were randomly divided into control, AngII, and AngII + SO2groups. Mice in AngII group and AngII + SO2group received a capsule-type AngII pump implanted under the skin of the back at a slow-release dose of 1000 ng·kg-1·min-1. In addition, mice in AngII + SO2received intraperitoneal injections of SO2donor. Arterial blood pressure of tail artery was determined. The thickness of the aorta was measured by elastic fiber staining, and proliferating cell nuclear antigen (PCNA) and phosphorylated-extracellular signal-regulated kinase (P-ERK) were detected in aortic tissues. The concentration of SO2 in serum and aortic tissue homogenate supernatant was measured using high-performance liquid chromatography with fluorescence determination. In the in vitro study, VSMC of A7R5 cell lines was divided into six groups: control, AngII, AngII + SO2, PD98059 (an inhibitor of ERK phosphorylation), AngII + PD98059, and AngII + SO2 + PD98059. Expression of PCNA, ERK, and P-ERK was determined by Western blotting.</p><p><b>RESULTS</b>In animal experiment, compared with the control group, AngII markedly increased blood pressure (P < 0.01) and thickened the aortic wall in mice (P < 0.05) with an increase in the expression of PCNA (P < 0.05). SO2, however, reduced the systemic hypertension and the wall thickness induced by AngII (P < 0.05). It inhibited the increased expression of PCNA and P-ERK induced by AngII (P < 0.05). In cell experiment, PD98059, an ERK phosphorylation inhibitor, blocked the inhibitory effect of SO2on VSMC proliferation (P < 0.05).</p><p><b>CONCLUSIONS</b>ERK signaling is involved in the mechanisms by which SO2inhibits VSMC proliferation in AngII-induced hypertensive mice via ERK signaling.</p>
Assuntos
Animais , Masculino , Camundongos , Angiotensina II , Farmacologia , Proliferação de Células , MAP Quinases Reguladas por Sinal Extracelular , Metabolismo , Hipertensão , Tratamento Farmacológico , Músculo Liso Vascular , Biologia Celular , Transdução de Sinais , Dióxido de Enxofre , Usos TerapêuticosRESUMO
OBJECTIVES@#To study the differential genes expression in the early stage of acute renal ischemia-reperfusion injury and explore potential molecular mechanisms.@*METHODS@#The ischemia-reperfusion model was made via clamping renal artery of rat. The microarray detection and bioinformatics analyzing of the genes expression were performed. Differentially expressed genes were screened and related cellular activities and signaling pathways were analyzed in early stage of acute kidney injury. Meanwhile, molecules closely relative to acute kidney injury were explored by establishing a biological network of the differentially expressed genes, and the results were verified by real-time PCR.@*RESULTS@#A total of 151 genes showed differential expression in this study, including 132 up-regulated and 19 down-regulated genes. Cell proliferation, cytokines mediated signaling transduction and immune responses were greatly enriched by GO and KEGG analysis. The results of real-time PCR showed that compared with control groups, three selected genes (ANXA1, PHLDA1 and KLF6) which related to the acute kidney injury had an obvious differential expression in the early stage of disease. The multiple of increase was essentially the same as the multiple detected by microarray.@*CONCLUSIONS@#This study shows differential gene expression profile, related biological processes and signaling pathways involved in the early stage of acute kidney injury. ANXA1, PHLDA1 and KLF6 may play a role in the pathogenesis of acute kidney injury.
Assuntos
Animais , Ratos , Injúria Renal Aguda/genética , Anexina A1/genética , Proteínas Reguladoras de Apoptose/genética , Expressão Gênica , Rim/patologia , Fator 6 Semelhante a Kruppel/genética , Reação em Cadeia da Polimerase em Tempo Real , Traumatismo por Reperfusão/genética , Transdução de SinaisRESUMO
<p><b>OBJECTIVE</b>To construct the eukaryotic expression vector of HBEBP1 gene and express HBEBP1 recombinant protein in yeast.</p><p><b>METHODS</b>PCR was performed to amplify the gene of HBEBP1 from the cDNA template origining from HepG2, and the gene was cloned into pGEM-T vector. After sequencing, the correct DNA fragment was cut from pGEM-T-HBEBP1 and inserted into yeast expression plasmid pGBKT7. The reconstructed plasmid pGBKT7-HBEBP1 was transformed into yeast cell AH109 and screened on the synthetic dropout nutrient medium (SD/-Trp/Kana). The yeast protein was isolated and analyzed with SDS-PAGE and Western Blot.</p><p><b>RESULTS</b>The eukaryotic expressive vector was constructed successfully. The results of Western Blot showed HBEBP1 protein was existed within yeast cells and the molecular weight of it was about 33 x 10(3).</p><p><b>CONCLUSIONS</b>The successful expression of HBEBP1 protein in yeast cells lay the foundation for studying biological function of HBEBP1.</p>
Assuntos
Western Blotting , Proteínas de Transporte , Genética , Antígenos E da Hepatite B , Metabolismo , Plasmídeos , Proteínas Recombinantes , Saccharomyces cerevisiae , GenéticaRESUMO
<p><b>OBJECTIVE</b>To observe the effect of DNA Sema4D gene silencing by RNA interfering on the proliferation, migration and invasion of human ovarian cancer SKOV3 cells, and to study the effect of pshRNASema4D on the growth of SKOV3 cells in transplanted tumor in nude mice.</p><p><b>METHODS</b>Recombinant plasmid pshRNASema4D-A, B and C were respectively transfected into SKOV3 cells by lipofetamine 2000, while cells transfected by plasmid vector pcDNA3.1 and cells untreated as control groups. RT-PCR was adopted to select the recombinant plasmid which showed the most optimal inhibition effect. RT-PCR and Western blot were used to detected the mRNA and protein expression of Sema4D in SKOV3 cells tranfected for 24, 48 and 72 hours. MTT assay was used to investigate the proliferation of the SKOV3 cells after trasnsfection. Transwell cell migration and invasion assays were used to investigate the migration and invasion abilities of the SKOV3 cells after trasnsfection. Human ovarian cancer model was established in nude mice, and the nude mice were treated with pshRNASema4D-B once every 3 days for 3 weeks. The bulk of the transplanted tumor was measured.</p><p><b>RESULTS</b>Three Sema4D-targeted short hairpin RNA (shRNA) A, B and C were successfully inserted into the plasmid vector pshRNA, and the coding sequences of the obtained shRNA were consistent with the designed fragment. The results indicated that both recombinant plasmid pshRNASema4D-A and B could effectively knock down the expression of Sema4D gene in human ovarian cancer cells, of which pshRNASema4D-B was the better choice, while no effect of pshRNASema4D-C was seen. RT-PCR results showed that the relative mRNA expression of Sema4D gene in SKOV3 cells transfected with pshRNA-Sema4D for 24, 48 and 72 hours were 0.401 ± 0.051, 0.120 ± 0.035 and 0.014 ± 0.015, respectively, which were significantly lower than that in SKOV3 cells transfected by empty vector and non-transfected cells at 72 hours after transfection. (0.521 ± 0.019, 0.536 ± 0.040,respectively, P < 0.05). The Westen blot analysis manifested that the relative expression of Sema4D protein of SKOV3 cells transfected by pshRNASema4D for 24, 48 and 72 hours were 0.196 ± 0.023, 0.074 ± 0.015 and 0.040 ± 0.014, respectively, which were significantly lower than that in SKOV3 cells transfected by empty vector and non-transfected cells at 72 hours after transfection. (0.275 ± 0.009, 0.282 ± 0.015, respectively, P < 0.05). Comparing with the empty vector-transfected and non-transfected cells, the proliferation, invasion and migration ability of SKOV3 cells transfected with pshRNA-Sema4D were obviously weakened. The pshRNASema4D-B significantly suppressed the growth of the SKOV3 cells-transplanted tumors in nude mice, and the IR (inhibitory rate) of pshRNASema 4D-B group was (61.0 ± 3.3)% (P < 0.05).</p><p><b>CONCLUSIONS</b>Sema4D can be successfully silenced by RNA interfering in human ovarian cancer SKOV3 cells. Downregulation of Sema4D can inhibit the proliferation, migration and invasion of ovarian cancer cells. The pshRNASema4D can significantly suppress the growth in transplanted tumor of human ovary cancer in nude mice. Sema4D may become a candidate gene of gene therapy of human ovarian cancer.</p>
Assuntos
Animais , Feminino , Humanos , Camundongos , Antígenos CD , Genética , Metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Vetores Genéticos , Camundongos Nus , Invasividade Neoplásica , Transplante de Neoplasias , Neoplasias Ovarianas , Genética , Metabolismo , Patologia , Plasmídeos , Interferência de RNA , RNA Mensageiro , Metabolismo , RNA Interferente Pequeno , Genética , Proteínas Recombinantes , Genética , Metabolismo , Semaforinas , Genética , Metabolismo , Transfecção , Carga TumoralRESUMO
<p><b>OBJECTIVE</b>To evaluate the effects of PTEN on invasive and migration ability of human ovarian cancer cell line A2780 and related mechanisms.</p><p><b>METHODS</b>The plasmid including WT-PTEN and mutant PTEN were transferred into A2780 cells. The invasive and migration ability were measured before and after transfection by transwell chamber and wound-healing assays. The expression of PTEN protein and related proteins in the cells were detected by Western blot analysis. Empty plasmid-transfected A2780 and normal A2780 cells were used as control (the different four groups were named as WT-PTEN/A2780, C124A-PTEN/A2780, GFP/A2780 and A2780).</p><p><b>RESULTS</b>The number of penetrating cells was significantly less in WT-PTEN/A2780 cells (24.3 ± 2.5) than those in C124A-PTEN/A2780, GFP/A2780 and A2780 cells (43.7 ± 3.8, 44.7 ± 2.1 and 45.0 ± 3.0) (P < 0.05). The migration distance was markedly shorter in WT-PTEN/A2780 cells (54.1 ± 3.7) than those in C124A-PTEN/A2780, GFP/A2780 and A2780 cells (78.7 ± 3.4, 78.1 ± 3.1 and 76.8 ± 3.5) (P < 0. 05).</p><p><b>CONCLUSIONS</b>Transfection with PTEN may suppress the invasive and migration ability of ovarian cancer cell line A2780 depending on its phosphatase activity, and the suppressive effect may be due to the down-regulation of MMP-9 in the cancer cells.</p>
Assuntos
Feminino , Humanos , Linhagem Celular Tumoral , Movimento Celular , Regulação para Baixo , Vetores Genéticos , Metaloproteinase 9 da Matriz , Metabolismo , Mutação , Invasividade Neoplásica , Neoplasias Ovarianas , Metabolismo , Patologia , PTEN Fosfo-Hidrolase , Genética , Metabolismo , Fisiologia , Plasmídeos , TransfecçãoRESUMO
<p><b>OBJECTIVE</b>To investigate the involvement of apoptosis inducing factor (AIF) in caspase-independent pathway mediating apoptosis of cultured renal tubular epithelial cells induced by cisplatin (CP).</p><p><b>METHODS</b>Western Blot analysis and real-time PCR were performed to detect cytosol AIF (cAIF), nuclear AIF (nAIF) and AIF mRNA expression in cultured renal epithelial cells (HK-2) treated with cisplatin (CP) at various concentrations (0 - 200 micromol/L) and time courses (0 - 12 h). Immunofluorescence analysis was used to detect the AIF protein distribution in HK-2 cells. Pan-caspase inhibitor (Z-VAD-FMK) and AIF-siRNA treatment, TUNEL and flow cytometer were used to measure the suppression of apoptosis induced by CP in HK-2 cells.</p><p><b>RESULTS</b>The expressions of cAIF, nAIF protein and AIF mRNA were all increased to some extent in HK-2 cells treated with CP at various concentrations and time points. cAIF expression was 2.3-fold (P < 0.05) increased after 25 micromol/L CP treatment for 12 h and 1.7-fold (P < 0.01) increased after 50 micromol/L CP treatment for 3 h, compared with that of control groups, and showed a concentration- and time-dependent increment. The nAIF expression reached a peak (4.3-fold increase) (P < 0.005) after 150 micromol/L CP treatment for 12 h and 3.7-fold incease (P < 0.05) after 50 micromol/L CP treatment for 9 h, compared with that of the 25 micromol/L group and 3 h group, respectively. The expression of nAIF was approximately consistent with cleaved-PARP expressive pattern. Real-time PCR showed that AIF mRNA increased gradually with prolonged treatment with 50 micromol/L CP and reached a peak at 9 h. Immunofluorescence assay showed AIF translocation from cytosol to nuclei in some cultured HK-2 cells treated with CP. Applying pan-caspase inhibitor (Z-VAD-FMK) and AIF-siRNA to CP-treated HK-2 cells, the apoptotic rates were decreased by 60.1% and 39.2%, respectively. The inhibitory effect on HK-2 cell apoptosis was even more significant with combination of both Z-VAD-FMK and AIF-siRNA.</p><p><b>CONCLUSION</b>The AIF activation and translocation to nuclei with the increment of its mRNA expression mediates CP-induced apoptosis of renal tubular epithelial cells in vitro. It may provide a new therapeutic target for protecting from nephrotoxciity of cisplatin.</p>
Assuntos
Humanos , Clorometilcetonas de Aminoácidos , Farmacologia , Antineoplásicos , Farmacologia , Apoptose , Fator de Indução de Apoptose , Genética , Metabolismo , Inibidores de Caspase , Núcleo Celular , Metabolismo , Células Cultivadas , Cisplatino , Farmacologia , Citosol , Metabolismo , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Células Epiteliais , Biologia Celular , Metabolismo , Túbulos Renais , Biologia Celular , Transporte Proteico , Interferência de RNA , RNA Mensageiro , Metabolismo , RNA Interferente Pequeno , GenéticaRESUMO
<p><b>OBJECTIVE</b>To investigate the biological functions of TTG1A in liver fibrosis.</p><p><b>METHODS</b>Yeast two-hybrid system was used to screen proteins associated with TTG1A. Briefly, the coding sequence of TTG1A was cloned into pGBKT7 vector, and the recombinant plasmid was transformed into yeast cells AH109 ( a type), then these cells were mated with yeast cells Y187 (a type) transformed with human leukocyte cDNA library plasmid pACT2. The obtained diploid yeast cells were plated on synthetic dropout nutrient medium containing X-alpha-gal for double selection. The plasmids from positive colonies were transformed into E.coli and sequenced.</p><p><b>RESULTS</b>The recombinant yeast expression vector pGBKT7-TTG1A was successfully constructed. Nineteen TTG1A binding proteins, including Homo sapiens major histocompatibility complex, class II DP beta 1 (HLA-DPb1), Homo sapiens ribosomal protein L30 (RPL30), Homo sapiens nucleophosmin Homo sapiens nucleobindin 2 (NUCB2), Homo sapiens ash2, variant Gaucher disease and variant metachromatic leukodystrophy, MORF4L1, Homo sapiens ubiquitin-conjugating enzyme E2L3 (UBE2L3), APOA1, Homo sapiens lectin, and galectin 1, were identified.</p><p><b>CONCLUSIONS</b>This study may help to elucidate the molecular function of TTG1A.</p>
Assuntos
Humanos , Proteínas de Transporte , Genética , Clonagem Molecular , DNA Complementar , Genética , Biblioteca Gênica , Genes Reguladores , Vetores Genéticos , Células Estreladas do Fígado , Cirrose Hepática , Genética , Análise de Sequência com Séries de Oligonucleotídeos , Plasmídeos , Genética , Proteínas Ribossômicas , Genética , Ativação Transcricional , Fator de Crescimento Transformador beta1 , Genética , Técnicas do Sistema de Duplo-Híbrido , Leveduras , GenéticaRESUMO
<p><b>OBJECTIVES</b>To construct a cDNA subtractive library of genes transactivated by TGF beta 1 in LX02 hepatic stellate cells (HSC); to screen and to clone the regulated genes transactivated by TGF beta 1; and to elucidate the molecular biological mechanism of hepatic fibrosis mediated by TGF beta 1.</p><p><b>METHODS</b>mRNA was isolated from HSC treated with TGF beta 1 or with PBS (as controls). Suppression subtractive hybridization (SSH) technique was employed to analyze the differentially expressed DNA sequence between the two groups. After restriction enzyme Rsa I digestion, small size cDNAs were obtained. Then tester cDNA was divided into two groups and ligated to specific adaptor 1 and adaptor 2, respectively. After tester cDNA was hybridized with driver cDNA twice and underwent polymerase chain reaction twice it then was subcloned into pGEM-Teasy plasmid vectors to set up the subtractive library. Amplification of the library was carried out with E. coli strain DH5a. The cDNA was sequenced and analyzed in GenBank with Blast search.</p><p><b>RESULTS</b>The subtractive cDNA library of genes transactivated by TGF beta 1 in HSC was constructed successfully. The amplified library contained 146 positive clones, which contained 200-1000 bp of inserts. Randomly, thirty clones were analyzed by sequencing and bioinformatics, consisting of 28 known genes and 2 unknown genes.</p><p><b>CONCLUSIONS</b>The subtractive cDNA library of genes transactivated by TGF beta 1 in HSC using SSH technique was constructed successfully. Some gene coding proteins are those involved in cell growth regulation, protein synthesis, signal transduction, extracellular matrix metabolism, and anti-lipid peroxidative, which gives us some new clues for the study of the mechanism of liver fibrosis.</p>
Assuntos
Animais , Ratos , Linhagem Celular , Clonagem Molecular , Biblioteca Gênica , Vetores Genéticos , Células Estreladas do Fígado , Metabolismo , Hibridização de Ácido Nucleico , Métodos , RNA Mensageiro , Genética , Homologia de Sequência , Fator de Crescimento Transformador beta1 , GenéticaRESUMO
<p><b>OBJECTIVE</b>To investigate the effects of over-expression of decorin (DCN) gene on apoptosis of cultured rat mesangial cells (MsC).</p><p><b>METHODS</b>PcDNA3.1A-DCN plasmid was transfected into cultured rat MsC by the induction of liposome and positive clones were selected by treating the cells with G418. The MsC clones stably expressing DCN (MsC/DCN) were confirmed by cellular immunofluorescence, reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot. The DCN-siRNA was used for blocking DCN expression in MsC/DCN, and was confirmed by Western blot. The apoptosis of MsC was assayed by flow cytometry and Hoechst staining. Expression of Caspase-3 was assayed by Western blot.</p><p><b>RESULTS</b>Positive clones with DCN over-expression were established. The apoptotic rate in MsC/DCN was (20.40 +/- 8.01)% and was much higher than the (2.07 +/- 0.99)% in MsC (P < 0.01). Some of the MsC/DCN cells showed typical morphologic changes of apoptosis. The protein expression of active Caspase-3 was also significantly increased in MsC/DCN compared to MsC (P < 0.01). DCN-siRNA transfection not only significantly blocked the expression of DCN and reduced the rate of apoptotic cells, but also down-regulated the expression of active Caspase-3.</p><p><b>CONCLUSIONS</b>Over-expression of DCN induces apoptosis of cultured rat MsC in vitro. This effect of DCN inducing apoptosis suggests a novel strategy for regulating the proliferation of MsC in glomerular diseases.</p>
Assuntos
Animais , Masculino , Ratos , Apoptose , Células Cultivadas , Decorina , Proteínas da Matriz Extracelular , Farmacologia , Células Mesangiais , Proteoglicanas , Farmacologia , Ratos Sprague-DawleyRESUMO
<p><b>OBJECTIVE</b>To study the effect of interferon-gamma (IFN-gamma) on the proliferation of mesangial cells (MsC) and transforming growth factor (TGF)-beta/Smad signal pathway, the mRNA and protein expression of matrix metalloproteinase-2 (MMP-2) and tissue inhibitors of matrix metalloproteinase-2 (TIMP-2), and to provide an experimental basis for IFN-gamma treatment of renal fibrosis.</p><p><b>METHODS</b>Cultured MsC were treated with IFN-gamma at different concentrations and the proliferation of MsC was examined by MTT. Protein and RNA samples were extracted from MsC at 0, 0.5, 1, 2, 4, 6, 12, 24 h after treated by 100 IU/ml IFN-gamma. The mRNA and protein expression of Smad3, Smad7, MMP-2 and TIMP-2 were analyzed by real-time RT-PCR and Western blot, respectively.</p><p><b>RESULTS</b>The expression of Smad7 mRNA and protein were promptly elevated at 0.5 hour after the IFN-gamma treatment and lasted for 6 hours, but the proliferation of MsC was not altered. The elevated expression of Smad3, MMP2 mRNA and proteins persisted after 6 hours, whereas the expression of TIMP-2 mRNA and protein decreased.</p><p><b>CONCLUSION</b>The therapeutic effect of IFN-gamma of renal fibrosis may be mediated by TGF-beta/smads signal pathway through up-regulation of MMP-2 expression, coupled with down-regulation of TIMP-2 expression.</p>
Assuntos
Animais , Ratos , Proliferação de Células , Células Cultivadas , Interferon gama , Farmacologia , Metaloproteinase 2 da Matriz , Genética , Metabolismo , Células Mesangiais , Biologia Celular , Metabolismo , RNA Mensageiro , Metabolismo , Proteínas Recombinantes , Transdução de Sinais , Proteína Smad3 , Genética , Metabolismo , Proteína Smad7 , Genética , Metabolismo , Inibidor Tecidual de Metaloproteinase-2 , Genética , Metabolismo , Fator de Crescimento Transformador beta , MetabolismoRESUMO
<p><b>OBJECTIVE</b>To study the clinicopathologic features of microscopic polyangitis (MPA), and to compare the differences in anti-neutrophil cytoplasmic autoantibody (ANCA)-positive and ANCA-negative patients, as well as in ANCA-positive cases with or without glomerular immunoglobulin deposition.</p><p><b>METHODS</b>Thirty-four biopsy-proven cases of MPA were retrieved from the archival files of the Department during the past 7 years. The clinicopathologic characteristics between ANCA-positive and negative patients, as well as between ANCA-positive cases with and without glomerular immunoglobulin deposition, were compared.</p><p><b>RESULTS</b>Amongst the 34 MPA patients studied, about one-fifth to one-half were accompanied by various extrarenal symptoms. Serum ANCA was positive in 26 patients (76.5%). A slight to moderate increase in urinary protein was demonstrated in 31 patients, while 3 patients had nephrotic syndrome. Elevated serum creatinine was detected in 32 cases. Renal biopsy revealed crescentic glomerulonephritis in 24 cases, focal segmental glomerulonephritis in 8 cases, vascular fibrinoid necrosis with inflammation in 7 cases, intimal thickening of arterioles in 24 cases, interstitial inflammatory cells, including neutrophil infiltration (21 cases), in 29 cases. Crescentic formation was more common in the ANCA-positive group than in the ANCA-negative group (P < 0.05). Amongst the 26 ANCA-positive cases, 10 had glomerular immunoglobulin deposits (including 1 case with IgA nephropathy). In general, these cases had a greater degree of proteinuria than those without glomerular immunoglobulin deposits (P < 0.05).</p><p><b>CONCLUSIONS</b>The diagnosis of MPA relies on histologic examination of renal biopsy and clinicopathologic correlation. Serum ANCA seems important for glomerular crescent formation. Glomerular immunoglobulin deposition may also play a significant role in the exacerbation of proteinuria.</p>
Assuntos
Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Anticorpos Anticitoplasma de Neutrófilos , Metabolismo , Biomarcadores , Biópsia , Glomerulonefrite , Metabolismo , Patologia , Isotipos de Imunoglobulinas , Metabolismo , Rim , Patologia , Nefropatias , Metabolismo , Patologia , Síndrome Nefrótica , Metabolismo , Patologia , Proteinúria , Patologia , Estudos Retrospectivos , Vasculite , Metabolismo , PatologiaRESUMO
<p><b>OBJECTIVE</b>To study the role of connective tissue growth factor (CTGF) in the development of glomerulosclerosis by experimental alteration of fibronectin (FN) and Type IV collagen (Col IV) expression in cultured rat mesangial cells (MsC).</p><p><b>METHODS</b>CTGF expression vector was transfected into MsC by Lipofectimine method. Protein and mRNA expression levels of CTGF, FN and Col IV were studied by Western blot and reverse transcription-polymerase chain reaction (RT-PCR) respectively.</p><p><b>RESULTS</b>Two of MsC clones (MCT-1 and MCT-2) with CTGF overexpression were successfully established and found to have significant increases of FN and Col IV at both protein and mRNA levels. Compared with the controls, the expression of FN protein and mRNA in the two clones were 3.2 times (P < 0.05) and 2.9 times (P < 0.05) higher respectively. The expression of Col IV protein and mRNA was 3.8 times (P < 0.01) and 2.4 times (P < 0.01) higher respectively.</p><p><b>CONCLUSION</b>CTGF up-regulates FN and Col IV expression in MsC and may play an important role in the development of glomerulosclerosis.</p>
Assuntos
Animais , Ratos , Western Blotting , Células Cultivadas , Colágeno Tipo IV , Genética , Metabolismo , Fator de Crescimento do Tecido Conjuntivo , Genética , Metabolismo , Fibronectinas , Genética , Metabolismo , Vetores Genéticos , Genética , Células Mesangiais , Biologia Celular , Metabolismo , RNA Mensageiro , Genética , Metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
Objective To examine expression of PTEN gene in ovarian cancer cisplatin-sensitive cell line OV2008 cells and cisplatin-resistant cell line C13K cells,and evaluate the effect of wild-type PTEN gene on reversing cisplatin-resistance of C13K cells and underlying mechanisms.Methods The expression of PTEN mRNA and protein in OV2008 and C13K cells were detected by semi-quantitative RT-PCR and western blot.Recombinant eukaryotic expression plasmid containing human wild-type PTEN gene was transfected into C13K cells by lipofectamine 2000.The expression of PTEN mRNA was monitored by RT- PCR and the expression of PTEN,protein kinase B(AKT),phospho-AKT(p-AKT)protein were analyzed by western blot in PTEN transfected and untransfected C13K cells.Proliferation and chemosensitivity of cells to cisplatin were measured by methyl thiazolyl tetrazolium(MTT),and cell apoptosis was detected by flow cytometry after treatment with cisplatin.Results(1)The expression of PTEN mRNA and protein(1.02 ?0.05,1.02?0.07)in OV2008 cells were significantly higher than those in C13K cells,which were 0.45 ?0.03 and 0.55?0.03 respectively(P