目的:分析肾间质纤维化(RIF)早期的蛋白质组表达变化情况,为探讨干预RIF的新靶点奠定基础.方法:(1)48只雄性Wistar大鼠,随机分为正常对照(control)组(6只)、假手术(sham)组(6只)和单侧输尿梗阻(UUO)模型组(36只).UUO组行右侧输尿管结扎,并分别于术后第3、7、11、14、21和28天各处死6只,取肾脏标本行HE染色和Masson染色,光镜下观察病理变化;control组不作任何处理,sham组开腹后只分离右输尿管但不结扎,两组均于第28天取肾脏标本作病理观察;(2)取UUO术后第7天大鼠梗阻侧和健侧肾组织的蛋白质提取液行双向凝胶电泳,两侧对照找出差异蛋白质,选取部分差异蛋白点进行质谱鉴定;(3)Western blot检测UUO大鼠肾脏组织膜联蛋白A5(annexin A5)表达的动态变化.结果:(1)UUO各组梗阻肾脏逐渐出现肾小管扩张、炎症细胞浸润、肾小管上皮细胞萎缩、间质胶原纤维沉积等变化,其中,肾梗阻后第7天开始出现间质纤维化;(2)与健侧肾比较,UUO术后第7天的梗阻侧肾出现≥1.5倍的差异蛋白质166个,其中上调81个,下调85个,质谱成功鉴定出19种差异蛋白质;(3)Western blot显示,各时段UUO组大鼠梗阻侧肾组织中annexin A5表达均明显高于sham组(P<0.05),其中以梗阻第3天组的表达量最高,随后逐渐下降,但仍高于sham组.结论:(1)RIF早期存在大量表达差异的蛋白质;(2)RIF的annexin A5表达显著增加,可能与某些病理变化存在密切关系.

目的:探讨沙利度胺(thalidomide,THD)和转化生长因子β1(transformation growth factor-β1,TGF-β1)对人胚肺成纤维细胞(human embryonic lung fibroblasts,HELF)中结缔组织生长因子(connective tissue growth factor, CTGF)基因启动子的作用及其分子机制.方法:利用CTGF 基因启动子驱动的萤光素酶报告基因系统观察TGF-β1 和THD 对HELF 细胞CTGF 基因启动子活性的影响;同时以带有生物素标记的CTGF 基因启动子序列作为探针,采用DNA pull-down 技术分析TGF-β1 和THD 对CTGF 基因启动子结合蛋白的影响.结果:TGF-β1 能显著增强HELF 细胞中报告基因的活性(P <0.01),而THD 以剂量依赖方式显著抑制TGF-β1 上调报告基因活性的效应(P <0.01).同时,TGF-β1 引起的CTGF 基因启动子结合蛋白变化也能被THD 所抑制(P <0.05).结论:CTGF 基因启动子结合蛋白调节可能参与TGF-β1 对HELF 细胞中CTGF 基因启动子的激活过程,而THD 能有效拮抗此过程.

AIM:To investigate the effects of thalidomide ( THD) on the activation of connective tissue growth factor ( CTGF) gene promoter induced by transforming growth factor β1 ( TGF-β1 ) in human embryonic lung fibroblasts ( HELF) .METHODS:DNA sequence of CTGF gene promoter was cloned into luciferase reporter gene vector to construct the recombinant eukaryotic expression vector pGL 3-CTGFP, and the recombinant vector was transfected into HELF cell line.The effects of TGF-β1 and THD on the activation of CTGF gene promoter were detected by dual-luciferase analysis . RESULTS:TGF-β1 increased the reporter gene activity dose-dependently (P<0.05), with a plateau at 5 μg/L being 2.16 folds as high as the control .TGF-β1-induced increase in the reporter gene activity was also time-dependent ( P<0.05).After exposure to TGF-β1(5 μg/L), the level of luciferase activity reached its peak at 12 h and was 2.52 folds as high as the control .THD significantly inhibited TGF-β1-induced increase in the reporter gene activity in a dose-dependent manner , but its basal activity was not changed .CONCLUSION: TGF-β1 stimulates the transcriptional activity of CTGF gene promoter in HELF cells in a dose-and time-dependent manner , while THD may inhibit the effects dose-dependently .

Sepsis remains a major cause of mortality in intensive care units, better therapies are urgently needed. Gram-negative bacterial lipopolysaccharide (LPS) is an important trigger of sepsis. We have demonstrated that berberine (Ber) protects against lethality induced by LPS, which is enhanced by yohimbine (Y) pretreatment, and Ber combined with Y also improves survival in septic mice. However, the precise mechanisms by which Y enhances protection of Ber against LPS - induced lethality remain unclear. The present study confirmed that simultaneously administered Y also enhanced protection of Ber against LPS-induced lethality. Ber or/and Y attenuated liver injury, but not renal injury in LPS-challenged mice. Ber or/and Y all inhibited LPS-stimulated IκBα, JNK and ERK phosphorylation, NF-κB activation as well as TNF-α production. Ber also increased IL-10 production in LPS-challenged mice, which was enhanced by Y. Furthermore, Ber or/and Y all suppressed LPS-induced IRF3, TyK2 and STAT1 phosphorylation, as well as IFN-β and IP-10 mRNA expression in spleen of mice at 1 h after LPS challenge. Especially, Y enhanced the inhibitory effect of Ber on LPS-induced IP-10 mRNA expression. In vitro experiments further demonstrated that Y significantly enhanced the inhibitory effect of Ber on TNF-α production in LPS-treated peritoneal macrophages, Ber combined with Y promoted LPS-induced IL-10 production and LPS-stimulated IκBα, JNK, ERK and IRF3 phosphorylation and NF-κB activation were also suppressed by Ber or/and Y pretreatment in peritoneal macrophages. Taken together, these results demonstrate that Y enhances the protection of Ber against LPS-induced lethality in mice via attenuating liver injury, upregulating IL-10 production and suppressing IκBα, JNK, ERK and IRF3 phosphorylation. Ber combined with Y may be an effective immunomodulator agent for the prevention of sepsis.

利用Blackboard网络教学平台开设病理生理学病例讨论教学,培养医学生自主学习、综合分析解决问题能力,融会贯通各基础学科知识,了解临床病例的多样性和复杂性,培养缜密的临床思维,为医学生的临床学习打下坚实的理论基础和思想基础。

AIM:To explore the effect of hyperhomocysteinemia on the Alzheimer-like pathological changes in the brain of aged rat.METHODS: The rats were treated with homocysteine(Hcy) by intravenous injection through vena caudalis with or without a simultaneous supplementation of folate and Vitamin B12(FB) in the drinking water for 28 weeks.The distribution and aggregation of tau protein were detected by Bielschowsky silver staining.Western blotting was used to determine the protein levels of tau phosphorylation,glycogen synthase kinase 3β(GSK-3β) and protein phosphatase 2A(PP2A).RESULTS: Treatment with Hcy induced hyperhomocysteinemia in aged rats and resulted in the formation of neurofibrillary tangles in the brain.Supplementation of FB effectively reduced the tangles.Hyperhomocysteinemia also induced Alzheimer-like tau hyperphosphorylation at multiple sites(Ser199/202and Ser396).Hyperhomocysteinemia activated GSK-3β and inhibited the activity of PP2A.Supplementation of FB alleviated the changes of tau and the activities of GSK-3β and PP2A.CONCLUSION: Supplementation of FB ameliorates the hyperhomocysteinemia-induced Alzheimer-like pathological changes of tau protein possibly through regulating the activity of GSK-3β and PP2A in aged rats.

目的:探讨川芎嗪对肺心病肺动脉压的影响及其机制.方法:将肺心病患者随机分为常规治疗组和常规治疗+川芎嗪治疗组(川芎嗪组),测定两组治疗前、后的右室射血前期时间与肺动脉血流加速时间比值(RVPEP/AT)的变化,作为衡量肺动脉压的指标;此外,测定健康组及两治疗组治疗前、后的血浆血管性假血友病因子(vWF)、内皮素-1(ET-1)和一氧化氮(NO)含量的变化.结果:治疗前两治疗组血浆vWF和ET-1明显高于健康组,而血浆NO明显低于健康组(P<0.01).两治疗组治疗后RVPEP/AT比值、血浆vWF和ET-1含量均低于治疗前,而血浆NO高于治疗前(p<0.01);但所有这些变化均以川芎嗪组的变化程度更明显(P<0.01).两治疗组血浆ET-1含量与RVPEP/AT比值呈正相关(r=0.591,P<0.01);两治疗组血浆NO含量与RVPEP/AT比值呈负相关(r=-0.617,P<0.01).结论:川芎嗪有助于肺心病患者肺动脉高压的缓解,其机制可能与保护肺血管内皮细胞、重建血管活性因子平衡有关.

目的:观察川芎嗪对慢性肺心病急性发作期患者血浆血管性假血友病因子(vWF)含量、丙二醛(MDA)含量和超氧化物岐化酶(SOD)活性的影响,探讨川芎嗪缓解肺心病的机制。方法:将100例肺心病患者随机分为常规治疗组和川芎嗪治疗组各50例,测定两治疗组治疗前后血浆vWF含量、MDA含量和SOD活性的变化,并以健康组作对照。结果:与健康组相比,肺心病急性发作期患者血浆vWF含量、MDA含量均明显升高(P<0.01);两治疗组治疗后vWF含量、MDA含量均明显低于治疗前(P<0.01),且川芎嗪组的下降程度比常规组更明显(P<0.01)。而两治疗组治疗前的SOD活性明显低于健康组(P<0.01),治疗后血浆SOD活性均高于治疗前(P<0.05,P<0.01);且治疗后血浆SOD活性的升高程度,川芎嗪组比常规组更明显(P<0.01)。结论:川芎嗪可能通过减轻脂质过氧化反应及提高机体抗氧化能力,从而达到保护肺血管内皮细胞、缓解病情的作用。

目的探讨肺癌患者血清单核细胞趋化蛋白-1(MCP-1)含量和单核细胞趋化活性(MCA)的变化规律。方法采用ELISA测定肺癌组和健康对照组血清MCP-1的含量,并以Boyden小室趋化实验测定两组血清MCA。结果肺癌组血清MCP-1含量明显高于健康对照组(P<0.01),但肺癌组血清MCA却明显低于健康对照组(P<0.01)。肺癌组血清MCP-1含量与MCA的相关系数(r=0.39)明显低于健康对照组(r=0.87)。结论肺癌患者存在单核细胞趋化活性不足。

Objective To construct eukaryotic cell expression plasmid of pmCherry-C2 fusion protein and analysis its expression and intracellular localization in NIH3T3 cell. Methods Septin 8 coding region was amplified by PCR from human glioma cells cDNA library and subcloned into pmCherry-C2 vector. After comfirmed by PCR, enzyme digestion and DNA sequencing,the construct was then transfected into NIH3T3 cells and analyzed by fluorescence microscopy. Results pmCherry-C2/septin 8 fusion protein was highly expressed in NIH3T3 cell. Fluorescence microscope analysis revealed that the fusion protein is mainly distributed in the cytoplasm. Conclusion The pmCherry-C2 / septin 8 fusion protein plasmid has been successfully constructed and expressed in NIH3T3 cell, which laid the foundation for the functional analysis of septin 8 in the future.

OBJECTIVE To construct pNTAP-MK2 eukaryotic expression plasmid and establish a HEK293 cell line stably expressing tandem affinity purification (TAP)-tagged MK2. METHODS The MK2-encoding region was subcloned into the vector pNTAP to construct the recombinant plasmid pNTAP-MK2, which was subsequently transformed into DH5 alpha.E.coli. After identification by PCR, digestion with restriction endonuclease and sequencing, the recombinant expression plasmid was transfected into HEK293 cells via liposome, and the cell line with stable expression of exogenous TAP tag-MK2 gene was selected by antibiotic G418. The expression and localization of the fusion protein TAP tag-MK2 were detected by Western blotting and immunofluorescence assay. RESULTS The results of PCR, restriction endonuclease digestion and sequencing all confirmed the correct construction of the recombinant eukaryotic expression plasmid pNTAP-MK2. Western blotting showed that the recombinant plasmid was expressed stably in HEK293 cells after transfection with G418 selection. Immunofluorescence assay identified the expression product TAP tag-MK2 mainly in the cell nuclei. CONCLUSION The eukaryotic expression vector pNTAP-MK2 has been successfully constructed, and in the established cell line with stable expression of TAP tag-MK2, TAP tag does not influence the localization of exogenous MK2.

AIM: To construct pNTAP-PRAK eukaryotic expression plasmid and to establish a stable HEK293 cell line expressing tandam affinity purification ( TAP)-tagged PRAK. METHODS: Human PRAK coding region was subcloned into pNTAP vector to construct a recombinant plasmid called pNTAP-PRAK,then DH5α E. coli was transformed with the recombinant plasmid. After identified by PCR,digestion with restriction endonuclease and sequencing,the correct recombinant expression plasmid was transfected with PolyFect liposome transfection reagent to HEK293 cells. The cell line with stable expression of exogenous TAP tagged-PRAK gene was established by screening of antibiotic G418. The expression and localization of the fusion protein TAP tagged-PRAK were detected by Western blotting and immunofluorescence assay. RESULTS: All the results of identification by PCR,digestion with restriction endonuclease and sequencing indicated that the recombinant eukaryotic expression plasmid pNTAP-PRAK was constructed correctly. The result of Western blotting showed that the recombinant plasmid was expressed stably in HEK293 cells after transfection followed by G418 screening. The result of immunofluorescence assay showed that the expression product TAP tagged-PRAK distributed mainly in the nucleus. CONCLUSION: The eukaryotic expression vector pNTAP-PRAK was successfully constructed and the cell line stably expressing TAP tagged-PRAK was established. TAP tag didn't influence the localization of exogenous PRAK.

BACKGROUND:The actin and Tau protein are important components of microfilament and microtubule,whether actin and Tau protein can be served as observation indexes of microfilament and microtubule? OBJECTIVE:To construct eukaryotic cell expression vectors of actin and Tau fused with enhanced green fluorescent protein(EGFP)or red fluorescent protein(RFP),further more,to study their expressions and intracellular localization in eukaryotic cells.DESIGN,TIME AND SETTING:A single sample experiment was conducted at the Department of Pathophysiology of Southern Medical University and Key Function Proteomics Laboratory of Guangdong Province from March to August 2008.MATERIALS:The eukaryotic vectors pcDNA3-actin,pcDNA3-Tau,pmCherry-C2 and pEGFP-C2,as well as DH5αand NIH3T3 cell lines were preserved at the Department of Pathophysiology of Southern Medical University.METHODS:The coding sequences of actin and Tau in pcDNA3 vectors were sub-cloned into pEGFP-C2 or pmCherry-C2 vectors,followed by transfected into NIH3T3 cells,the intracellular localization of their expression products were observed with fluorescence microscope.MAIN OUTCOME MEASURES:The expressions and localization of four recombinant plasmids were detected by Leica fluorescence microscope at 24 hours after transfected with NIH3T3.RESULTS:The four recombinant plasmids were verified by enzyme digestion,PCR and sequencing,and the fusion proteins were highly expressed in NIH3T3 cells.With fluorescence microscope observation,EGFP-or RFP-tagged actin distributed mainly in the cytoplasm as short,filamentous structures,while EGFP-or RFP-tagged Tau was radial around the nucleus.CONCLUSION:The fusion expression vectors of pEGFP-Tau,pEGFP-actin,pmCherry-Tau and pmCherry-actin proteins are successfully constructed and expressed efficiently in eukaryotic cells,which providing an important tool for studying the effect of cytoskeleton on signaling molecules translocation.

AIM:To investigate the growth-inhibiting and apoptosis-inducing effect of Kou on human glioma cell line U251 in vitro.METHODS:MTT assay and trypan blue exclusion method were applied to observe the growth-inhibiting effect of Kou on human glioma U251 cells.The apoptosis-inducing effects of Kou on U251 were examined with flow cytometry(FCM),laser scanning confocal microscope(LSCM) and electron microscope(EM).RESULTS:MTT assay showed that Kou had remarkable inhibitory effect on U251 in the concentration ranges of 5-50 mg/L,and cell curve measurement results indicated the inhibitory effect displayed in a time-dependent manner.FCM assay showed a evident apoptosis peak and the apoptotic rate was 22.4%.Marked morphological changes such as karyopycnosis and nuclear fragmentation of cell apoptosis were observed very clearly by Hoechst 33342 fluorescence staining.Typical early apoptotic appearance was also seen under EM.CONCLUSION:Kou exhibits dose-and time-dependent growth-inhibiting effects on U251 cells in a certain concentration range,its antineoplastic characteristics are related with tumor its apoptosis-inducing effect on cells.

AIM:To clone the promoter sequence in 5' non-coding region(NCR) of murine manganum superoxide dismutase gene(MnSOD,SOD2),and to identify the transcription activity of SOD2 promoter at rest or induced by 1ipopolysaccharide(LPS),NaAsO2,etc in murine embryonic fibroblasts(MEF) by the reporter gene technology.METHODS:Genomic DNA was extracted from mouse liver,and the promoter sequence of SOD2(-1 554～+48) was amplified by polymerase chain reaction(PCR) method.The red fluorescent protein reporter gene vector driven by SOD2 promoter was constructed by gene recombination technique.The recombinant vector was transiently transfected into MEF cells.The expression of the red fluorescent protein was observed by fluorescent microscopy in MEF cells at rest or stimulated by NaAsO2,LPS or phorbol-12-myristate-13-acetate(PMA).RESULTS:SOD2 promoter(-1 554～+48) of mouse was correctly amplified and the red fluorescent protein reporter gene vector was successfully constructed.It was observed that the MEF cells transfected with reporter gene vector only expressed few and weak red fluorescent protein at rest.However,red fluorescent signals were evidently enhanced after the cells were stimulated by NaAsO2,LPS or PMA.CONCLUSION:SOD2 promoter(-1 554～+48) of mouse has the transcriptional activity at rest,and the expression of SOD2 has enhanced after the cells are stimulated by inflammatory substance or oxidative stress.The reporter gene vector driven by SOD2 promoter will provide an experimental tool for the further study on the regulatory mechanism of the SOD2 expression.

p38 regulated/activated protein kinase (PRAK) plays a key role in cell senescence and tumor suppression. The aim of this study was to investigate if PRAK had effect on cell proliferation. The growth of PRAK +/+ and PRAK −/− mouse embryonic fibroblast (MEF) cells was measured by methylthiazoletetrazolium (MTT) colorimetric assay, and the proportion of the cell number in different phases of the cell cycle was analyzed by flow cytometry. The growth curves showed that the growth rate was notably decreased, and cell double time was elongated in PRAK −/− cells; moreover, the number of PRAK −/− cells was decreased by 44.5% compared with that of PRAK +/+ cells cultured for 96 h, suggesting that G 2 /M transition is inhibited in PRAK −/− cells. Meanwhile, G 1 /S transition was also inhibited in PRAK −/− cells, observed with flow cytometry analysis. The ratios of G 0 /G 1 , G 2 /M, and S phases of PRAK +/+ cells were 44.9%, 12.2%, and 42.9%, respectively, while those of PRAK −/− cells were 55.3%, 7.3%, and 37.4%, respectively. There were 23.1% increase and 12.7% decrease of the number of PRAK −/− cells in G 1 and S phases in comparison with that of PRAK +/+ cells, respectively. Taken together, PRAK gene knockout in MEF cells leads to cell cycle arrest and proliferation inhibition.

Objective:To study the intracellular localization of MAPK-activated protein kinase 2(MAPKAPK2/MK2) and its relation with the function.Methods:Eukaryotic cell expression vectors of green fluorescent protein fused with MAPK-activated protein kinase 2(MK2) mutants were constructed.Wild-type MK2 cloned in the green fluorescent protein vector,pEGFP-C2,was site-mutated to produce dominant negative mutant MK2(320A) and constitutively active mutant MK2(320E).Then the mutants were transfected into NIH3T3 cells,and the intracellular localizations of these mutants were observed with fluorescence microscope.Results:After identification by sequencing,the GFP-fused MK2 mutants were highly expressed in NIH3T3 cells.The green fluorescence of fusion proteins showed that in resting cells,MK2(WT) were mainly in the nuclei,and MK2(320E) was mainly in the cytosol,while MK2(320A) dispersed all over the cell.Once the cells were stimulated by UV,MK2(WT) translocated into the cytosol,while the intracellular localizations of MK2(320E) showed no stress-dependent redistribution.Conclusions:Expression vectors with different mutants of MK2 fused with GFP are successfully constructed and highly expressed in eukaryotic cells.Some functional point mutations have effects on the intracellular localizations of MK2.

AIM:To investigate the role of p38 mitogen-activated protein kinase(MAPK)in the cell apoptosis induced by arsenite.METHODS:After the treatment with or without arsenite,p38+/+ and p38-/-cells were fixed with paraformaldehyde and stained with DAPI.The nuclear morphological changes were observed under a fluorescence microscope.p38+/+ and p38-/-cells were double-stained with Annexin V-FITC and PI,and the ratios of apoptotic cells were detected by flow cytometry.RESULTS:With the stimulation of arsenite,typical apoptotic morphological changes appeared in most p38-/-cells,yet only a few p38+/+ cells showed nucleus condensation.Consistently,the ratio of apoptotic p38-/-cells induced by arsenite significantly increased in comparison with that in the untreated cells(P0.01),and also much higher than that in p38+/+ cells stimulated with arsenite(P0.01).CONCLUSION:p38 gene knockout leads to a decrease in cellular resistance to stress of arsenite,which produces a proapoptotic state.p38 MAPK may play an important role on the enhancement of cell adaptive capability to harmful stresses.

Objectives: To observe the relation of serum supersensitivity C reactive protein and different syndromes of cerebral infarction,and the degree of nervous system defect of different syndromes of cerebral infarction,and to analyse the relations.Offer the gist of clinical diagnosis and syndrome differentiation of TCM of cerebral infarction.Methods: As for 82cases of cerebral infarction,the different syndromes were differed,serum hs-CRP,serum TG,TC and HDL-C were detected.The value of TC/HDL was calculated,and the degree of nervous system defect was evaluated.The relation of different syndromes of TCM was analysed.Results: The hs-CRP is manifest abnormality and the degree of nervous system defect are manifest in the patients with wind-phlegm stasis,qi deficiency with blood stasis,and have significant deviation with those in patients with yin deficiency and liver wind.The values of TC,TG and TC/HDL have no significant deviation in the three syndromes.The value of hs-CRP has positive correlation with that of TG,TC and TC/HDL.The value of hs-CRP has correlation with the degree of patients with nervous system defect.Conclusions: The rise of the value of hs-CRP is the important index of phlegm syndrome,blood stasis of cerebral infarction.The rise of the value of hs-CRP maybe become one of the index of appraisal of patient's condition.

OBJECTIVETo construct different mutants of human p53 for expression in eukaryotic cells and investigate the effects of these mutants on stress-induced cell apoptosis.METHODSHuman p53 cDNA was amplified by PCR and cloned into pcDNA3/HA vector following the routine procedures. The Ser15 and Ser46 of p53 were mutated to Ala and identified by enzyme digestion and PCR, and these mutants were expressed in NIH3T3 cells and detected by Western blotting. After transfection with the plasmids of different p53 mutants, the NIH3T3 cells were double-stained with AnnexinV-FITC and propidium iodide for apoptotic analysis using flow cytometry.RESULTSThe recombinant plasmids of HA-tagged wild-type p53, HA-p53(WT), and its mutants, HA-p53(S15A) and HA-p53(S46A), were successfully constructed and expressed efficiently in NIH3T3 cells. The apoptotic ratio of p53(WT)-transfected cells induced by arsenite increased and that of p53(S15A)-transfected cells decreased significantly after arsenite stimulation, but no significant changes occurred in the apoptosis of p53(S46A)-transfected cells.CONCLUSIONThe phosphorylation on Ser15 of p53 plays an important role in mediating arsenite-induced cell apoptosis.