Objective To investigate the correlation between plasma proteasome and endothelial dysfunction in patients with uremia. Methods Forty-five uremic patients who did not receive hemodialysis were defined as A group; seventy-five uremic patients who had received hemodialysis for 6 to 12 months were divided into sufficient hemodialysis group (44 cases,B group)and insufficient hemodialysis group (31 cases,C group).The primary disease of these patients was chronic glomerulonephritis.Fifteen healthy people were defined as healthy control group (D group).The diameter of radial artery lumen (DRL),intima-media thickness (IMT),intima-media area (IMA),endothelium-dependent or independent dilation (EDD or EID) of radial artery in right forearm were detected by diasonography.The levels of 20S proteasome,tumor necrosis factor α (TNF-α),C-reaction protein (CRP) and transforming growth factor β 1 (TGF-β1) of plasma and supernatant of cultured human umbilical veins endothelium (HUVEC) were determined by enzyme linked immunosorbent assay (ELISA).20S proteasome activity was analyzed by special substrate.Results Compared with D group,the level and activity of 20S proteasome,as well as TNF-α,CRP and TGF-β1 in A,B and C groups were significantly increased.Compared with A group,these plasma indices levels were significantly decreased in B group but strongly increased in C group.IMT and IMA were elevated,while DRL,EDD and EID were decreased significantly in A,B and C groups when compared with D group.These parameters were worse in C group than those in A and B groups.After co-culture of HUVEC with above mentioned human uremic serum,the level and activity of 20S proteasome and TNF-α were higher in A,B,C groups than that in D group.In A and C groups,there were negative correlations of EDD with the level or activity of 20S proteasome,TNF-α,CRP and TGF-β1,and there were positive correlations of 20S proteasome level or activity with TNF-α,CRP and TGF-β1. Conclusions 20S proteasome level and activity are significantly increased in uremic patients.There is a close correlation between 20S proteasome and endothelial dysfunction of radial artery.

Objective To study the effect of adenine nucleotide translocase 1(ANT1) gene over-expression on apoptosis of vascular smooth muscle cells(VSMC) in a rat carotid balloon injury model induced by adenovirus plasmid.Methods Seventy-two SD rats were randomly divided into normal group,non-transfection group,Ad-GFP transfection group and Ad-ANT1 transfection group(18 in each group).ANT1 gene was transfected into rat carotid arteries with Ad-ANT1 adenovirus after a rat carotid balloon injury model was established.The arteries were harvested on days 7,14 and 28 after operation.Expression of ANT-1,BAX,and Bcl-2 in arteries was detected by RT-PCR,Western blotting,and immunohistochemistry,respectively,with HE staining.Apoptosis of VSMC in tunica intima and tunica media of the model was assayed with TUNEL staining.Results The ANT1 gene was significantly expressed in rat carotid arteries after Ad-ANT1 transfection and reached its peak level on day 14,which was significantly higher in Ad-ANT1 transfection group than in Ad-GFP transfection group and balloon injury group(P<0.01).The expression level of BAX was significantly higher in Ad-ANT1 transfection group than in Ad-GFP transfection group and balloon injury group on days 7 and 14 after transfection(P<0.05).The expression level of Bcl-2 was higher in Ad-ANT1 transfection group than in normal group(P<0.05).However,no significant difference was found in expression level of Bcl-2 between the two groups(P>0.05).The apoptosis rate of VSMC in tunica intima and tunica media of the model was significantly higher in Ad-ANT1 transfection group than in the other 3 groups(P<0.05).The tunica intima/ tunica media area ratio in the model was lower in Ad-ANT1 transfection group than in Ad-GFP transfection group and balloon injury group on days 14 and 28 after transfection(P<0.05).Conclusion Adenovirus plasmid-induced over-expression of ANT1 gene induces apoptosis of VSMC in tunica intima/ tunica media of the model by up-regulating the expression of BAX.

Objective To cultivate the medical students' better clinical competence,we explored the effection of clinical probation of diagnostics with the PBL teaching method.Methods Two groups of 120 students in grade 2007 were devided randomly into PBL and tradition teaching method on average,and to compared discrepancy of their teaching effection.Results The datas shows that the students in PBL group has better grasp of theory,clinical operated skill and other skill than that of tradition teaching group(P<0.01).Conclusion PBL improve the students' enthusiasm of study,the ability of clinical thought and problem-solving.It means that PBL is an effective teaching method.

Objective To construct the recombinant adenovirus of rattus ANT1 gene and observe its expression in vascular smooth muscle cells(VSMCs) in rat.Methods Rattuse ANT1 gene fragment was cloned into the shuttle plasmid pShuttle-GFP-CMV to form the transfer vector by the method of homogenous recombination in bacteria.Then the recombinant adenovirus was transfected into 293 cells using liposome DOTAP.The target gene was detected by polymerase chain reaction(PCR).The titer and infection rate of recombinant adenovirus were determined using green fluorescent protein(GFP) expression in shuttle plasmid.The expression of ANT1 protein in VSMCs after transfection with Ad-ANT1 by Western blotting.Results Restriction endonuclease and PCR analyses confirmed that the ANT1 gene was successfully inserted into the adenovirus vector.The titer of the recombinant adenovirus was 2×1011 pfu/ml.The adenovirus had a more strong effect on VSMCs in rat than that in control cells(P<0.05).Conclusions The recombinant adenovirus containing ANT1 is constructed successfully and ANT1 could be expressed effectively in VSMCs after transfection,which provides an experimental basis for studying the role of ANT1 gene in VSMC apoptosis.

Objective To identify the effects of adenovirus mediated adenine nucleotide translocase 1(ANT1) gene transfection on the apoptosis of rat vascular smooth muscle cells(VSMCs).Methods After adenovirus-mediated ANT1(Ad-ANT1) or adenovirus(Ad-GFP) infected rat primarily cultured VSMCs,Hoechst 33258 staining for the VSMCs was observed by laser confocal microscopy for cell apoptosis.Flow cytometric analysis was used to detect the proportion of VSMCs apoptosis with Annexin V staining.The expressions of Bcl-2 and Bax were also detected by Western blot analysis.Results Laser confocal microscopy showed a progressive increase in the percentage of apoptotic VSMCs after Ad-ANT1 infection [(13.40±1.14)% in Ad-ANT1 group vs(1.80±0.84)% in Ad-GFP group,P<0.01].Flow cytometric analysis indicated a significant increase in the percentage of apoptotic VSMCs after that Ad-ANT1 infection [(10.66±1.75)% in Ad-ANT1 group vs(3.13±0.37)% in Ad-GFP group,P<0.05].The results of Western blotting indicated that the Bax expression was upregulated in VSMCs transfected with adenovirus-mediated ANT1(Ad-ANT1) compared with cells of adenovirus-empty(Ad-GFP)group.Meanwhile,there is no significant difference in the Bcl-2 expression in two groups.Conclusion Overexpression of ANT1 gene may induce apoptosis of rat VSMCs through upregulating Bax expression.

Objective To determine the effect of costimulatory molecules V-set and immunoglobulin domain-containing protein 4(VSIG4) which is exclusively expressed in the macrophages on the proliferation and activation of T lymphocytes.Methods Mouse peritoneal macrophages from 8-week-old VSIG4 knockout(VSIG4-/-) mice and healthy wide-type(VSIG4+/+) mice(C57BL/6 mice) were collected,and then co-cultured with T cells respectively under the stimulation of CD3 monoclonal antibody.T cells cultured without macrophages but in the stimulation of CD3 monoclonal antibody served as control,and those without macrophages and CD3 monoclonal antibody as the blank control.Purified T cells was evaluated by -TdR incorporation assay.Activity of CD69 was tested by FACS.Levels of IL-2 and IFN-γ protein in the supernatants of the co-cultured cells were measured by enzyme-linked immunosorbent assay(ELISA),and mRNA expression of IL-2 and IFN-γ were detected by RT-PCR.Results In the two co-culture groups,the -thymidine incorporation,activity of CD69,cell mRNA and protein levels of IL-2 and IFN-γ were significantly lower than those in the control groups(P<0.05,P<0.01).And these indicators in the VSIG4+/+/T group were significantly declined when compared with those of VSIG4-/-/T group(P<0.05).Conclusion Costimulatory molecule VSIG4 specifically expressed in the macrophage might play a role in the process of inhibiting the proliferation and activation of T lymphocytes co-cultured macrophage.

腺嘌呤核苷酸转位酶(adenine nucleotide translocase,ANT)是位于线粒体内膜上的一类转运蛋白,既能将细胞的产能和耗能过程耦联起来,也与线粒体膜通透性转换孔(mitochondrial permeability transition pore MPTP)密切相关,MPTP是位于线粒体内外膜之间的多蛋白组成的高导离子通道,它的开放可诱导细胞凋亡。所以ANT是一种能量转运和凋亡诱导的双重功能蛋白。本文主要就其与细胞凋亡的关系做一综述。

Objective To investigate the effect of retinoic acid (RA) on chemokines and inflammation in kidney of UUO rats. Methods One hundred and thirty five healthy male SD rats were randomly divided into 3 groups (45 each):sham operation group,UUO group and RA treatment UUO group. Left ureters of the rats were ligated in both UUO and RA treatment group,while in sham group the ureters were isolated only without further manipulation. Each group was divided into 3 subgroups (15 each). Three RA subgroups were treated with daily subcutaneous injection of RA in a dose of 5,10 and 20mg/kg,respectively. The subgroups of the sham operation and UUO rats received same volume of dimethyl salfoxide and arachis oil. Five rats of each subgroup were sacrificed to get the kidneys on day 3,7 and 14 after operation. The area of renal tubular lesion and interstitial inflammation,and expression of chemokine were determined. The expressions of RANTES,CCR5,MCP-1,MIP-1α and TGF-β1 in kidney were assayed by immunohistochemistry and RT-PCR methods. Results No significant pathological change and expression of chemokine (RNTES,CCR5,MCP-1,MIP-1α) were detected in the renal tissues of sham operation group. Rats in UUO group showed progressive tubular cell damage,interstitial inflammation and fibrosis. Interstitial infiltration of macrophages and lymphocytes were found (P<0.05),and the protein content and mRNA expression of chemokine increased since the 3rd day up to 14th day (P<0.01). The interstitial inflammation and tubular lesion of RA group rats were dramatically reduced as compared with that in UUO group (P<0.05),and the protein and mRNA expression of chemokine were significantly decreased compared with those in UUO group (P<0.01). However,no significant difference in renal histology and chemokine expression was found among animals receiving different RA dosages. Conclusion RA may reduce renal interstitial inflammation and tubular lesion in UUO rats by down-regulating the expression of RANTES,CCR5,MCP-1 and MIP-1α.

Objective To explore the effects of retinoic acid(RA) on the expression of regulating activation of normal T expressed and secreted(RANTES),and to investigate the mechanism of RA on tubulointerstitial inflammation and fibrosis of unilateral ureteral obstruction(UUO) rat.Methods A total of 45 healthy male SD rats were randomly divided into three groups:the sham operation group rats received sham operation without ureteral obstruction;RA treated group and UUO group rats received left ureteral obstruction operation.Then,RA group rats were treated with daily subcutaneous injection of 5mg/kg of RA,other two group rats received same volume vehicle.There were 15 rats in each group.Five rats were killed at 3d,7d and 14d post operation for every group.The percentage of renal tubular lesion and interstitial inflammation,the protein and mRNA expression of TGF-β1,RANTES and its receptor CCR5 were assessed.Results In RA treatment group rats the interstitial inflammation and tubular lesion were significantly reduced(P0.05),and RANTES,CCR5 and TGF-β1 protein and mRNA expression were significantly lightened than those of UUO group rats(P0.01).Conclusion RA reduced interstitial inflammation and tubular lesion of UUO rat,this role might be through reducing the expression of RANTES,CCR5 and TGF-β1.