To evaluate the clinical characteristics of percutaneous coronary interventional (PCI) treatment in patients with chronic renal insufficiency, 116 patients with serum creatinine ≥141?mol/L and 352 patients who had normal serum creatinine level were included for the stady. It was found that the ineidences of using alcohol, acute myocardial infarction, hypertension, diabetes mellitus, as well as hospital stay days after PCI and the entire hospital stay days, and mean number of diseased vessels were significantly higher in renal insufficiency group compared with the control group. On the other hand, the value for HDL, the time for PCI operation, and the dose of contrast medium were lower in renal insufficiency group than in control group. The results suggested that patients with renal insufficiency were able to tolerate PCI if proper peri-operative care was observed, though they had more predisposing factors of arteriosclerosis.

Objective To improve disadvantages such as asynchrony during embryoid bodies(EBs)preparation,and to establish a novel method to prepare EBs by using attached mouse embryonic stem Cells.Methods Mouse R1 embryonic stem(ES)cells were trypsinized to a single cell suspension when reaching a sub-confluent state of 70 %～80 %,then 1?106 ES cells were plated into 100 mm tissue culture dishes.After being cultured in medium containing low concentration leukemia inhibitory factor(LIF,1 ?g/L)for 3 days,EBs were collected and suspended in EBs culture medium for further development.Morphology studies were performed on suspended EBs and their cryosections,then immunofluorescent staining of ?-fetoprotein(AFP),platelet endothelial cell adhesion molecule-1(PECAM-1),and neurofilament 68 KD(NF-68)were performed on suspended and attached EBs.Results EBs obtained by this method were homogeneous in size,synchronous in developmental stage;typical in structure and could well display the developmental process from simple EBs to mature cystic EBs.Immunofluorescent staining showed that AFP,PECAM-1 and NF-68 were positive.Conclusion Compared with present methods of preparing EBs,this novel method have advantages of simple manipulation,high efficiency of EBs formation and obtaining EBs at synchronous developmental stage,furthermore,these EBs have typical structures and the potential to differentiate into derivatives of all three embryonic germ layers.Thus this method can be used as an ideal tool for studies on early embryonal development,ES cell differentiation and so on.

Objective The aim of this investigation is to explore the effects of hypoxia on the biological activity of endothelial progenitor cells(EPC)and to improve the efficacy of EPC transplantation.Methods Rat bone marrow derived EPC were isolated and cultured either under normoxic or hypoxic conditions.The proliferation,migration and angiogenic ability of EPC were observed.Results In hypoxic group,the number of attached cells per high power field(hpf)was significantly more than that in normoxic group (91.0?8.0)vs(42.5?5.3),P

AIM: To investigate the effect of fibrinogen (Fg), fibrin (Fb) and fibrin degradation products (FDPs) on the proliferation and migration of human vascular smooth muscle cells (VSMC).METHODS: The effects of Fg, Fb and FDPs on the proliferation of VSMC were observed by means of cell counting and MTT test, migration assays were performed using the wounding model and the transwell cell culture apparatus.RESULTS: Fg itself did not stimulate the proliferation of VSMC, but stimulated VSMC migration. Fb and FDPs both stimulated the proliferation and migration of VSMC, meanwhile the effect of Fb was in a dose-dependent manner.CONCLUSION: Fb, in particular FDPs, may play an important role by stimulating the proliferation and migration of VSMC in restenosis and atherogenesis.

AIM: To confirm that the inflammation response after mechanical arterial injury correlates with the neointimal hyperplasia in animal model. METHODS: Male Wistar rats underwent left common carotid balloon angioplasty were injected twice with a bacterial lipopolysaccharide (LPS, 50 ng/rat) before and after surgery. Next, just after neointima formation, the animals were sacrificed for the evaluation by morphometric analysis, histological observation and immunostaining. Western blot was used to investigate the protein expression of several known mediators of apoptosis. RESULTS: Serum interleukin-1 beta levels as a marker of inflammation were increased after LPS treated. Early neotimal lesions were characterized by intimal thickening and the presence of SMCs. Neointima with smooth muscle alpha-actin negative were observed at 7 days after injured. These areas of neointima demonstrated a relatively high proliferation index by proliferating cell nuclear antigen (PCNA) antibody staining, whereas the proliferation index in media was low. Neointimal thickness was significantly increased at 4 weeks after injury in LPS treated animals compared with controls, from (151.2?14.5 to 173.9?15.3) ?m2. Activation of caspase-3 was observed, indicating that smooth muscle cells of neointima was associated with apoptosis. Immunofluorescence analysis revealed NF-?B expression located to the adventitia. CONCLUSIONS: Our results indicate that nonspecific stimulation of low-level LPS facilitates neointimal formation and may be an important factor in the restenosis of angioplasty.

OBJECTIVEThe purpose of this study was to develop RT- PCR assay for Rapidly detect and distinguish between Norovirus genogroup I and genogroup II with a pair of primers.

METHODSA pairs of primers specific to capsid prote in ORF2 gene of G I and G II Norovirus were dsigned according to the published complete genome sequence, with which the RNA of Norovirus was extracted and RT-PCR amplification. The sensitivity, specificity of the RT- PCR assay was estimated and apply it to the detection of Norovirus in clinical specimens.

RESULTSThe results showed that the assay possessed high specificity for Norovirus detection and without any evident cross-reaction with other viruses, including rotavirus, enteric adenovirus and hepatitis A virus. The detection limit of RT-PCR assay for Norovirus G I and G II were up to 100 pg/ml and 10 pg/ml respectively.

CONCLUSIONThe RT- PCR assay provide rapid and sensitive detection of Norovirus G I and G II and should prove to be useful for Norovirus diagnosis in the outbreaks of acute gastroenteritis.

Caliciviridae Infections ; diagnosis ; virology ; DNA Primers ; genetics ; Gastroenteritis ; diagnosis ; virology ; Humans ; Norovirus ; classification ; genetics ; isolation & purification ; Reverse Transcriptase Polymerase Chain Reaction ; instrumentation ; methods

Objective To explore if B lymphocyte stimulator (BlyS) stimulates B lymphocytes from patients with chronic idiopathic urticaria (CIU) to produce anti-high affinity IgE receptor (FcεRI) or anti-IgE antibodies.Methods Totally,300 CIU patients and 300 health controls were enrolled in this study.Blood samples were obtained from these subjects.Peripheral blood B lymphocytes were isolated and cultured in vitro for 72 hours.Then,BlyS of various concentrations (2,4,8,16 ng/ml) was added to the culture medium of B lymphocytes followed by another 72-hour culture.Enzyme-linked immunosorbent assay was performed to determine the serum levels of BlyS,anti-FcεRI and anti-IgE antibodies,as well as the supernatant levels of anti-FcεRI and anti-IgE antibodies.The relationship between BlyS and anti-FcεRI and anti-IgE antibody production was assessed.SPSS software version 16.0 was used for statistical analysis.Chi-square test was performed to compare the positivity rate of antibodies,and analysis of variance and least significance difference-t test to assess numerical data.Results The CIU patients showed higher levels of serum BlyS (t =3.04,P ＜ 0.01),anti-FcεRI antibodies (t =3.51,P ＜ 0.01),and anti-IgE antibodies (t =3.29,P ＜ 0.01) compared with the health controls.The serum level of BlyS was positively correlated with that of anti-FcεRI antibodies (r =0.93,P ＜ 0.01) and anti-IgE antibodies (r =0.91,P ＜ 0.01).The levels of anti-FcεRI antibodies and anti-IgE antibodies were significantly increased in the culture supematant of patient-derived B lymphocytes treated with BlyS compared with those remaining untreated (t =3.67,3.56,respectively,both P ＜ 0.01),and the concentration of BlyS was positively correlated with the levels of both anti-FcεRI antibodies and anti-IgE antibodies (r =0.96,0.91,respectively,both P ＜ 0.01).The coincidence rate between the serum and supernatant was 94.76％ and 87.84％ in the detection of anti-FcεRI antibodies and anti-IgE antibodies respectively.Conclusions BlyS level is upregulated in the serum of patients with CIU,which may play an important role in the pathogenesis of CIU by stimulating B lymphocytes to produce anti-FcεRI antibodies or anti-IgE antibodies.

OBJECTIVE:To investigate the possible association between the gene ALOX5AP encoding 5-lipoxygenase activating protein (FLAP)and coronary artery disease(CAD)in the Han population of North China.METHODS:A total of 680 cases underwent selective coronary angiography(SCA)from Shenyang General Hospital of Chinese PLA was recruited from January 2006 to September 2007.According to the results of SCA.680 cases were divided into CAD group with angiography positive(n=336)and control group with angiography negative or the stenosis of coronary arteries<50%(n=344)without evidence of cardiac ischemia.Single nucleotide polymorphisms of ALOX5AP gene was screened in 48 unrelated Han individuals of North China by polymerase chain reaction fPCR)-Re-sequencing method and 7 polymorphisms were found.The genotype and allele distribution of T(-1340)G polymorphism between two groups was determined by polymerase chain reaction and restriction fragment Iength polymorphism(PCR-RFLP)analysis in CAD and controI subjects.RESULTS:The genotype frequencies of TT,TG and GG in the ALOX5AP T(-1 340)G polymorphism were 26.79%,51 179%and 21.43%in CAD patients,33.72%,47.38%and 18.90%in the controls,respectively(x~2=3.90,P>0.06).The genotype distribution between two groups was in accordance with hardy-weinberg equilibrium.There are no significant differences in the distribution of three genotypes between the two groups.The frequencies of ALOX5AP G allele in cases and controls were 47.32%,42.59%,respectively(x~2=3.08,P>0.05).Subsequent stratified analysis by gender also showed no statistical significance in the genotype frequencies and allele frequencies between the two groups.CONCLUSION:The result suggests that T(-1340)G polymorphism of the ALOX5AP gene might not be associated with CAD in the Han population of North China.

BACKGROUND:Embryonic stem cells (ESCs) serve as a major cell source for smooth muscle cells,but the heterogeneity of cells derived from ESCs result in difficulty to obtain high purity smooth muscle cells.OBJECTIVE:To construct a double expression vector of puromycin resistance (pac) gene and enhanced green fluorescence protein (EGFP) gene driven by smooth muscle specific SM22α promoter (pSM22α-PAC-IRES2-EGFP),in addition,to detect its availability and specificity in ESCs.DESIGN,TIME AND SETTING:The observational experiment of gene level was performed at the Cardiovascular Institute,General Hospital of Shenyang Military Region from April 2007 to September 2008.MATERIALS:ESCs line R1 with number SCRC-1011TM was purchased from American ATCC Company.The pSM22α-EGFP vector was constructed by our laboratory.And the pIRES2-EGFP,pSM2C and pSuper.basic vectors were purchased from Invitrogen Company.METHODS:SM22α promoter was cloned from pSM22α-EGFP by polymerase chain reaction.CMV promoter of pIRES2-EGFP vector was replaced by SM22 promoter to establish pSM22α-IRES2-EGFP.Pac gene,excised from pSM2C by HindⅢ/Clal digestion,was sub-cloned into pSuper.basic to establish pSuper-PAC.After BgⅢ/Accl enzyme digestion of pSuper-PAC,pac gene fragment was obtained,which was further sub-cloned into pSM22α-IRES2-EGFP to produce pSM22α-PAC-IRES2-EGFP.ESCs were transfected with pSM22α-PAC-IRES2-EGFP using lipofectamine.Positive clones were selected by G418 and induced to differentiate and further identified by amplification of pac gene by RT-PCR.Differentiated cells were immunostained by SM α-actin,and expression of SM α-actin and EGFP was observed simultaneously under fluorescence microscope.MAIN OUTCOME MEASURES:Sequencing result of pSM22α-PAC-IRES2-EGFP;Amplification of pac gene;EGFP expression;as well as SM α-actin immunostaining.RESULTS:Three segments of 261 bp,664 bp,and 5000 bp were obtained by HindⅢ/Clal digestion,which was coincident with expectation,and the sequencing results showed that pSM22α-PAC-IRES2-EGFP vector was successfully constructed.Amplification of pac gene identified 4 ESCs clones successfully transfected.After induction of differentiation,partial portion of differentiated cells expressed EGFP,accompanied by positively stained by SM α-actin antibody.CONCLUSION:pSM22α-PAC-IRES2-EGFP vector was successfully constructed.ESCs clones transfected with this vector expressed pac gene and EGFP gene,and the expression of EGFP is smooth muscle specific.

Objectives The cellular repressor of E1A-activated genes (CREG), a novel gene, was recently found to play a role in inhibiting cell growth and promoting cell differentiation. The purpose of this study was to obtain antibody against CREG protein and to study the expression of CREG protein in human internal thoracic artery cells (HITASY) which express different patterns of differentiation markers after serum withdrawal. Methods The open reading frame of CREG gene sequence was amplified by PCR and cloned into the pGEX-4T-1 vector. Glutathione-S-transferase (GST)-CREG fusion protein was expressed in E. Coli BL21 and purified from inclusion bodies by Sephacryl S-200 chromatography. Rabbits were immunized with the purified GST-CREG protein. Western blot examined with immunohistochemistry staining and the protein expression level was analyzed by Western blot in HITASY cells after serum removal. Results It was confirmed by using endonuclease digesting and DNA sequencing that the PCR product of CREG was correctly inserted into the vector. The GST-CREG protein was purified with gel filtration chromatography. Polyclonal antibody against GST-CREG was obtained from rabbits. CREG protein immunohistochemistry staining displayed a perinuclear distribution in the cytoplasm of HITASY cells. Results from Western blot suggested that comparing with the untreated cells upregulation of CREG polyclonal antibody against CREG was comfirmed. Using this antibody, the changes of CREG protein expression was observed in the process of phenotypic modulation of HITASY cells. These results provide basic understanding on the relationship of CREG gene with the cell phenotypic conversion.