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

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.

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.

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.

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.

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.

AIM: To evaluate the effects of over-expression of cellular repressor of E1A-stimulated genes(CREG) mediated by retrovirus on neointima formation in injured rat carotid.METHODS: The pluronic F127 containing pLNCX/CREG or pLNCX/GFP retroviral vectors was placed around the injured rat carotid.The neointima,media areas and the intima to media ratio were calculated.Expressions of CREG,SM ?-actin and Ki-67 were detected.RESULTS: The GFP expression was observed at day 2 in pLNCX/GFP groups.The expression of exogenous CREG was also significantly increased in arteries at day 2 after pLNCX-CREG infection.Over-expression of CREG significantly suppressed neointima formation,attenuated the expression of Ki-67 and up-regulated SM ?-actin expression.CONCLUSION: Over-expression of CREG inhibits VSMCs proliferation and promotes VSMCs differentiation after vascular injury.It suggests that modulation of CREG expression or activity may be a viable approach to treat neointimal restenosis after percutaneous coronary intervention.

【Objective】 A framework to support nurses and midwives making the clinical decision and providing the written instruction for blood transfusion has been developed and implemented in the United Kingdom as a response to the changing needs of the patient and in recognition that blood transfusion services to patients could be improved by using the untapped knowledge and expertise of experienced nurses and midwives.Special education and training program for this role development are provided jointly by the national blood and nurse management authority, higher education institutions and transfusion societies.The British government has issued and implemented a compulsory professional indemnity which cover nurses and midwives as well.The development and implementation of the framework, policies and procedures for this role development is based on the regulatory compliance and the collaboration of, and beneficial to the multiple stakeholders, with the gaps left by doctors being fillled, work load of doctors reduced, nurses and midwives achieving professional development, hospitals performing more efficiently, and most importantly, the patients having a better transfusion services.At present, there is no similar policy or program for nurses and midwives in China.Therefore, this paper introduces the policy framework and implementation for this role development in UK, which would be a valuable reference for the role development and extension of nurses and the organization, education and training for transfusion professional teams as well in China in the near future.