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Through defining the connotation of division and cooperation among medical institutions, this paper introduces the current status for division and cooperation among medical institutions of Britain, Germany, Singapore and USA. The advantages include excellent general practices, optimal health resource allocation, effective first treat-ment in community health facilities, and effective two-way referral. We analyzed the influence of some new policies on the division and cooperation among medical institutions such as medical association, managed care and the General Practices’ service contract. This provided a lot of experience and revelations, including the dynamics to promote the division and cooperation, the separation of outpatient services and inpatient services, privatization of primary medical institutions, division and cooperation between medical institutions and nursing, rehabilitation institutions, distinguis-hing the division and cooperation from hospitals alliance, and division and cooperation among medical institutions can not resolve the all issues in the medical care supply system.
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A summary of the reform for public hospitals'internal operating mechanism in 17 pilot cities identified its four main patterns,the internal decision-making machanism,human resource management mechanism,compensation allocation system,and business mechanism.It also analyzed the initial outcomes and problems encountered in the reform.On this basis,the authors proposed the direction for the reform pattern in designing the internal operating mechanism for reference of a wholescale reform of public hospitals in China.
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An analysis of the present development of general practitioners in China,probed into the bottlenecks found.The paper proposed to set forth from the training,use and incentives of GPs to promote the systematic design of China's GP system.These measures center on the regulation of GPs development,while building a multi-channel and multi-level GP development system.Other recommendations include raising the professional standing and attractiveness of GPs as a discipline,and enhancing GPs capacity building to promote general practitioners development.
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The carboxyl-terminal amino acids 272-299-truncated apoE4 (delta272-299) is the main fragments of apoE4 hydrolysate in neurons. The effects of truncated-ApoE4 (delta272-299) overexpression on tau phosphorylation in cultured N2a cells were investigated. The truncated-apoE4 (delta272-299) cDNA was subcloned into pEGFP-c3 to form recombinant pEGFP-T-apoE4. pEGFP-c3, pEGFP-T-apoE4 and pEGFP-apoE4 were transfected into N2a cells respectively by lipofectamine 2000 method. After 24--48 h, tau phosphorylation was detected by Western blot assay and glycogen synthase kinase-3 (GSK-3) activity by using GSK-3 activity assay. The results showed that the overexpression of both full length-apoE4 and truncated apoE4 fragments in N2a cells induced a dramatic increase in phosphorylation of tau at Ser202 sites and the activation of GSK-3 as compared with untransfected cells, most significantly in the cells transfected with pEGFP-T-apoE4 (P < 0.05). It was concluded that in vitro overexpression of truncated-ApoE4 (delta272-299) can result in tau hyperphosphorylation in N2a cells by activating GSK-3, suggesting truncated-ApoE4 (delta272-299) might contribute the pathogenesis of Alzheimer disease.
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The carboxyl-terminal amino acids 272-299-truncated apoE4 (△272-299) is the main fragments of apoE4 hydrolysate in neurons. The effects of truncated-ApoE4 (△272-299) overexpression on tau phosphorylation in cultured N2a cells were investigated. The truncated-apoE4 (△272-299)cDNA was subcloned into pEGFP-c3 to form recombinant pEGFP-T-apoE4. pEGFP-c3, pEGFP-TapoE4 and pEGFP-apoE4 were transfected into N2a cells respectively by lipofectamine 2000 method. After 24-48 h, tau phosphorylation was detected by Western blot assay and glycogen synthase kinase-3 (GSK-3) activity by using GSK-3 activity assay. The results showed that the overexpression of both full length-apoE4 and truncated apoE4 fragments in N2a cells induced a dramatic increase in phosphorylation of tau at Ser202 sites and the activation of GSK-3 as compared with untransfected cells, most significantly in the cells transfected with pEGFP-T-apoE4 (P＜0.05). It was concluded that in vitro overexpression of truncated-ApoE4 (△272-299) can result in tau hyperphosphorylation in N2a cells by activating GSK-3, suggesting truncated-ApoE4 (△272-299) might contribute the pathogenesis of Alzheimer disease.
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BACKGROUND: Leptin is a kind of polypeptide hormone secreted by fatty tissue, previous studies have shown that leptin plays a certain role in the formation of atherosclerosis.OBJECTIVE: To investigate the effects of leptin on the expression of tumor necrosis factor-alpha (TNF-α) in RAW264.7 cells, and investigate the possible mechanism from the angle of the change of nuclear factor-κB (NF-κB) activity.DESIGN: A controlled observational experiment.SETTING: Department of Biochemistry and Molecular Biology, Tongji Medical College, Huazhong University of Science and Technology.MATERIALS: The experiments were carried out in the Department of Biochemistry and Molecular Biology, Tongji Medical College, and the Department of General Surgery, Union Hospital affiliated to Huazhong University of Science and Technology between April 2005 and February 2006.The cultured RAW264.7 cells were divided into leptin treated groups treated with different concentration (12.5, 25, 50, 100 μg/L), I kappa B kinase inhibitor group and blank control group. Each group had 3 sub-wells,and the experiments were repeated for 3 times.METHODS: Mouse macrophage RAW264.7 cells were incubated into a 6-well plate at the density of 109 cells L-1, cultured in RPMI-1640 culture medium containing bovine serum of 0.1 in volume fraction. When the cells grew to 80%, serum-free culture medium Opti-MEM was changed to culture for another 24 hours, and then the cells were divided into leptin groups treated with different concentration (12.5, 25, 50, 100 μg/L), I kappa B kinase inhibitor group and blank control group. After the cells were incubated with leptin for 4 hours, the expression of TNF-α mRNA expression in RAW264.7 cells was detected with reverse transcription-polymerase chain reaction (RT-PCR). After the RAW264.7 cells were incubated with leptin for 1, 3, 6 and 9 hours, the expression of TNF-c protein expression was detected with double antibody sandwich enzyme-linked immunosorbent assay (ELISA). After the RAW264.7 cells were incubated with leptin for different times, the activity of NF-κB was detected analyzed with electrophoretic mobility shift assay. Another, the RAW264.7 cells were treated with or without 50 μmol/L leptin and/or 100 μmol/L PS1145 (I kappa B kinase specific inhibitor)divided into four groups: blank control group, I kappa B kinase specific inhibitor PS1145 (10 μmol/L) treated group, leptin (50 μmol/L) treated group, leptin (50 μmol/L) + PS1145 (10 μmol/L) group. Aftere incubated for 6 hours, the activity of NF-κB and expression TNF-α mRNA were detected respectively.MAIN OUTCOME MEASURES: ① Effect of leptin of different concentration on the expression of TNF-α mRNA and protein in RAW264.7cells; ② Effect of leptin of different concentration on the activity of NF-κB in RAW264.7 cells; ③ Influence of inhibition I kappa B kinase activity inhibition on expression of TNF-a induced by leptin in RAW264.7cells.RESULTS: ① After the RAW264.7 cells were treated with leptin of different concentration, the TNF-α mRNA level was elevated in a dose-dependent manner, and it reached the peak value emerged in the 50 μg/L leptin treated group. ② The expression of TNF-α protein increased in dose-dependent and time-dependent manners, and it reached the peak val ue at 6 hours in the 50 μg/L leptin treated group. ③ The activity of NF-κB was also positively correlated with the leptin concentration, and it was the highest value at 6 hours treated with 50 μg/L leptin (P ＜ 0.05). ④ I kappa B kinase activity inhibition only partially suppressed the leptin induced elevation of TNF-α expression induced by leptin.CONCLUSION: Leptin can increase the expression and secretion of TNF-α in RAW264.7 cells directly in both dose-dependent and time-dependent manners, and the mechanism may be correlated with the activated NF-κB induced by leptin. It may be one of the mechanisms of atherosclerosis induced by leptin.
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BACKGROUND: The degree of neurofilament (NF) phosphorylation is closely correlated with the occurrence of Alzheimer disease (AD), and apolipoprotein E4 (apoE4) is a generally acknowledged liability factor for AD, but the effect and mechanism of apoE4 on the NF phosphorylation in neurons are not very clear. It has been reported that in the neurons cultured in vitro and in brain tissue of AD patients, the amino acid residues of apoE4 protein C terminal (272-299) could be truncated by hydrolysis,and produce truncated-apoE4 fragment. The latter interacts with the NF phosphorylation in neurofibrillary tangles (NFTs), which are the characteristic pathological changes of AD.OBJECTIVE: To observe the effect of truncated-apoE4 overexpression on the NF phosphorylation in the cultured neurons.DESIGN: A non-randomized controlled observation.SETTING: Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Tongji Medical College, Huazhong University of Science and Technology.MATERIALS: The experiment was carried out in the Laboratory of Biochemistry and Molecular Biology, Tongji Medical College, Huazhong University of Science and Technology in December 2005. The mice neuroma cell strain N2a was provided by Dr. Xu.METHODS: The truncated-apoE4(△272-299) cDNA was subcloned into pEGFP-c3 to form pEGFP-T-apoE4 recombinant. Then pEGFP-c3, pEGFP-apoE4 and pEGFP-T-apoE4 were transfected into N2a cells by lipofectamine2000 respectively. NF phosphorylation was detected by Western blot assay. The activities of glycogen synthase kinase-3 (GSK-3) and cyclin-dependent kinase 5(CDK-5) were measured.MAIN OUTCOME MEASURES: The degree of NF phosphorylation and the activities of GSK-3 and CDK-5 were mainly observed.RESULTS: In the transfected groups, the contents of phosphorylated NF were significantly increased, the GSK-3 activities were significantly increased, which were the most significant in the pEGFP-T-apoE4 transfected group (P＜0.05), but the CDK-5 activities were not significantly different from that in th e control group (P＞0.05).CONCLUSION: These results indicate that in vitro overexpression of truncated-apoE4(△272-299) can lead to NF hyperphosphorylation by activating of GSK-3 but not CDK-5, which may be the underline mechanism of AD induced by truncated-apoE4(△272-299).
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In this study, we studied the effect of glycogen synthase kinase-3 (GSK-3) overactivation on neurofilament phosphorylation in cultured cells. After N2a cells were treated with the specific inhibitor (wortmannin) of phosphoinositol-3 kinase (PI-3K) or treated with wortmannin and the specific inhibitor (LiCl) of glycogen synthase kinase-3 (GSK-3), GSK-3 activity and neurofilament phosphorylation were detected by using GSK-3 activity assay, Western blots and immunofluoresence. Our results showed that after treatment of N2a cells with wortmannin for 1 h, overactivation of GSK-3 caused a reduced staining with antibody SMI32 and an enhanced staining with antibody SMI31. When N2a cells were treated with wortmannin and LiCl, the activity of GSK-3 was reduced substantially. At the same time, the phosphorylation of neurofilament was also reduced. The study demonstrated that overactivation of GSK-3 induced hyperphosphorylation of neurofilament and suggested that in vitro overactivation of GSK-3 resulted in neurofilament hyperphosphorylation and this may be the underlying mechanism for Alzheimer's disease.
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BACKGROUND: One of the key neuropathological changes in Alzheimer disease is that neurofibrils over phosphorylated cytoskeletal protein (such as r and neurofilaments) composed of entwist together, and the phosphorylation of τ protein can be catalyzed by cyclin dependent kinase 5 (CDK5),however whether the phosphorylation of neurofilaments can be catalyzed by CDK5, as well as its role in the pathogenesis of Alzheimer diseases is less acknowledged.OBJECTIVE: To explore the role of over-expression of intracellular CDK5 in the phosphorylation of neurofilamentsDESIGN: Randomized controlled study.SETTING: Biochemical and Molecular Biological Department of Tongji Medical College, Huazhong University of Science and Technology.MATERIALS: This study was conduced at Biochemical and Molecular Biological Department of Tongji Medical College, Huazhong University of Science and Technology between February and May 2001. In vitro cultured rat neuroblastoma cell strain (N2a) was adopted as subjects.METHODS: In vitro cultured N2a cells were divided into 2 groups, namely transfection group and non-transfection group. In transfection group,CDK5 gene was transfected into N2a cell line by using liposome transfection technique so as to obtain N2a/CDK5 cell line stably expressing CDK5, immune-precipitation and enzyme activity assay was used to detect the CDK5 activity, meanwhile immunofluorescence technique and immuneblot assay was used to detect CDK5 expression and phosphorylation of neurofilaments.MAIN OUTCOME MEASURES: Phosphorylation of neurofilaments in both groups.RESULTS: In transfection group of N2a cell line, CDK5 expression increased presented by deep coloration of SMI31 antibody and weak coloration of SMI32 antibody, implying hyper-phosphorylation of neurofilaments. Meanwhile, the activity of CDK5 was 3.5 times higher than that in non-transfection group.CONCLUSION: Intracellular over-expressison of CDK5 would lead to hyperactivity of CDK5 and hyper-phosphorylation of neurofilaments, however the hyper-phosphorylation of neurofilamentsmight invlove in the pathological development of AD.
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BACKGROUND: Puerarin functions to relieve the injury caused by ischemia reperfusion, improve the microcirculation, and inhibit the agglutination of platelet. But it is not clear yet that whether its protection on neurons relates to the apoptosis of cells.OBJECTIVE: To observe the protection of puerarin on neurons injured by mimic ischemia reperfusion in vitro.DESIGN: A random control study. SETTING: Laboratory of Biochemistry and Moleculobiology Department of Preclinical Medicine College of Tongji Medical College of Huazhong University of Science and Technology.PARTICIPANTS: The experiment was done on March 11, 2002. The cultured N2a cells of rat neuromablast were observed.METHODS: Ninety minutes after the cultured N2a cells were put into the 37 ℃ incubator with 0.05 CO2 and 0.95 N2(v/v), puerarin 0.5 mmol/L was added for cuiture for 24 hours. The methyl thiazolyl tetrazolium (MTT)method was used to observe the surviving ability of cells. The Annexin-V staining was adopted to exam the severity of apoptosis in the early stage and the supernate was collected to analyze the activity of lactate dehydrogenase (LDH) to reflect the permeability of cytomembrane. The immunoblotting method was applied to analyze the expression of caspase-3, at the same time, its activity was measured.MAIN OUTCOME MEASURES: The severity of cell apoptosis, the activity of LDH, the expression and activity of caspase-3.RESULTS: Puerarin could improve significantly the surviving rate of N2a cells 24 hours after reperfusion, decrease remarkably the activity of LDH in the culture fluid, lessen obviously the N2a apoptosis (P ＜ 0.01), at the same time, reduce tremendously the activity and expression of caspase-3 induced by ischemia reperfusion (P ＜ 0.01).CONCLUSION: Puerarin can protect nerves, inhibit obviously the N2a apoptosis induced by ischemia reperfusion. The mechanism is that it inhibits greatly the expression and activity of caspase-3.
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To explore the intracellular signal pathways for beta-VLDL induced very low density lipoprotein receptor (VLDL-R) transcription up-regulation and their effects on lipid accumulation in macrophages, Western Blot was used to examine phosphorylated ERK1/2 protein and regulated effects by different singal kinase inhibitants. It was found that beta-VLDL induced an increase in ERK1/2 activity in a protein kinase C (PKC)-dependent manner in murine RAW264.7 macrophages. By using different protein kinases inhibitors or activators, it was observed that the effect of beta-VLDL induced VLDL receptor transcription, which was monitored by RT-PCR analysis of VLDL receptor mRNA, was not affected by the inhibitor of p38 kinase and cAMP analog, but extremely abolished by pretreating cells with PD98059, an inhibitor of ERK and GF 109203X, an inhibitor of PKC. These results demonstrated that the PKC-ERK1/2 cascade is the essential signaling pathway by which beta-VLDL activated VLDL-R mRNA expression. Inhibition of the ERK1/2 signaling cascade resulted in suppression of the cellular lipid accumulation induced by beta-VLDL in macrophages.
Subject(s)
Cells, Cultured , Lipoproteins, VLDL , Metabolism , Macrophages , Cell Biology , Metabolism , Mitogen-Activated Protein Kinase 1 , Metabolism , Physiology , Mitogen-Activated Protein Kinase 3 , Metabolism , Physiology , Protein Kinase C , Metabolism , Receptors, LDL , Genetics , Signal Transduction , Transcription Factors , Metabolism , Transcription, Genetic , Up-RegulationABSTRACT
To observe the inhibitory effects of an antisense u-PAR vector on invasion of highly invasive PC-3M cell subclones, the effects of the antisense u-PAR on activity of MMP-9 in those highly invasive cell subclones were detected by a quantitative RT-PCR and zymography. The monolayer invasion assay and colony formation assay in soft agar were used. And tumorigenesis rate and invasions by the cell subclones with or without the antisense u-PAR were observed in nude mice. It was found that in vitro growth of highly invasive PC-3M cell subclones transfected with the antisense u-PAR was declined, and the ability of anchorage-independent growth of those cell subclones was found decreased sharply, with the inhibiting rate becoming 79% and 60%, respectively. Although the antisense u-PAR didn't change MMP-9 gene transcription, they could inhibit the activation of MMP-9 of highly invasive PC-3M cell subclones. Moreover, the tumorigenesis rate of the cell subclones with the antisense u-PAR decreased and the growth of a neoplasm also slowed down. The t tests showed the difference between experimental and control groups was statistically significant (P < 0.01). The antisense u-PAR vector could not only inhibit the invasion ability of highly invasive PC-3M cell subclones in vitro but also restrain the growth of those cell subclones in vivo.
Subject(s)
Animals , Humans , Male , Mice , Antisense Elements (Genetics) , Genetics , Pharmacology , Cell Division , Cloning, Molecular , Matrix Metalloproteinase 9 , Genetics , Metabolism , Mice, Inbred BALB C , Mice, Nude , Neoplasm Invasiveness , Prostatic Neoplasms , Metabolism , Pathology , RNA, Antisense , Receptors, Cell Surface , Genetics , Metabolism , Receptors, Urokinase Plasminogen Activator , Reverse Transcriptase Polymerase Chain Reaction , Transfection , Tumor Cells, CulturedABSTRACT
To evaluate the specific inhibition of antisense u-PAR on the u-PAR expressions in highly invasive cell subclones and to determine its blocking function in the invasion by those cells, a cDNA fragment of u-PAR obtained by RT-PCR was inserted into a plasmid vector named pcDNA3 in antisense orientation. Then the antisense u-PAR recombinant was transfected into highly invasive cell subclones. The u-PAR expression in neo-resistant cells was examined by RT-PCR and immunohistochemical assay. Compared to the control cells, the content of mRNA and protein of u-PAR in transfected cells decreased sharply, and the rate of inhibition was 53% and 73%, respectively, indicating that an antisense u-PAR might have played a specific inhibitory role in its expression in the cells, which may provide a good cell model for making further investigation of the inhibitory effects of the antisense u-PAR on invasion in highly invasive cell subclones of human prostate carcinoma.