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Objective To investigate whether chemokine CC motif 2 (CCL2) is involved in the high residual platelet response,and the mechanism of CCL2 being involved in the regulation of platelets.Methods Forty patients with ST elevation myocardial infarction (STEMI) were admitted.P2Y12 reaction unit (PRU) was detected by VerifyNow.Forty patients were divided into high platelet reactivity group (high reactivity group,n=24) and normal platelet reactivity group (normal reactivity group,n=16) according to the results of PRU detection.Plasma CCL2 concentration of the STEMI patients was examined by ELISA.The expressions of CCL2 and CCR2 in the platelets were detected by Western blotting.After CCL2 stimulation,the kinases of which phosphorylation was changed in the platelets were screened by ARY003B protein chips.The phosphorylation of p38MAPK and HSP27 in the platelets was tested by Western blotting after CCL2 stimulation in the presence or absence of CCR2 antagonist (RS 102895) or p38MAPK signal pathway inhibitor (SB 203580).Results The plasma CCL2 concentration of high reactivity group was markedly higher than that of normal reactivity group.Moreover,compared with normal reactivity group,the expressions of CCL2 and CCR2 in the platelets of high reactivity group significantly increased.After the platelets were stimulated by CCL2,the phosphorylation of p38α and HSP27 enhanced in the platelets by protein chips screening.When RS 102895 or SB 203580 was treated before CCL2 stimulation,the phosphorylation of p38MAPK and HSP27 decreased.Conclusions CCL2 participates in high residual platelet response in an autocrine/paracrine way.CCL2/CCR2 might affect the function ofplatelets through p38MAPKHSP27 signal pathway.
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Objective:To investigate the influence of different salt concentration on the renal fibrosis and macrophages infiltration in salt sensitive hypertension.Methods:Dahl salt sensitive rats were randomly divided into the normal salt (0.3 ％ nacl) group,4 ％ high salt,8 ％ high salt groups at six weeks continuously feeding for 8 weeks,each group contained 15 rats.Tail-cuffmethod was used to value rat blood pressure at 8 weeks,Masson trichromatic method was used to detect renal fibrosis of the three groups at 8 week.Immunohistochemistry and Western blot method were used to depict the renal macrophage infiltration at 8 week.Results:1) The blood pressure of 4 ％ salt and 8％ high salt group rats were significantly higher than those of the normal salt group at 8week,meanwhile the blood pressure of 8 ％ high salt was further increased than that of 4 ％ high salt group at 8 week.2) The relative kidney weight and renal fibrosis of 4 ％ salt and 8 ％ high salt group rats were obviously higher than that of normal salt group at 8week,meanwhile the relative kidney weight and renal fibrosis of 8 ％ high salt were further increased than those of 4 ％ high salt group at 8 week.3) The macrophage infiltration of 4 ％ salt and 8％ high salt group rats were higher than that of the normal salt group at 8week,meanwhile the macrophage infiltration of 8 ％ high salt was further increased than that of 4 ％ high salt group at 8 week.Conclusion:Different high salt concentrations had different effect on the renal fibrosis and macrophage infiltration in the salt sensitive hypertension,high salt concentration could exacerbate the renal fibrosis and macrophage infiltration.
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BACKGROUND:It has been found that cel ular repressor of E1A-stimulated genes (CREG) is a lysosomal protein binding directly to the mannose-6-phosphate (M6P)/insulin-like growth factor II receptor (IGFIIR) and depends on the interaction with M6P receptors for efficient delivery to lysosomes OBJECTIVE:To study the interactions between the exogenous CREG protein and cathepsins and M6P/IGFIIR and to confirm the effect of CREG protein on expression and distribution of M6P/IGFIIR. METHODS:Double-stained immunofluorescence and coimmunoprecipitation were applied to observe the interactions between the exogenous CREG protein and cathepsin B, cathepsin L and M6P/IGFIIR. Using gain-of-function and loss-of-function approaches, the effect of CREG on expression and distribution of M6P/IGFIIR were studied by western blot assay and immunofluorescence staining. RESULTS AND CONCLUSION:Double-stained immunofluorescence and coimmunoprecipitation analyses confirmed the direct interactions between the exogenous CREG protein and cathepsin B, cathepsin L and M6P/IGFIIR. It was verified that CREG plays a critical role not in the expression but in the distribution of M6P/IGFIIR using gain-of-function and loss-of-function approaches. These findings provide evidence that exogenous CREG protein is located in lysosomes and has interactions with cathepsins and M6P/IGFIIR, also CREG plays a critical role in the distribution of M6P/IGFIIR.
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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.