Effects of insulin on cardiac fibroblast proliferation and cardiac myocyte hypertrophy

AIM:To study the effect of insulin on proliferation and hypertrophy of cardiac myocytes and its role in the induction of cardiac hypertrophy. METHODS:1. The neonatal rat cardiac myocytes and cardiac fibroblasts were cultured respectively and identified with light microscopy, electron microscopy and immunocytochemistry. 2. Cell proliferation was measured with cell number, metabolic activity and DNA synthesis (with WST-1, BrdU enzyme-linked immunosorbent assay ) and the percentage of S+G₂+M in cell cycle (by flow cytometry ). 3.Cell hypertrophy was evaluated by cell protein content (Coomassie Briliant Blue's method). RESULTS:1. The cultured cells showed the characteristic of cardiac myocytes and cardiac fibroblasts, respectively. 2. After being treated with insulin, the cell number, absorbance of BrdU incorporation and WST-1 cleavage products and the percentage of S+G₂+M of cardiac fibroblasts increased significantly (P＜0.01 orP＜0.05), while the above parameters of cardiac myocytes remained unchanged (P＞0.05). 3. Protein content of cardiac myocytes increased significantly in a dose-dependent manner (P＜0.01 orP＜0.05) in insulin treated groups (10^-10 mol/L-10^-7 mol/L). CONCLUSION:Insulin promoted cardiac fibroblast proliferation and increased myocytes protein content(induced myocyte hypertrophy)in vitroand may play an important role in pathogenesis of cardiac hypertrophyin vivo.     

Effects of advanced glycation end products on protein and mRNA expression of macrophage inflammatory protein-1α in cultured human umbilical vein endothelial cells

AIM: To investigate the effects of advanced glycation end products (AGEs) on protein and mRNA expression of macrophage inflammatory protein-1α (MIP-1α) in cultured human umbilical vein endothelial cells(HUVECs). METHODS: HUVECs were cultured with AGEs at different concentrations for 24 h and at a concentration of 400 mg/L for different time.The levels of mRNA and protein expression of MIP-1α in cultured HUVEC were detected by in situ hybridization and Western blot, respectively. RESULTS: In situ hybridization showed that after exposure of HUVECs to AGEs at different concentrations (100 mg/L, 200 mg/L, 400 mg/L) for 24 h, the average integrated optical density values (18.76±3.17, 26.58±1.61, 34.23±2.25) of MIP-1α mRNA expression in HUVECs were higher than that in control group (13.83±1.24, P0.05). After exposure of HUVECs to AGEs at a concentration of 400 mg/L for 12 h, 24 h and 36 h, the average integrated optical density values of MIP-1α mRNA expression in HUVECs were 22.67±1.46, 34.23±2.25 and 42.28±3.14, higher than that in 0 h group (12.56±1.24, P0.05). Western Blot showed that exposure of HUVECs to AGEs at different concentrations(100 mg/L, 200 mg/L, 400 mg/L) for 24 h resulted in a 1.34-fold, 1.87-fold and 2.46-fold increase in the expression of MIP-1α protein in HUVECs compared with BSA control group (P<0.05). Meanwhile, exposure of HUVECs to AGEs at a concentration of 400 mg/L for 12 h, 24 h and 36 h resulted in a 1.82-fold, 2.71-fold and 3.34-fold increase in MIP-1α protein expression in HUVECs compared with 0 h group (P<0.05). CONCLUSION:These data suggest that AGEs could induce a high expression of MIP-1αmRNA and protein in cultured HUVECs in a dose-dependent and time-dependent manner.     

Effects of cholecystokinin octapeptide on changes in rabbit thoracic aortic reactivities induced by lipopolysaccharides in vitro

AIM and METHODS: To elucidate the mechanism of anti-endotoxic shock of cholecystokinin octapeptide(CCK-8), the effects of CCK-8 on changes in rabbit thoracic aortic reactivities induced by lipopolysaccharides(LPS) in vitro were studied, and the ultrastructure of the endothelial cells was observed under scanning electron microscope. RESULTS: Incubation of thoracic aortic rings(TARs) with LPS(100 mg/L) resulted in an time-dependent impairment of the endothelium-dependent relaxations to acetylcholine(incubation for 3, 7, 14 h), a reduction of contractive response to phenylphrine(incubation for 14 h) and ultrastructural injury in endothelial cells(incubation for 7 h), all of which were alleviated by concomitant incubation with CCK-8(1 mg/L). In contrast, neither the vascular contractions nor the relaxations were affected by CCK-8 (1 mg/L) alone. CONCLUSION: CCK-8 improved the vascular reactivities in the presence of LPS, which may be one of the anti-endotoxic shock mechanisms of CCK.     

Antagonistic effect of captopril on activation and injury of vascular endothelial cells induced by lipopolysaccharide

AIM: To investigate the antagonistic action of Captopril (Cap) on the activation and injury of human umbilical vascular endothelial cells (HUVECs) induced by lipopolysaccharide(LPS) and the possible mechanisms. METHODS: After 18 h exposure of the cultured HUVECs to LPS (1 mg/L), or LPS (1 mg/L) plus Cap at the concentration of 10^-7mol/L, 10^-5mol/L and 10^-3mol/L, the expression of vWF protein in the conditioned media was tested by enzyme-linked immunosorbent assay (ELISA), the expression of ICAM-1 protein in HUVECs was determined by indirect immunofluorescence technique with flow cytometry as well. In addition, the expression of TNFα mRNA was determined by in situ hybridization. RESULTS: The results of ELISA and indirect immunofluorescence technique showed that exposure to LPS at a concentration of 1 mg/L led to a significant increase in the vWF and ICAM-1 expression in HUVECs as compared to the control (P＜0. 05), whereas they were somewhat decreased when exposed to Cap at three increasing concentrations mentioned above, especially in the Cap (10^-3mol/L) plus LPS group (P＜0.05). Cap inhibited vWF secretion and ICAM-1 expression of HUVECs caused by LPS in a concentration-dependent manner. In situ hybridization revealed that the expression of TNFα mRNA was inhibited by Cap both in a concentration of 10^-3mol/L, and in a lower concentration of 10^-5mol/L. CONCLUSION: Cap antagonizes the activation and injury of HUVECs induced by LPS, which may be related to the decrease in TNFα mRNA expression.     

Effect of calcitonin gene-related peptide on proliferation of vascular smooth muscle cells

AIM: To elucidate the effect of calcitonin gene-related peptide (CCRP) in the therapy of atherosclerosis.METHODS:Effect of CGRP on cell cycle kinetics of cultured vascular smooth muscle cells(HA-VSMC) was investigated by flow cytometry. The expression of cyclins D₁ and E required for initiation of S phase were also studied by immunochemistry method. RESULT: CGRP was shown to arrest VSMC in the G₀/G₁ phase of cell cycle and reduced expression of cyclins D₁ and E. CONCLUSION:CGRP inhibits proliferation of HA-VSMC by arresting cells in G₁ phase via limiting accumulation of cyclin D₁ and E. It might play a role in the therapy of atherosclerosis.     

中国园艺学会第七届青年学术讨论会

中国园艺学会第九届第8次常务理事扩大会决定,“中国园艺学会第七届青年学术讨论会”由山东农业大学园艺科学与工程学院和山东省园艺学会承办,将于2006年7月或8月在山东泰安举行。会议交流主题:(1)园艺作物种质资源、遗传育种与生物技术;(2)园艺作物有机、无公害及标准化安全生     

Effects of changing PKC activity on proliferation and telomerase expression in human hepatocellular carcinoma cells

AIM: To investigate whether protein kinase C (PKC) is involved in the proliferation and the telomerase expression in human hepatocellular carcinoma cells. METHODS: Human hepatocellular carcinoma cells (BEL-7402) were treated with exogenous phorbol-12-myristate-13-acetate (PMA, PKC activator) and staurosporine (SP, PKC inhibitor) for 48 hours. The techniques of cell culture and the telomeric repeat amplification protocol silver staining in combination with computer image scanning system in vitro were used to observe the variations of the growth and the telomerase expression. RESULTS: The proliferative potential of BEL-7402 cells was decreased by the action of PMA as well as SP, and the telomerase expression was also inhibited by PMA and SP. CONCLUSION: Our findings suggest that the proliferation of human hepatocellular carcinoma cells and the telomerase expression may be related to PKC.     

Inhibiting role of polydatin to expression of intercellular adhesion molecule-1 induced by lipopolysaccharide on vascular endothelial cells

AIM and METHODS:To investigate expression of intercellular adhesion molecule-1(ICAM-1) on human umbilical vein endothelial cells (HUVEC) induced by lipopolysaccharide (LPS) , and inhibiting role of polydatin by cellular immune fluorescent staining and laser confocal microscope scanning technology. RESULTS: Compared with basic expression of ICAM-1 on HUVEC, the ICAM-1 expression was enhanced significantly after stimulated by LPS from 8 h to 36 h, dose-dependent relation appeared between expression of ICAM-1 and LPS. ICAM-1 expression on endothelial cells treated only by polydatin had no abvious change,but inducing role of LPS to expression of ICAM-1 was inhibited significantly by polydatin pretreating endothelial cells. CONCLUSION:The expression of ICAM-1 on endothelial cells can be promoted by LPS , and polydatin can inhibit LPS-induced ICAM-1 expression.     

Effect of lipopolysaccharide on viability and secretion function of human umbilical vein endothelial cells

AIM: To observe the direct effect of lipopolysaccharide (LPS) on secretion of endothelin-1 (ET-1) and nitric oxide by human umbilical vein endothelial cell and cell viability of the secretor. METHODS: The third passage of human umbilical vein endothelial cells were incubated with different concentrations of LPS (1 g/L, 100 mg/L, 10 mg/L, 1 mg/L, 100 μg/L, 10 μg/L, 1 μg/L) for 6 hours, and the culture supernatants were collected. The concentrations of ET-1 were determined by radioimmunoassay, the concentrations of nitric oxide were determined using Greiss's method. The viabilities of cells were measured by MTT method. RESULTS: The concentration of ET-1 (pg/L) of normal control group was 251.64±10.90. The concentrations of ET-1 (pg/L) of LPS treated groups were 220.85±19.14, 278.67±15.45, 306.40±11.60, 312.87±33.50, 324.38±17.02, 291.49±14.30, 282.11±13.38, respectively (each group compared with normal control group, P<0.05 or P<0.01). The concentration of NO_x (μmol/L) of normal control group was 629.46±13.36. The concentrations of NO_x (μmol/L) of LPS treated groups were 732.58±23.21, 669.87±9.32, 661.24±16.80, 650.33±13.24, 606.59±12.94, 626.75±9.83, 627.61±5.61, respectively (each group compared with normal control group, P<0.05 or P<0.01). The viabilities of endothelial cells of LPS treated groups were 74%, 81%, 86%, 88%,91%, 93%, 93%, respectively. CONCLUSION: LPS of lower concentrations had no significantly lethal effect on human umbilical vein endothelial cells, but enhanced secretion of ET-1 and inhibited NO production. LPS in higher concentrations showed significant lethal effect on human umbilical vein endothelial cells, inhibited secretion of ET-1 and enhanced NO production.     

Effects of amlodipine on apoptosis of vascular endothelial cells induced by oxyeterols

AIM:To investigate the apoptosis of endothelial cells (EC) induced by oxysterols and to examine the effect of amlodipine on the apoptosis induced by oxysterols.METHODS:Light microscope, electron microscope, DNA agarose gel electrophoresis and flow cytometry were used to detect the apoptosis of EC.RESULTS:The characteristic morphological features of apoptosis were observed under light and electron microscope; DNA electrophoresis showed"DNA Ladder"; Flow cytometry showed the sub-G1 peak, apoptotic rate is 32.25% and 23.04% in Triol-treated and 25-OH-treated groups, respectively. While treated EC with amlodipine at the same time, the apoptotic rate decreased significantly.CONCLUSION:Both Triol and 25-OH can induce apoptosis of EC, which can be inhibited by amlodipine.     

Influence of caveolin-1 on the ET-1-induced VSMC proliferation

AIM: To investigate the effect of caveolin-1 on the endothelin-1 (ET-1)-induced vascular smooth muscle cells (VSMC) proliferation. METHODS: The [³H]-thymidine ([³H]TdR) incorporation, immunofluorescence assays and western blotting were used in this study. RESULTS:The ETA receptor specific antagonist BQ123 inhibited the increase in[³H]TdR incorporation in response to ET-1 on VSMC.Immunofluorescence assays showed that caveolin-1 was mostly distributed in plasmalemma of VSMC.After 24 h treatment of VSMC with ET-1,the expression of caveolin-1 in VSMC was significantly decreased.Western blotting showed that ET-1 inhibited the expression of caveolin-1 in VSMC,BQ123 reversed the effect of ET-1.CONCLUSION: Caveolin-1 was mostly distributed in plasmalemma of VSMC. ET-1 downregulated caveolin-1 expression in VSMC via ETA receptor.     

Effects of probucol on the proliferation of rat vascular smooth muscle cells stimulated by basic fibroblast growth factor and hydrogen peroxide

AIM: To investigate the effect of probucol on proliferation of rat vascular smooth muscle cells(VSMC) stimulated by basic fibroblast growth factor (bFGF) and/or hydrogen peroxide(H₂O₂). METHODS: Effects of probucol on VSMC proliferation and DNA synthesis stimulated by bFGF and/or H₂O₂ were observed by means of MTT test, cell number count and [3H]-TdR incorporation. RESULTS: ①Probucol significantly inhibited proliferation and DNA synthesis in VSMC stimulated by bFGF and/or H₂O₂, with dosage-dependent manner. Cell number, A value and [3H]-TdR incorporation in group probucol+bFGF and group probucol+H₂O₂ were reduced by 40.0%, 39.1%, 45.5% and 46.9%, 45.0%, 39.5%, respectively, compared with group bFGF and group H₂O₂ (P＜0.05, P＜0.01, respectively). ②Pretreatment of VSMC with probucol for 24 h prior to bFGF and/or H₂O₂ stimulation exhibited significant inhibiton of VSMC proliferation and DNA synthesis, but after prestimulation by bFGF and/or H₂O₂ for 24 h, probucol had no influence on VSMC proliferation and DNA synthesis (P＞0.05). CONCLUSION: Probucol dramatically inhibited proliferation and DNA synthesis in VSMC stimulated by bFGF and/or H₂O₂, but had no inhibitory effect on the cell proliferation prestimulated by bFGF and /or H₂O₂.     

Effects of component of some Chinese herbs on proliferation of human umbilical vein endothelial cells in vitro

AIM: To observe the effects of some component of Chinese herbs for external use on proliferation of human umbilical vein endothelial cells (HUVEC) and investigate the mechanism of promoting tissue repair. METHODS: The method of MTT was used to examine the effects of Rg1, Rh1, perlolyrine, cinnamyl aldehyde, muscone, astragaluspolysaccharin (APS), velver antler polypeptide (VAP) and soluble extract of boswellia carterii birdw (BCB) on proliferation of HUVEC. RESULTS: APS did not promote proliferation of HUVEC at 9.75 mg/L-2.5 g/L; Rh1 promoted proliferation of HUVEC at 1.94 mg/L-0.5 g/L (P<0.05 or P＜0.01), and Rg1 inhibited proliferation of HUVEC at 31 mg/L (P<0.05); VAP promoted proliferation of HUVEC at 1 mg/L-0.5 g/L with optimal dose of 10 mg/L (P<0.01), Cinnamyl aldehyde promoted proliferation of HUVEC at 2 g/L(P＜0. 05); Muscone and soluble extract of BCB inhibited proliferation of HUVEC at 1 g/L, 0.5-2.5 kg/L(P＜0. 01), respectively; Perlolyrine inhibited proliferation of HUVEC at 0.125 g/L-0.5 g/L(P＜0. 01). CONCLUSION: The external herbs for supplementing Qi and warming Yang can promote HUVEC proliferation and improve angiogenesis during tissue repair. The external herbs for promoting blood circulation and accelerating capillary movement may have influence upon other stages of tissue repair.     

The stimulating effects of neuropeptide Y on cultured arterial smooth muscle cell proliferation and losartan treatment

AIM: To observe expression of proliferating cell nuclear antigen (PCNA), platelet derived growth factor (PDGF) and c-myc in cultured vascular smooth muscle cells which is effected by neuropeptide Y (NPY) and losartan (the receptor blocking agent of angiotensin Ⅱ) therefore exploring effects of NPY on the generation of hypertension and its relationship with losartan reverse treatment in molecular biology. METHODS: Applied the method of quantitative immunocytochemistry through ACAS570. RESULTS: 24 hours exposure of vascular smooth muscle cell to NPY caused an increase in expression of PCNA, PDGF and c-myc respectively. But losartan could reverse these expressions by NPY, decreased the expression of PCNA, PDGF and c-myc.CONCLUSION: NPY is closely related to the generation hypertension. But losartan could reverse these effects of NPR.     

Protective effect of heat-stress preconditioning on anoxic endothelial cells and its mechanism

AIM:To observe the protective effect of heat stress preconditioning on endothelial cells under anoxia and explore its mechanism. METHODS:The endothelial cells were divided into 4 groups: (1) anoxia; (2) heat stress; (3) heat stress preconditioning + hypoxia; (4) control. LDH activity was measrued by using Automatic Biochemistry Analysis-Meter. Cell death rate was determined by trypan blue, NO production was tested by measuring NO₂^-/NO₃^- content in cellular culture medium by using Griess assay. RESULTS:LDH release and cell death rate of the anoxia endothelial cells significantly increased compared with control; 39℃ heat stress preconditioning reduced those increment by 29.47%, 33.67% respectively. 41℃ heat stress preconditioning has no protection against the anoxia-induced injury in endothelial cells. The NO production in anoxic endothelial cells decreased markedly. 39℃ heat stress preconditioning induced the increase in NO production in endothelial cells, but 41℃ heat stress preconditioning made the NOS activity decrease. The NO production was correlated negatively with LDH release and cell death rate in anoxic endothelial cells. CONCLUSION:The heat stress preconditioning within the limits can protect the endothelial cells from anoxia injury. The increase in NO in endothelial cells may play an important role in the mechanism of the protective effect.     

氮和磷对膜荚黄芪不定根芒柄花苷积累的影响

以膜荚黄芪不定根为试材,通过改变B5培养基成分,依次研究了总氮含量、NH⁺₄∶NO^-₃比例、磷酸盐含量对不定根生物量以及芒柄花苷积累的影响,以期得到最佳氮、磷配比。结果表明:当培养基中总氮浓度为3.0 mmol·L^-1、NH⁺₄∶NO^-₃比例为1.5∶1.5、PO₄^3-浓度为0.625 mmol·L^-1时,黄芪不定根生物量及芒柄花苷含量均依次达到了最大值,且与初始培养基相比芒柄花苷的含量分别增加了7、14、19倍。因此,该方法简单可靠,且配比结果为进一步利用膜荚黄芪不定根规模化生产芒柄花苷奠定了理论基础。     

Effects of two EBV-LMP1 variants on proliferation characteristics of CNE1 cell line

AIM: To investigate the possible effect of different Latent membrane protein 1 (LMP1) variants on proliferation characteristics of a human well-differentiated nasopharyngeal carcinoma (NPC) cell line CNE1. METHODS: The plasmids which carried EBV-LMP1 gene cloned from B95-8 lymphocyte (B95-8-LMP1) and NPC tissues (CAO-LMP1) were introduced into CNE1 by liposomal transfection. Expression of LMP1 in CNE1 was identified by RT-PCR and Western blot, respectively. Different Effects of the two EBV-LMP1 variants on proliferation characteristics of CNE1 including growth curve, cell cycle distribution and clony-forming efficiency were investigated by means of crystal violet staining proliferation assay, flow cytometry and plastic plate clony-forming assay, respectively. RESULTS: Two transfected cell lines stably expressing different LMP1 variants were established successfully. Either of the two LMP1 variants increased CNE1 growth rate, proliferation index (PI) and clony-forming rate significantly and CAO-LMP1 had more significant growth-promoting effect on CNE1 than B95-8-LMP1. CONCLUSION: It can be concluded from the study that CAO-LMP1 promotes CNE1 proliferation more effectively than B95-8-LMP1.     

Effect of fibrinogen,fibrin and fibrin degradation products on the proliferation and migration of human vascular smooth muscle cells

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.     

Effect of lipopolysaccharide on apoptosis of human umbilical vein endothelial cells

AIM: To explore the mechanism of lipopolysaccharide (LPS) on vascular endothelial cell(VEC) damage. METHODS: By using cytometry techniques, we studied the effects of LPS on apoptosis of human umbilical vein endothelial cells (HUVECs) in vitro. RESULTS: LPS was able to induce apoptosis of HUVECs in a time-dose-dependent fashion.CONCLUSION:Apoptosis might play a role in LPS-induced damage of vascular endothelial cells.