Losartan对血管平滑肌细胞增殖和血小板衍化生长因子及c-myc基因表达的影响

目的　探讨血管紧张素受体拮抗剂 Ｌｏｓａｒｔａｎ 治疗高血压、动脉粥样硬化等心血管疾病的细胞作用机制。方法　运用免疫荧光组织化学技术和激光共聚焦显微镜定量观察 Ｌｏｓａｒｔａｎ 作用下的血管平滑肌细胞（ Ｖ Ｓ Ｍ Ｃ） 中细胞核增殖抗原（ Ｐ Ｃ Ｎ Ａ） 、 Ｐ Ｄ Ｇ Ｆ 和ｃｍｙｃ 原癌基因表达的平均荧光值。定量研究 Ｌｏｓａｒｔａｎ 对体外培养血管平滑肌细胞增殖及血小板生长因子（ Ｐ Ｄ Ｇ Ｆ） 和ｃｍｙｃ 癌基因表达变化的影响。结果　 Ｌｏｓａｒｔａｎ 作用下, Ｖ Ｓ Ｍ Ｃ 中的 Ｐ Ｃ Ｎ Ａ、和ｃｍｙｃ 癌基因的平均荧光值均低于对照组,而 Ｐ Ｄ Ｇ Ｆ 的平均荧光值与对照组相比无显著差异。结论　 Ｌｏｓａｒｔａｎ 抑制 Ｖ Ｓ Ｍ Ｃ 的增殖及ｃｍｙｃ原癌基因的表达。该作用与癌基因调控的蛋白产物作用机制有关,是 Ｌｏｓａｒｔａｎ 治疗高血压,参与血管重塑的重要的机制     

An in vitro model for videoimaging of human bladder smooth muscle cell contractions

Knowledge regarding human bladder smooth muscle cell (SMC) physiology is very limited. Only a few specific medical therapies for bladder disorders have therefore been established. The objective of this study was to develop a model for videomicroscopy of bladder SMC contractions. Cells were isolated from human cystoprostatectomy specimens and cultured in a modified EMEM medium. These cells were identified as SMCs by means of immunohistochemistry. For videomicroscopy, the culture flasks were coated with a viscous agent to allow cell contraction. Contractions were visualized by means of a cell culture microscope with a time-lapse videosystem. For cholinergic stimulation of the cells, acetylcholine, in concentrations ranging from 100 μM to 10 mM, was applied. The percentage of contracting cells within the observation field was evaluated for quantitative analysis. In control experiments without contractile stimulant 6% of the cells were observed to contract. Stimulation with acetylcholine induced a significant dose-dependent increase to 47% in contracting cells. These results demonstrated that videomicroscopy is an appropriate tool to investigate the contraction mechanisms of bladder SMCs. This model offers the possibility of studying drug effects on the human detrusor in vitro. Received: 16 September 1999 / Accepted: 1 May 2000     

RNA干扰沉默雷帕霉素靶蛋白基因表达对血管平滑肌细胞增殖活性的影响

目的 构建靶向雷帕霉素靶蛋白（mTOR）的RNA干扰表达载体，观察其对血管平滑肌细胞（VSMC）增殖活性的影响。方法 根据大鼠mTOR基因序列设计2个短发夹环RNA（shRNA）序列，化学法合成单链寡核苷酸序列，将cDNA序列插入逆转录病毒载体pLXIN，脂质体介导转染包装细胞PT67后获得mTOR的重组逆转录病毒shRNA表达载体，感染VSMC，Northern blot和Western blot法检测mTOR及其下游底物真核细胞启动子4E结合蛋白（4E-BP1）、p70s6k等表达的变化，流式细胞仪检测VSMC细胞周期的变化，噻唑蓝（MTT）法检测VSMC增殖活性的改变。结果 shRNA序列插入pLXIN载体并感染VSMC，证实其能够显著抑制mTOR的mRNA和蛋白产物表达，mTOR通路下游的核糖体蛋白S6激酶（p70s6k）表达相应减少，而4E-BP1的表达却显著增强；感染前VSMC细胞G1／Go期比例为71％，S期为15％，凋亡细胞为2％；转染后72h，G1／岛期比例为87％，S期为6％，凋亡细胞为6％（P＜0．01）；表明G0／G1→S过程受阻，VSMC的分裂、增殖受到抑制，凋亡机制启动，更多细胞停滞在G0／G1期。结论 成功构建靶向mTOR基因的shRNA表达载体，能够明显抑制VSMC分裂、分化和增殖。     

The effect of N-methyl-D-aspartate receptor antagonist (memantine) on esophageal and gastric smooth muscle: functional investigation in a rat hydrocephalus model

Object

The purpose of this study was to investigate the effect of N-methyl-d-aspartate (NMDA) receptor antagonist on esophageal and gastric smooth muscle reactivity in a rat hydrocephalus model.

Material and Methods

Hydrocephalus was induced in rats by injection of kaolin into the cisterna magna. Two weeks after the procedure, memantine (20 mg/kg per day, 2 weeks) was given to rats with hydrocephalus in the memantine group (MG). The rest of the rats with hydrocephalus received serum physiologic (hydrocephalus group, HG). The control group (nonhydrocephalic rats, CG) was sham operated. The fourth group consisted of nonhydrocephalic rats with treated memantine (memantine control group, MC). Contractile (KCl, carbachol) and relaxant (isoprenaline, papaverine) esophageal and gastric smooth muscle reactivity were determined by in vitro muscle technique.

Results

No significant difference was found in the KCl (nonreceptor-mediated)-induced esophageal smooth muscle reactivity among the groups. Carbachol (receptor-mediated)-induced smooth muscle reactivity significantly decreased in HG compared to other groups. The isoprenaline (receptor-mediated)-induced smooth muscle reactivity significantly decreased in HG compared to other groups. No significant difference was found in smooth muscle reactivity to papaverine (nonreceptor-mediated) among the groups.Gastric smooth muscle reactivity to KCl significantly increased in HG compared to other groups. Also, KCl-induced smooth muscle reactivity significantly increased in MG compared to CG and MC. Carbachol-induced smooth muscle reactivity significantly decreased in HG compared to MG, CG, and MC. No significant difference was observed in isoprenaline- and papaverine-induced smooth muscle reactivity among the groups.

Conclusion

Our findings suggest that memantine may influence esophageal and gastric smooth muscle reactivity in hydrocephalus.     

Inhibition of diabetic bladder smooth muscle cell proliferation by endothelin receptor antagonists

Urinary bladder hypertrophy and hyperplasia are well recognised in diabetic cystopathy. The urinary bladder is known to synthesise endothelin-1 (ET-1), a potent vasoconstrictor peptide with mitogenic properties. Using diabetic New Zealand White (NZW) rabbits, we investigated the potential role of ET receptor subtypes (ET_A and ET_B) on the proliferation of bladder smooth muscle cells (SMC). Diabetes mellitus was induced in adult male NZW rabbits. After 6 months, control (n=6) and diabetic (n=6) bladders were removed and SMC from the dome and bladder neck were grown using standard explant methodology. At passage two, the cells were made quiescent and then further incubated in foetal calf serum (FCS), control age-matched rabbit serum (CRS) or diabetic rabbit serum (DRS) in the presence or absence of ET_A-antagonist (BQ123) or ET_B-antagonist (BQ788). SMC proliferation was then measured with 5-bromo-2′deoxy-uracil 24 h later and by cell counting (using a haemocytometer) at 48 h. Neither BQ123 nor BQ788 influenced detrusor or bladder neck SMC proliferation in FCS or CRS. However, in the presence of DRS, BQ123 and BQ788 significantly inhibited diabetic detrusor and bladder neck SMC proliferation at 30 and 100 nmol/l (P < 0.03 and P < 0.01, respectively). Cell counts were also significantly reduced from the diabetic detrusor and bladder neck (P < 0.01 and P < 0.03 with BQ123 and BQ788, respectively). These results suggest that ET may play a pathophysiological role in the bladder SMC hyperplasia associated with diabetes mellitus. Received: 24 November 1999 / Accepted: 21 March 2000     

兔阴蒂海绵体平滑肌细胞的体外培养方法及生物学特性

目的：探索新西兰白兔阴蒂海绵体平滑肌细胞的体外培养方法及生物学特性。方法：取兔阴蒂海绵体，采用酶消化法进行平滑肌细胞体外培养；在倒置显微镜下观察培养细胞的生长状况、形态特征及贴壁过程；用计数法测定细胞贴壁率；用MTT法描绘原代培养细胞的生长曲线；利用逆转录聚合酶链反应(RT—PCR)方法检测平滑肌细胞特征性标记物α-actin表达。结果：培养的兔阴蒂海绵体平滑肌细胞具有典型的平滑肌细胞形态特征，为梭形，呈长轴平行排列，具有明显的方向性；体外贴壁快，生长迅速，体外培养的阴蒂海绵体平滑肌细胞在合适的传代条件和比例下能够生存并保持其稳定的生物学特性。结论：体外培养的兔阴蒂海绵体平滑肌细胞模型可用于进一步研究阴蒂组织细胞学、分子生物学机制以及受体介导的平滑肌收缩信号转导机制及药理学作用等。     

血管壁重塑与平滑肌细胞表型改变及调亡的研究

目的 研究血管平滑肌细胞(VSMC)表型改变和调亡与血管壁重塑的关系。方法 建立兔髂动脉粥样硬化再狭窄模型，分析再狭窄血管壁形态和结构变化，研究中膜、内膜VSMC的表型改变，检测各个时间段内膜和中膜层VSMC调亡。结果 再狭窄血管腔面积与血管壁重塑指数呈正相关(r＝0．9250，P＝0．0024)，再狭窄血管的管径较对侧明显缩小(P＝0．0001)。中膜VSMC表型主要以收缩型表型为主，血管壁内膜的VSMC的表型发生动态变化。中膜与内膜VSMC调亡峰值基本一致，中膜VSMC调亡指数明显高于内膜(P＝0．0061)。结论 血管壁重塑在血管重建再狭窄中起着重要的作用，在血管壁重塑过程中调控VSMC的表型转化和调亡可能控制血管壁的不适重塑，减少再狭窄的发生率。     

家兔阴茎海绵体平滑肌细胞体外培养方法对比研究

目的　探索更好、更方便的家兔阴茎海绵体平滑肌细胞体外培养方法。方法　分别采用组织块法、酶消化法、组织块 酶消化法对家兔阴茎海绵体平滑肌细胞进行培养 ,在倒置显微镜下对培养过程的细胞分别作了生长情况及形态学观察。用HE染色、MTT法分别描绘原代培养细胞的生长曲线、细胞分裂指数曲线 ;用倒置显微镜观察活细胞的贴壁过程 ,计数法测定细胞贴壁率。结果　组织块法及酶消化法培养的细胞其生长状况和形态学各有自己的特点。组织块法的细胞生长慢 ,培养时间相对较长 ,纯度相对较高 ,原代培养细胞生长速度快 ,但由于该法消化时间不易掌握 ,常影响其成功率 ,多数细胞呈梭形 ,细胞的密度高 ,原代培养时间为 7～ 10d。结论　每种培养方法都有各自的优缺点。我们可根据实验的需要而选择不同的方法或联合培养     

苯肾上腺素对体外培养人前列腺平滑肌细胞增殖及凋亡的作用

目的探讨苯肾上腺素（PE）对体外培养人前列腺平滑肌细胞增殖及凋亡的作用。方法将原代培养得到的3—5代人前列腺平滑肌细胞随机分组，各组加入浓度为0．1μmol／L～100μmol／L的PE和／或10μmol／L a1受体阻滞剂酚妥拉明（Ph），以噻唑蓝（MTT）检测细胞增殖情况．原位凋亡法（TUNEL）检测细胞凋亡情况。结果PE浓度大于1μmol／L时对前列腺平滑肌细胞有诱导增殖和抗凋亡作用（P均〈0.05），并且存在浓度依赖性。r分别为0．90和-0．893（P均〈0．05）；10μmol／L Ph能够拮抗PE的诱导增殖和抗凋亡作用（P〈0．01）。结论PE可以通过a1受体信号传导途径诱导体外前列腺平滑肌细胞增殖增加和凋亡减少。     

Potassium channels and human corporeal smooth muscle cell tone: further evidence of the physiological relevance of the Maxi-K channel subtype to the regulation of human corporeal smooth muscle tone in vitro

PURPOSE: Recent evidence indicates that the large conductance, voltage dependent, Ca2+ sensitive K channel or Maxi-K has an important role in the modulation of human corporeal smooth muscle tone and, thus, in erectile capacity. We further clarified the contribution of the Maxi-K channel subtype to the generation of contractile responses in isolated human corporeal tissue strips. MATERIALS AND METHODS: We performed pharmacological studies of phenylephrine contracted isolated corporeal tissue strips in the presence and absence of the 2 Maxi-K channel blockers tetraethylammonium chloride (TEA) and charybdotoxin, and the Maxi-K opener NS1619. K channel treatment effects were evaluated using 2 parameters, including 1) the steady state parameter of the empirically determined peak magnitude of the steady state contractile response and 2) the kinetic parameter of time required to achieve half of the peak steady state contractile response or half-time. Electrophysiological studies in freshly isolated and cultured myocytes were performed in parallel to corroborate findings further at the tissue level. RESULTS: Pre-incubating isolated human corporeal tissue strips with 1 mM. TEA and 1 microM. charybdotoxin was associated with an approximate 20% increase in the peak steady state contractile response and a corresponding approximate 20% decrease in the half-time of the phenylephrine induced contractile response. Conversely, pre-incubation with 10 microM. NS1619 produced a significant, approximately 20% decrease in the peak steady state contractile response and an approximate 38% increase in the half-time of the phenylephrine induced contractile response. Adding 30 to 180 microM. NS1619 to phenylephrine pre-contracted smooth muscle strips resulted in a 30% to 50% reduction in steady state contractile tension. No detectable effect of NS1619 was observed in 120 mM. KCl or 100 mM. TEA pre-contracted corporeal tissue strips. Whole cell recordings of freshly isolated and cultured corporeal myocytes confirmed that 30 microM. NS1619 induced a charybdotoxin sensitive hyperpolarizing current mediated by the Maxi-K channel. CONCLUSIONS: These in vitro studies confirm and extend previous observations indicating the importance of the Maxi-K channel for regulating human corporeal smooth muscle tone, and by extension, erectile capacity and function.     

Smooth excimer laser coronary angioplasty (SELCA) and conventional excimer laser angioplasty: Comparison of vascular injury and smooth muscle cell proliferation

Although the excimer laser, which utilizes ‘non-thermal ablation effects’, has achieved encouraging results in early clinical trials, the long-term results have failed to show any advantage over conventional percutaneous transluminal coronary angioplasty (PTCA). A new system, Smooth Excimer Laser Coronary Angioplasty (SELCA), has been developed to reduce the tissue damage in the vessel wall caused by shock waves and vapour bubbles.SELCA (wavelength 308 nm, pulse duration 115 ns, repetition rate 150 Hz and energy density 50 mJ mm^-2) lowers the amount of shock wave formation and pressure peak amplitude in the surrounding tissue by about eight times when compared to the conventional 308 nm excimer laser (ELCA). In this preclinical evaluation, this new system was compared to ELCA. Fifty New Zealand White rabbits were stimulated by repeated weak DC impulses for a period of 28 days in order to form an atherosclerotic plaque in the right carotid artery. The vessels were excised 3, 7,14 and 28 days after laser irradiation for immunohistochemical analysis. SELCA and ELCA laser treatment lead to a decrease in maximal intimal wall thickness 3 days after intervention (control: 177±4 μm; SELCA: 131±22μm; ELCA: 120 ±33μm). In the period between 3 and 28 days, a moderate increase in intimal wall thickness was observed after SELCA treatment compared to a significant increase after ELCA (28 days after intervention: SELCA: 157±22μm; ELCA: 274 ±28μm). Bromodeoxyuridine (BrdU) was applied 18 and 12 h before excision of the vessels in order to determine the percent of cells undergoing DNA synthesis. The percent of BrdU labelled SMC in the intima (control: 13 ± 2 cells mm_-2) increased in both groups after 3 days (SELCA: 248 ± 107 cells mm^-2; ELCA: 162 ± 41 cells mm^-2) and 7 days (SELCA: 162± 55 cells mm^-2; ELCA: 279 ± 119 cells mm^-2). The present results demonstrate that vascular wall injury and increase in intimal wall thickness following SELCA are reduced in comparison to the results achieved with the conventional technique. Further trials are necessary to assess whether these improvements will lead to more favourable long-term results after excimer laser angioplasty.     

细胞外信号调节激酶对人血管平滑肌细胞的作用及其机制

目的观察磷酸化细胞外信号调节激酶(ERK)对离体培养的平滑肌细胞增殖、凋亡和表型改变的影响。方法培养人大隐静脉平滑肌细胞,实验组培养液中加入PD98059对磷酸化ERK进行阻断后,观测平滑肌细胞的增殖活性,凋亡细胞所占比例以及细胞表型,并与对照组比较。结果实验组与对照组相比,细胞增殖活性明显低于对照组(P<0.01),电镜和荧光染色测定凋亡细胞所占比例明显高于对照组(P<0.01),电镜检测合成型细胞所占比例明显低于对照组(P<0.01),收缩型细胞所占比例较对照组明显为高(P<0.01),α肌动蛋白免疫荧光化学检测显示处理组荧光表达强于对照组。结论对离体培养的人大隐静脉平滑肌细胞磷酸化ERK进行阻断后,平滑肌细胞的增殖受抑,凋亡增加,细胞由合成型向收缩型转变。     

Clinical and experimental studies on the role of platelet-activating factor (PAF) in the pathogenesis of septic DIC

Various toxic factors induced by endotoxin (Et) are thought to be deeply involved in the pathogenesis of severe infections. In this study, particular attention was paid to the role of the platelet-activating factor (PAF) in these conditions, and clinical and experimental studies were conducted on the relationship between PAF and the changes observed in the general parameters after surgical infections. In the clinical study, changes in the PAF concentration in the blood of seven patients with disseminated intravascular coagulation (DIC), five of whom were septic and two non-septic, were monitored by gas/mass spectrometry. The mean PAF level in the septic DIC group tended to be higher than that in the non-septic DIC group. Moreover, in the septic DIC group, the relationship between the increase in the PAF level and platelet count was analyzed with the lapse of time and we surmised a negative correlation between these parameters.Experimentally, we also investigated the role of PAF in Et shock and the effect of an anti-PAF agent and protease inhibitor. The Et-induced fall in blood pressure was similarly prevented by both the anti-PAF agent and protease inhibitor. However, the decrease in the platelet count was more significantly inhibited by the anti-PAF agent than by the protease inhibitor, whereas the parameters of the blood coagulation/fibrinolysis system were more affected by the protease inhibitor than by the anti-PAF agent.     

RNA干扰沉默蛋白激酶B基因表达对血管平滑肌细胞增殖活性的影响

目的 构建靶向蛋白激酶B（PKB／Akt）基因的逆转录病毒RNA干扰表达载体，观察其对血管平滑肌细胞（VSMC）增殖活性的影响。方法 针对大鼠PKB／Akt基因序列（NM_033230）设计多个短发夹环RNA（shRNA）序列，化学合成法合成单链寡核苷酸序列，pGEM—T载体克隆后双酶切，将cDNA序列插入逆转录病毒载体pLXIN，脂质体介导转染包装细胞PT67后获得PKB／Akt的重组逆转录病毒RNA干扰载体，感染血管平滑肌细胞（VSMC），Northern blot和Western blot法检测Akt及其下游底物-核糖体蛋白S6激酶（p70s6k）等表达的变化，流式细胞仪检测VSMC细胞周期的变化，噻唑盐（MTT）比色法检测VSMC增殖活性的改变。结果 shRNA序列经T载体克隆，酶切确定该片段为PKB／Akt基因shRNA序列；感染VSMC，证实其能够显著抑制PKB／Akt的mR—NA和蛋白产物表达，下游的p70s6k表达相应减少；被感染VSMC的分裂、增殖过程受阻，更多细胞停滞在C0／G1期。结论 成功构建PKB／Akt基因逆转录病毒shRNA载体，感染VSMC能够明显抑制其分裂、分化和增殖。     

雷帕霉素靶蛋白反义RNA真核表达载体的构建及其在血管平滑肌细胞中的表达

目的构建雷帕霉素靶蛋白（mTOR）的反义RNA真核表达载体，观察其对血管平滑肌细胞（VSMC）功能的影响。方法提取人VSMC总RNA，逆转录．聚合酶链反应（RT-PCR）扩增mTOR基因cDNA序列，经pGEM-T载体克隆后双酶切，将cDNA序列反向插入绿色荧光蛋白表达载体pEGP-C1，转染VSMC，Westernblot法检测反义表达载体对mTOR蛋白表达的影响，流式细胞仪检测细胞周期的变化。结果经RT-PCR获得664bp产物，T载体克隆后，酶切确定该片段为mTOR基因cDNA，进而构建反义RNA真核表达载体pEGFP-C1-mTOR，测序证明序列正确后转染VSMC，证实其能够显著抑制mTOR蛋白产物表达，转染72h的mTOR蛋白产物表达抑制率达82％，S期细胞由15％降低为7％，凋亡细胞增至9％。VSMC增殖过程在Gx／Go期→S期受阻。结论成功构建mTOR基因的反义RNA真核表达载体。     

雌二醇对大鼠血管平滑肌细胞细胞周期和周期相关蛋白的影响

目的研究17β雌二醇(E2)对大鼠血管平滑肌细胞(VSMC)生长的影响。方法应用流式细胞仪检测不同浓度E2在有或无血清刺激下对传代VSMC细胞周期及其相关蛋白Cy-clinD1和CDK4表达的影响。结果在有血清的条件下,E2(1、10、100nmol/L)促进VSMC从G1期向S过渡,其S期细胞比率分别为(31.89±9.14)%、(35.90±4.59)%、(30.77±1.20)%,显著高于对照细胞(21.63±1.80)%(P<0.05),并伴随CyclinD1和CDK4蛋白表达量的显著增高;而在无血清的环境中,高浓度(10、100nmol/L)E2则抑制VSMC增殖,其S期细胞比率分别为(9.93±1.43)%、(8.76±1.80)%,显著低于对照细胞(16.58±3.04)%(P<0.05),且伴随Cy-clinD1和CDK4蛋白表达量降低。结论E2在有或无血清条件下分别对VSMC增殖起促进或阻滞作用,其机制可能是通过影响CyclinD1和CDK4蛋白的表达而作用于VSMCG1期。     

靶向Pik3cb shRNA表达载体的构建及其对大鼠血管平滑肌细胞凋亡的影响

目的构建大鼠Pik3cb shRNA真核表达载体，并检测其下调大鼠血管平滑肌细胞Pik3cb mRNA表达的效应。方法根据Genbank中大鼠Pik3cb序列设计并合成两条shRNA寡核苷酸片段，退火形成双链并克隆导入载体pGenesil-1，酶切鉴定和测序无误后分别命名为pU6-Pik3cb—shRNA．1和pU6-Pik3cb—shRNA-2。通过脂质体转染大鼠胸主动脉平滑肌细胞，确定转染效率；通过荧光定量聚合酶链反应（PCR）检测Pik3cb mRNA的表达；TUNEL法检测细胞凋亡。结果pU6-Pik3cb—shRNA-1和pU6-Pik3cb-shRNA-2经测序和PCR扩增与原序列相同，转染大鼠平滑肌细胞48h时转染率为15．7％，荧光定量PCR结果显示转染组Pik3cb mRNA表达明显减少，与对照组比较差异有统计学意义（P〈0．05），错配质粒组与对照组比较Pil（3cb mRNA表达差异无统计学意义（P〉0．05）。TUNEL结果显示转染组凋亡细胞明显增加，与对照组比较差异有统计学意义（P〈0．05）。结论成功构建重组真核表达质粒pU6-Pik3cb—shRNA，并有效下调大鼠胸主动脉平滑肌细胞Pik3cb mRNA的表达，为靶向Pik3cb防治移植血管再狭窄的基因治疗奠定了基础。     

Protease-mediated human smooth muscle cell proliferation by urokinase requires epidermal growth factor receptor transactivation by triple membrane signaling

Background

Urokinase (uPA) modulates cellular and extracellular matrix responses within the microenvironment of the vessel wall and has been shown to activate the epidermal growth factor receptor (EGFR). This study examines the role of the protease domain of uPA during EGFR activation in human vascular smooth muscle cells (VSMC).

Methods

Human coronary VSMC were cultured in vitro. Assays of cell proliferation and EGFR phosphorylation were examined in response to the carboxyterminal fragment of uPA (CTF) in the presence and absence of the plasmin, metalloprotease and a disintegrin and metalloproteinase (ADAM) inhibitors, heparin-bound epidermal growth factor (HB-EGF), and EGFR inhibitors, and small interfering RNA to EGFR and ADAMs.

Results

CTF produced a dose-dependent increase in DNA synthesis and cell proliferation in human VSMC, which was blocked in a dose-dependent manner by both plasmin inhibitors and the EGFR inhibitor, AG1478. CTF induced time-dependent EGFR phosphorylation, which was blocked by inhibitors of plasmin and metalloproteinases activity. The presence of urokinase plasminogen activator receptor was not required. Inhibition of ADAM-10 and -12, and of HB-EGF blocked EGFR activation in response to CTF. CTF-mediated activation of EGFR was mediated through Gβγ, src, and NAD(P)H oxidase.

Conclusions

In human coronary VSMC, uPA induces uPAR-independent, domain-dependent smooth muscle cell proliferation through transactivation of EGFR by a plasmin-mediated, ADAM-induced, and HB-EGF–dependent process, which is mediated by the intracellular pathways involving Gαi, Gβγ, src, and NAD(P)H oxidase.     

体外不同培养方法对血管平滑肌细胞生物学性状影响的比较研究

目的　探讨体外不同培养方法对血管平滑肌细胞生物学性状的影响。方法　分别用酶消化及组织块法分离、培养血管平滑肌细胞 ,对细胞的形态、生长增殖及平滑肌α-肌动蛋白表达情况进行观察与检测。结果　组织块法原代培养得到的细胞数高于酶消化法 ,前者较后者细胞生长速度快 ,两者细胞均表达平滑肌α -肌动蛋白。结论　组织块法是适合血管组织工程获取血管平滑肌种子细胞的培养方法