肾上腺髓质素及其受体系统抑制溶血磷脂酸诱导的大鼠主动脉平滑肌细胞增殖

目的观察溶血磷脂酸对肾上腺髓质素及其受体系统生成的影响和肾上腺髓质素在溶血磷脂酸促进血管平滑肌细胞增殖中的作用。方法贴块法培养大鼠胸主动脉血管平滑肌细胞,H3-TdR掺入测定血管平滑肌细胞DNA合成,γ-32P-ATP标记的同位素法测定丝裂原活化蛋白激酶活性,放射免疫法测定血管平滑肌细胞中肾上腺髓质素的含量。结果溶血磷脂酸促进大鼠血管平滑肌细胞肾上腺髓质素生成,上调血管平滑肌细胞肾上腺髓质素及其受体CRLR、RAMP2和RAMP3mRNA表达;肾上腺髓质素可抑制溶血磷脂酸刺激大鼠血管平滑肌细胞3H-TdR掺入,抑制溶血磷脂酸诱导的丝裂原活化蛋白激酶激活。结论溶血磷脂酸上调血管平滑肌细胞肾上腺髓质素及其受体系统,肾上腺髓质素及其受体抑制血管平滑肌细胞增殖的作用与其抑制丝裂原活化蛋白激酶激活有关。     

血小板衍化生长因子对血管平滑肌细胞增殖及胶原蛋白合成的影响

目的 :观察血小板衍化生长因子 （PDGF）对培养的血管平滑肌细胞 （VSMC）增殖及胶原蛋白合成的影响。方法 :采用培养的兔动脉 VSMC,应用 3H- Td R的 3H-脯氨酸掺入方法 ,观察 PDGF- BB对兔 VSMC DNA合成以及胶原蛋白合成的影响。结果 :PDGF- BB可促进处于静止状态的兔 VSMC DNA及胶原蛋白的合成 ,并呈现明显的浓度依赖关系 ,在 40 μg/ L 的浓度时 DNA及胶原蛋白的合成达到高峰 ,DNA及胶原蛋白分别处于 36 h和 48h合成最为显著。结论 :PDGF- BB可明显促进培养的 VSMC增殖及胶原蛋白的合成。     

Mitogenic activity of endothelin-1 and -3 on vascular smooth muscle cells is inhibited by atrial natriuretic peptides

The effect of endothelin-1 (ET-1) and endothelin-3 (ET-3) on the proliferation of aortic vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) was assessed by the measurement of (3H)-thymidine incorporation into DNA. ET-1 and ET-3 stimulated the DNA synthesis in a dose-dependent manner. ET-3 was less potent than ET-1 in its stimulatory activity. The increase in (3H)-thymidine incorporation is clearly attenuated by Atriopeptin III (ANP 5-28). Atriopeptin I (ANP 5-25), a ANP fragment with the reduced biological activity compared to ANP 5-28, has no effect on the endothelin mediated stimulation of DNA-synthesis.     

Early changes in proteoglycans production by resistance arteries smooth muscle cells of hypertensive rats

Several functional and structural modifications at the vascular level have been described in spontaneously hypertensive rats (SHR) during the early development of hypertension. In this study, we hypothesize that changes in the extracellular matrix (ECM) could precede the development of hypertension. Synthesis of secreted and membrane-bound sulfated proteoglycans (S-PG) by cultured vascular smooth muscle cells (VSMC) obtained from young spontaneously hypertensive rats (pSHR) mesenteric resistance arteries, during the period preceding the elevation of blood pressure (BP) was tested. After 24 h of stimulation with angiotensin II (Ang II), 10% fetal calf serum (FCS), or 0.1% FCS as control, medium and cell layer S-PG synthesis was evaluated by labeling sulfated disaccharides with [35S] sodium sulfate. To relate this variable with cell proliferation, DNA synthesis was measured by incorporation of [3H]thymidine in the cell lysate. The VSMC from pSHR synthesized more secreted and membrane-bound S-PG than age-matched Wistar rat (pW) cells in the nonstimulated (0.1% FCS) and stimulated (Ang II or 10% FCS) experimental groups. When data were expressed as percent of their own control value, both Ang II and 10% FCS lowered basal secreted and cell-associated S-PG content in VSMC from pSHR, whereas in pW rat cells, these agents produced a small increase or no change. An inverse relationship between proliferation and total S-PG production (secreted plus membrane-bound) was found in pSHR cells, but not in pW cells. In conclusion, the present study demonstrates that changes in S-PG synthesis by VSMC of resistance arteries precede the vascular dysfunction associated with the development of hypertension in SHR.     

胰岛素对血管平滑肌细胞增殖和组织型纤溶酶原激活剂及抑制物活性的影响

以体外培养的猪主动脉平滑肌细胞为实验对象，研究了不同浓度胰岛素对细胞增殖和组织型纤溶酶原激活剂琢抑制物活性的影响。细胞增殖检测采用氚标脱氧胸腺嘧啶核苷掺入法，组织型纤溶酶原激活剂及其抑制物活性测定采用底物显色法。结果发现胰岛素促进血管平滑肌细胞增殖，刺激纤溶酶原激活剂抑制物活性增加，均呈剂量依赖性。而组织型纤溶酶原激活剂活性在猪血管平滑肌细胞体外培养液中不存在，且与胰岛素的刺激与否及作用浓度、时间     

Vascular smooth muscle cell proliferation is effectively suppressed by the non-specific growth factor inhibitor suramin

The purpose of this study was to investigate the effects of the non-specific growth factor inhibitor suramin on smooth muscle cell proliferation in vitro and in vivo. Cultured vascular smooth muscle cells (VSMC) were stimulated by platelet-derived growth factor (PDGF) and cellular DNA synthesis assessed by [3H]-thymidine uptake. Suramin dose-dependently inhibited DNA synthesis in VSMC, and 100 microM of suramin completely suppressed the PDGF-AB-induced cellular DNA synthesis. Rabbit carotid arteries were injured by the balloon catheter, and then suramin locally delivered using a porous balloon catheter over ten minutes. Three weeks after the vascular injury, the extent of intimal thickening was compared between the suramin-treated and control rabbits. The neointimal formation triggered by balloon-mediated vascular injury was suppressed significantly and dose-dependently by locally infused suramin, and the intima to media area ratios of the control and 1 mM suramin-treated animals were 48.8+/-14.9 and 12.2+/-6.0%, respectively (p < 0.01. n = 6 for each group). These results suggest that one time local administration of suramin was sufficient to suppress neointimal formation after balloon-mediated vascular injury, and that pharmacological intervention targeting the growth factor's signaling pathways could be a promising approach to prevent smooth muscle cell proliferation in various proliferative vascular diseases.     

阿魏酸钠抑制内皮素-1介导高血压病血管平滑肌细胞增殖的研究

目的研究阿魏酸钠(SF)对高血压痛患者血管平滑肌细胞(VSMC)在体外培养条件下内皮素-1(ET-1)介导增殖的影响.方法采用体外培养的VSMC,选用3～8代VSMC分为:ET-1组;SF组;ET-1与SF组.在ET-1组中加入6个不同浓度的SF(0,20,40,80,100,120μg/ml),培养24h后用苔酚蓝法(MTT)和氚-胸腺嘧啶核苷(3H-TdR)掺入法测定细胞含量及其培养液中一氧化氮(NO).结果SF可使细胞MTT含量和3H-TdR掺入量明显减少,ET-1可使细胞MTT含量和3H-TdR掺入量明显增加.同时,SF可使ET-1所致的细胞MTT含量增加和3H-TdR掺入量增加明显减少,并随SF含量增加而NO升高.结论SF可抑制VSMC增殖,SF抑制ET-1诱导VSMC增殖,并呈剂量依赖型.其作用可能与通过拮抗ET-1、增高NO活性有关.     

莲心碱对血管平滑肌细胞增殖及热应激蛋白70和P53表达的影响

采用培养的内皮素所致血管平滑肌细胞增殖模型。用氚-胸腺嘧啶核苷掺入法、流式细胞术、Westernblot及Northernblot分析方法，观察了莲心减对血管平滑肌细胞增殖的作用及对热应激蛋白70及其mRNA和抑癌基因P53表达的影响。结果发现，遂心碱能逆转内皮素所致的~3H－TdR掺入量增多，阻止血管平滑肌细胞由Go／G1期进入DNA合成期（S期）和有丝分裂期（G2／M期）．并能使效应激蛋白70及mRNA表达减弱，P53抑癌基因及mRNA表达增强。以上结果提示，莲心碱能抑制血管平滑肌细胞增殖，这可能与热应激蛋白70及P53的调控有关。     

多巴胺D3受体对胰岛素受体介导的促血管平滑肌细胞增殖作用的影响

目的研究多巴胺D3受体对胰岛素受体介导的促血管平滑肌细胞增殖作用的影响。方法以胚胎大鼠胸主动脉血管平滑肌细胞株(A10)为研究对象,观察在D3受体激动剂作用下,胰岛素受体促血管平滑肌细胞增殖作用的变化。利用[3H]-TdR细胞掺入实验观察细胞增殖状况,并利用免疫印迹法观察D3受体对胰岛素受体蛋白表达的影响,初步探讨D3受体影响胰岛素受体介导的促血管平滑肌细胞增殖作用的机制。结果D3受体激动剂(PD128907)本身对血管平滑肌细胞增殖没有影响,但可抑制胰岛素受体介导的促血管平滑肌细胞增殖作用。刺激D3受体可降低胰岛素受体的表达,提示D3受体可能通过影响胰岛素受体的表达,从而影响胰岛素受体促血管平滑肌细胞增殖的过程。结论D3受体对胰岛素受体介导的促血管平滑肌细胞增殖具有一定的抑制作用。     

Autocrine Effects of Endothelin on In Vitro Proliferation of Vascular Smooth Muscle Cells from Spontaneously Hypertensive and Normotensive Rats

According to previous studies, endothelin-1 (ET-1) is the most potent growth factor in the regulation of vascular smooth muscle cell (VSMC) proliferation in spontaneously hypertensive rats (SHR). To evaluate if the dominant effect of ET-1-induced VSMC proliferation is achieved by autocrine regulation, aortic smooth muscle cells from four-week-old SHR and WKY (Wistar-Kyoto) rats were cultured in 24-well dishes, and the effects of ET-1 on VSMC proliferation were determined by (a) ³H-thymidine incorporation assays with different ET-1 blocking treatments, including a specific anti-ET-1 antibody; BQ-123, an ETA receptor blocker; and BQ-788, an ETB receptor blocker; and (b) examining the ET-1 blockade on the effects of treatment with other growth factors, including thrombin and angiotension II (AT-II). These results demonstrated that the anti-ET-1 antibody, BQ-123, BQ-788, and BQ-123 plus BQ-788 all caused dose-dependent inhibition of proliferation. A 90% inhibitory effect was observed at the maximum doses used except for BQ-123. The ET-1 receptor blockers inhibited thrombin-induced VSMC growth; however, they did not efficiently inhibit AT-II-induced VSMC growth. These results indicate that the autocrine effects of ET-1 play a predominant role in the proliferation of VSMCs from SHR and WKY rats. They also suggest that thrombin-induced VSMC growth is mediated by the autocrine effects of ET-1, and angiotensin II-induced VSMC growth is mediated by other signal pathways.     

PD098059及N-乙酰半胱氨酸在缓激肽引起血管平滑肌细胞增殖反应中的调节作用

目的 :研究丝裂原活化蛋白激酶 （MAPK ）信号途径在缓激肽 （BK）介导的大鼠血管平滑肌细胞 （VSMC）增殖中的作用。方法 :通过 3H -胸苷 （3H- Td R）掺入率与 SMC3H-亮氨酸掺入率分别反映 VSMC的 DNA代谢与蛋白质合成代谢速率 ;并通过给予 PD0 980 5 9及 N-乙酰半胱氨酸预处理 ,观察其对细胞增殖的影响。结果 :1BK（10 nm ol/L ）处理 30 min,VSMC3H-胸苷掺入率和 3H-亮氨酸掺入率均明显增高 ;2 BK增高 3H -胸苷掺入的作用可明显被PD0 980 5 9所抑制 ,对 3H -亮氨酸掺入的作用可部分被 PD0 980 5 9所抑制 ;3BK增高 3H -胸苷掺入和 3H -亮氨酸掺入的作用均可部分被 N-乙酰所抑制 ,完全被 N-乙酰 +PD0 980 5 9所抑制。结论 :细胞外信号调节激酶 （ERKs）激活在缓激肽介导的 VSMC增殖中具有重要作用 ,并可通过 ERKs信号途径的特异性抑制剂影响血管平滑肌细胞的增殖效应。     

Effects of nicorandil on cell proliferation and cholesteryl ester accumulation in arterial smooth muscle cells in culture

Summary Ca²⁺ regulates a variety of cellular mechanisms in vascular cells as well as in platelets. Nicorandil interacts with the intracellular Ca²⁺-activated processes in vascular smooth muscle cells, while Ca²⁺ channel blockers such as verapamil and diltiazem block voltage-dependent Ca²⁺ channels. The effects of nicorandil are due to the hyperpolarization of the membrane, interference with mobilization of Ca²⁺ from the intracellular storage sites, and blockade of receptor-operated Ca²⁺ channels. In the present study, the effects of nicorandil on cell proliferation and cholesteryl ester accumulation in rat arterial smooth muscle cells in culture were compared to Ca²⁺ channel blockers. Smooth muscle cells were prepared from rat thoracic aorta, and the rate of proliferation was determined by measuring the cell number and by [³H]-thymidine incorporation into cellular DNA. The effect of nicorandil on cholesteryl ester content in smooth muscle cells was determined by thin-layer chromatography of the cell extracts. Nicorandil at concentrations of 10^–6 to 10^–4 M, as well as Ca²⁺ channel blockers (verapamil and diltiazem) inhibited the proliferation and DNA synthesis of cultured smooth muscle cells. The acute inhibitory effects on cell proliferation were observed significantly 16 hours after the addition of the three agents in serum-stimulated cells. These effects were dose dependent, both in acute and in chronic treatment with the three agents. Addition of 10^–5 M nicorandil to medium supplemented with 10% serum resulted in a decrease of the net cholesteryl ester content by 18±1%, while cellular free cholesterol content was the same as control. Similar results were also obtained in the presence of verapamil and diltiazem. These data suggest that nicorandil may suppress atheroma formation, not only by inhibiting cell proliferation but also by decreasing cholesteryl ester accumulation in arterial smooth muscle cells.     

Dipeptidyl Peptidase IV Regulates Proliferation of Preglomerular Vascular Smooth Muscle and Mesangial Cells

The purpose of this study was to investigate the role of dipeptidyl peptidase IV in regulating the effects of 2 of its substrates, neuropeptide Y(1-36) and peptide YY(1-36), on proliferation of and collagen production by preglomerular vascular smooth muscle and glomerular mesangial cells from spontaneously hypertensive and normotensive rats. In cells from hypertensive rats, neuropeptide Y(1-36) and peptide YY(1-36) stimulated [(3)H]-thymidine incorporation (cell proliferation index), cell number, and [(3)H]-proline incorporation (index of collagen synthesis); and sitagliptin (dipeptidyl peptidase IV inhibitor) significantly enhanced most of these effects. Neuropeptide Y(3-36) and peptide YY(3-36) (products of dipeptidyl peptidase IV) had little effect on [(3)H]-thymidine incorporation, and sitagliptin did not enhance the effects of either peptide. BIBP3226 (Y(1) receptor antagonist) blocked the effects of neuropeptide Y(1-36) and peptide YY(1-36) on [(3)H]-thymidine incorporation in the absence and presence of sitagliptin. Neuropeptide Y(1-36) and peptide YY(1-36) stimulated [(3)H]-thymidine and [(3)H]-proline incorporation and cell number in cells from normotensive rats; however, the effects were weak and mostly not affected by sitagliptin. Real-time PCR and Western blotting showed similar dipeptidyl peptidase IV mRNA and protein levels in cells from hypertensive versus normotensive rats, with greater levels in smooth muscle versus mesangial cells. Both cell types converted peptide YY(1-36) to peptide YY(3-36) in a concentration-dependent manner that was attenuated by sitagliptin, and dipeptidyl peptidase IV activity was greater in smooth muscle versus mesangial cells. In conclusion, dipeptidyl peptidase IV inhibitors might entail a risk of renal dysfunction because of abnormal proliferation of cells in the preglomerular microcirculation and glomeruli.     

葛根素抑制凝血酶诱导的血管平滑肌细胞增殖

目的　观察葛根素对T诱导的血管平滑肌细胞(VSMC)增殖的影响。方法　建立凝血酶(T)诱导的大鼠血管平滑肌细胞增殖模型,以细胞计数法和流式细胞仪DNA含量测定的方法观察T及葛根素对VSMC增殖和DNA合成的影响。结果　T对VSMC有明显促增殖作用,促增殖效应在2 4小时末达峰,且T浓度在0 . 1U/L～1 . 0U/L之间有剂量依赖关系;葛根素呈剂量依赖性地抑制T诱导的细胞增殖与DNA合成。结论　葛根素能抑制T诱导的VSMC增殖。     

Coculture of Vascular Endothelial Cells and Smooth Muscle Cells from Spontaneously Hypertensive Rats

The effect of endothelium‐released vasoactive factors on vascular smooth muscle cell (VSMC) proliferation was studied in a coculture system. Isolated aortic endothelial cells and smooth muscle cells from 4‐week‐old spontaneously hypertensive rats (SHR) and age‐matched Wistar–Kyoto (WKY) rats were cocultured. After coculture, the VSMC proliferation rate was examined by ³H‐thymidine incorporation assay and the levels of the vasoactive factors in medium were determined by enzyme immunoassay (EIA). The results indicate that the proliferation rate of VSMCs in SHR was significantly higher than in WKY rats when VSMCs were cultured alone. When SHR vascular endothelial cells (VECs) were cocultured with VSMCs, the proliferation rate of SHR VSMCs was enhanced; however, there was no growth promoting effect in WKY VSMCs. When WKY VECs were cocultured with VSMCs, no VSMC proliferation effect was observed. When VSMCs were cultured alone, the endothelin‐1 (ET‐1) secretion in SHR was significantly higher than in WKY rats. When VECs and VSMCs were cocultured, the ET‐1 concentration increased in both SHR VEC and WKY VEC coculture groups in a similar manner; but the SHR VECs tended to release more thromboxaneA₂ (TXA₂) and less PGI₂ than WKY VECs. These results suggest that some kind of interaction between SHR VSMCs and SHR VECs is responsible for the high proliferation of SHR VSMCs but not the effects of SHR VECs per se.     

卡托普利对血管平滑肌细胞增殖和PDGF—B、bFGF及相关癌基因的影响

采用内皮素建立培养的血管平滑肌细胞增殖模型。用氚-胸腺嘧啶核苷（［3H］－TdR）参入法，流式细胞术，免疫细胞化学及NorthemBlot杂交方法，观察卡托普利（Cap）对VSMC增殖的作用及对生长因子PDGF－B，bFGF及其相关癌基因c－sis，c－myc表达的影响。结果发现：Cap能逆转内皮素所致的［3H］－TdR参入量增多，阻止血管平滑肌细胞由静止期（G0／G1期）进入DNA合成期（S期）和有丝分裂（G2／M期），并能逆转内皮素引起的PDGF—B，bFGF抗原，c－sis，c－mycmRNA表达增强。提示：aop有抑制血管平滑肌细胞增殖作用，与生长因子及癌基因调控的分子生物学机制有关。     

RNAi技术在抑制血管平滑肌细胞增殖中的研究进展

移植血管狭窄是影响冠脉旁路移植术后远期疗效的主要原因,其机理包括血管中膜平滑肌细胞表型转化、增殖、迁移至内膜并合成大量的细胞外基质。RNA干扰(RNAi)技术是一种沉默特异基因序列的基因治疗方法,现对其机理及在抑制血管平滑肌细胞增殖中的应用进展进行综述。     

大黄素通过p53途径抑制血管平滑肌细胞增殖的实验研究

目的探讨p53途径在大黄素抑制血管平滑肌细胞增殖作用中的地位。方法通过细胞计数、老化相关β-半乳糖苷酶染色、Annexin V标记等方法观察大黄素抑制血管平滑肌细胞增殖的特点。^3H-胸苷掺入法测定DNA合成、流式细胞仪了解细胞周期变化、Western blot检测p53蛋白表达变化、基因芯片观察mRNA表达水平。结果（1）1．6～3．1μg／ml大黄素延缓细胞生长,6．3～12．5μg／ml大黄素促进细胞老化,25．0μg／ml大黄素则可显著诱导细胞凋亡。（2）大黄素干预24h后,出现非计划性DNA合成现象,这是DNA损伤的敏感性标志。p53基因和蛋白表达水平呈大黄素浓度依赖性上调。除了细胞增殖基因表达下调,其他基因表达均上调,如细胞老化基因、细胞凋亡基因、DNA损伤修复基因。（3）大黄素能够迅速渗透进入细胞,在细胞内的分布具有明显的选择性,绝大多数以颗粒形态分布于细胞胞浆中,细胞核中也有少量分布。结论大黄素通过损伤DNA激活p53途径。随着大黄素浓度升高,p53途径激活程度也随之增强并产生多种细胞增殖抑制效应,即生长停滞、细胞老化和细胞凋亡。     

L-精氨酸和牛磺酸对同型半胱氨酸诱导平滑肌细胞增殖的抑制作用

目的　观察Ｌ－精氨酸和牛磺酸对同型半胱氨酸（ＨＣＹ）诱导的血管平滑肌细胞（ＶＳＭＣ）增殖的影响。方法采用同位素技术测定ＶＳＭＣ３Ｈ－ＴｄＲ参入。结果ＨＣＹ可促进ＶＳＭＣ增殖。Ｌ－精氨酸及牛磺酸均可呈剂量依赖性抑制ＨＣＹ诱导的ＶＳＭＣ增殖,且合用Ｌ－精氨酸和牛磺酸的对ＶＳＭＣ增殖的抑制作用明显强于单纯应用Ｌ－精氨酸或牛磺酸组。结论Ｌ－精氨酸和牛磺酸均可抑制ＨＣＹ诱导的ＶＳＭＣ增殖。