大鼠主动脉内皮剥脱后平滑肌细胞增殖与c-myc基因表达的研究

用Fogarty导管对25只大鼠行主动脉内皮细胞剥脱，在剥脱后的24h,5天，10天，15天时观察，发现主动脉平滑肌细胞增殖和内膜增厚随时间延长而降低，20天接近基础水平，结果表明，c-myc基因表达可能在启动平滑肌细胞增殖和内膜增厚中起重要作用。     

内皮剥脱后家兔胸主动脉舒张反应的变化

本实验在家兔胸主动脉内皮剥脱模型上，观察血管舒张反应的变化及其与血管平滑肌细胞增殖的关系。结果发现．内皮剥脱后血管对乙酰胆碱的内皮依赖性舒张反应明显降低。术后２周和４周时分别为对照组的２７．７±９．９％和５１．１±１１．４％；实验组和对照组血管对去甲肾上腺素的收缩反应和硝普钠的舒张反应均无显著性差异。内皮剥脱后血管平滑肌细胞显著增殖，内膜增厚。这些结果提示，内皮剥脱后血管的内皮依赖性舒张功能受损，内皮源性舒张因子产生可能减少；血管平滑肌细胞增殖可能与这些变化有关．     

血清应答因子在血管新生内膜形成过程中的表达变化

目的探讨SRF与VSMC表型转化及增殖之间的关系。方法建立大鼠颈总一胸腹主动脉内皮剥脱后内膜增生模型，应用Northern印迹、Western印迹及免疫组化等方法从RNA和蛋白质水平上检测新生内膜形成过程中SRF表达的动态变化。结果血管内皮剥脱后3d，可见局部内膜增生，中膜平滑肌细胞排列不齐；术后7～14d，内膜呈进行性弥漫增厚，管腔出现狭窄。在增生的内膜中，SRF的mRNA和蛋白水平明显升高，于术后7d达高峰，其动态变化模式与血管内膜增厚速率相一致，但与收缩型VSMC的标志物SM22α的表达却呈反相关变化。结论血管内皮剥脱后SRF表达活性升高与VSMC去分化和新生内膜形成具有直接关系。     

钙稳态失衡在内皮损伤后血管平滑肌增生中的作用

目的探讨内皮剥脱后血管平滑肌细胞钙稳态的变化及其在内皮损伤诱导血管平滑肌细胞的增殖中的作用。方法在大鼠球囊内皮剥脱模型上，用３Ｈ－胸腺嘧啶核苷和３Ｈ－亮氨酸掺入及４５Ｃａ转运的方法。结果内皮损伤可导致血管平滑肌细胞增殖、内膜增厚；血管平滑肌细胞钙内流增多，剥脱后３天时为４．１２±０．２８、对照组为３；２８±０．１４ｎｍｏｌ／×１０６细胞（Ｐ＜０．０５）；１０天时为４．０９±０．２１、对照组３．３１±０．０９ｎｍｏｌ／×１０６细胞，（Ｐ＜０．０１）、钙外移减少、钙含量升高（剥脱后３天时为９９５±５４、对照组为６９５±３３ｎｍｏｌ／×１０６细胞（Ｐ＜０．０１）；１０天时为１０２２±９４对照组、７０９±３２ｎｍｏｌ／×１０６细胞（Ｐ＜０．０１））、肌浆网和线粒体钙摄取功能增强；应用钙通道阻断剂治疗可以调整内皮剥脱后血管平滑肌细胞的钙稳态失衡，还可以抑制内皮损伤诱导的血管平滑肌细胞增殖。结论钙稳态失衡可能是内皮损伤引起血管平滑肌细胞增殖的细胞学机制之一。     

黄芪、当归对血管内皮剥脱后内膜增生的影响及作用机制

目的 研究黄芪和当归防治血管内皮剥脱后再狭窄与血管平滑肌细胞(VSMC)表型转化之问的关系。方法 建立大鼠主动脉内皮剥脱模型，分别采用形态学和Northern印迹分析技术，观察大鼠血管内皮剥脱后内膜增生情况及VSMC表型标志基因SM α—肌动蛋白、平滑肌胚胎型肌聋蛋白重链(SMemb)表达活性。结果 术后7d模型组内膜明显增生，14～21d内膜呈进行性弥漫性增厚，与模型组相比，两种中药治疗组的内膜增生程度均明显藏轻1分化标志SM α—肌动蛋白基因表达增高，去分化标志SMemb表达降低。结论 黄芪和当归通过抑制VSMC表型转化而减缓血管内膜增生。     

全反式维甲酸对球囊损伤大鼠胸主动脉内皮后P16及增殖细胞核抗原表达的影响

为研究全反式维甲酸对球囊损伤大鼠胸主动脉内皮后内膜增生过程、P16及增殖细胞核抗原表达的影响，球囊剥脱大鼠胸主动脉内皮，并随机将大鼠分为手术组、全反式维甲酸治疗组及对照组，各组均于术前4天灌胃至实验结束，除对照组于术后14天处死大鼠外，全反式维甲酸治疗组和手术组分别在术后2天、7天、14天和28天处死大鼠并摘除大鼠胸主动脉，通过组织学检查和免疫组织化学技术检测术后14天和28天的内膜增生情况及术后2天、7天、14天和28天P16和增殖细胞核抗原的表达及全反式维甲酸(每天30mg／kg)灌胃对它们的影响。结果发现，对照组及内皮损伤后2天均无血管内膜增厚，7天内膜开始增生，28天血管平滑肌细胞增殖减弱，但细胞外基质增加。在手术组各时间点P16的表达变化不显著，增殖细胞核抗原于球囊损伤后2天在中膜明显表达，术后7天表达达到高峰，且主要在内膜表达，14天后逐渐下降。使用全反式维甲酸治疗后，内膜增生程度及增殖细胞核抗原的表达明显降低，而P16的表达在术后2天开始升高，14天达高峰。以上结果提示，全反式维甲酸可有效抑制血管内皮损伤后内膜的增生，其机制可能与促进P16表达和抑制增殖细胞核抗原表达，从而抑制血管平滑肌细胞的增殖有关。     

动脉粥样硬化形成中平滑肌细胞增殖与c-myc、hsp 70基因表达的研究

动脉粥样硬化形成中平滑肌细胞增殖与ｃ－ｍｙｃ、ｈｓｐ７０基因表达的研究万腊香，杨和平，杨永宗（衡阳医学院分子生物学研究中心，衡阳４２１００１）本实验用高胆固醇饲料加兔主动脉内皮剥脱方法复制了实验性兔动脉粥样硬化模型，观察了在动脉粥样硬化形成过程中平滑...     

C-sis反义寡脱氧核苷酸抑制兔主动脉平滑肌细胞增殖的时效关系

为观察局部处理Ｃ－ｓｉｓ反义寡脱氧核苷酸对平滑肌细胞增殖及新生内膜肥厚的抑制作用，球囊导管损伤６０只新西兰白兔胸主动脉后，经导管局部处理每只兔１ｍｇ（２ｍＬ）Ｃ－ｓｉｓ反义或正义寡脱氧核苷酸或２ｍＬ生理盐水，于损伤后第３、７、１４天和第２８天，用免疫组织化学方法结合图象分析，评价损伤动脉内膜平滑肌细胞增殖及新生内膜改变。结果发现，Ｃ－ｓｉｓ反义寡脱氧核苷酸明显抑制内膜平滑肌细胞增殖，最高抑制作用出     

高脂血症对内皮剥脱术后血管炎症反应的影响

目的探讨高脂血症对内皮剥脱术后血管炎症反应的影响。方法建立大鼠主动脉内皮剥脱后内膜增生模型,利用免疫组化及Western blot方法检测血管壁NF-κB和iNOS的表达变化,并结合形态学分析研究高脂血症大鼠内皮剥脱后血管炎症反应及新生内膜增生情况。结果主动脉内皮剥脱后内膜呈弥漫性增厚,管腔变小,增厚的内膜以VSMC为主,中膜VSMC排列紊乱。高脂血症明显促进了增生的内膜中NF-κB和iNOS的表达。结论高脂血症通过促进NF-κB表达进而诱导iNOS的表达,加剧了内皮剥脱后的血管炎症反应。     

C-sis反义寡脱氧核苷酸抑制兔主动脉平滑肌细胞增殖的时效关系

为观察局部处理C-sis反义寡脱氧核苷酸对平滑肌细胞增殖及新生内膜肥厚的抑制作用、球囊导管损伤60只新西兰白兔胸主动脉后。经导管局部处理每只兔1mg（2mL）C-sis反义或正义寡脱氧核苷酸或2mL生理盐水,于损伤后第3、7、14天和第28天，用免疫组织化学方法结合图象分析，评价损伤动脉内膜平滑肌细胞增殖及新生内膜改变。结果发现．C-sis反义寡脱氧核苷酸明显抑制内膜平滑肌细胞增殖，最高抑制作用出现于损伤后第3天；抑制百分率达69．8％，第7、14天和第28天的抑制率依次降低，分别为59％、51.9％和47.8％；而新生内膜面积的抑制百分率分别是48．9％、77．8％、72．6％和70．8％，与生理盐水处理组相比，平滑肌细胞数及新生内膜面积均有明显降低。研究结果表明，局部处理C-sis反义寡脱氧核苷酸，通过反义途径，可明显抑制球囊损伤主动脉内膜平滑肌细胞增殖，其抑制作用随时间而降低，同时，对新生内膜面积肥厚的最大抑制出现于第7天，随后降低，提示局部处理C-sis反义寡脱氧核苷醚，有可能降低再狭窄的发生。     

c—myc反义寡核苷酸对缺氧内皮细胞条件培养液刺激的肺血？…

探讨ｃ－ｍｙｃ反义寡核苷酸对缺氧内皮细胞条件培养液刺激的肺血管平滑肌细胞的增殖的影响。方法 制备ＨＥＣＣＭ，用其刺激肺动脉ＳＭＣ后，Ｎｏｒｔｈｅｒｎ杂交分析ｃ－ｍｙｃ基因表达，＾３Ｈ－胸腺嘧啶核苷掺入试验及细胞生长计数分析ＳＭＣ增殖情况。     

Vascular smooth muscle phenotype and growth behaviour can be influenced by macrophages in vitro

The effect of macrophages on rabbit vascular smooth muscle phenotype and proliferative ability was examined using ultrastructural morphometry. The volume fraction of myofilaments (Vv myo) in smooth muscle cells (SMC) from 9-week-old rabbit aorta in vivo was 39.5 +/- 1.2%. After seeding the enzymatically isolated SMC at 4 X 10(5) cells/ml in primary culture, the Vv myo was 38.9 +/- 1.2% on day 3 dropping to 29.9 +/- 2.0% by day 5. On day 6 the Vv myo was 29.2 +/- 1.8%, and the cells began to proliferate. Confluency was reached after less than 24 h proliferation and the Vv myo rose abruptly on day 7 to 36.9 +/- 1.9%. When the SMC were co-cultured with macrophages, the Vv myo fell to 31.2 +/- 0.9% on day 3 and to 25.9 +/- 0.5% on day 5 at which time cells commenced proliferation. Confluency occurred on day 6 but the SMC Vv myo did not rise throughout the rest of the culture period (27.4 +/- 1.8% and 26.9 +/- 1.3% on days 7 and 9, respectively) and the cells, unlike the controls, continued to proliferate, becoming multi-layered. Early phenotypic modulation in sparsely seeded SMC (8 X 10(4) cells/ml) co-cultured with macrophages was also found using fluorescent labelled antibodies to smooth muscle myosin. Measurement of proliferation by cell counts (and tritiated thymidine autoradiography) showed that macrophages stimulated SMC in primary culture to proliferate at a significantly greater rate than control cells grown alone in 5% whole blood serum (WBS). Proliferation of subcultured SMC co-cultured with macrophages was also stimulated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specific induction of 68 KD protein synthesis in rabbit smooth muscle cells by growth stimuli in platelets: comparison of intimal and medial SMC

We examined the influence of growth stimuli in heat-treated platelet extract on specific protein synthesis in the early phase of the cell cycle prior to the initiation of DNA synthesis in cultured rabbit aortic smooth muscle cells (SMC). The extract preferentially stimulated the synthesis of a cytoplasmic protein with a molecular weight of 68000 (p68) and an isoelectric point of around 6.3. Stimulation of p68 synthesis occurred within 1 h after the addition of heat-treated platelet extract to growth-arrested and quiescent SMC, continued until 4 h after stimulation, and then returned to the baseline level. Actinomycin D preferentially inhibited p68 synthesis. In SMC prepared from atheromatous plaques from the aorta of hyperlipidemic rabbits (I-SMC), the amount of DNA synthesis and of p68 synthesis by heat-treated platelet extract were less than those of SMC prepared from normal media (M-SMC), suggesting that the decreased capacity for cell proliferation of I-SMC in response to heat-treated platelet extract was due to the down-regulation of signal transduction in the early G0/G1 phase of the cell cycle.     

高脂血症兔主动脉内皮剥脱后c-myc基因的表达

本实验在喂高胆固醇饲料的基础上行兔主动脉内皮剥脱术，分别于内皮剥脱术后1、2、4和6周处死动物．观察动脉粥样硬化形成过程中兔主动脉形态、平滑肌细胞超微结构的变化和c－nryc基因表述规律．实验结果发现：内皮剥脱加高胆固醇饲料能在6周内形成较典型的粥样斑块，斑块肉细胞成分主要为平滑肌细胞源性泡沫细胞，巨噬细胞源性泡沫细胞少见。在动脉粥样硬化斑块形成过程申，平滑肌细胞发生表型改变，术后一周新生内膜的平滑肌细胞中肌丝成分减少，细胞器增多，此时C－myc基因表达最高．术后2、4周内膜平滑肌细胞内含有大量细胞器，肌丝成分少见，成为典型的的合成型细胞，从2周开始合成型细胞内已开始出现数个脂滴，且随时间延长而增加，至6周时这些细胞已充满大量脂满，细胞器亦减少。成为肌源性泡沫细胞，2周后，c-myc基因表达逐渐降低。这些结果提示：兔主动脉内皮细胞剥脱能诱导c-myc基因高表达，加速实验性动脉粥样硬化形成。     

Arterial injury-induced smooth muscle cell proliferation in rats is accompanied by increase in polyamine synthesis and level

Proliferation of smooth muscle cells (SMC), enhancement of polyamine biosynthesis and increase in polyamine level in response to deendothelialization in the rat aorta were studied. [3H]Thymidine incorporation into SMC in aortas denuded with a balloon catheter began 25 h after injury, and maximal incorporation occurred 33-37 h after injury. Afterwards, [3H]thymidine incorporation declined, approaching the baseline level, but was slightly higher than that of sham-operated controls until 14 days after injury. Intimal thickening started 7 days after injury, and peaked at 21 days. Prior to these proliferative changes in aortic SMC, a rapid and transient increase in ornithine decarboxylase (ODC) activity was observed within 8 h after injury. There was no significant difference in ODC activity between injured and intact aortas after 4 days. The levels of polyamines, putrescine, spermidine, and spermine increased and were maximal at 48 h after injury, 8.1, 3.4 and 1.4 times the control levels, respectively. Increased levels of polyamines, in particular spermidine, continued until 7 days after injury. These results suggest that the enhancement of polyamine synthesis and the increased polyamine content of the aorta play important roles in the proliferation of SMC and in the development of intimal thickening, particularly in the initial proliferative response of medial SMC after deendothelialization.     

Studies on aorta during development. I. Fetal rabbit aorta under ex vivo and in vitro conditions: rapid changes in smooth muscle cell phenotype, cell proliferation and cholesterol content with organ culture

The structural development of the already well defined fetal rabbit aortic wall from 22 to 31 days of gestation in vivo consists of increasing aortic wall thickness, elastic lamina continuities, extracellular matrix deposition, and maturing of the fine structure of the medial smooth muscle cells. In vivo at term (31 days), the mature aortic smooth muscle cells demonstrated the characteristic thin, thick and intermediate filaments, dense plaques, endoplasmic reticulum, golgi, plasmalemma vesicles and an incomplete basal lamina. The fetal aorta rapidly responded to organ culture with various changes. Fetal smooth muscle cells modified their phenotype to the synthetic state when cultured in both serum-supplemented and serum-free media. This smooth muscle cell modification occurred after 3 days of culture in fetal explants. The synthetic type smooth muscle cells (fetal) began to proliferate after 6 days of culture. This proliferation resulted in a peripheral outgrowth after 9 days of 10-20 layers in fetal cultures from serum-supplemented media and of 2-4 layers in serum-free media. The orderly arrangement of the internal elastic lamina and alternating medial layers of smooth muscle cells and elastic lamina seen in vivo was disrupted along with increased matrix after 9 days of fetal explant culture. Significant numbers of 'modified' synthetic phenotype smooth muscle cells were not observed in adult aortic explants until after 15 days in culture in serum supplemented media. The mature contractile phenotype smooth muscle cell predominated in adult explants cultured in serum-free media. Significant synthetic phenotype smooth muscle cell proliferation only occurred in adult explants after 15 days culture in serum-supplemented media. When compared to aorta in vivo evidence for increases in cholesterol esterification were observed in both fetal (9 days) and adult (15 days) explants cultured in both serum-supplemented and serum-free media. The fetal aorta in organ culture appeared to be more susceptible than the adult aorta to (a) phenotypic modulation of smooth muscle cells to the synthetic state, (b) smooth muscle cell proliferation, and (c) early cholesteryl ester accumulation.     

Role of apoptosis in the closure of neonatal ductus arteriosus

Apoptosis regulates the remodeling of tissue during embryonic development by eliminating unwanted cells and structures. The present study investigated smooth muscle cell (SMC) proliferation and apoptosis in the neonatal ductus arteriosus (DA) during closure. In the DA of 39 swine neonates and 5 autopsy human neonates, apoptosis was detected using in situ end-labeling and electron microscopy, and proliferation was evaluated using proliferating cell nuclear antigen. In swine, apoptosis of SMC was first observed at 24h after birth. After 48h, both apoptosis and proliferation quickly increased and became most prominent at 3 days, mainly in the intima and inner media. From 5 days, both apoptosis and proliferation quickly disappeared, and were present to a minor extent at the 2 weeks after birth. During these processes, there was no sign of inflammation or necrosis. In humans, apoptosis was found in tissue specimens obtained from 2 term neonates who died at 1 and 5 days after birth. These findings suggest that SMC contribute to the functional closure of the DA by active constriction, and soon after, they switch to proliferation and apoptosis, which may contribute to the anatomical closure of the DA.     

舒心益脉煎对气滞血瘀型大鼠动脉内膜损伤后细胞凋亡及基因蛋白产物的影响

探讨气滞血瘀型大鼠胸主动脉内膜剥脱后内膜增生中，舒心益脉煎对血管平滑肌细胞凋亡及相关基因Fas抗原和Fas配体的影响。将58只大鼠随机分为假且10只，舒心益脉煎组和对照组各24只。后两组大鼠行胸主动脉内膜剥脱术，舒心益脉煎组动物于术前7d至术后14d每日接受舒心益脉煎治疗。术后14d处死动物，用免疫组织化学法测定Fas抗原与Fas配体在血管中的表达，原位末端标记法测定凋亡细胞。术后14d血管新生内膜中有平滑肌细胞凋亡；舒心益脉煎显著增加新生内膜中Fas抗原与Fas配体的表达，增加血管平滑肌细胞凋亡，减少新生内膜面积。说明舒心益脉煎可能通过Fas系统调节动脉血管内皮损伤修复过程中血管平滑肌细胞凋亡，从而抑制新生内膜形成。     

Differences in the glutathione system of cultured aortic smooth muscle cells from young and aged rats

We studied the relation between the glutathione (GSH) system and cell proliferation in a model of smooth muscle cells (SMC) derived from the thoracic aorta of 4–6-week-old (young) and 15-month-old (aged) rats. SMC from aged rats showed greater levels of total non-protein thiol compounds (T-SH), increased glutathione transferase (GST) and increased glutathione reductase (GSSG-Red) activities compared with cells from young rats. These changes were associated with an increased proliferation rate of SMC from aged rats. To evaluate the role of GSH on cell proliferation better, a specific inhibitor of gamma-glutamyl-cystein synthetase, -buthionine-SR-sulphoximine (BSO) was used. BSO showed a dose-dependent inhibition of cell growth, with an IC₅₀ of 10⁻⁴ M, after 48–72 h of incubation. Removal of BSO restored cell growth, further suggesting a link between GSH levels and vascular cell proliferation. The inhibitory effect of BSO was about two times greater on SMC from young than on SMC from aged rats. BSO showed 56% inhibition on the proliferation of SMC from young rats and 32% inhibition on SMC from aged rats (10⁻⁴ M, 72 h of incubation). A parallel reduction of GSH levels of 38% and 19% for SMC from young and aged rats, respectively, was observed, suggesting that age-related factors may influence the involvement of GSH system in cell proliferation.