培养的正常及动脉粥样硬化兔主动脉平滑肌细胞的超微结构比较研究

将正常及动脉粥样硬化兔主动脉病灶处平滑肌细胞进行体外培养,应用培养细胞的原位包埋技术及超微结构的定量分析,观察并比较了两者的超微结构。结果表明,动脉粥样硬化兔平滑肌细胞内肌丝成分显著较正常兔平滑肌细胞少,粗面内质网及线粒体较正常者丰富。本文对其意义进行了讨论。     

前列腺素与动脉平滑肌细胞的增殖

动脉平滑肌细胞(SMC)发生增殖反应是动脉粥样硬化(AS)发生的中心环节。本文综述了前列腺素(PG)对(SMC)增殖的影响。体外和动物实验证实,PG(主要是PGE_1、PGI_2和PGD_2)能抑制动脉SMC的增殖,从而对AS的发生可能起负相关作用。     

深低温保存兔颈总动脉体外灌注后的结构和力学性能研究

 目的 探讨深低温保存对动脉血管组织学和力学性能的影响。方法 取 20 只新西兰兔的颈总动脉，用含 1.5 mol/L 1, 2-丙二醇的低温保护剂深低温（－196℃）保存 10 ~ 15 d，并在预冷的冰袋内缓慢复温；以新鲜血管作为对照。对新鲜和冷冻-复温血管的平滑肌细胞（SMC）进行体外培养，观察和分析新鲜、冷冻-复温和冷冻复温后体外灌注 6、12 和 24 h血管的内皮细胞、SMC、血管壁组织构和力学性能（轴向、周向弹性模量和断裂应力）的变化。结果 冷冻-复温后血管的 SMC 在体外培养中开始生长时间与新鲜血管基本相同（24 ~ 36 h），生长速度接近。深低温保存后，血管内皮细胞数量减少，电镜观察SMC数量和形态无明显变化，但血管的轴向弹性模量（5.82 ± 0.55）、轴向断裂应力（58.78 ± 6.96）和周向弹性模量（1.44 ± 0.22）、周向断裂应力（3.45 ± 0.40）均明显低于新鲜血管（分别为6.45 ± 0.80、146.96 ± 23.60 和 1.83 ± 0.17、5.33 ± 0.50， P < 0.05或 P < 0.01）。体外灌注后，冷冻-复温血管的内皮细胞脱落，SMC 变性、坏死，内弹力纤维和胶原出现不同程度崩裂，力学性能指标中除了轴向弹性模量外，均进一步降低（P < 0.05 或 P < 0.01）。结论 兔颈总动脉 SMC 深低温保存效果满意，但体外灌注对经深低温保存血管的内皮细胞、SMC 和血管壁结构具有明显损伤作用，导致血管力学性能显著降低。     

内皮细胞条件培养液对平滑肌细胞合成胶原的影响

本实验采用胶原酶消化法分离、培养兔主动脉内皮细胞(EC)及平滑肌细胞(SMC),以[~3H]-脯氨酸掺入SMC合成的[~3H]-羟脯氨酸中的量作为测定胶原的指标,观察了兔主动脉内皮细胞条件培养液(EC-CM)对动脉SMC合成胶原的影响,结果发现融合的EC-CM能促进SMC的胶原合成,但SMC数无明显变化;1:2稀释的EC-CM促进SMC合成胶原的作用最大。可见EC通过促进SMC的胶原合成而参与了动脉粥样硬化过程。     

红景天甙对缺氧状态下兔肺动脉平滑肌细胞增殖的影响

目的 :高原缺氧性肺动脉高压形成的主要因素有缺氧性肺动脉收缩和缺氧性动脉壁构型重建。肺动脉平滑肌细胞的增殖是缺氧性肺动脉壁构型重建的重要环节。药用植物红景天甙具有抗缺氧、提高机体对高原的适应能力等作用 ,在我国西北、西南高原缺氧地区被用于防治高原适应不全症 ,取得满意效果 ,但对其作用机理尚缺乏研究。方法 :本实验应用细胞培养、ＭＴＴ实验、[3Ｈ]-ＴｄＲ掺入实验、流式细胞仪测定、Ｆｌｕｏ - 3和激光扫描共聚焦显微术以及斑点杂交等方法研究缺氧状态下红景天甙 (Ｓａｌ)对兔肺动脉平滑肌细胞 (ＰＡＳＭＣ)增殖、ＤＮＡ合…     

阳离子脂质体介导TIMP-3基因对兔血管平滑肌细胞增殖和凋亡的影响

目的：通过阳离子脂质体介导含有TIMP-3基因的质粒转染体外兔平滑肌细胞，观察瞬时表达的TIMP-3对细胞增殖及凋亡的影响。方法：构建兔TIMP-3基因质粒表达载体。按照正常对照组、空质粒组、重组质粒载体组将72孔兔血管平滑肌细胞分为3组，每组又分为24、48、72h 3个亚组。阳离子脂质体介导质粒分别转染相应组细胞，正常对照组无质粒加入。转染后进行细胞计数，描绘生长曲线；将24孔细胞分为3组进行基因转染，转染后48h检测细胞凋亡率。结果：正常对照组细胞数、细胞凋亡率与空质粒组相比均无明显差别；重组质粒组细胞数与前二者相比明显减少（P〈0．05），重组质粒组细胞凋亡率均明显高于前二者（P〈0．05）。结论：TIMP-3对体外血管平滑肌细胞具有明显抑制增值及促进细胞凋亡的作用；阳离子脂质体作为新型、高效转染介质对细胞生长无明显不良反应，使用安全，应用于TIMP-3基因治疗支架后再狭窄前景广阔。     

含辛伐他汀药物血清对兔血管平滑肌细胞增殖的直接作用及意义

目的 :探讨辛伐他汀对体外培养兔血管平滑肌细胞增殖的影响及意义。方法 :16只雄性新西兰兔随机分为血清对照组和三个不同剂量的辛伐他汀亚组 (每日分别给予辛伐他汀 5mg/kg、10mg/kg、15mg/kg) ,7天后采血并混合每组 4只兔血 ,无菌分离制备三亚组的辛伐他汀含药血清。采用内皮素 1(ET 1)刺激正常喂饲原代培养兔主动脉血管平滑肌细胞的方法 ,建立血管平滑肌细胞增殖模型。采用MTT及3H TdR法检测各组辛伐他汀含药血清对血管平滑肌细胞增殖的作用。结果 :与不含药的正常对照组相比 ,不同亚组辛伐他汀含药血清呈剂量依赖性抑制血管平滑肌细胞增殖 (P <0 .0 1～0 .0 5 )。结论 :兔口服辛伐他汀后的血清具有抑制血管平滑肌细胞增殖的作用     

阿新蓝一钌红方法显示平滑肌细胞中粘多糖

本室在实验性动脉粥样硬化研究过程中,建立了体外培养动脉壁平滑肌细胞(SMC)高脂损伤模型。为了观察SMC中粘多糖的存在及性质及其与动脉粥样硬化病变的关系,采用阿新蓝一钌红法,显示SMC内羟基酸性粘多糖和硫酸基粘多糖。一材料 1.标本制备:取新小牛主动脉平滑肌。(1)在无菌条件下,剔除动脉内外膜,将中膜剪成小块,置于胶原酶、弹性蛋白酶1∶1混合液中,在37℃条件下进行孵育,边孵边搅动,孵育6小时后,弃除溶液中胶原组织,900转/分离心5分钟。Hank’s液清洗沉淀物三次,然后加入含小牛血清(cs)的IMDM培养液,在37℃封闭培养,每隔四日换液一次,分别培养15天,72天。(2)刮下SMC,用蒸馏     

瓜蒌注射液对兔血管平滑肌细胞增殖细胞核抗原表达的影响

目的:观察瓜蒌注射液对血管平滑肌细胞(SMC)增殖细胞核抗原(PCNA)表达的影响。方法:用免疫组织化学LSAB法检测培养的兔主动脉SMC中PCNA的表达,液闪法测定SMC氚-胸腺嘧啶核苷([³H]-TdR)掺入量,同时测定培养液中超氧化物歧化酶(SOD)、脂质过氧化物(LPO)、前列环素(PGI₂)及环磷酸腺苷(cAMP)的含量。结果:瓜蒌注射液能增加SOD活性,降低LPO和升高PGI₂、cAMP水平,抑制SMC的[³H]-TdR掺入量和PCNA的表达(P均＜0.05,0.01)。结论:瓜蒌有抑制SMC增殖的作用。     

兔主动脉平滑肌细胞摄取N-VLDL的受体途径

泡沫细胞的来源和形成是动脉粥样硬化(AS)发病机制的关键问题之一。细胞化学和超微结构分析证实细胞来源有二:即平滑肌细胞(SMC)和单核巨噬细胞(Mφ)。八十年代初我室较为系统的研究了N-VLDL使巨噬细胞转变为泡沫细胞的机理,对于平滑肌细胞摄取N-     

Composition and metabolism of lipid in macrophages from normally fed and cholesterol-fed rabbits

The composition and metabolism of lipid in peritoneal macrophages obtained from normally fed rabbits were compared with those of macrophages obtained from cholesterol-fed rabbits. Macrophages from cholesterol-fed rabbits had a higher cholesterol content and a markedly higher cholesterol ester content than normal macrophages. The increase in cholesterol ester content was most marked for cholesterol oleate and cholesterol linoleate, with the cholesterol ester fatty acid composition of the cholesterol-fed macrophages resembling that of foam cells derived from aortic lesions of similarly cholesterol-fed rabbits. Metabolic differences were also demonstrated between the cells obtained from normal and cholesterol-fed rabbits. In the latter, incorporation of ¹⁴C-labeled acetate into cholesterol was almost completely suppressed whereas in macrophages from normally fed rabbits, ¹⁴C-labeled acetate was incorporated predominantly into cholesterol. Incubation in vitro of normal macrophages for periods up to 20 hr with hyperlipemic serum, however, was not associated with any appreciable suppression of cholesterol synthesis.     

Stimulating effect of intermediate-density lipoproteins (IDL) from hyperlipemic plasma on hepatic lipase

Summary The main lipoprotein density classes, namely very-low-density lipoproteins (VLDL), intermediate-density lipoproteins (IDL), low-density lipoproteins (LDL), high-density lipoproteins₂ (HDL₂) and HDL₃ were investigated with respect to their influence on hepatic lipase (HTGL) activity in vitro.Lipoproteins from pooled normal plasma (NP) and from pooled hyperlipemic plasma (HP) were prepared by means of sequential ultracentrifugation. Hepatic lipase was determined radioenzymatically after preincubation with protamine sulfate. It could be demonstrated that IDL from HP were able to stimulate HTGL activity by approximately 100% above the baseline value. HDL₃ from both NP and HP revealed an inhibiting effect on HTGL activity. VLDL, LDL, and HDL₂ exhibited no significant effect on HTGL activity.It is speculated that HTGL could possibly represent a second pathophysiological pathway for the catabolism of IDL in hyperlipemia but this presumption is supported by only a few investigations in vivo.

---

Abbreviations HDL₂ High-density lipoproteins₂ - HDL₃ High-density lipoproteins₃ - HP Hyperlipemic plasma pool - HTGL Hepatic lipase - IDL Intermediate-density lipoproteins - LDL Low-density lipoproteins - NP Normal plasma pool - VLDL Very low-density lipoproteins     

Lipoprotein degradation and cholesterol esterification in primary cell cultures of rabbit atherosclerotic lesions.

Lipoprotein metabolism and cholesterol accumulation in atherosclerotic lesions was studied using enzymatically isolated primary cell cultures from aortas of rabbits made atherosclerotic by cholesterol feeding. The cultures consisted of macrophages and smooth muscle cells, thus resembling, in composition, fatty streak lesions. The mean (+/- SD) cholesteryl ester content of the dispersed cells was 1059 +/- 445 micrograms/mg cell protein, but it declined steeply during 1 week in primary culture. The uptake of low-density lipoprotein (LDL), beta-migrating very low-density lipoprotein (beta-VLDL), and acetylated LDL (acetyl-LDL), labeled with 125I or with the fluorescent probe 1,1'-dioctadecyl-3,3,3',3'- tetramethylindocarbocyanine (DiI), was studied in 2-day-old primary cultures. DiI-acetyl-LDL was avidly taken up by the macrophages and, to a lesser extent, by some smooth muscle cells. The uptake of DiI-beta-VLDL by the macrophages was weaker and less homogeneous than that of DiI-acetyl-LDL. The degradation rates of 125I-labeled beta-VLDL, LDL and acetyl-LDL were 135 +/- 54, 195 +/- 20, and 697 +/- 14 ng/mg cell protein/8 hours, respectively. Incubation with unlabeled acetyl-LDL enhanced the incorporation of [3H]oleate into cholesteryl esters and increased the cellular cholesteryl ester content. These results suggest that arterial macrophages and, to some extent, smooth muscle cells from cholesterol-fed rabbits actively metabolize acetyl-LDL and are thus capable of accumulating cholesteryl esters by uptake of modified forms of LDL.     

Lipid lowering activity of Citri unshii pericarpium in hyperlipemic rats

The inhibitory effect of the MeOH extract of Citri unshii pericarpium (CU) and its fractions were tested in hyperlipemic rats using for animal models induced by high cholesterol-diet. We measured plasma levels of triglyceride, total cholesterol, low-density lipoprotein (LDL)-cholesterol and high-density lipoprotein (HDL)-cholesterol as measures of its hyperlipemic effects. We demonstrated that CU decreases plasma levels of triglyceride, total cholesterol and LDL cholesterol. There was also no elevation of plasma ALT and AST levels, which indicate CU did not cause liver injury. These results indicate that CU is a good candidate for the treatment on high cholesterol diet-induced blood circulatory disorders, obesity and hyperlipidemia.     

Lipid Lowering Activity of Citri Unshii Pericarpium in Hyperlipemic Rats

The inhibitory effect of the MeOH extract of Citri unshii pericarpium (CU) and its fractions were tested in hyperlipemic rats using for animal models induced by high cholesterol-diet. We measured plasma levels of triglyceride, total cholesterol, low-density lipoprotein (LDL)-cholesterol and high-density lipoprotein (HDL)-cholesterol as measures of its hyperlipemic effects. We demonstrated that CU decreases plasma levels of triglyceride, total cholesterol and LDL cholesterol. There was also no elevation of plasma ALT and AST levels, which indicate CU did not cause liver injury. These results indicate that CU is a good candidate for the treatment on high cholesterol diet-induced blood circulatory disorders, obesity and hyperlipidemia.     

Stimulation of proliferation in stationary primary cultures of monkey and rabbit aortic smooth muscle cells. I. Effects of lipoprotein fractions of hyperlipemic serum and lymph.

Medial explants of thoracic aorta from rhesus monkey or rabbit show outgrowth within 5–10 days when cultured in 90% basal Eagle's medium and 10% normal serum. After 5–6 weeks of rapid growth, cell colonies around the explant reach a stationary phase with low mitotic activity. Homologous hypercholesterolemic serum and lymph stimulate these stationary cultures into another proliferative phase, when 5% of the normal serum is replaced in the culture medium. Measurement of increase in colony size and evaluation of [³H]thymidine incorporation by autoradiography serve to indicate increased proliferation. Low density lipoproteins (LDL) from hyperlipemic serum and lymph have the greatest stimulatory effect, while high density lipoproteins (HDL) and the lipid-free bottom fraction, as well as LDL from normal serum and lymph, have no effect. These stationary cultures are valuable for the study of agents suspected of having a stimulatory effect on cell proliferation of aortic lesions in vivo.     

The effect of estradiol on the proliferation of rabbit aortic medial tissue culture cells induced by hyperlipemic serum

The outgrowths of medial explants of thoracic aorta from New Zealand rabbits were used to study the influence of estrogen on cell proliferation. After 5-6 weeks of rapid growth in Basal Eagle Medium (BME) supplemented with 10% normal rabbit serum, such cultures reached a stationary phase during which they showed little mitotic activity and little further increase in surface area. Replacement of 5% of the normal serum with hyperlipemic rabbit serum resulted in a stimulation of these stationary cultures into a phase of renewed proliferation, which was measured directly as increase in cell culture size and by [3H]thymidine incorporation visualized by autoradiography. The addition of estrogen (estradiol, Progynon, Schering Corp.) in a concentration of 0.02 microgram/ml to the culture medium inhibited the proliferative effect induced by the hyperlipemic serum. On the other hand it had no effect on the growth rate of such explant cultures during their rapid growth phase if added at the time of explantation for 6 weeks. This would indicate that the inhibition of the hyperlipemic serum-induced proliferation by estrogen is not due to a toxic effect on mitosis in general. Cells exposed to estrogen tended to have larger amounts of intracellular lipid as visualized by oil red O staining. Moreover, prolonged exposure to estrogen resulted in a significant decrease in stainable collagen and elastin in these cultures.     

Consequences of current lipid guidelines for the Federal Republic of Germany

Summary According to the results of the first nationally representative population study in the Federal Republic of Germany (FRG) conducted in 1984–1986, a vast majority of the population have elevated cholesterol levels and undesirable low density lipoprotein (LDL) cholesterol levels. This problem increases with age in two dimensions, more individuals become hyperlipemic and those who were hyperlipemic develop more extreme pathologic values. Preliminary analyses suggest that the national profile in the FRG is worse than that of the USA. Based on the current recommendations of the national cholesterol education program of the USA, 23 million adults in the FRG should be receiving dietary advice for elevated lipid levels and 18 million persons should be in regular treatment programs. Consequently, implementation of such a national strategy and program would require the assembly and training of an adequate professional body of physicians and support staff in the diagnosis and treatment of hyperlipidemias.Abbreviations HDL high density lipoprotein cholesterol - LDL low density lipoprotein cholesterol - CVD cardiovascular disease     

Immunohistochemical localization of apoprotein B in aortas from hyperlipemic swine. Preferential accumulation in lesion-prone areas

In hyperlipemic swine, areas of the aortic arch that accumulate intravenously injected Evans blue dye (blue areas) appear to be more susceptible to early atherogenesis than adjacent areas that are devoid of dye uptake (white areas). We used immunofluorescence microscopy to determine the localization of apoprotein-B (apoB) in these blue and white areas, and in intimal cell masses (ICMs) of the abdominal aortas obtained from hyperlipemic and normolipemic swine. The results showed that before aortic lesions were visible grossly or microscopically, extracellular accumulations of apoB occurred preferentially in the thickened intima of blue areas and in ICMs of the abdominal aorta. Normal white areas in the aortic arch and abdominal aortas, and in the aortas obtained from control swine showed negligible immunoreactivity. Thus, the accumulation of apoB at anatomic sites predilected to early atherogenesis lends further evidence linking these lipoproteins with atherogenesis.     

Effects of low-density lipoprotein from normal rabbits on hypercholesterolemia and the development of atherosclerosis

To clarify whether or not normal LDL separated from normal rabbits is atherogenic, 0.75 mg of normal LDL-cholesterol in 6 ml of 0.85% saline and 6 ml of saline alone were injected every day for 5 weeks into the auricular vein of two groups of rabbits, respectively. The inoculated rabbits were fed a standard diet containing 0.5 and 1% cholesterol. Blood was drawn before injection and at 1, 2, 3 and 5 week intervals thereafter. After 5 weeks, all rabbits were sacrificed. Following exsanguination, the aorta was stained by Sudan III. The Sudan III staining was more extensive in rabbits injected with saline than in those that received LDL. In addition, plasma cholesterol, plasma phospholipid, VLDL-cholesterol and LDL-cholesterol levels were lower in LDL-injected rabbits than in those injected with saline. But HDL-cholesterol levels were not significantly different between LDL-cholesterol and saline-injected rabbits. Although the exact cause of antiatherogenic effect of normal LDL is not clear, it seems reasonable to suggest that at least some normal LDL acts as a 'good' lipoprotein, namely antiatherogenic lipoprotein, in our experimental protocol.