大鼠血管壁中膜平滑肌细胞的原代培养及鉴定

目的探讨一种方便、快捷、大量原代培养大鼠血管壁中膜平滑肌细胞的方法,为心血管疾病的体外研究提供可靠的实验材料。方法利用改良的组织贴块法进行血管壁中膜平滑肌细胞原代培养,使用倒置相差显微镜观察细胞形态,并用免疫荧光染色鉴定分离培养的细胞。结果改良的组织贴块法成功培养出大鼠血管壁中膜平滑肌细胞,3 d~5 d可见细胞爬出,7 d~9 d即可传代。镜下见细胞以长梭形为主,呈典型"峰-谷"样生长,免疫荧光染色鉴定阳性细胞率在95%以上。结论本研究介绍的改良组织贴块法技术成熟,具有操作简单、方便等优点,使用该法不仅可以获得纯度高、活性好的血管中膜平滑肌细胞,而且缩短了培养周期。     

大鼠胸主动脉血管平滑肌细胞的原代培养和鉴定

目的探讨大鼠胸主动脉血管平滑肌细胞的原代培养方法,了解生长特性,为血管增生性疾病的治疗研究提供试验材料。方法采用组织贴块法培养大鼠胸主动脉血管平滑肌细胞,用倒置相差显微镜观察其生长情况,用免疫组化染色做鉴定,台盼蓝染色进行活力检测。结果85%的组织块接种存活,原代培养后2周左右即可传代,至少可以传8代以上,并且细胞形态、生长特点不发生明显的改变,6代的血管平滑肌细胞纯度达97%以上。台盼蓝染色约有94%以上的细胞为活细胞。镜下培养的血管平滑肌细胞呈典型的"谷峰状"生长,免疫组化染色显示胞浆内α平滑肌肌动蛋白阳性表达。结论正确地利用组织贴块法可以简单、经济、高效地培养出血管平滑肌细胞,为血管增生性疾病的治疗研究提供了理想的细胞模型。     

SD大鼠胸主动脉血管平滑肌细胞组织贴块法培养及鉴定

目的建立SD大鼠胸主动脉血管平滑肌细胞(vascular smooth muscle cells,VSMC)组织贴块培养法并进行鉴定。方法无菌条件下取大鼠胸主动脉,4⁶h后加入含20%胎牛血清的DMEM培养液,原代培养2周并用免疫荧光染色对培养的VSMC进行鉴定。结果组织贴块法成功培养了大鼠胸主动脉VSMC,56h后加入含20%胎牛血清的DMEM培养液,原代培养2周并用免疫荧光染色对培养的VSMC进行鉴定。结果组织贴块法成功培养了大鼠胸主动脉VSMC,5⁷d细胞迁出,107d细胞迁出,10¹4d细胞融合至80%进行传代。体外培养的VSMC可传至20代,培养的细胞呈典型的"峰谷"样生长,免疫荧光染色鉴定细胞纯度达95%。结论组织贴块法培养大鼠胸主动脉VSMC培养体系稳定,操作简单、方便,纯度较高,免疫荧光是鉴定VSMC的较佳方法。     

酶消化组织块法培养人心房成纤维细胞

目的 建立人心房成纤维细胞体外培养的有效方法。方法 无菌条件下将人右心耳标本剪成1 mm3大小组织块,经 1.25 g/L胰蛋白酶预消化15 min后,用组织块贴壁法培养。通过倒置显微镜观察培养细胞生长及形态学特点,使用免疫荧光法进行细胞鉴定。结果 培养3~4 d后可见细胞自组织块爬出,7~9 d后细胞可传代,传代后的细胞呈长纺锤形或多边形。免疫荧光染色发现其抗波形蛋白（vimentin）及抗α-平滑肌肌动蛋白（α-SMA）染色阳性,抗血管性血友病因子（vWF）染色阴性,鉴定为肌成纤维细胞。结论 酶消化组织块法培养人心房成纤维细胞/肌成纤维细胞简便、高效,方法可行。     

脐动脉平滑肌细胞体外培养方法的改进

目的:建立并优化的人脐动脉血管平滑肌细胞(VSMCs)的体外培养方法。方法:人脐动脉VSMCs的体外原代培养应用优化的组织块贴壁法进行。在分别应用传统细胞培养法和优化后细胞培养法贴壁培养提取的原代细胞10 d后,计数6孔板每孔细胞数目,并做统计比较分析相同时间内原代细胞的数量。以抗α-平滑肌肌动蛋白(α-SMA)抗体和抗CD31抗体进行免疫荧光染色和倒置相差显微镜观察鉴定培养的细胞。结果:与传统细胞培养法相比,在相同的时间周期内优化实验方法后培养的原代细胞数量较传统多,我们可以在较短时间内得到实验所需的细胞量,因此优化后可缩短细胞的培养周期。原代和传代培养的脐动脉VSMCs生长良好,细胞经冻存、复苏后状态依然良好。光镜下细胞呈典型的"峰-谷"样生长,细胞多为长梭形,具有VSMCs的特征,且传代培养至第7代,细胞生长特性不变。经抗α-SMA、CD31抗体进行免疫荧光染色鉴定证实为VSMCs。结论:优化的组织块贴壁法可缩短原代人脐动脉VSMCs的培养周期,且细胞的生物学特性较稳定,为与平滑肌相关的疾病进展及心血管生长发育的研究奠定了实验基础。     

大鼠胸主动脉平滑肌细胞的培养与鉴定

目的探讨以简便、高效的方法获取血管平滑肌细胞,为相关研究提供实验材料。方法采用组织贴块法进行原代培养,胰酶消化法传代,并应用相差显微镜和平滑肌肌动蛋白对细胞进行形态学和免疫组化鉴定。结果90%的组织块接种存活,培养5代的平滑肌细胞纯度达98%以上。镜下可见培养细胞呈典型的“峰-谷状”样生长,免疫组化染色显示胞浆内平滑肌肌动蛋白阳性表达。结论本法简便、可靠,短期内可获大量高纯度、功能良好的血管平滑肌细胞,可作为研究心血管疾病良好的体外模型。     

大鼠胸主动脉平滑肌细胞的培养与鉴定

目的 改进大鼠血管平滑肌细胞(VSMC)的培养方法.方法 采用机械刮除、差异贴壁等手段进行组织贴块法原代培养,胰蛋白酶消化传代,并应用相差显微镜和即用型SABC免疫组化染色试剂盒对细胞进行形态学和免疫组化鉴定.结果 镜下培养细胞呈典型的"谷峰状"生长,免疫组化染色显示胞浆内αactin阳性表达.结论 本法可获纯度高、结构和功能良好的VSMCs.     

大鼠冠状动脉平滑肌细胞的原代培养及鉴定

目的探讨大鼠冠状动脉平滑肌细胞的原代培养方法,为冠状动脉疾病的研究提供理想的细胞模型。方法无菌条件下分离大鼠冠状动脉,采用酶消化法分离培养平滑肌细胞,进行形态学观察及台盼蓝染色测定细胞活力,采用免疫荧光染色技术对平滑肌α肌动蛋白进行鉴定。结果形态学、免疫荧光染色鉴定表明培养的细胞为血管平滑肌细胞,细胞存活率达97%。原代培养14d左右即可进行传代,可以传6代以上,且细胞形态及生长状态良好。结论酶消化法分离大鼠冠状动脉平滑肌细胞,方法简单、高效,细胞纯度高、且活性好、生长迅速。     

小鼠主动脉平滑肌细胞培养及钙化模型的优化

目的 通过改良后的酶消化法进行小鼠主动脉血管平滑肌细胞（vascular smooth muscle cells,VSMCs）原代培养，提高VSMCs原代培养效率及质量，进而建立更快速有效的体外钙化模型。方法 剥离小鼠主动脉中膜并将其剪碎，分别采用改良组织贴块法及改良酶消化法进行小鼠VSMCs原代培养，免疫荧光染色鉴定细胞纯度后，分组（经典钙化组、改良钙化组）进行体外钙化诱导，采用vonKossa染色法评估各组钙化差异。结果 与改良组织贴块法相比，改良后的酶消化法培养原代VSMCs可在短时间内获得大量细胞，鉴定VSMCs纯度>98%，且细胞不易老化，传代存活率高。与传统体外钙化方法比，改良组的钙化更容易且稳定。结论 本研究通过改良的酶消化法提高了原代平滑肌细胞的存活率及培养效率，同时通过改良体外钙化诱导液配方，建立了良好的体外钙化模型。     

组织块贴壁法培养SD大鼠腹主动脉平滑肌细胞及鉴定

目的培养、鉴定SD大鼠腹主动脉平滑肌细胞。方法采用组织块贴壁法分离培养SD大鼠腹主动脉平滑肌细胞,胰蛋白酶消化传代,并应用相差显微镜和平滑肌肌动蛋白对细胞进行形态学和免疫组化鉴定。结果培养出的平滑肌细胞呈长梭形生长,镜下可见培养细胞呈典型的"峰-谷"状生长,高倍镜下可见胞质内大量棕色、与细胞长轴平行的纤维细丝,免疫组化染色显示胞浆内平滑肌肌动蛋白阳性表达。结论组织块贴壁法体外培养SD大鼠腹主动脉平滑肌细胞操作简单、纯度较高、可行性高。     

Regulation of renin-like enzyme in cultured human vascular smooth muscle cells

Cultured human arterial smooth muscle cells produced an immunologically specific renin-like enzyme. The renin-like enzyme in the culture medium was mostly an inactive form; the proportion of the active form in the cell was 30 to 75%. Phorbol 12-myristate 13-acetate, N'-O'-dibutylyladenosine 3', 5'-monophosphate and isoproterenol with theophylline increased the renin-like enzyme in the medium and in the cell, dose dependently. Endothelial cell growth supplement also increased the renin-like enzyme produced by cultured vascular smooth muscle cells, and heparin promoted the effects of endothelial cell growth supplement. The existence of the regulation of the renin-like enzyme produced by cultured vascular smooth muscle cells strongly suggests the existence of a local renin angiotensin system in human vascular walls.     

体外培养成人脂肪间充质干细胞及向平滑肌细胞诱导分化

目的 体外培养成人脂肪间充质干细胞,并应用转化生长因子β将脂肪间充质干细胞诱导分化为平滑肌细胞.方法 采用酶消化法和贴壁培养法分离培养脂肪间充质干细胞,流式细胞仪对第3代和第5代细胞进行表面抗原检测,对第5代细胞进行转化生长因子β诱导,于诱导后第10天进行免疫化学鉴定.结果 体外培养的脂肪间充质干细胞呈扁平的长梭形,细胞形态均一,传代稳定.干细胞相关标志CD29和CD44表达阳性,造血干细胞相关标志CD34随传代次数的增加由弱阳性逐渐转为阴性,内皮细胞相关标志CD31表达阴性.流式细胞仪检测结果 发现脂肪间充质干细胞中G0/G1、S和G2/M期的细胞分别占90.14%、3.77%和6.09%.转化生长因子β定向诱导后倒置显微镜下观察细胞呈"峰"、"谷"样形态,免疫荧光化学检测发现诱导组细胞α平滑肌肌动蛋白表达阳性.结论 成人脂肪组织中含有间充质干细胞,且可在转化生长因子β诱导后分化为平滑肌细胞.     

Platelet-derived growth factor suppresses and fibroblast growth factor enhances cytokine-induced production of nitric oxide by cultured smooth muscle cells. Effects on cell proliferation.

Stimulation of thymidine incorporation by basic fibroblast growth factor or epidermal growth factor treatment of cultured quiescent smooth muscle cells (rat and human) was attenuated by the concomitant treatment with interleukin-1 beta in the presence of indomethacin. Platelet-derived growth factor-AB and -BB-induced thymidine incorporation was not inhibited by the presence of the cytokine under similar experimental conditions. Elevation of nitrite levels in the conditioned medium of cultures exposed to interleukin-1 beta correlated with the inhibition of thymidine incorporation. Platelet-derived growth factor-AB and -BB inhibited the production of nitric oxide (measured as nitrite levels in conditioned medium) by cells treated simultaneously with interleukin-1 beta and growth factor. However, platelet-derived growth factor-AA neither affected nitrite production nor thymidine incorporation by smooth muscle cells. Levels of cytokine-stimulated nitrite production by smooth muscle cells were increased synergistically by the presence of fibroblast growth factors or epidermal growth factor. The inhibition of thymidine incorporation and concomitant elevation of nitrite production was abolished in the presence of nitro-L-arginine. Cultures maintained in the presence of low levels of the cytokine for 9 days were growth-inhibited, and this was reversed when culture medium was supplemented with nitro-L-arginine. The treatment of smooth muscle cells, which were grown in coculture inserts with the cytokine to induce nitric oxide production, before their combination with other quiescent layers of cells resulted in the inhibition of thymidine incorporation by this second layer of cells regardless of the growth factor used for stimulation. Nitric oxide may act as an endogenous inhibitor of smooth muscle cell proliferation in the vessel wall, and impairment of its production may be one action of potent vascular mitogens such as platelet-derived growth factor.     

Exposure to platelet-derived growth factor modulates the porcine aortic smooth muscle cell response to somatomedin-C

Platelet-derived growth factor (PDGF) is a potent mitogen for smooth muscle cells, but to detect maximal stimulation by PDGF, the cells must be incubated with plasma. Somatomedin-C (Sm-C), a peptide growth factor that is present in plasma, has been shown to interact with PDGF synergistically to stimulate DNA synthesis in cultured fibroblasts. These studies were designed to test the hypothesis that PDGF interacted with Sm-C to stimulate smooth muscle cell replication and to compare the response of this cell type to that of fibroblasts. When PDGF or Sm-C was added individually to smooth muscle cell cultures, each growth factor induced only minimal increases in [3H]thymidine incorporation into DNA (i.e. PDGF, 3,400 +/- 1,120 to 54,900 +/- 1,550 cpm; Sm-C, 3,400 +/- 1,120 to 10,950 +/- 980 cpm). In contrast, addition of increasing concentrations of Sm-C to cultures that were continuously exposed to PDGF in the presence of Sm-C-deficient plasma resulted in a synergistic increase in [3H]thymidine incorporation (3,400 +/- 1,120 to 54,500 +/- 1,800 cpm; P less than 0.001). To determine if Sm-C was required for smooth muscle cell replication, cultures were sequentially exposed to PDGF, followed by Sm-C-deficient plasma. The rate at which the non-Sm-C-exposed cells synthesized DNA was retarded compared to that of cells exposed to Sm-C-containing plasma; however, 68% nuclear labeling was present after 44 h of incubation. To exclude the possibility that some cellular secretory product was substituting for Sm-C, the medium was changed every 2 h and replaced by fresh Sm-C-deficient medium. Using these test conditions, exposure to PDGF and Sm-C-deficient plasma induced only 11% labelling. Readdition of pure Sm-C to this medium restored nuclear labeling to 82% at 44 h. Other variables that appeared to modulate the cellular response to Sm-C were culture density and simultaneous PDGF exposure. Sm-C and PDGF both appear to be potent mitogens for porcine aortic smooth muscle cells, and when added together to quiescent cultures, their effects are synergistic. Smooth muscle cells appear to require Sm-C to initiate DNA synthesis, and in its absence produce a Sm-like factor that can partially compensate for Sm-C deficiency and allow replication.     

Isolation and culture of arterial smooth muscle cells from human placenta

A simple and economical technique was developed to isolate and culture human arterial smooth muscle cells from chorionic plate vessels. Placentas from healthy women were collected at the time of term delivery. Chorionic plate arteries were identified, excised, and cut into small pieces. An explant technique was used to grow cultures of placental arterial smooth muscle (PASM) cells. Small pieces of vessel with lumens down were placed in 100-mm culture plates and grown in Dulbecco modified eagle medium and 10% fetal bovine serum. Cells appeared from explants within 1 week and grew to confluence in approximately 4 weeks. At confluence, PASM cell cultures had a uniform cell morphology that was characterized by elongated cells in parallel rows, typical of smooth muscle cells. Smooth muscle cell phenotype was evaluated by morphology and by immunoblotting and immunofluorescence of smooth muscle myofilament proteins. All PASM cell cultures expressed alpha-smooth muscle actin, beta-tropomyosin, and h-caldesmon. Expression was similar to that of human aortic smooth muscle cells, but not to endothelial cells or fibroblasts. PASM cells stained uniformly for alpha-smooth muscle actin and lacked staining for a fibroblast-specific antigen. PASM cells were evaluated for their response to inflammatory mediators, tumor necrosis factor-alpha, and interleukin-1beta by measurement of interleukin-8 production. Cells cultured for 18 hours showed a progressive increase in interleukin-8 production with time. Treatment with inflammatory mediators increased interleukin-8 production by 3-fold as compared with media control. This technique provides a simple method to obtain normal human arterial smooth muscle cells for in vitro studies of physiology and pathophysiology.     

Isolation, characterization, and long-term cultivation of porcine and murine cerebral capillary endothelial cells

We present a simple method for isolation and long-term cultivation of porcine and murine cerebral capillary endothelial cells (cEC). Two major points are made. First, that the "characteristic" morphology of the endothelial cells depends mainly on the presence of endothelial cell growth factors in the culture medium and second, that the identification of the cells as endothelial cells requires a special lectin instead of criteria used for large vessel endothelial cells, such as factor VIII staining or LDL uptake. Pure cerebral capillaries were isolated by means of a series of centrifugation steps; endothelial cells were released by collagenase treatment and cultivated on plastic petri dishes, which proved to be better for cell attachment than collagen or gelatin coating. The microvascular cells were cultivated in either the presence or absence of growth factors. Medium 199 + 10% FCS produced mainly spindle-shaped cells, growing in the "hills and valleys" pattern, which, if not passaged for weeks, showed three dimensional tubular structures. Cells of the "cobblestone" phenotype were promoted in medium 199 + 10% FCS, enriched with endothelial cell growth supplement (ECGS) and heparin (referred to as complete medium). These cells retained their phenotype for months and could be passaged up to 35 times till now. If ECGS and heparin were omitted from these cultures, the cells became elongated and resembled smooth muscle cells. This effect was reversible when the cells were transferred to complete medium. With cEC, cloned by limiting dilution, we noticed this reversal phenomenon as well. We used several markers to characterize the microvascular cells and could show that the lectin of Bandeiraea simplicifolia is a highly reliable marker for endothelial cells and that the monoclonal antibody alpha-sm-1 (anti-smooth muscle cell actin) is excellent for determining smooth muscle cells.     

Variables controlling the secretion of a somatomedin-like peptide by cultured porcine smooth muscle cells

Somatomedin-C, a peptide growth factor that is also termed insulin-like growth factor I, stimulates smooth muscle cell replication; however, these cells proliferate in somatomedin-C-deficient medium. We postulated, therefore, that these cells might release a somatomedin-like molecule into the surrounding culture medium. Porcine aortic smooth muscle cells that were exposed to serum-free Dulbecco's modified minimum essential medium for 24 hours were found to release a somatomedin-like molecule that could be detected by a specific radioimmunoassay for somatomedin-C. Several variables in the design of tissue culture conditions were found to regulate the secretion of the somatomedin-like peptide. There was an inverse relationship between somatomedin-like peptide secretion and culture density. Cultures grown to a density of 110,000 cells/well secreted 0.18 +/- 0.02 U/ml per 10(5) cells, whereas cultures grown to 38,000 cells/well secreted 0.59 +/- 0.06 U/ml per 10(5) cells (P less than 0.001). Serum deprivation and days of preincubation before initiation of an experiment were found to influence somatomedin-like peptide secretion significantly. Platelet-derived growth factor, which stimulates smooth muscle cell replication, was a potent stimulant of somatomedin-like peptide secretion. Exposure to a concentration of 500 ng/ml platelet-derived growth factor induced a 2.8-fold increase in somatomedin-like peptide secretion over basal levels, (i.e., 0.37 +/- 0.05 to 0.98 +/- 0.7 U/ml, P less than 0.001). The effect of platelet-derived growth factor was enhanced (41% increase) by concomitant incubation with 1% somatomedin-C-deficient platelet poor plasma.(ABSTRACT TRUNCATED AT 250 WORDS)     

Smooth muscle cell migration induced by shear-loaded platelets and endothelial cells. Enhanced platelet-derived growth factor production by shear-loaded platelets.

BACKGROUND: Uncontrolled migration and proliferation of smooth muscle cells (SMC) are the main mechanisms of development of atherosclerotic neointima. However, it has not yet been elucidated how mechanical stress is involved in this process. We investigated smooth muscle cell mitogenic activity induced by shear-loaded platelets and endothelial cells. METHODS: Experimental design: in vitro experimental study. We devised four types of conditioned media; supernatant of mixed culture of platelets and endothelial cell (ST), supernatant of shear-loaded mixed culture of platelets and endothelial cell (SH), ST medium neutralised with anti-PDGF antibody (ST+), and SH medium neutralised with anti-PDGF antibody (SH+). Smooth muscle cells were cultured in each conditioned medium, and their spreading activity was determined under a microscope. RESULTS: Smooth muscle cells spreading activity in the SH group was significantly greater than that in the ST group. Their spreading activity was suppressed by anti-PDGF antibody under shear conditions (SH+), but it was not by anti-PDGF antibody under static conditions (ST+). CONCLUSIONS: Our results demonstrate that platelet-derived growth factor is produced by shear-loaded platelets and endothelial cells, and local mechanical forces may play an important role in cardiovascular disease.