氧化修饰HDL剌激培养人主动脉平滑肌细胞增殖

平滑肌细胞（smooth muscle cell, SMC）增殖在动脉粥样硬化（atherosclerosis, AS）形成中起着重要作用.氧化修饰HDL（oxidized HDL, OX-HDL）可刺激 ³H-TdR掺入培养人动脉SMC的DNA,促进SMC增殖.以四甲基偶氮唑盐(MTT)法直接观察OX-HDL对培养人动脉SMC增殖细胞数的影响.结果显示,天然HDL（native HDL N-HDL）对SMC增殖没有影响,而OX-HDL则显著刺激SMC增殖（P＜0.01）,N-HDL显著抑制OX-LDL刺激SMC增殖作用（P＜0.01）,而OX-HDL则显著增加OX-LDL刺激SMC增殖作用（P＜0.01）     

三种不同内皮细胞／平滑肌细胞共培养模式的渗流率及其对LDL的吸收／沉积

采用3种不同的内皮细胞／平滑肌细胞共培养模式模拟血管内膜层,实验研究了共培养细胞层的渗流率和对LDL的吸收／沉积．3种细胞共培养模式分别为：（1）内皮细胞单独培养（EC／Φ）;（2）内皮细胞／平滑肌细胞直接共培养（EC—SMC）;（3）内皮细胞／平滑肌细胞间接共培养（EC／SMC）,即把两种细胞分别培养在Millicell—CM透析膜的不同面上．研究结果表明,同等条件下,EC／SMC模式的渗流率最低,EC／Φ模式最高;EC／Φ模式对LDL的吸收／沉积最低,EC-SMC模式对LDL的吸收／沉积最高．同时表明,体外培养的细胞层对LDL的吸收／沉积随渗流率的增加而增加．研究结果提示,动脉血管壁内LDL的吸收／沉积与血管壁面LDL的浓度极化成正相关;在致动脉粥样性脂质渗入血管壁的过程中,血管壁内皮层的完整性起着至关重要的作用．     

脂蛋白(a)的赖氨酸结合异质性对人动脉SMC增殖的影响

为探讨脂蛋白 ( a) [Lp( a) ]的赖氨酸结合功能在致动脉粥样硬化中的作用 ,利用短时超速离心结合凝胶层析分离纯化 Lp( a) ,以 Lysine- Sepharose4B亲和层析分离出能与柱结合的 Lp( a)Lys+ 和不能与柱结合的 Lp( a) Lys-.采用 MTT比色法、流式细胞仪分析细胞增殖状况 ,同时以ELISA法检测细胞培养液中转化生长因子β( TGF-β)的活化水平 ,观察了两种 Lp( a)对培养的人动脉平滑肌细胞 ( SMC)增殖的影响 .结果表明 :Lp( a)、Lp( a) Lys+ 较 Lp( a) Lys-能有效地促进SMC增殖 ,刺激细胞从 G0 /G1期进入 S和 G2 /M期 ,并显著降低培养液中 TGF- β活化量 .提示 :Lp( a) Lys+ 可能是 Lp( a)促人 SMC增殖的主要组分 ,其机制可能与干扰纤溶酶原活化 ,从而抑制TGF-β活化有关 .     

兔主动脉平滑肌细胞低密度脂蛋白受体相关蛋白(LRP)的诱导表达调节

在兔主动脉平滑肌细胞 ( SMC)培养基中分别加入正常低密度脂蛋白 ( N- LDL)、氧化低密度脂蛋白 ( ox- LDL)、正常极低密度脂蛋白 ( N- VLDL)、氧化极低密度脂蛋白 ( ox- VLDL)和 β-极低密度脂蛋白 (β- VLDL )培养 2 4 h后 ,用定量 RT- PCR和配体结合实验检测平滑肌细胞 LRP的m RNA和蛋白质水平的表达 .结果表明 :五种脂蛋白均能在转录和翻译水平诱导兔主动脉平滑肌细胞的 LRP表达 ,尤以富含胆固醇的 N- LDL ,ox- LDL和β- VLDL的刺激作用更明显 .用胆固醇单独或与脂蛋白共同温育 SMC后 ,发现胆固醇本身可促进 SMC的 LRP蛋白水平的表达 ,脂蛋白与胆固醇的共同刺激作用更为显著 .结果提示 :上述五种脂蛋白对 SMC上 LRP的表达有上调作用 ,其机制可能主要是通过其中的胆固醇来实现的 .     

豚鼠耳蜗螺旋动脉平滑肌细胞和内皮细胞静息膜电位特性

目的:多种内耳疾病和内耳微循环障碍有关,但目前对提供内耳主要血供的耳蜗螺旋动脉平滑肌(SMC)和内皮细胞(EC)的生理学特性还不十分清楚,需要进一步研究。方法:本研究采用双细胞内微电极记录技术和细胞荧光染色技术,研究耳蜗螺旋动脉平滑肌和内皮细胞的膜电位特性和细胞间的通讯联系。结果:研究发现耳蜗螺旋动脉SMC和EC具有高、低两种静息膜电位(RP)状态,两种静息膜电位状态的细胞对乙酰胆碱和高K+的反应完全不同。双微电极可同时记录到EC-ECS、MC-SMC和SMC-EC不同类型的细胞,两个细胞的静息膜电位也可以是双高RP、双低RP和一高一低RP。实验所记录的一高一低RP均是SMC-EC类型,而且EC初始膜电位均为高电位,SMC初始膜电位均为低电位。而双高RP和双低RP可以是SMC-SMC或EC-EC或SMC-EC类型。结论:结果表明耳蜗螺旋动脉的SMC和EC在0.3~0.5 mm的范围内,同类细胞之间有很好的通讯联系,能很好的保持功能的协同和一致,血管壁异类细胞则不同。     

氧化修饰脂蛋白刺激人动脉平滑肌细胞DNA合成

动脉平滑肌细胞(SMC)是动脉粥样硬化(As)斑块中的主要细胞, 它的增殖在As的形成过程中极为重要. 在建立人主动脉SMC体外培养法的基础上, 通过 ³H-TdR掺入实验观察了人低密度脂蛋白(LDL)、极低密度脂蛋白(VLDL)及高密度脂蛋白(HDL)和相应的氧化修饰型脂蛋白对培养人SMC DMA合成的影响.结果发现,HDL对 ³H-TdR掺入SMC DNA无影响(P>0.05); LDL和VLDL ³H-TdR掺入量明显增加(P<0.05);OX-LDL, OX-VLDL及OX-HDL均使 ³H-TdR掺入DNA显著增加(P<0.01).结果表明,LDL和OX-LDL, OX-VLDL及OX-HDL均能刺激SMC DNA合成,促进SMC增殖.     

淫羊藿苷促进宫颈癌TC-1细胞凋亡作用的研究

目的:研究淫羊藿苷体外对致宫颈癌TC-1细胞的增殖抑制及促凋亡作用。方法:利用细胞培养,用不同浓度的淫羊藿苷在一定的时间处理致宫颈癌TC-1细胞,光学显微镜直接观察药物对细胞的作用;MTT法检测淫羊藿苷对TC-1细胞的增殖抑制作用;Dapi核染色、Annexinv-FITC/PI流式细胞学检测细胞凋亡。结果:淫羊藿苷对TC-1细胞有显著的抑制作用,且呈时间、剂量依赖,20μg/ml作用72小时后,细胞抑制率达99%;DAPI核染色和流式细胞术检测可发现典型细胞凋亡特征。结论:淫羊藿苷对TC-1细胞增殖有抑制和促凋亡作用,并呈时间浓度依赖性。     

淫羊藿苷促进宫颈癌TC-1细胞凋亡作用的研究

目的：研究淫羊藿苷体外对致宫颈癌TC-1细胞的增殖抑制及促凋亡作用。方法：利用细胞培养,用不同浓度的淫羊藿苷在一定的时间处理致宫颈癌TC-1细胞,光学显微镜直接观察药物对细胞的作用;MTT法检测淫羊藿苷对TC-1细胞的增殖抑制作用;Dapi核染色、Annexinv-FITC/PI流式细胞学检测细胞凋亡。结果：淫羊藿苷对TC-1细胞有显著的抑制作用,且呈时间、剂量依赖,20μg/ml作用72小时后,细胞抑制率达99%;DAPI核染色和流式细胞术检测可发现典型细胞凋亡特征。结论：淫羊藿苷对TC-1细胞增殖有抑制和促凋亡作用,并呈时间浓度依赖性。     

胰岛素和IGF-I对离体犊牛小肠上皮细胞增殖和吸收功能的影响

本试验选择离体犊牛小肠上皮细胞,以细胞增殖率和葡萄糖吸收率作为细胞生长发育与功能成熟的指标,研究了胰岛素与胰岛素样生长因子-I(IGF-)对细胞生长发育的影响。结果表明,胰岛素浓度为10μg/ml时,明显促进小肠上皮细胞的增殖和对葡萄糖的吸收,浓度达到50μg/ml时则抑制细胞的增殖和吸收(P<0.01)。IGF-I浓度为100ng/ml时,对促进小肠上皮细胞增殖和吸收葡萄糖的作用最强(P<0.01),但100ng/ml、500ng/ml和1000ng/ml三种不同浓度的IGF-I对刺激细胞增殖和提高吸收功能无显著差异(P>0.05)。     

胰岛素样生长因子-1对血管平滑肌细胞PI3K/PTEN信号通路的影响

目的:探讨胰岛素样生长因子-1(IGF-1)促血管平滑肌细胞(VSMC)增殖的细胞内信号转导机制.方法:体外培养的兔血管平滑肌细胞分3组处理,以细胞计数、噻唑盐比色法测定细胞增殖能力,以磷脂酰肌醇-3激酶(PI3K)特异性抑制剂渥漫青霉素(WT)孵育细胞间接反映PI3K作用.Western Blot定量磷酸酶PTEN表达水平,免疫沉淀、特异底物diC16PIP3绿色试剂法测定PTEN脂质磷酸酶活性.结果:IGF-1(100 μg/L)使细胞计数及MTT 比色A值分别增加至对照组的2.8倍和3.8倍,WT抑制VSMC增殖,并完全逆转IGF-1的作用(均P<0.01).各浓度IGF-1对PTEN蛋白表达水平无明显影响,其对PTEN活性的抑制呈浓度(10～100 μg/L)及时间(3 min～24 h)依赖性(均P<0.01).结论:IGF-1促VSMC增殖作用与活化PI3K蛋白激酶的促生长活性及抑制PTEN脂质磷酸酶的负性调节细胞生长作用有关.     

Dynamic connexin43 expression and gap junctional communication during endoderm differentiation of F9 embryonal carcinoma cells

Gap junctional communication permits the direct intercellular exchange of small molecules and ions. In vertebrates, gap junctions are formed by the conjunction of two connexons, each consisting of a hexamer of connexin proteins, and are either established or degraded depending on the nature of the tissue formed. Gap junction function has been implicated in both directing developmental cell fate decisions and in tissue homeostasis/metabolite exchange. In mouse development, formation of the extra embryonal parietal endoderm from visceral endoderm is the first epithelial-mesenchyme transition to occur. This transition can be mimicked in vitro, by F9 embryonal carcinoma (EC) cells treated with retinoic acid, to form (epithelial) primitive or visceral endoderm, and then with parathyroid hormone-related peptide (PTHrP) to induce the transition to (mesenchymal) parietal endoderm. Here, we demonstrate that connexin43 mRNA and protein expression levels, protein phosphorylation and subcellular localization are dynamically regulated during F9 EC cell differentiation. Dye injection showed that this complex regulation of connexin43 is correlated with functional gap junctional communication. Similar patterns of connexin43 expression, localization and communication were found in visceral and parietal endoderm isolated ex vivo from mouse embryos at day 8.5 of gestation. However, in F9 cells this tightly regulated gap junctional communication does not appear to be required for the differentiation process as such.     

SRC utilizes Cas to block gap junctional communication mediated by connexin43

The Src tyrosine kinase phosphorylates Cas (Crk-associated substrate) to confer anchorage independence and invasive growth potential to transformed cells. Gap junctional communication is often lower between aggressive tumor cells compared with normal or benign precursors. The gap junction protein connexin43 (Cx43) is a tumor suppressor that can inhibit tumor cell growth. Src can phosphorylate Cx43 to block gap junctional communication between transformed cells. However, mechanisms by which this event actually closes intercellular channels have not been clearly defined. Here, we report that Src and Cas associate with each other at intercellular junctions. In addition, Cas is required for Src to reduce dye transfer and electrical coupling between cells expressing Cx43. Thus, Src utilizes Cas to inhibit gap junctional communication mediated by Cx43. This finding introduces a novel role of the Cas focal adhesion linker protein in the gap junction complex. This observation may help explain how gap junctional communication can be suppressed between malignant and metastatic tumor cells.     

间隙连接蛋白在卵泡发育过程中的作用

间隙连接广泛分布于各种组织细胞中,由其构成的通道允许小分子信号物质在相邻细胞间直接传递,在细胞间的通讯方面起着非常重要的作用。间隙连接由连接蛋白(Cx)组成,目前已经发现Cx家族有20多个成员[1],它们在相邻细胞间组成同种或异种间隙连接,调控着细胞的增殖和分化。在哺乳动物卵泡发育过程中,卵母细胞与周围的颗粒细胞之间形成的缝隙连接,介导胞间通讯,对生殖细胞迁移、卵母细胞减数分裂能力恢复、颗粒细胞分层、卵泡成腔、黄体形成、促性腺激素信号传递有非常重要的调节作用。本文根据近年来相关的研究报道,对卵泡发育过程中间隙连接的作用进行综述。     

日本沼虾生精细胞与支持细胞之间的连接关系

用透射电镜技术研究了日本沼虾精子发生过程中不同细胞之间的连接关系。结果表明,从精原细胞期到次级精母细胞期,在生精细胞之间存在间隙连接与分隔连接与分隔连接,并且两种连接相互邻接,桥粒仅在精原细胞之间发现;从精原细胞期到精细胞期,在生精细胞与支持细胞之间也存在相互邻接的间隙连接与分隔连接,两类细胞之间有大量桥粒,形成血淋巴－精巢屏障,这种屏障可保持生精细管内环境的稳定性;精子发生的不同时期,支持细胞之     

Blood vessel maturation in a 3-dimensional spheroidal coculture model: direct contact with smooth muscle cells regulates endothelial cell quiescence and abrogates VEGF responsiveness.

Paracrine interactions between endothelial cells (EC) and mural cells act as critical regulators of vessel wall assembly, vessel maturation and define a plasticity window for vascular remodeling. The present study was aimed at studying blood vessel maturation processes in a novel 3-dimensional spheroidal coculture system of EC and smooth muscle cells (SMC). Coculture spheroids differentiate spontaneously in a calcium-dependent manner to organize into a core of SMC and a surface layer of EC, thus mimicking the physiological assembly of blood vessels with surface lining EC and underlying mural cells. Coculture of EC with SMC induces a mature, quiescent EC phenotype as evidenced by 1) a significant increase in the number of junctional complexes of the EC surface layer, 2) a down-regulation of PDGF-B expression by cocultured EC, and 3) an increased resistance of EC to undergo apoptosis. Furthermore, EC cocultured with SMC become refractory to stimulation with VEGF (lack of CD34 expression on VEGF stimulation; inability to form capillary-like sprouts in a VEGF-dependent manner in a 3-dimensional in gel angiogenesis assay). In contrast, costimulation with VEGF and Ang-2 induced sprouting angiogenesis originating from coculture spheroids consistent with a model of Ang-2-mediated vessel destabilization resulting in VEGF responsiveness. Ang-2 on its own was able to stimulate endothelial cells in the absence of Ang-1 producing SMC, inducing lateral sheet migration as well as in gel sprouting angiogenesis. Taken together, the data establish the spheroidal EC/SMC system as a powerful cell culture model to study paracrine interactions in the vessel wall and provide functional evidence for smooth muscle cell-mediated quiescence effects on endothelial cells.     

The supramolecular architecture of junctional microdomains in native lens membranes

Gap junctions formed by connexons and thin junctions formed by lens-specific aquaporin 0 (AQP0) mediate the tight packing of fibre cells necessary for lens transparency. Gap junctions conduct water, ions and metabolites between cells, whereas junctional AQP0 seems to be involved in cell adhesion. High-resolution atomic force microscopy (AFM) showed the supramolecular organization of these proteins in native lens core membranes, in which AQP0 forms two-dimensional arrays that are surrounded by densely packed gap junction channels. These junctional microdomains simultaneously provide adhesion and communication between fibre cells. The AFM topographs also showed that the extracellular loops of AQP0 in junctional microdomains adopt a conformation that closely resembles the structure of junctional AQP0, in which the water pore is thought to be closed. Finally, time-lapse AFM imaging provided insights into AQP0 array formation. This first high-resolution view of a multicomponent eukaryotic membrane shows how membrane proteins self-assemble into functional microdomains.     

Co-culture of endothelial cells and smooth muscle cells affects gene expression of angiogenic factors

Endothelial cells (EC) are in contact with the underlying smooth muscle cells (SMC). The interactions between EC and SMC in the vessel wall are considered to be involved in the control of growth and function of blood vessels. A co-culture system of EC and SMC and a method for separation of these cells was developed in order to investigate whether the presence of physical contact between EC and SMC affected the gene expression of angiogenic factors. Human EC and SMC were prepared from the great saphenous veins. Autologous EC were added on top of the confluent layer of SMC. After 72 h in co-culture, the EC were magnetically separated from SMC with the use of superparamagnetic beads. RT-PCR products for bFGF, bFGFR, VEGF, PDGF-AA, PDGF-BB, TGF-beta, and beta-actin were analyzed to study the mRNA expressions. The protein level of selected factors was studied by ELISA technique. In co-cultured SMC there was a statistically significant higher gene expression of VEGF, PDGF-AA, PDGF-BB, and TGF-beta and significant lower gene expression of bFGF and its receptor than in single cultured SMC. The protein level of PDGF-BB and TGF-beta was also significantly higher in co-cultured SMC. In co-cultured EC there were no significant differences in gene expression of PDGF-AA, PDGF-BB, and TGF-beta compared with single cultured EC. The gene expression and protein synthesis of VEGF was significantly higher in co-cultured EC. The findings from the present study suggest that cell-cell interactions of EC and SMC affect the gene and protein expression of angiogenic factors.     

Expression of connexin 37, 40, and 43 mRNA and protein in renal preglomerular arterioles

Gap junctions allow direct intercellular coupling between many cells including those in the vascular wall. Studies of connexin expression in cells of the microcirculatory system are very few in number. However, cell-to-cell communication between cells of the arteriolar wall may be particularly important in microcirculatory control. We investigated the expression of connexins 43, 40, and 37 (Cx43, Cx40, Cx37) mRNA and proteins in primary cultures of smooth muscle cells (SMC) from rat renal preglomerular arterioles and in the aortic cell line A7r5. Furthermore protein expression in preglomerular arterioles in frozen sections was evaluated. SMC were isolated from kidneys using an iron oxide sieve method and explant technique. Total RNA from these cultures was tested by RT-PCR analysis for the expression of the three connexins mRNA. Using immunofluorescence we examined whether the expression pattern of connexin protein in the cell culture and frozen sections corresponded to the mRNA expression. The data show that A7r5 and preglomerular SMC express mRNA for Cx37 in addition to Cx43 and Cx40. In A7r5 cells the mRNA for Cx43, Cx40, and Cx37 are translated to protein, whereas cultured preglomerular SMC and the media of afferent arterioles in frozen sections only showed Cx40 immunoreactivity.     

Direct coupling through gap junctions is not involved in urethral neurotransmission

Interstitial cells of Cajal (ICC) are believed to participate in urethral neurotransmission and it was proposed that direct coupling of ICC and smooth muscle cells (SMC) through gap junctions (GJ) is involved, although this still remains unclear. Hence, we investigated the distribution of different connexins (Cx 43, Cx40, and Cx37) in the sheep and rat urethra, as well as their possible role in neurotransmission. Conventional PCR confirmed that three Cxs are expressed in the urethra. Moreover, both Cx43 and Cx37-immunoreactivity (-ir) were present in SMC, ICC, and the urothelium, although Cx37-ir was significantly weaker and Cx40-ir was limited to the endothelium. While these results indicate that GJ intercellular communication could occur between SMC and ICC, neither the contractile (noradrenergic) nor the relaxant (nitrergic) responses of the rat and sheep urethra to electrical field stimulation were significantly modified by two different GJ inhibitors: 18α-glycyrrhetinic acid and a cocktail of Cx mimetic peptides ((Cx43)Gap 26, (Cx37, Cx43)Gap 27, and (Cx40)Gap 27). By contrast, contractions induced by high K(+) were effectively reduced by both blockers, evidence that they effectively inhibit intercellular communication. These results indicate that GJ are not implicated in urethral neurotransmission, although the question of whether ICC modulate neurotransmission through some other mechanism remains to be determined.     

ATP抑制人胃腺癌(MGC-803)细胞增殖的机理研究(简报)

ATP已是临床治疗心血管疾病的常用药,目前国内外对ATP的抗癌作用也十分重视。我们曾报道过ATP对人胃癌细胞(SGC-7901、MGC-803)增殖的抑制效应,Rapaport和Fang等也相继报道ATP对小鼠结肠移植癌及人前列腺癌的增殖有抑制作用,但对ATP抗癌作用的机理除了报道膜上嘌呤受体的改变和cAMP第二信使调节作用外,其他方面未见报道。本实验研究了ATP抑制MGC-803细胞增殖与细胞周期时相分布、间隙连接通讯功能、c-Ha-ras基因表达之间的关系,为临床应用ATP治疗癌症提供了实验依据。