脂质体介导hHCN2基因转染HEK293细胞

目的建立一种研究离子通道的有效模型。方法采用Lipofacta mine2000脂质体将人的超极化激活的环核苷酸门控（HCN）基因转染人胚胎肾（HEK）293细胞,利用全细胞膜片钳技术检测克隆人HCN2基因的表达。结果pcDNA3-hHCN2真核表达载体转染HEK293细胞3d后,全细胞膜片钳技术记录到克隆人HCN2基因编码的通道电流。结论全细胞膜片钳技术稳定、可靠,可为开展克隆离子通道结构和功能关系研究提供基础。     

粉防己碱促进和敏化肾性高血压大鼠血管平滑肌细胞凋亡

目的：从大鼠血管平滑肌细胞凋亡角度探讨粉防己碱逆转肾血管性高血压血管重构的机制。方法：两肾-夹法制备肾性高血压模型,原位末端标记法检测主动脉和尾动脉动脉壁平滑肌细胞凋亡数量;[3H]-TdR掺入和流式细胞术分析粉防己碱对培养细胞凋亡率的影响。结果：粉防己碱能够诱导血管平滑肌细胞凋亡,并使低血清和TNFα、转化生长因子（TGFβ1）诱导的细胞凋亡率增加。结论：粉防己碱逆转肾性高血压大鼠（RHR）血管重构的机制可能与诱导和敏化RHR血管平滑肌细胞凋亡有关。     

血管内皮生长因子受体-3在血管内皮细胞和平滑肌细胞中的表达

目的探讨血管内皮细胞生长因子受体-3（VEGFR-3）在血管内皮细胞和平滑肌细胞中的表达。方法分离人脐带中的脐静脉和脐动脉,冰冻切片后以免疫荧光法检测VEGFR-3的表达。结果VEGFR-3荧光信号表达于脐静脉内皮细胞和平滑肌细胞,以及脐动脉内皮细胞、平滑肌细胞和外周细胞。结论血管内皮细胞和平滑肌细胞均表达VEGFR-3。     

富含微量元素矿泉水对人血管平滑肌细胞生长增殖的影响

目的研究富含微量元素矿泉水对体外培养血管平滑肌细胞（HVSMCs）生长增殖的影响。方法无菌取12例冠状动脉旁路移植术患者术中废弃的大隐静脉,应用金山矿泉水配制的DMEM培养液（MW—DMEM）和双蒸水配制的DMEM培养液（DDW—DMEM）分别培养平滑肌细胞,分为金川矿泉水组和双蒸水组。观察记录2种培养液培养的HVSMCs游出组织块的时间及细胞呈“峰”-“谷”样时间;建立血管紧张素Ⅱ诱导HVSMCs增殖模型,采用MTT比色法观察2种培养液对AngⅡ诱导HVSMCs增殖抑制作用;应用电镜和免疫组织化学染色法观察鉴定HVSMCs。结果金川矿泉水组培养的HVSMCs贴壁时间为（3．7±0．2）d,双蒸水组为（8．5±0．6）d;金川矿泉水组培养的HVSMCs自贴壁后到长成“峰”-“谷”样形态的时间为（8．3±0．2）d,双蒸水组为（7．1±0．5）d;MTT比色法测定金川矿泉水组培养的HVSMCs的增殖活度明显降低;电镜观察MW—DMEM培养的HVSMCs细胞形态完整,表面有丰富的微绒毛。HVSMCs胞浆内富含肌丝,纵向平行排列,胞内有密体;抗-actin平滑肌肌动蛋白免疫组织化学染色后可见胞浆内染成褐色呈细丝网状。结论富含微量元素和矿物质的金川矿泉水具有修复血管平滑肌细胞损伤和抑制血管紧张素Ⅱ诱导HVSMCs增殖的作用。     

酶消化法培养老龄SD大鼠血管平滑肌细胞

目的：采用酶消化法培养老龄SD大鼠血管平滑肌细胞,为血管疾病,特别是为动脉粥样硬化研究提供大量的原代平滑肌细胞。方法：无菌取老龄SD大鼠主动脉,0.2%Ⅱ型胶原酶消化分离细胞,采用自然纯化、差速贴壁纯化平滑肌细胞,免疫组化鉴定平滑肌细胞α肌动蛋白。结果：免疫组化染色显示细胞纯度在95%以上。结论：酶消化法分离获取SD大鼠平滑肌细胞方法简单,易掌握,采用本方法可稳定获得大量的平滑肌细胞供实验使用。     

大鼠Adrb3基因重组慢病毒干扰载体的构建及其对血管平滑肌细胞Adrb3表达的影响

目的 构建大鼠Adrb3 （rAdrb3） 基因慢病毒干扰载体, 筛选高效率rAdrb3基因干扰序列, 观察其对大鼠血管平滑肌细胞 （VSMC） Adrb3基因表达的影响, 为进一步研究Adrb3在心力衰竭中的作用机制提供实验工具。方法 设计2条rAdrb3基因shRNA寡核苷酸序列, 构建慢病毒干扰质粒并进行测序鉴定。三质粒法共转染293T细胞包装慢病毒, 测定慢病毒滴度。大鼠VSMC设置正常对照组 （Normal组）, 空载慢病毒组 （Lv-rAdrb3-shRNA-control组）,携带rAdrb3基因慢病毒干扰载体1组 （Lv-rAdrb3-shRNA-1组）, 携带rAdrb3基因慢病毒干扰载体2组 （Lv-rAdrb3- shRNA-2组）, 感染5 d后, 收集细胞。提取细胞总RNA和蛋白, Real-time PCR检测rAdrb3 mRNA表达, Western blot 检测rAdrb3蛋白表达。结果 构建2个携带rAdrb3基因慢病毒干扰载体, 滴度均为2×108 TU/mL。慢病毒感染大鼠 VSMC 72 h后, 感染效率可达80%。与Normal组比较, Lv-rAdrb3-shRNA-1、 Lv-rAdrb3-shRNA-2组Adrb3 mRNA水平沉默效率为87.18%、 65.27% （P＜0.05）; 与Normal组比较, Lv-rAdrb3-shRNA-1、 Lv-rAdrb3-shRNA-2组rAdrb3蛋白水平沉默效率为85.57%、 70.04% （P＜0.05）。结论 成功构建并筛选出有效携带rAdrb3基因慢病毒干扰载体, 可显著抑制大鼠VSMC Adrb3的表达。     

糖尿病大鼠冠状动脉平滑肌细胞体外培养方法的探讨

目的探讨糖尿病大鼠冠状动脉平滑肌细胞体外培养方法,建立糖尿病大鼠冠状动脉平滑肌细胞模型,为糖尿病性冠心病的研究奠定基础。方法建立糖尿病大鼠模型,用酶消化法体外培养冠状动脉血管平滑肌细胞。结果糖尿病大鼠造模成功后分离冠状动脉,用酶消化法培养糖尿病大鼠血管平滑肌细胞,24h更换培养液液,培养7~10d细胞重叠生长达多层,高低起伏呈“峰—谷”状。细胞α-actin免疫组织化学染色鉴定为平滑肌细胞。结论糖尿病大鼠血管平滑肌细胞增殖速度快,培养条件要求严格,在形态学上与正常大鼠平滑肌细胞相同。     

趋化因子受体CCR6的克隆及其在HEK293细胞中的表达

目的：构建人趋化因子受体6（CCR6）cDNA序列的真核表达载体，并了解其在HEK293细胞中的表达。方法：提取人淋巴结总RNA，通过逆转录PCR扩增出CCR6基因片段，并构建真核表达载体pcDNA3，1（＋）-CCR6；重组载体通过脂质体转染HEK293细胞，免疫荧光法鉴定CCR6的表达。结果：酶切鉴定和序列分析证实重组质粒含有CCR6编码序列．转染实验表明重组质粒能在HEK293细胞中表达出具有活性的CCR6片段。结论：CCR6真核表达载体构建及表达成功，为下一步CCR6拈抗剂的筛选奠定了基础。     

hi FGF2真核表达载体的构建及其高表达对细胞凋亡的影响

目的构建hi FGF2(high molecular weight isoform fibroblast growth factor-2,hi FGF2)真核表达载体,并观察其过表达后对细胞凋亡的影响。方法设计合成hi FGF2 cDNA模板引物,Nhel和Hind III双酶切pDsRed1-N1质粒,T4DNA连接酶重组hi FGF2质粒,PCR扩增目的基因,琼脂糖凝胶电泳检测及测序鉴定。将重组hi FGF2质粒瞬时转染HEK293细胞,荧光倒置显微镜检测转染效率。AnnexinV-FITC/PI双染法流式细胞仪检测细胞凋亡。结果 hi FGF2真核表达载体符合设计要求,瞬时转染HEK293细胞的转染率达70%以上。过表达hi FGF2,HEK293细胞FITC/PI双染阳性率达(29.12±2.81)%,与正常组、转染空载体组差别有显著性(P<0.01或P<0.05)。结论成功构建hi FGF2真核表达载体,过表达hi FGF2导致细胞凋亡。     

雌激素对血管平滑肌细胞凋亡的影响

目的 :观察雌激素对血管平滑肌细胞 (SMC)凋亡的影响。方法 :以大鼠为对象 ,观察正常对照组、去卵巢组及去卵巢 +雌二醇治疗组大鼠血管平滑肌细胞凋亡的关系。结果 :雌二醇治疗组血管平滑肌细胞凋亡较去卵巢组相比明显减少(P>0 .0 5 ) ,与正常对照组比较差别无显著性意义 (P >0 .0 5 )。结论 :雌二醇对血管平滑肌细胞的凋亡有抑制作用 ,可能是其预防动脉粥样硬化的机制     

家兔下腔静脉血栓模型建立的实验研究

目的：探讨通过手术在家兔下腔静脉内置入螺旋铜丝建立下腔静脉血栓模型的可行性及成功率。方法将30只家兔分为血栓组（25只）与对照组（5只）。血栓组实验兔通过手术暴露下腔静脉,将自制长约3 cm直径约3 mm螺旋铜丝穿刺置入下腔静脉,止血后将自制U形铜丝夹于螺旋铜丝近心端下腔静脉收紧,留缝隙约2 mm,缝合腹膜、肌肉及皮肤。对照组进行与血栓组相同的手术过程,但下腔静脉不置入螺旋铜丝。1d后处死解剖2组实验兔,观察血栓组下腔静脉血栓形成情况,计算造模成功率,对照组观察下腔静脉血流情况。血栓组取血栓行HE染色病理检查确定血栓性质。结果血栓组25只实验兔下腔静脉管壁完好。23只实验兔下腔静脉内可见血栓形成,红色血栓与白色血栓沿螺旋铜丝间或存在,造模成功率92％（23/25）。2只实验兔造模失败。对照组5只实验兔下腔静脉内未见血栓形成。血栓组血栓病理证实均为新鲜混合血栓。结论本方法建立下腔静脉血栓模型成功率高,血栓病理符合深静脉血栓特点,可用于静脉血栓的实验研究。     

Expression and localization of C-type natriuretic peptide in human vascular smooth muscle cells

OBJECTIVES: C-type natriuretic peptide (CNP) released by vascular endothelium relaxes smooth muscle and is important in the maintenance of vascular tone. Since it is not known whether other human vascular cell types produce CNP, we investigated its expression in human vascular smooth muscle. METHODS: CNP expression was examined by RT-PCR in vascular smooth muscle cells (SMC) cultured from human saphenous vein (SV), internal mammary artery (IMA) and radial artery (RA), and CNP protein was probed using immunostaining, in tissue sections and in SMCs cultured from these vessels, respectively. RESULTS: PCR for CNP produced a 334 bp product in all SMC cultures, as expressed in endothelial cells, although the band intensity was markedly less in SMCs. Myocardium from CNP-knockout mouse did not express CNP, while there was expression in wild-type mouse. CNP protein was detected by immunostaining in 100% of SMC cultures. By immunostaining of tissue sections, CNP was detected throughout the medial layer, but not adventitia, of all vessel types. CONCLUSIONS: Expression of CNP at gene and protein level by human vascular SMCs suggests that CNP may have the capacity to regulate vascular tone independently of the endothelium.     

线粒体融合蛋白2在原发性高血压患者血管组织和平滑肌细胞中表达的研究

目的 观察线粒体融合蛋白2(Mfn2)在人血管组织和平滑肌细胞(VSMCs)中的表达情况.方法 选取2006年10月至2007年10月在北京安贞医院行冠状动脉旁路移植术的患者60例,分为高血压组和正常血压组各30例,收集术中剩余桥血管(乳内动脉).取1例正常产妇产新生婴儿脐带,进行人乳内动脉和VSMCs培养.提取乳内动脉组织和VSMCs总RNA,RNA的反转录-cDNA的聚合酶链式扩增(RTPCR),观察2组患者Mfn2的表达情况.用不同浓度血小板源性生长因子(PDGF-BB)诱导VSMCs增殖并检测Mfn2表达情况.结果 2组患者血压水平、体质指数差异有统计学意义(P＜0.05).人乳内动脉血管组织和VSMCs均有Mfn2表达,高血压组Mfn2表达水平均低于正常血压组(人乳内动脉血管组织:0.42±0.10比0.49 ±0.12,P=0.014;VSMCs:0.29 ±0.07比0.33 ±0.08,P=0.041).高血压组原代VSMCs细胞计数高于正常血压组(0.48 ±0.15比0.38 ±0.11,P=0.005).与对照组相比,随PDGF-BB浓度增加VSMCs计数增加(对照组:0.36 ±0.09,10 μg/L组:0.53 ±0.13,100 μg/L组:0.54±0.12),VSMCs的Mfn2表达减少(对照组:0.31 ±0.08,10μg/L组:0.22±0.06,100μg/L组:0.21±0.05,P＜0.05);100 μg/L组与10 μg/L组相比,VSMCs细胞计数和VSMCs的Mfn2表达差异无统计学意义(P值分别为0.862和0.755).结论 在人血管组织和培养的VSMCs中验证了高血压和正常血压入选者存在Mfn2的表达差别.     

蕲蛇酶联合肝素治疗对兔下腔静脉血栓血管内膜的影响研究

目的:研究蕲蛇酶联合肝素治疗对兔下腔静脉血栓血管内膜的影响。方法:复制下腔静脉血栓模型后均分为3组,即肝素、尿激酶联合肝素、蕲蛇酶联合肝素组。复制模型3d后给药,分别在用药后第3、7、10天观察病变血管内膜增生程度、静脉壁胶原纤维沉积量、内皮细胞形态学变化。结果:与肝素组比较,尿激酶联合肝素、蕲蛇酶联合肝素组胶原染色显著减少(P<0.05);在用药后的第3天尿激酶联合肝素、蕲蛇酶联合肝素组内皮细胞损伤程度较轻(P<0.05);第7、10天时,尿激酶联合肝素、蕲蛇酶联合肝素组内皮细胞损伤程度显著减轻(P<0.01)。结论:蕲蛇酶联合肝素在治疗兔下腔静脉血栓方面具有保护病变血管内皮细胞、减轻血管壁炎症反应的功效,对静脉血栓有较好的治疗及预后效果。     

Pharmacological endothelin receptor interaction does not occur in veins from ET(B) receptor deficient rats

Heterodimerization of G-protein coupled receptors can alter receptor pharmacology. ET A and ET B receptors heterodimerize when co-expressed in heterologous expression lines. We hypothesized that ET A and ET B receptors heterodimerize and pharmacologically interact in vena cava from wild-type (WT) but not ET B receptor deficient (sl/sl) rats. Pharmacological endothelin receptor interaction was assessed by comparing ET-1-induced contraction in rings of rat thoracic aorta and thoracic vena cava from male Sprague Dawley rats under control conditions, ET A receptor blockade (atrasentan, 10 nM), ET B receptor blockade (BQ-788, 100 nM) or ET B receptor desensitization (Sarafotoxin 6c, 100 nM) and ET A plus ET B receptor blockade or ET A receptor blockade plus ET B receptor desensitization. In addition, similar pharmacological ET receptor antagonism experiments were performed in rat thoracic aorta and vena cava from WT and sl/sl rats. ET A but not ET B receptor blockade or ET B receptor desensitization inhibited aortic and venous ET-1-induced contraction. In vena cava but not aorta, when ET B receptors were blocked (BQ-788, 100 nM) or desensitized (S6c, 100 nM), atrasentan caused a greater inhibition of ET-1-induced contraction. Vena cava from WT but not sl/sl rats exhibited similar pharmacological ET receptor interaction. Immunocytochemistry was performed on freshly dissociated aortic and venous vascular smooth muscle cells to determine localization of ET A and ET B receptors. ET A and ET B receptors qualitatively co-localized more strongly to the plasma membrane of aortic compared to venous vascular smooth muscle cells. Our data suggest that pharmacological ET A and ET B receptor interaction may be dependent on the presence of functional ET B receptors and independent of receptor location.     

PROCEEDINGS OF THE HBPRCA DIFFERENTIAL EFFECT OF RENAL WRAP HYPERTEN SION ON AORTIC SMOOTH MUSCLE POLYPLOIDY IN THE RAT AND RABBIT

1. The incidence of aortic smooth muscle cell polyploidy was investigated in rabbits and rats with renal wrap induced (cellophane perinephritic) chronic hypertension. 2. Bilateral renal cellophane wrapping was performed in young adult animals. Blood pressure was measured intra-arterially in the rabbits twice during the experimental period and tail-cuff blood pressure measured twice weekly in the rats. At 8 weeks post-surgery the incidence of aortic smooth muscle cell polyploidy was determined in enzymatically isolated cells by flow cytometric DNA analysis. 3. Systolic blood pressure was significantly increased in the bilateral renal wrapped rabbits and rats compared to the shams, such that at 8 weeks post-surgery, systolic blood pressure was 139 ± 2 mmHg and 84±2 mmHg, respectively, in the rabbits and 188±6 mmHg and 155±4 mmHg, respectively, in the rats. 4. The incidence of polyploid smooth muscle cells was significantly higher in the hypertensive renal wrapped rat compared to the sham (20.9±1.5% and 8.1±0.5%, respectively). However, the incidence of polyploid cells was low in the rabbit aortae with no significant difference in the incidence of aortic smooth muscle polyploidy in the hypertensive rabbit compared to the sham (2.6 ± 0.6% and 2.7 ± 0.6%, respectively). 5. This study demonstrates a species difference in the induction of polyploidy during the same model of experimental hypertension in aortic smooth muscle derived from the rabbit and rat.     

Role of Rho kinase signalling in healthy and varicose human saphenous veins

1. The present study was performed to determine the role of Rho-Rho kinase signalling pathway in smooth muscle cells from both healthy and varicose human saphenous vein. 2. The Rho kinase inhibitor Y-27632 inhibited the noradrenaline (NA)-induced contraction in human saphenous veins with IC(50) corresponding to 0.5 microM and 10.9 microM in control and varicose veins, respectively. The maximal amplitude of the NA-induced contraction was smaller in varicose vein compared to control (1263+/-172 mg versus 1974+/-245 mg, P<0.05). 3. In beta-escin permeabilized strips, GTPgammaS induced a rise in tension that was inhibited by Y-27632. The amplitude of the GTPgammaS-induced contraction was smaller in varicose compared to control veins (23.1+/-2.4% versus 41.3+/-2.2%, P<0.002). 4. In smooth muscle cells, Y-27632 induced disassembly of both actin cytoskeleton and extracellular fibronectin matrix. In comparison to control cells, varicose vein smooth muscle cells show decreased actin cytoskeleton organization and reduction of fibronectin matrix deposition. 5. The Rho proteins Rnd1 and RhoA, and Rho kinase 1 are expressed in human saphenous veins. A 2.6 fold reduction of Rho kinase expression was found in varicose veins. 6. These results indicate that RhoA-Rho kinase mediated Ca(2+) sensitization of the contraction and regulated actin cytoskeleton and extracellular fibronectin matrix assembly in human saphenous smooth muscle. The decrease of Rho kinase expression and Rho kinase-dependent functions detected in smooth muscle from varicose veins supports a role of this signalling pathway in the functional alterations of the vein wall occurring in the course of the disease.     

A regional difference in the distribution of postsynaptic alpha-adrenoceptor subtypes in canine veins

The responsiveness of helical venous strips isolated from fifteen different sites in the body of dogs to relatively selective alpha 1- and alpha 2-adrenoceptor agonists was studied, as well as to a non-selective alpha-adrenoceptor agonist. Longitudinal strips of the portal and mesenteric veins and the inferior vena cava between the liver and the renal vein (segment C) were also investigated. All veins contracted to noradrenaline or phenylephrine whereas only seven veins responded significantly to clonidine: the saphenous, cephalic, jugular and femoral veins and longitudinal strips of the portal and mesenteric veins and the segment C of the inferior vena cava. The brachiocephalic, azygos, pulmonary and splenic veins and the superior vena cava and the supradiaphragmatic portion (segment A) and the infrarenal portion (segment D) of the inferior vena cava responded little to clonidine. Unlike the longitudinal strips, the helical strips of the portal and mesenteric veins and the segment C of the inferior vena cava did not respond to clonidine. According to the relative sensitivities to phenylephrine and clonidine, those veins which responded to clonidine could be divided into three groups. (1) The veins in which the sensitivity to phenylephrine was higher than to clonidine: longitudinal strips of the portal vein and segment C of the inferior vena cava, (2) the veins whose sensitivity to phenylephrine was lower than to clonidine: the saphenous, cephalic, femoral and external jugular veins, (3) the vein whose sensitivity to the two agonists was comparable: longitudinal strips of the mesenteric vein. Subtype characteristics were further analyzed in the saphenous vein and in the portal vein using prazosin, phentolamine and yohimbine as antagonists. Analysis of Schild plots to noradrenaline suggested that a mixed population of alpha-adrenoceptor subtypes might be present in the saphenous vein, whereas a rather homogeneous population of a single subtype might occur in the portal vein. The results of the antagonism experiment against phenylephrine and clonidine suggested that contractions of the saphenous vein are mediated by both alpha 1- and alpha 2-adrenoceptors whereas contractions of the portal vein are exerted mainly through alpha 1-adrenoceptors. The results suggest that there may be a distinct regional difference with respect to postsynaptic alpha- adrenoceptor subtypes in the canine venous system.     

MG132诱导人血管平滑肌细胞凋亡及对caspase3表达的影响

目的观察蛋白酶体抑制剂MG132对人脐静脉血管平滑肌细胞(VSMC)的致凋亡作用及其对凋亡相关的天冬氨酸特异的半胱氨酸蛋白酶3(caspase3)表达的影响。方法采用多个浓度(2.5,5,10μmol/L)的蛋白酶体抑制剂MG132处理人脐静脉VSMC48h;DNA琼脂糖凝胶电泳检测细胞凋亡;流式细胞术分析细胞周期和细胞凋亡率;反转录-聚合酶链反应(RT-PCR)检测caspase3基因转录水平;免疫细胞化学检测细胞caspase3蛋白表达。结果蛋白酶体抑制剂MG132处理48h后细胞凋亡率增加;RT-PCR检测发现细胞内凋亡相关基因caspase3mRNA表达上调;免疫细胞化学检测细胞caspase3蛋白表达水平升高。结论蛋白酶体抑制剂MG132能够诱导人脐静脉VSMC凋亡,其作用呈量-效关系;MG132诱导VSMC凋亡的机制可能与MG132抑制泛素-蛋白酶体途径(UPP)活性,促进caspase3基因转录,使细胞内caspase3增加而促进细胞凋亡。