采用组织块法培养心房颤动患者心房成纤维细胞

目的建立心房颤动(简称房颤)患者心房肌成纤维细胞培养模型。方法通过心脏外科手术获得房颤患者右心耳组织30 mg,对经典的大鼠组织块培养法作改进,采用组织块放入75%酒精中浸泡1 m in,培养基配方和植块方法改进,第二代传代时采用差速贴壁法除去内皮细胞,培养房颤患者心房肌成纤维细胞。结果形态学观察显示,心肌成纤维细胞生长较慢,3天左右开始从组织块边缘爬出,10天呈接合状态,细胞呈梭形,细胞排列较紧密,有的交叉重叠生长,胞核较大,呈椭圆形,通常含1~3个核,胞浆透明,胞体较大,无搏动;免疫细胞化学染色可见,心肌成纤维细胞波形蛋白呈强阳性反应,第八因子呈阴性反应;成功培养的心肌成纤维细胞纯度达95%以上。结论采用组织块法可成功培养人心房的心肌成纤维细胞,且简单、易行。     

风湿性心脏病患者心房成纤维细胞的原代培养

目的建立一种人心房成纤维细胞原代培养简单有效的方法。方法新鲜右心耳组织取自心外科风湿性心脏病手术患者,采用0.25%胰蛋白酶和0.2%Ⅱ型胶原酶消化法分离细胞,用含2O%胎牛血清的DMEM培养液进行培养并传代,显微镜下观察心房成纤维细胞生长状况。波形蛋白(Vimentin)、α平滑肌肌动蛋白(α-SMA)、Ⅷ因子(VWF)抗体对细胞进行免疫荧光鉴定。结果获取大量细胞,形态典型;Vimentin、α-SMA表达阳性,VWF阴性,鉴定为心房肌成纤维细胞。结论胰蛋白酶和Ⅱ型胶原酶消化法是一种简单有效的人心房成纤维细胞原代培养方法。     

用包皮成纤维细胞培养弓形虫速殖子的研究

目的 建立用包皮成纤维细胞(Human foreskin fibroblasts，HFF)培养弓形虫速殖子的方法。方法 将包皮环切术切下的包皮在含青、链霉素的Hank’s液中充分洗涤后，用眼科剪子剪成0．5cm。的组织小块，移入培养瓶中培养，待HFF细胞长满瓶底后，用胰酶消化并将其接种到培养皿中的盖玻片上培养，HFF长满盖玻片后，用弓形虫速殖子感染HFF，镜下观察并于1、2．5、4、5．5、10、23和32 h取出其中一皿中的盖玻片，经姬氏染色、二甲苯透明后，镜下观察速殖子生长情况。结果 弓形虫速殖子大多在感染后3h～5h侵入HFF，32h左右，假包囊破裂，释放出虫体。结论 成功建立了用HFF培养弓形虫的方法。     

心房颤动患者心房组织成纤维细胞增殖与心房纤维化的关系

目的 检测碱性成纤维细胞生长因子(bFGF)、α平滑肌肌动蛋白(α-SMA)和增殖细胞核抗原(PCNA)在心房颤动(房颤)患者心房组织中的基因表达,探讨成纤维细胞增殖与心房纤维化关系的分子机制.方法 75例风湿性心脏瓣膜病接受换瓣手术者分为三组,窦性心律组34例,阵发性房颤组11例,持续性房颤组30例;于术中获取右心耳组织,采用苦味酸天狼猩红染色法对心房组织胶原沉积量及分布情况进行分析,分别采用半定量逆转录聚合酶链反应和免疫组化技术测定bFGF、α-SMA和PCNA的mRNA和蛋白含量.结果 阵发性房颤组、持续性房颤组心房组织胶原容积分数(CVF)明显高于窦性心律组(均为P<0.01);持续性房颤组增加更明显(P<0.01).心房组织CVF与左心房内径(r=0.320,P=0.005)、房颤持续时间呈正相关(r=0.390,P=0.010).与窦性心律组相比,bFGF、α-SMA和PCNA的mRNA和蛋白表达水平在阵发性房颤患者(均为P<0.05)、持续性房颤患者(P<0.01)中均显著上调,持续件房颤组相对于阵发性房颤组亦明显增高(P<0.05～0.01).同时bFGF的mRNA和蛋白表达与CW(r=0.330,P=0.004和r=0.292,P=0.013)、房颤持续时间(r=0.330,P=0.005和r=0.299,P=0.010)及左心房内径(r=0.342,P=0.003和r=0.285,P=0.015)呈正相关.结论 心房组织中bFGF、α-SMA和PCNA的基因表达上调可能是成纤维细胞增殖导致心房纤维化的分子机制之一,与房颤的发生和维持有关.     

用包皮成纤维细胞培养弓形虫速殖子的研究

目的　建立用包皮成纤维细胞 (Humanforeskinfibroblasts ,HFF)培养弓形虫速殖子的方法。　方法　将包皮环切术切下的包皮在含青、链霉素的Hank’s液中充分洗涤后 ,用眼科剪子剪成 0 .5cm3 的组织小块 ,移入培养瓶中培养 ,待HFF细胞长满瓶底后 ,用胰酶消化并将其接种到培养皿中的盖玻片上培养 ,HFF长满盖玻片后 ,用弓形虫速殖子感染HFF ,镜下观察并于 1、2 .5、4、5 .5、10、2 3和 3 2h取出其中一皿中的盖玻片 ,经姬氏染色、二甲苯透明后 ,镜下观察速殖子生长情况。　结果　弓形虫速殖子大多在感染后 3h～ 5h侵入HFF ,3 2h左右 ,假包囊破裂 ,释放出虫体。　结论　成功建立了用HFF培养弓形虫的方法。     

血管紧张素Ⅱ通过抑制人心房成纤维细胞BKCa通道诱导心房纤维化

目的 探讨在血管紧张素Ⅱ(AngⅡ)诱导心房纤维化的过程中,大电导钙激活钾通道(BKCa通道)的作用机制。方法 通过组织块贴壁法获取原代人心房成纤维细胞,使用免疫荧光染色进行鉴定。用浓度为500 nmol/L的AngⅡ处理人心房成纤维细胞24 h,实时荧光定量PCR与蛋白质印迹法用于检测处理前后纤维化标志基因α-平滑肌肌动蛋白(α-SMA)、胶原蛋白Ⅰ(collagenⅠ)和胶原蛋白Ⅲ(collagenⅢ),以及BKCa通道的α与β亚基的mRNA和蛋白表达水平,全细胞膜片钳技术检测AngⅡ处理前后的BKCa通道的电流变化。结果 (1)人心房成纤维细胞经AngⅡ处理后,α-SMA、collagenⅠ和collagenⅢ的mRNA和蛋白表达水平升高;(2)经过AngⅡ处理后,BKCa通道α及β亚基mRNA和蛋白表达水平降低;(3)人心房成纤维细胞存在功能正常的BKCa通道,具有电压依赖性;(4)人心房成纤维细胞BKCa通道的宏观电流幅度在经AngⅡ处理后降低;(5)在人心房成纤维细胞上过表达BKCa通道α亚基后,纤维化标志物α-SMA、collagenⅠ和collagenⅢ的表达受到了明显...     

体外原代培养脐带基质间充质细胞和人皮肤成纤维细胞

目的探讨体外原代培养脐带基质间充质细胞(UMC)和人皮肤成纤维细胞(HSF)的方法。方法用Ⅳ型胶原蛋白酶、Ⅰ型脱氧核糖核酸酶和0.25%胰蛋白酶消化法原代分离培养UMC细胞,用组织块贴壁法原代分离培养HSF细胞,镜下观察细胞的培养过程和生长状态。结果脐带组织分离培养第3天,有少量UMC贴壁生长,细胞膜周围有折光性,形态类似于成纤维细胞;皮肤组织块贴壁培养第7天,有HSF爬出,呈长梭形、不规则三角形。随着细胞培养时间延长,UMC和HSF数量逐渐增多,细胞生长状态良好。结论应用酶消化法和组织块贴壁法可成功分离培养出UMC和HSF。     

人皮肤成纤维细胞分离培养的研究

目的：探讨人包皮成纤维（human foreskin fibroblast，HFF）细胞分离培养方法，通过分析HFF细胞体外培养条件，建立稳定的HFF细胞系。方法：用0．25％dispaseⅡ酶消化包皮环切术后皮肤组织，分离表皮和真皮，真皮组织用0．1％Ⅰ型胶原酶消化，收集细胞于含20％胎牛血清的DMEM培养液中，置CO2培养箱孵育，待细胞长成单层后消化传代。结果：消化法得到的人皮肤成纤维细胞24h内贴壁，刚开始贴壁时细胞多数呈圆形，HFF细胞随贴壁时间延长，逐渐伸展成梭形或多边形。传代后的成纤维细胞12h开始贴壁，24h内完全贴壁。HFF细胞采用SABC法染色细胞浆呈棕色，前10代细胞活力均在98％以上。结论：该方法可获得高产量、高活力的HFF细胞，是一种可靠的HFF细胞培养方法。     

氨基胍延缓人胚肺二倍体成纤维细胞复制性衰老的实验研究

目的探讨人工合成单体抗氧化剂氨基胍（2-AG）对人胚肺二倍体成纤维细胞（2BS）复制性衰老的影响及其分子机制。方法观察2-AG对2BS细胞衰老表型、细胞代龄、传代速度、细胞周期、细胞增殖能力、晚期糖基化终末产物（AGEs）水平及衰老相关β半乳糖苷酶（SA-β-Gal）阳性率的影响。结果2-AG可维持细胞的非衰老表型，增加2BS细胞代龄19～20代，MTT法分析发现用2-AG孵育细胞84h，细胞增殖能力较对照组升高36％～40％。2-AG显著加快了细胞的增殖速度，其培养细胞S期的比例较对照细胞升高约1倍。另外，2-AG连续培养的细胞在老龄阶段AGEs、SA-β-Gal阳性率均显著低于老年对照细胞，而与年轻2BS细胞相似。结论2-AG可有效延缓2BS细胞的复制性衰老。     

Prolactin expression and secretion by human breast glandular and adipose tissue explants

Prolactin (PRL) is a 23-kDa hormone produced by the pituitary and extrapituitary sites. The main target of PRL is the breast, where it affects cellular growth, differentiation, and milk production. Recent evidence suggests that locally produced PRL plays a role in breast tumorigenesis. Our objective was to examine PRL synthesis/release in different tissues of the human breast and determine the effect of ovarian steroids. Breast tissue, obtained from women undergoing mastectomy or breast reduction, was separated into glandular (nonmalignant) and adipose explants and incubated for 10 d. Conditioned media were analyzed for PRL by a bioassay. PRL release from glandular explants decreased by 60% from d 1-3, followed by a 4-fold increase on d 10. PRL release from adipose explants was unchanged from d 1-3 and increased more than 10-fold by d 10. PRL gene expression, determined by RT-PCR, was low on d 0 and markedly increased on d 10 in both types of explants. De novo synthesis of PRL was confirmed by metabolic labeling. Progesterone suppressed PRL release from glandular explants without affecting adipose explants. Estradiol did not alter PRL release from either tissue. In conclusion, the human breast produces and releases bioactive PRL, with a higher release rate by adipose than glandular tissue. The time-dependent rise in PRL release suggests removal from inhibitory control. Progesterone may be one of the factors that suppresses PRL production in the glandular compartment, whereas the factor(s) that regulate adipose PRL are unknown. These data suggest an autocrine/paracrine role for PRL in human glandular and adipose breast tissue.     

人心房肌细胞的培养与鉴定

目的　探索人心房肌细胞的原代及传代培养方法。方法　取心外科手术患者 (1 5～6 0岁 ,平均 2 5岁 )常规切除的右心耳 ,利用组织贴块法原代培养心房肌细胞并进行传代培养 ,对培养细胞 (取第 3代 )进行光镜、透射电镜形态学观察和免疫细胞化学鉴定 ,并绘制生长曲线。结果原代培养 1 0d左右时细胞数目可达 1 0 6～ 1 0 7/ml;此后每 4～ 5d可传代一次 ,大多可传至第 8代。透射电镜观察到典型心肌细胞的超微结构 ,免疫细胞化学分析显示 90 %以上的细胞呈α 肌动蛋白及心肌特异性肌钙蛋白Ⅰ抗体染色阳性。细胞生长曲线显示第 3代细胞在密度为 1× 1 0 6～ 8× 1 0 6/ml呈对数生长 ,倍增时间约 2 4h。结论　利用组织贴块法成功培养出人心房肌细胞 ,所得心房肌细胞纯度高并能传代培养 ,为深入研究人心房肌细胞的病理生理及分子生物学奠定了基础。     

Arrhythmogenic effects of theophylline in human atrial tissue

We studied the effects of theophylline on the transmembrane action potential and the contractile force of human atrial fibers obtained from the hearts of 15 patients, undergoing corrective open-heart surgery. Atrial fibers were perfused with Tyrode solution and driven electrically at a constant rate of 60 beats per min. Theophylline (0.1-1 mM) steepened the diastolic depolarization, increased the amplitude of oscillatory potential during diastole and facilitated the development of spontaneous slow response action potentials. These arrhythmogenic effects of theophylline were suppressed after diltiazem (0.1-0.3 microM) pretreatment. The present findings provide the electrophysiologic evidence that abnormal atrial automaticity as a result of triggered activity may be the underlying cause for atrial ectopic activity and multifocal atrial tachycardia in patients taking theophylline.     

Myosin isoenzyme distribution in overloaded human atrial tissue

Using nondenaturing polyacrylamide gel electrophoresis, we have identified two distinct myosin isoenzymes in human atrial tissue that correspond to the V1 and V3 isomyosins found in rat ventricular tissue. Normal left and right atrial appendages have approximately 50% V3. When the left atrium was exposed to hemodynamic overload secondary to mitral stenosis, the percent V3 increased to 77 +/- 10% (n = 10); exposure to hemodynamic overload secondary to mitral regurgitation caused an increase to 70 +/- 14% (n = 6). Changes in the isoenzyme pattern were seen in the right atria of patients with mitral stenosis and markedly elevated pulmonary arterial pressures compared with control subjects and patients with mitral stenosis without severe pulmonary hypertension. Several clinical variables were examined to determine which factors might influence isoenzyme expression. Age, sex, the presence of atrial fibrillation, and pulmonary capillary wedge pressure did not predict the isoenzyme pattern. However, patients with mitral valvular disease and only slightly enlarged left atria tended to have a higher percent V3 than those with massively enlarged atria. These data confirm that human atrial tissue, like rat ventricular tissue, can alter its isomyosin composition in response to a hemodynamic load. The data further suggest that the isoenzyme shift is an early adaptation to the imposed load.     

Part of the activating cross-linked immunoglobulin G is internalized by human platelets to sites not accessible for enzymatic digestion

The differential uptake of tritium-labeled immunoglobulin G (IgG) cross- linked with bisdiazonium-benzidine (BDB) (3H-BDB-IgG) by washed, pooled human platelets to sites inaccessible to pronase digestion was tested. Up to 52% of the 3H-BDB-IgG associated with platelets at 37 degrees C resisted pronase treatment, whereas only 23% of the cross-linked IgG associated with platelets at 4 degrees C, or at 37 degrees C but in the presence of deoxyglucose/antimycin A, remained refractory to pronase. This effect was not due to platelet agglutination. Pronase resistance reached a maximum after a 60-minute incubation period at 37 degrees C. With increasing 3H-BDB-IgG input, both the total cross-linked IgG associated with platelets and the fraction resistant to pronase digestion approached saturation at 4 degrees C, but not at 37 degrees C. The proportion of 3H-BDB-IgG bound to platelets at 4 degrees C that was resistant to pronase treatment increased by 13% within five minutes of warming the platelets to 37 degrees C. Pretreatment of platelets with 10 mmol/L acetylsalicylic acid (or 10 mumol/L prostaglandin E1) prior to the addition of 3H-BDB-IgG led to a 74% (95%) inhibition of the 3H-BDB-IgG-induced 14C-serotonin release, but to only a 44% (49%) inhibition of pronase-digestible bound ligand. In contrast, pretreatment with 10 mumol/L cytochalasin B led to a mere 17% reduction of 14C-serotonin release, whereas acquisition of resistance to pronase digestion by the bound 3H-BDB-IgG was inhibited by 90%. Incubation of platelets at 37 degrees C with 3H-BDB-IgG and removal of unbound material prior to the addition of prostaglandin E1 or deoxyglucose/antimycin A had little effect on the susceptibility of platelet-associated 3H-BDB-IgG to pronase, whereas the addition of cytochalasin B to 3H-BDB-IgG-treated platelets resulted in greatly increased susceptibility of the platelet-associated ligand to pronase. Thus, after binding, 3H-BDB-IgG becomes transferred in an energy- dependent process to pronase-resistant cellular sites, most likely to the open canalicular system.     

Regulation of monocyte chemoattractant protein 1 (MCP-1) release by explants of human visceral adipose tissue

BACKGROUND: Monocyte chemoattractant protein-1 (MCP-1) is a chemokine involved in monocyte recruitment during inflammation whose plasma level is elevated in obesity. OBJECTIVE: The present studies were designed to examine the release of MCP-1 in primary culture by explants of visceral adipose tissue from morbidly obese women. RESULTS: Most of the MCP-1 released by adipose tissue explants was derived from the nonfat cells in adipose tissue. The release of MCP-1 by adipose tissue explants was upregulated almost five-fold between 3 and 48 h of incubation. Approximately half of this upregulation was due to the release of endogenous tumor necrosis factor alpha (TNFalpha) and IL-1beta based on the ability of a combination of a soluble TNFalpha receptor (etanercept) and a blocking antibody against IL-1beta to reduce MCP-1 release. The release of MCP-1 over 48 h was unaffected by insulin or dexamethasone but significantly reduced by the combination of both agents. MCP-1 release was reduced by 60% in the presence of an inhibitor of the nuclear factor kappaB (NF-kappaB) pathway. There were no significant effects of inhibitors of p44/42 mitogen-activated protein kinase (ERK), Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK) pathways on MCP-1 release. However, inhibition of MCP-1 release in the presence of inhibitors of both the p38 MAPK and NF-kappaB pathways was greater than that seen with only the NF-kappaB inhibitor. DISCUSSION: The present data shows that MCP-1 formation is upregulated over a 48-h incubation of primary explants of visceral adipose tissue. Half of this upregulation is dependent upon endogenous TNFalpha and Il-1beta and involves the p38 MAPK and NF-kappaB pathways.     

Inhibition of microvascular endothelial apoptosis in tissue explants by serum albumin

Plasma factors appear to inhibit endothelial cell (EC) apoptosis in vivo so that flow influences microvascular form. The identity of these factors has not, however, been established. Earlier, we reported that apoptosis in isolated, serum-deprived human EC is inhibited by albumin (Alb). Here, we demonstrate likely biological relevance of this to vascular remodelling in experiments with tissue explants. Rat skin explants were incubated in medium M199 with or without serum, bovine Alb, or human Alb. EC in paraffin sections of explants were labelled by lectin histochemistry and the relative proportion of apoptotic was EC determined. Apoptosis was confirmed by transmission electron microscopy and terminal deoxynucleotidyl transferase labelling. Serum-free culture induced EC apoptosis (P < 0.02) and this was strongly inhibited by Alb at physiological concentrations (P < 0.01). This was not a nonspecific protein effect, as mercaptoethanol denaturation destroyed the activity and ovalbumin was not protective. Also, protection was not due to serum contaminants, as recombinant human Alb had activity identical to that of native material. The dose response was identical for all Alb preparations tested, with maximal activity at physiological concentrations. Protection was not limited to rat tissue as similar results were obtained with human gingival explants. These data support a role for Alb as a plasma antiapoptotic factor for EC in tissues.     

Plasma concentration of human atrial natriuretic hormone in patients with connective tissue diseases

Summary Atrial natriuretic peptide (ANP), a peptide released from the cardiac atria, compensates blood volume expansion by its diuretic, natriuretic and vasoactive properties. We measured human plasma ANP(hANP) levels in patients with systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and progressive systemic sclerosis (PSS) and found that their values were higher than those of healthy controls. In SLE patients, hANP levels correlated with serum creatinine concentration and the patients with proteinuria showed high levels of hANP. Administration of large amount of corticosteroid as a remission induction of the patients with SLE caused high levels of hANP. In patients with PSS, %FEV1 showed strong inverse correlations between hANP levels, and the patients with an enlarged second curvature of the heart had high levels of hANP. In patients with RA, no significant correlation was found between hANP levels and clinical variables including patients' age.