Effect of thrombin on proliferation, contraction and prostaglandin production of rat glomerular mesangial cells in culture.

The effect of thrombin on mesangial cell function was investigated. Thrombin caused a dose-dependent increase in [3H] thymidine incorporation (EC50 = 0.36 +/- 0.09 U/ml), intracellular calcium [(Ca++)i] mobilization (EC50 = 1.9 +/- 0.5 U/ml) and prostaglandin E2 (PGE2) production (EC50 = 0.25 +/- 0.02 U/ml) in rat glomerular mesangial cells. These effects were blocked by the thrombin inhibitor, hirudin (KB = 10.4 +/- 0.2 nM). The role of (Ca++)i mobilization and arachidonate metabolism in thrombin-stimulated proliferation was tested by the addition of the calcium channel blocker, nifedipine, and the cyclooxygenase inhibitor, indomethacin, to mesangial cell cultures. Indomethacin, at doses that completely inhibited the thrombin-mediated production of PGE2, had no significant effect on proliferation. The Ca++ channel blocker, nifedipine, inhibited both PGE2 production and [3H] thymidine incorporation in a dose-dependent fashion, but only at concentrations considered nonspecific. In addition to its effects on PGE2, thymidine incorporation and Ca++ mobilization, thrombin caused mesangial cell contraction as determined by a substrate distortion technique. This effect was not inhibited by indomethacin. These results indicate that thrombin can alter mesangial cell function in vitro.     

Amrinone effects on electromechanical coupling and depolarization-induced automaticity in ventricular muscle of guinea pigs and ferrets

The effects of the cardiotonic agent, amrinone (0.05-4 mM), on electrical and mechanical activities of ferret and guinea-pig papillary muscles were studied using current and voltage clamp (single sucrose gap) techniques. In current clamp studies, amrinone increased, in a dose-dependent manner, contractile force elicited by action potential in both species. Depolarization-induced automaticity was facilitated in ferret muscles at all maximum diastolic potentials between -70 and -15 mV. Facilitation of automaticity in guinea-pig muscles occurred only at potentials more negative than -35 mV and was suppressed at more positive potentials. Cimetidine (10 microM) partially reversed the effects of amrinone on automaticity in both species. In voltage clamp studies, amrinone increased the slow inward current. Steady-state outward current was increased in guinea-pig but not in ferret muscles. A dual effect of amrinone on tension was observed. Amrinone was found to increase phasic tension of ferret papillary muscles only for depolarizations lasting less than 250 to 300 msec. For longer depolarizations, amrinone decreased the phasic tension (in a dose-dependent manner), whereas the tonic tension was not modified. The decrease as well as the increase in tension was associated with an increase of the slow inward current. The results suggest that amrinone may be arrhythmogenic and may have an intracellular action at the sarcoplasmic reticulum level (partial inhibition) in addition to its action on the calcium current.     

The effect of acoustic cavitation on the contraction force and membrane potential of rat papillary muscle

In experiments on isolated rat papillary muscles an acoustic cavitation induced by continuous wave focused ultrasound (543 kHz with intensity up to 3 W/cm2) was found to result in reversible membrane depolarization by 54.0 +/- 1.4 mV (n = 5), loss of excitability and rise in resting tension up to 53.1 +/- 4.3% (n = 15) of contractile response in the control. It was supposed that the rapid recovery of excitability (69.3 +/- 10.3 s, n = 15) might be a result of Ca2+ pump activation and/or alterations of intercellular coupling when cavitation ends.     

Effect of dantrolene sodium on muscle contraction

The force of contraction of the adductor pollicis before and after dantrolene sodium was evaluated in 5 paraplegic patients. The muscle was indirectly stimulated through supramaximal stimulation of the ulnar nerve at the wrist, with single pulse and variable number of stimuli at frequency of 50 Hz. There was a marked and significant reduction (an average of 64%) of the single twitch response in all patients while on medication. When the muscle, however, was stimulated with tetanic frequency no significant difference was evident in the force of the sustained contraction with or without medication. The amplitude of evoked electrical potentials, simultaneously recorded from the adductor pollicis remained unchanged irrespective of the status of medication. Decrement of the twitch response, with no effect on the electrical activity, tends to substantiate the distal effects of dantrolene on muscle contraction. The drug, however, does not seem to prevent the muscle from achieving its normal maximal tetanic force when activated with frequencies close to the physiological frequencies of maximal, voluntary effort (50 Hz.) These effects point to the usefulness and limitations of the drug in clinical situations.     

Effect of insulin on the lysosomal function of rat aortic smooth muscle cells and subcutaneous fibroblasts in tissue culture

Extracellular and intracellular activities of N-acetyl-beta-glucosaminidase (NAG), a representative lysosomal enzyme, in rat aortic smooth muscle cells and subcutaneous fibroblasts cultured for 6 days with an insulin-enriched (200 microU/ml of insulin) or an insulin-deprived medium were measured. The culture medium was changed every 2 days. The net release of NAG into the medium between the 4th and the 6th day was significantly inhibited in the insulin-enriched culture of both smooth muscle cells and fibroblasts. The inhibition was more marked in the fibroblasts series than in the smooth muscle cell series. On the other hand, the intracellular NAG activity of both cell series was almost similar in the insulin-enriched culture and in the insulin-deprived culture. The results indicate that insulin has an inhibitory effect on the net extracellular release of NAG and, therefore, regulates the lysosomal function in these mesenchymal cells.     

Effects of iron loading and bacillus Calmette-Guerin on a glycoprotein recognition system on rat hepatic sinusoidal cells

Experiments were performed to determine the effects of agents that modify Kupffer cells on the mannose-N-acetylglucosamine-glycoprotein receptor on hepatic sinusoidal cells. Cells were prepared by collagenase liver perfusion, centrifugation on Percoll gradients, and centrifugal elutriation. The uptake of 125I-labeled agalacto-orosomucoid (125I-AGOR), an N-acetylglucosamine-terminated glycoprotein, was greatest (53% of total uptake) by elutriator fractions containing equal proportions of endothelial and Kupffer cells ("mixed cell" fraction). Uptake was specific and time and concentration dependent. The apparent Km (0.4 mumol/L) and the patterns of inhibition by monosaccharides were similar in all the elutriator fractions, suggesting that only one class of receptor was present. The highest apparent maximal velocity (18 pmol/hr/5 X 10(6) cells) was found in the mixed cell fraction, indicating this fraction contained the highest proportion of receptor-bearing cells. Latex (0.8 micron) and bacillus Calmette-Guerin pretreatments did not influence the hepatic uptake of the glycoprotein in vivo. Iron sorbitol significantly reduced hepatic glycoprotein uptake and caused a twofold increase in the proportion of the ligand remaining in the circulation. Uptake of 125I-agalacto-orosomucoid by cells from latex-treated rats was similar to controls, but uptake by bacillus Calmette-Guerin-treated rat cells was only 25% of control uptake. This was related to a marked increase in sinusoidal cell number caused by bacillus Calmette-Guerin. In contrast, iron sorbitol caused a selective suppression of 125I-agalacto-orosomucoid uptake (10% of control uptake) by cells in the mixed cell fraction. This study showed that maximal uptake of 125I-agalacto-orosomucoid was by elutriator fractions containing equal proportions of endothelial and Kupffer cells and that iron sorbitol suppressed ligand uptake by these cells, possibly by influencing the mannose-N-acetylglucosamine receptor on Kupffer cells.     

大鼠附睾脂肪垫脂肪源性干细胞的分离培养及分化潜能

背景:相对于骨髓基质干细胞,脂肪源性下细胞来源丰富,取材方便,干细胞丰度高,极易培养,具有多向分化潜能,有望成为组织工程、细胞治疗以及基因转染良好的源细胞,但目前对脂肪源性干细胞的研究尚处于初级阶段.目的:拟在体外分离培养大鼠脂肪源性干细胞,并探讨其分化潜能.设计、时间及地点:细胞学体外观察,于2008-06/2009-01在南方医科大学组胚教研室和南方医院临床医学实验中心完成.材料:SPF级成年雄性SD大鼠1只,由南方医科大学实验动物中心提供.方法:无菌条件下切取大鼠双侧附睾尾脂肪垫,胶原酶消化法分离培养脂肪源性干细胞,胰蛋白酶消化法传代扩增.取第4代脂肪源性干细胞接种到96孔板,5×104个细胞/皿,待细胞贴壁后分成3组:成骨诱导组加入成骨培养基,成脂诱导组加入成脂培养基,对照组仍用基础培养基,培养14 d.主要观察指标:脂肪源性干细胞的形态变化、体外增殖能力、细胞免疫化学鉴定结果.脂肪源性干细胞成骨和成脂分化能力.结果:体外培养的脂肪源性干细胞易扩增,传代后以长梭形细胞为主,呈旋涡状排列,克隆样生长;经多次传代仍能保持较强的增殖能力,细胞生长曲线争"S"形;细胞表面标志CD44和CD49d呈阳性表达,CDl06呈阴性表达.脂肪源性干细胞经成骨诱导7 d后,碱性磷酸酶染色呈阳性;成脂诱导3 d后,油红O染色示胞浆内有脂滴出现;对照组细胞碱性磷酸酶染色呈阴性,无脂滴存在.结论:从大鼠附睾脂肪垫内分离的脂肪源性干细胞易于培养和传代扩增,在特殊条件下可分化为成骨细胞和脂肪细胞.     

Resting transmembrane potential difference of skeletal muscle in normal subjects and severely ill patients

The resting membrane potential difference (Em) of skeletal muscle was measured in 26 normal human subjects, 7 patients with mild illness, and 21 patients with severe, debilitating medical disorders. A closed transcutaneous approach to the muscle was made by needle puncture and the Em was measured utilizing standard Ling electrodes. Measurements revealed an Em of -88 +/-3.8 mv in healthy subjects and -89 +/-2.1 mv in patients hospitalized for minor medical problems. The mean Em in 21 in-hospital patients, judged to be severely ill clinically from a variety of causes, was -66.3 +/-9.0 mv. Open deltoid muscle biopsies were performed in 7 of the healthy subjects and in 13 of the severely ill group. Estimation of the intra-extracellular water partition was made by calculating the chloride space from the previously measured Em. Analysis of the muscle samples revealed no significant difference in the intra-extracellular potassium ratios of the two groups biopsied. Intracellular Na(+) concentrations were uniformly increased in the muscle samples of the severely ill subjects and averaged 42.3% higher than those of the normal subjects. The mechanisms which might account for the elevation of intracellular Na(+) and a depression of Em independent of changes in intra-extracellular K(+) ratios are discussed and it is suggested that this defect may be a generalized cellular abnormality which is a common quality of serious illnesses.     

Effect of caffeine and zinc on DNA and protein synthesis of neonatal rat cardiac muscle cell in culture

The effect of caffeine and/or zinc on DNA and protein synthesis of purified neonatal-rat ventricular cardiac myocytes was studied. Caffeine (0.2–2 mM) inhibited both DNA and protein synthesis of the cells. Addition of EDTA in the growth medium inhibited both DNA and protein synthesis. Without caffeine and in the presence of lower concentrations of caffeine (0.2 mM) in the growth medium, 10μM of zinc concentration reversed DNA synthesis, which was inhibited by the chelating agent (EDTA). Higher concentrations of caffeine (2 mM) in the growth medium completely abolished sensitivity of cardiac myocytes to zinc. Additional zinc supplementation to the growth medium of cardiac myocytes did not alter the rate of protein synthesis. The present results suggest that the effect of caffeine on cardiac myocytes may be associated with the zinc-dependent enzymes involved in DNA synthesis.     

Effect of bisphosphonates on prostaglandin synthesis by rat bone cells and mouse calvaria in culture

Bisphosphonates are potent inhibitors of bone resorption and also inhibit prostaglandin (PG) E2 synthesis in bone cells. Therefore we have investigated whether a correlation exists between inhibition of bone resorption and inhibition of PGE2 formation. Initially, bisphosphonates were tested for their effect on the release of [14C]PGE2 from rat calvaria cells labelled with [14C]arachidonic acid and stimulated by bradykinin, thrombin and mechanical manipulation. The effect on [14C]-PGE2 synthesis was not correlated with the known inhibitory activity of bisphosphonates on bone resorption. Mouse calvaria were then treated with epidermal growth factor (EGF) to induce PGE2 synthesis and bone resorption, with or without bisphosphonates. The bisphosphonates either decreased, had no effect or increased PGE2 production, but all inhibited the release of calcium. Finally, the bisphosphonates were given in vivo to mice before explantation of the calvaria. Some of the bisphosphonates decreased the production of PGE2, suggesting that these compounds may have such an effect in vivo. But again no relationship between the effect on PGE2 synthesis and bone resorption was found. Thus, these experiments show the inhibitory effect of bisphosphonates on bone resorption is unlikely to be explained only by their effect on PGE2 synthesis.     

Effects of verapamil on ventricular tachycardias possibly caused by reentry, automaticity, and triggered activity.

To define the role of verapamil in the treatment of ventricular tachycardia (VT), we studied 21 patients with chronic recurrent VT. Electrophysiologic studies were performed before and during intravenous infusion of verapamil (0.15 mg/kg followed by 0.005 mg/kg per min). On the basis of the mode of VT initiation and termination, we identified three groups of patients: (a) 11 patients had VT suggestive of reentry, as VT could be initiated with ventricular extrastimulation and terminated with overdrive ventricular pacing. Verapamil did not affect the inducibility and cycle length of VT. (b) 7 patients had VT suggestive of catecholamine-sensitive automaticity as VT could not be initiated with programmed electrical stimulation but could be provoked by isoproterenol infusion. Moreover, the VT could not be converted to a sustained sinus rhythm with overdrive ventricular pacing and it resolved only with discontinuing isoproterenol infusion. Verapamil exerted no effects on VT. (c) 3 patients had VT with electrophysiologic characteristics suggestive of triggered activity related to delayed afterdepolarizations. Characteristically, after attaining a range of cycle lengths, the sinus, atrial or ventricular paced rhythm could initiate VT without ventricular extrastimulation. The first beat of VT invariably occurred late in the cardiac cycle with a premature coupling interval 0-80 ms shorter than the preceding QRS cycle length; the premature coupling interval gradually decreased as the sinus, atrial or ventricular paced cycle length progressively shortened. Of note, verapamil completely suppressed VT inducibility in these three patients. These observations lead us to suggest that verapamil does not affect VT caused by reentry and catecholamine-sensitive automaticity but is effective in suppressing VT caused by triggered activity related to delayed afterdepolarizations in humans.     

大鼠脂肪源性干细胞的分离培养及鉴定

背景：脂肪源性干细胞在体外易于培养,增殖快,具有多向分化潜能。目的：构建一种体外分离培养SD大鼠脂肪源性干细胞的方法,并对其部分生物学特性与表型进行分析。方法：切取SD大鼠腹股沟脂肪垫,应用胶原酶Ⅰ消化,分离大鼠脂肪源性干细胞,进行体外培养、传代,倒置显微镜观察细胞的生长增殖及形态变化,诱导成骨、成脂,分别行碱性磷酸酶、茜素红、von Kossa染色及油红O染色,绘制生长曲线及用流式细胞仪检测细胞表面标记。结果与结论：体外培养的脂肪源性干细胞呈梭形,增殖活跃,传代后形态均一,多次传代后细胞仍保持较强增殖能力,生长曲线呈＂S＂型。成骨诱导实验组碱性磷酸酶、茜素红、von Kossa染色阳性;成脂诱导实验组油红O染色阳性;对照组均为阴性。细胞CD29,CD44,CD105表达阳性,CD31,CD45表达阴性。提示SD大鼠腹股沟脂肪垫分离的脂肪源性干细胞在体外易于分离培养和传代扩增,特定条件下可诱导分化为成骨细胞和脂肪细胞,并表达间充质干细胞相关的表型。     

深吸气联合骨骼肌收缩对脑灌注的影响

目的：探讨深吸气联合全身骨骼肌收缩对颈动脉血流动力学的影响。方法：于2002-05／2003—06选择在解放军第四七六医院健康体检的男性军人32名，应用彩色多普勒血流显像测量深吸气联合骨骼肌收缩时颈动脉收缩峰l流速、收缩峰2流速、舒张峰流速、舒张末流速、平均流速、脑血流量、阻力指数和搏动指数等参数。结果：纳入受试者32名，均进入结果分析。①深吸气骨骼肌收缩状态时收缩晚期峰流速、舒张期峰流速、平均流速和血流量在颈总动脉较安静状态时分别提高了41％，31％，23％，26％；在颈内动脉较安静状态时分别提高了23％，31％，24％，25％。②深吸气骨骼肌收缩状态时血管阻力指数和血管搏动指数在颈总动脉较安静状态时降低了7％，26％；在颈内动脉降低了8％，24％。③平均流速和血流量有相关性（颈总动脉r=0．69，P〈0．05；颈内动脉r=0．72，P〈0．01）。④深吸气骨骼肌收缩状态时收缩早期峰流速、舒张末流速与安静状态时比较差异无显著性意义（P〉0．05）。结论：深吸气联合骨骼肌收缩动作主要影响并增大收缩晚期峰流速、舒张期峰流速和平均流速指标，降低脑血管阻力，增加脑供血。     

Effect of platelet-activating factor and serum-treated zymosan on prostaglandin E2 synthesis, arachidonic acid release, and contraction of cultured rat mesangial cells

The interaction of inflammatory cells and glomerular prostaglandins (PG) may be important during glomerulonephritis. We therefore examined the influence of platelet-activating factor (PAF), (a mediator of inflammation released from leukocytes) and of phagocytosis of zymosan on arachidonic acid metabolism and on cell contractility in rat glomerular mesangial cells in culture. PAF increased PGE2 synthesis (determined by radioimmunoassay) within minutes (threshold: 10(-10)M; maximal effect: 10(-7)M). Serum-treated zymosan also stimulated PGE2, but with a slower onset. In cells prelabeled with [14C]arachidonic acid both PAF and serum-treated zymosan released 14C from phospholipids and increased free [14C]arachidonate. The ratio of 14C-release to PGE2 was, however, different with PAF and serum-treated zymosan, indicating different phospholipid pools. Under phase-contrast microscopy, PAF caused contraction of mesangial cells with a dose-response and time-course parallel to that for PGE2 synthesis. Serum-treated zymosan caused no contraction. The PAF-induced contraction was enhanced by PG synthesis inhibition and was attenuated by addition of PGE2, indicating a feedback mechanism. The mesangial contraction by PAF may be important in favoring deposition of immune complexes, while the PGE2 synthesis stimulated by PAF and by phagocytosis of zymosan may counteract the deleterious effects of PAF during induction of glomerulonephritis.     

深吸气联合骨骼肌收缩对脑灌注的影响

目的:探讨深吸气联合全身骨骼肌收缩对颈动脉血流动力学的影响。方法:于2002-05/2003-06选择在解放军第四七六医院健康体检的男性军人32名,应用彩色多普勒血流显像测量深吸气联合骨骼肌收缩时颈动脉收缩峰1流速、收缩峰2流速、舒张峰流速、舒张末流速、平均流速、脑血流量、阻力指数和搏动指数等参数。结果:纳入受试者32名,均进入结果分析。①深吸气骨骼肌收缩状态时收缩晚期峰流速、舒张期峰流速、平均流速和血流量在颈总动脉较安静状态时分别提高了41%,31%,23%,26%;在颈内动脉较安静状态时分别提高了23%,31%,24%,25%。②深吸气骨骼肌收缩状态时血管阻力指数和血管搏动指数在颈总动脉较安静状态时降低了7%,26%;在颈内动脉降低了8%,24%。③平均流速和血流量有相关性(颈总动脉r=0.69,P<0.05;颈内动脉r=0.72,P<0.01)。④深吸气骨骼肌收缩状态时收缩早期峰流速、舒张末流速与安静状态时比较差异无显著性意义(P>0.05)。结论:深吸气联合骨骼肌收缩动作主要影响并增大收缩晚期峰流速、舒张期峰流速和平均流速指标,降低脑血管阻力,增加脑供血。