Age-dependent contribution of Rho kinase in carbachol-induced contraction of human detrusor smooth muscle in vitro

Aim： Activation of muscarinic receptors on the detrusor smooth muscle is followed by contraction, which involves both myosin light chain kinase （MLCK） and Rho kinase （ROCK）. The aim of this study was to determine the relative contributions of MLCK and ROCK to carbachol-induced contraction of human detrusor smooth muscle in vitro.
Methods： Detrusor smooth muscle strips were prepared from the macroscopically unaffected bladder wall of patients underwent cystectomy. The strips were fixed in an organ bath, and carbachol or KCl-induced isometric contractions were measured by force transducers.

Results： Addition of carbachol （0.4-4 μmol/L） into the bath induced concentration-dependent contractions of detrusor specimens, which was completely abolished by atropine （1 μmol/L）. Pre-incubation of detrusor specimens with either the MLCK inhibitor ML-9 or the ROCK inhibitors HA1100 and Y-27632 （each at 10 μmol/L） significantly blocked carbachol-induced contractions as compared to the time-control experiments. Moreover, MLCK and ROCK inhibition were equally effective in reducing carbachol-induced contractions. The residual carbachol-induced contractions in the presence of both MLCK and ROCK inhibitors were significantly smaller than the contractions obtained when only one enzyme （either MLCK or ROCK） was inhibited, suggesting an additive effect of the two kinases. Interestingly, ROCK-mediated carbachol-induced contractions were positively correlated to the age of patients （r=0.52, P〈0.05）.

Conclusion： Both MLCK and ROCK contribute to carbachol-induced contractions of human detrusor smooth muscle. ROCK inhibitors may be a new pharmacological approach to modulate human bladder hyperactivity.     

血吸虫病疫苗研究进展

在全球范围的传染病中 ,寄生虫的感染率可能要超过其它任何病原所致的感染 ,而血吸虫在许多方面都是一种不寻常的寄生虫 ,如雌雄异体、性染色体异配 ,寄生于宿主的门脉系统及有性和无性生殖的生活史等 ,这些特点 ,使其成为医学和兽医学中研究最为深入的虫种之一。据统计 ,目前大概有 2 - 3亿人有血吸虫感染 ,全球有 74个国家 6亿左右的人口受到血吸虫病的威胁 ,尽管已有不少血吸虫病的防治经验 ,但要把人畜共患的血吸虫病危害减至最少 ,仍有相当长的一段路要走。我国 5 0年来的血吸虫病防治工作虽然取得一定成效 ,但并未得到彻底控制 ,究其…     

温阳解毒化瘀颗粒对肠源性内毒素血症大鼠肠黏膜上皮紧密连接的影响

目的：研究温阳解毒化瘀颗粒对肠源性内毒素血症（ IETM ）模型大鼠结肠黏膜上皮紧密连接的影响,探索其抗肝衰竭的作用机制。方法：将大鼠随机分为正常组、模型组、温阳解毒化瘀颗粒（实验组）和对照组4组,采用D-半乳糖胺（D-gal）腹腔注射致肝衰竭ITEM大鼠模型。正常组在腹腔注射生理盐水24h后处死,模型组、实验组、对照组分别于造模后24h、48h、72h各取6只、7只、7只大鼠处死,检测各组肝功能、内毒素、结肠黏膜上皮咬合蛋白（occludin）及肌球蛋白轻链激酶（MLCK）。结果：模型组血清ALT/AST、内毒素、 MLCK表达水平均高于正常组, occludin表达低于模型组（ P＜0.01）;实验组血清ALT/AST、内毒素、 MLCK表达水平均低于模型组, occlu-din表达高于模型组（ P＜0.05）。结论：增强结肠粘膜上皮紧密连接功能,降低内毒素的吸收是温阳解毒化瘀颗粒抗肝衰竭的作用机制之一。     

磷酸化肌球蛋白轻链在大鼠慢性移植肾肾病早期病变中的作用及其机制

目的 探讨磷酸化肌球蛋白轻链(pMLC)在慢性移植肾肾病(CAN)中的作用与机制.方法 依照标准的CAN大鼠模型进行左肾原位移植,受者为LEW大鼠,供者为F344大鼠,另取雄性F344大鼠和LEW大鼠仅行单肾切除术,分别作为对照.分别于术后4、8及12周时,收集各组大鼠的24 h尿量,并检测其尿肌酐水平,计算肌酐清除率.然后取各组大鼠血液标本,测定血清肌酐水平.采用Banff分级标准评定各组肾小球肾病、肾小管萎缩、间质纤维化及血管内膜增厚程度.采用免疫组织化学法和采用蛋白质印迹法检测肾组织中磷酸化MLC(pMLC)和整合素连接激酶(ILK)的表达部位及表达水平.结果 移植组大鼠术后4周时肾间质可见单个核细胞浸润,12周时可见血管平滑肌细胞的移行与增殖.移植组大鼠各时相肾组织pMLC和ILK表达水平显著高于Lewis对照组及F344对照组,且随着移植时间的延长有逐渐增高趋势.大鼠移植肾组织中pMLC表达水平与24 h尿蛋白定量、血清肌酐水平、肾间质单个核细胞浸润、肾小动脉血管平滑肌细胞数量、Banff评分等呈显著正相关,相关系数(r)分别为0.273(P＜0.05)、0.434(P＜0.01)、0.525(P＜0.01)、0.676(P＜0.01)、0.570(P＜0.01),在移植后4周时,pMLC表达水平与肾小管间质ILK表达水平呈显著正相关,r=0.778(P＜0.01).结论 pMLC在慢性移植肾病早期病理变化中发挥重要作用,移植肾肾小管间质及肾小动脉中pMLC表达上调与ILK的作用机制相关.

Abstract：

Objective To investigate the role and mechanism of phosphate myosin light chain (pMLC) in the rat kidney of chronic allograft nephropathy (CAN) model. Methods The left donor kidneys from Fisher (F344) rats were orthotopically transplanted into Lewis recipients. Meanwhile, the F344 rats and LEW rats with resection of the right kidney served as control groups. Animals were harvested respectively at the 4th, 8th and 12th week after transplantation. The creatinine clearance rate (CCr) was calculated by urine creatinine of 24-h urine. Blood samples were collected from rats for determination of serum creatinine. The expression of pMLC was detected by using Western blotting and immunohistochernistry, and that of integrin-linked kinase (ILK) by using immunohistochemistry. Results Mononuclear cells infiltration of allografts was markedly aggravated as compared to the controls. Allografts got severe interstitial fibrosis and tubular atrophy at 12th week after transplantation. The expression of pMILC and ILK was up-regulated in the kidney of CAN rats after transplantation, and increased more significantly as the time went on. The expression of pMILC was significantly correlated with 24-h urine protein excretion (r= 0. 273, P＜0. 05), serum creatinine levels (r = 0. 434, P＜0. 01 ), the number of tubulointerstitial infiltrated mononuclear cells (r = 0. 525, P＜0. 01 ), the number of smooth muscle cells (SMC) in vascular wall (r= 0. 676, P＜0. 01 ) and the extent of interstitial fibrosis (r= 0. 570, P＜0. 01 ).There was a significantly positive correlation between ILK and pMLC in CAN rats at the 4th week after transplantation (r= 0. 778, P＜0. 01 ). Conclusion pMLC might play an key role in CAN, and the over-expression of ILK might be involve in the pathogenesis of CAN.     

温阳化饮益气活血法对慢性心功能不全大鼠心肌α-MHC、β-MHC基因表达的影响

目的：探讨温阳化饮益气活血法对慢性心功能不全大鼠心肌α-MHC、β-MHC基因表达的影响。方法：采用腹腔注射阿霉素法复制慢性心功能不全大鼠动物模型，利用RT—PCR技术检测心肌重构的相关调控因素α-MHC、β-MHC在慢性心功能不全发病中的作用，同时观察温阳化饮益气活血法对其干预作用。结果：慢性心功能不全大鼠模型组心肌组织α-MHC基因表达下调，β-MHC基因表达上调；温阳化饮益气活血法治疗组心肌组织α-MHC基因表达上调，β-MHC基因的表达下调。结论：温阳化饮益气活血法能上调慢性心功能不全大鼠α-MHC基因的表达，下调β-MHC基因的表达，为温阳化饮益气活血法的临床应用提供了实验依据，这可能是温阳化饮益气活血法防治慢性心功能不全的作用机制之一。     

慢性低频复合生理频率电刺激对肺气肿兔膈肌肌球蛋白重链及酶活性的影响

目的 研究不同频率慢性电刺激(chronic electrical stimulation,CES)后肺气肿兔膈肌肌球蛋白重链(MHC)亚型及酶活性的适应性变化,以了解间断低频复合生理频率电刺激对肺气肿兔膈肌结构及代谢的影响.方法 采用木瓜蛋白酶雾化吸入法建立肺气肿模型,测定正常对照组、肺气肿组和肺气肿CES组膈肌MHC亚型的适应性变化及肌浆网(SR)Ca2 -ATP酶、膈肌琥珀酸脱氢酶(SDH)、乳酸脱氢酶(LDH)的酶活性.结果 ①40、10、(10 40) Hz慢性电刺激后肺气肿兔膈肌MHC的相对含量都有所增加(P＜0.05).10 Hz组MHC-Ⅰ型比例明显增加,(10 40) Hz组MHC-Ⅱa型比例增加(P＜0.05).②(10 40)、40 Hz慢性电刺激后,膈肌SRCa2 -ATP酶活性增高(P＜0.05).40 Hz较(10 40) Hz Ca2 -ATP酶活性增高更明显(P＜0.05),10 Hz组Ca2 -ATP酶活性降低(P＜0.05).③间断低频复合生理频率电刺激(40 10 Hz)同肺气肿组比较,SDH酶活性明显升高(P＜0.05);与40 Hz组和10 Hz组比较,SDH亦明显升高(P＜0.05),40 Hz组和10 Hz组之间无显著性差异(P＞0.05).5组之间LDH酶活性均无差别(P＞0.05).结论 不同频率的CES可导致膈肌MHC亚型及酶活性产生不同的适应性变化,低频复合生理频率慢性电刺激可能是体外膈肌起搏对COPD患者膈肌康复治疗较好的频率模式.     

大鼠心脏非心肌细胞对心肌细胞收缩功能的影响及其与血管紧张素的关系

目的研究大鼠心脏非心肌细胞对心肌细胞α-肌球蛋白重链(α-MHC)及β-肌球蛋白重链(β-MHC)mRNA表达的影响及与血管紧张素受体的关系。方法分离、纯化、培养出生1~3 d的SD乳鼠心肌细胞(MC),同时制备非心肌细胞条件培养液(NMCM)。培养细胞的分组及药物干预:M组:MC+新鲜培养液;C组:MC+70%新鲜培养液+30%NMCM;L组:MC+新鲜培养液+AT1受体阻断剂〔氯沙坦(Losartan,10-6mol/L);〕CL组:MC+70%新鲜培养液+30%NMCM+Losartan;P组:MC+新鲜培养液+AT2受体阻断剂(PD123319,10-6mol/L);CP组:MC+70%新鲜培养液+30%NMCM+PD123319;LP组:MC+新鲜培养液+Losartan+PD123319;CLP组:MC+70%新鲜培养液+30%NMCM+Losartan+PD123319)。药物干预后24 h检测心肌细胞α-MHC及β-MHC mRNA的表达情况。结果C组、CP组的α-MHC表达明显低于其他组,差异有统计学意义(P<0.001),其余各组间差异无统计学意义。-βMHC mRNA表达的变化与此相反。结论非心肌细胞培养液可使心肌细胞MHC mRNA的表达从α亚型向β亚型转移。这种作用可能是非心肌细胞产生的AngⅡ通过AT1受体实现的。     

寄生蠕虫副肌球蛋白研究进展

副肌球蛋白（98-101kDa）是大多数无脊椎动物肌肉的主要结构蛋白，位于肌肉组织的粗肌丝中。大量研究表明人和家畜体内的众多寄生蠕虫都有副肌球蛋白的存在。对大多数长度而言，副肌球蛋白形成具重复疏水和亲水氨基酸序列的α螺旋盘绕结构，这使其对其它的蛋白分子有很高的亲和力。用曼氏血吸虫成虫组织匀浆免疫的小鼠能产生强烈的免疫应答，包括生成与97kDa成分结合的抗体，后经证实该97kDa成分为副肌球蛋白，由此可明显看出，副肌球蛋白的功能不止是作为寄生蠕虫的肌原纤维。     

间歇性外斜视病人与正常人眼外肌肌酶组织化学染色的比较(摘要)

目的研究间歇性外斜视病人与正常人眼外肌的肌酶组织化学染色的结果,探讨间歇性外斜视的可能发病机制·方法分别取间歇性外斜视病人和正常人近附着点处的内直肌进行HE染色和mATPase组织化学染色,光镜下观察并定量比较慢肌和快肌纤维的比例和分布·结果间歇性外斜视病人和正常人的慢肌纤维和快肌纤维的比例无差异·结论(1)间歇性外斜视病人内直肌的慢肌和快肌纤维比例的改变不是间歇性外斜视的可能病因·(2)人眼外肌肌球蛋白ATP酶染色酸性前孵育液的最适pH值是4·3~4·4,碱性前孵育似乎无法区分两型肌纤维(3)人眼外肌内直肌慢肌纤维所占…     

肌球蛋白ATP酶组织化学方法的改进

本文参照Guth介绍的酸碱预孵育肌球蛋白ATP酶法并加以改进，使之避免了肌纤维易断裂，组织块内产生冰晶，缓冲液种类过多等现象，结果表明，改进后的方法稳定可靠，在切片上能清楚显示不同类型的肌纤维。