扩张型心肌病与血管紧张素转换酶基因、chymase基因多态性的研究

目的 :探讨血管紧张素转换酶 (ACE)基因插入 /缺失 (I/D)多态性及chymase(CMA)基因B种A/G多态性是否是扩张型心肌病 (DCM )的易感性标志。 方法 :采用聚合酶链反应 (PCR)和限制性片段长度多态性方法 (RFLP)检测 4 3例DCM患者和 6 3例健康者中ACE基因I/D多态性和CMA/B基因A/G多态性的频率分布 ,同时超声心动图测量左心室内径 (LVDd、LVDs)大小 ,计算射血分数 (EF)值 ,测量心胸比、血压和心率等临床指标。结果 :①ACE基因II、ID、DD基因型及I、D等位基因频率在DCM组与对照组的分布差异无显著性意义。②CMA基因AA、AG、GG基因型及A、G等位基因频率在DCM组与对照组的分布差异无显著性意义。③ACE、CMA基因型间年龄、患病史、NYHA心功能分级、LVDd、LVDs、EF值、心胸比、心率、收缩压、舒张压差异无显著性意义。④ACE、CMA基因型与LVDd、LVDs、EF值不相关。结论 :ACE基因I/D多态性及CMA/B基因A/G多态性与DCM患者心脏大小及收缩功能不相关。     

Mast cell chymase is increased in chronic atopic dermatitis but not in psoriasis

Mast cell chymase is a chymotrypsin-like serine proteinase primarily stored in secretory mast cell granules. Mast cell chymase has various effects on angiotensin, metalloproteases, lipoproteins, procollagen, neuropeptides and cytokines. Recent studies have demonstrated that chymase inhibitors inhibit skin inflammation. In this study we sought to determine the role of mast cell chymase in atopic dermatitis (AD) in comparison with its role in psoriasis and normal skin. Skin biopsy specimens were obtained from non-lesional and lesional skin of patients with chronic AD and psoriasis and from normal skin of non-atopic and non-psoriatic controls. The number of mast cells containing chymase was determined by immunohistochemistry using a chymase-specific monoclonal antibody. A significantly (P<0.05) enhanced number of chymase-positive cells was found in lesional AD skin as compared to normal skin as well as to lesional and non-lesional skin of patients with psoriasis. A significant (P<0.05) increase in the number of chymase-positive cells was also found in non-lesional AD skin in comparison to psoriasis. An enhanced, albeit not statistically significant difference was noted in non-lesional AD skin as compared to normal skin. In conclusion, these results suggest that mast cell chymase may play an integral part in eliciting and maintaining cutaneous inflammation in AD but not in psoriasis. The increased proteinase activity of mast cell chymase may also be involved in promoting a skin barrier defect in AD, which subsequently enhances the skins permeability to allergens and microbes and thereby aggravates the eczema.     

Chymase-positive mast cells in small sized adenocarcinoma of the lung

Mast cells accumulate in angiogenesis-dependent situations of lung adenocarcinoma. Human mast cells are divided into two major subsets: MC_T (mast cells with immunoreactivity for tryptase but not chymase) and MC_TC (reactive for tryptase and chymase). Chymase is an important mediator of tissue remodeling, but research into chymase-containing mast cell subpopulations has been hampered by the lack of reagents suitable for use with formalin-fixed tissue. We stained chymase using CC1 antibody in 66 cases of small sized lung adenocarcinoma as well as CD34 and tryptase. There were significant positive correlations of microvessel counts with MC_T-type and MC_TC-type mast cell counts in lung adenocarcinomas. When analyzed according to Noguchi's classification, MC_T-type and MC_TC-type mast cells were significantly increased in Noguchi type-C tumors [localized bronchioloalveolar carcinoma (LBAC) with active fibroblastic proliferation] compared with in Noguchi type-A (LBAC) plus type-B tumors (LBAC with alveolar collapse). Members in the high-count group of MC_TC-type but not MC_T-type mast cells showed a significantly worse outcome than those in the low-count group in LBACs. Counting chymase-positive (MC_TC-type) mast cells in tumor stroma may be a good prognosis predictor for LBACs, especially Noguchi type-C tumors.     

Prospective single-arm observational study of human chymase inhibitor Polygonum hydropiper L in subjects with hypertension

ABSTRACT

Background and Purpose: Human chymase (h-chymase) is a serine protease that forms local angiotensin II and has been proven to be related to onset of hypertension, arteriosclerosis, and post myocardial infarction cardiac remodeling. Since no chymase inhibitor was clinically available, an extensive screening for inhibition of h-chymase in three different extracts (water, hot water, and ethanol) of approximately 800 food ingredients had been performed and we identified Polygonum hydropiper L (Polygonum). Using a dried and powdered Polygonum, we conducted a prospective, single-arm, pilot study to investigate its safety and antihypertensive effect in subjects with normal high blood pressure to moderate hypertension.

Methods: First, a single oral dose of Polygonum powder (4000 mg) was administered to assess acute toxicity. Then, a pilot study was conducted in 11 subjects using the sequence of placebo and Polygonum for 2 weeks each. The dose of Polygonum was increased sequentially (200–2000 mg/day). Home blood pressure and pulse rate were monitored.

Results: Oral administration of Polygonum (4000 mg) did not cause any adverse events. In the dose-escalation phase, evening systolic blood pressure was significantly decreased at 800 mg, 2000 mg doses post-treatment (p < 0.05, and p < 0.05, respectively). Depressor responders to Polygonum intake had significantly higher salt intake in spot urine (p < 0.05). No adverse events or reactions occurred.

Conclusion: This was the first investigation that an h-chymase inhibitory Polygonum intake for safety and tolerability was proven and, in addition, chymase inhibitory Polygonum appeared to have depressor effect especially in a hypertensive subject with excessive salt intake.     

Chymase在肺动脉高压疾病的肺动脉重构中的表达

目的探讨了慢性香烟暴露诱导的PAH病变过程中仓鼠肺组织chymase的表达变化。方法将12只雄性仓鼠随机分为：对照组、香烟暴露组;仓鼠暴露于香烟烟雾中4个月建立肺动脉高压模型;免疫组织化学染色法检测肺小动脉中chymase的蛋白表达。结果Chymase在香烟暴露组仓鼠的肺小动脉中的表达增加,尤其在肺小动脉外膜及增生的内皮细胞中,染色呈强阳性。结论随着肺结构的改变,香烟暴露可刺激chymase的表达增加,表明chymase在PAH中肺小动脉的重构病变中起作用,因此,抑制chymase的形成将有助于阻止香烟诱导的肺动脉高压疾病的发生、发展。     

Possible involvement of mast cells in renal fibrosis in patients with IgA nephropathy

Objective: The objective of the present study is to investigate whether human mast cells (MC) contribute to renal damage through local activation of the renin-angiotensin system, by assessing their numbers in renal biopsy specimens from patients with IgA nephropathy (IgAN) or minimal change nephrotic syndrome (MCNS). Methods: In patients with IgAN and MCNS, the numbers of tryptase-positive MC (MC(T)) and MC positive for both tryptase and chymase (MC(TC)) were examined histopathologically. Serum creatinine level, mean blood pressure and the severity of glomerular and tubulointerstitial lesions were also determined. Results: MC(TC) numbers differed between IgAN patients and MCNS patients. IgAN patients had more MC(TC) than MC(T). MC were found around but not in the conglomerate of the AngiotensinII (AngII)-positive cells. In the IgAN patients with the most severe pathology, the number of AngII-positive cells was correlated with that of MC(TC) and MC(T). Conclusion: Chymase-dependent AngII synthesis due to human MC may be involved in the inflammatory and fibrotic processes of IgAN. Received 4 September 2006; returned for revision 27 October 2006; returned for final revision 21 December 2006; accepted by M. Katori 30 May 2007     

糜酶介导自发性高血压大鼠心肌纤维化的作用机制

目的:观察糜酶对自发性高血压大鼠(SHR)心肌纤维化的影响及作用机制。方法:采用RT-PCR检测心肌组织Ⅰ、Ⅲ型胶原和糜酶mRNA表达,Western blot检测转化生长因子β1(TGF-β1)和过氧化酶体增殖物激活受体α(PPARα)蛋白表达。结果:SHR组和糜酶抑制剂(CHI)组Ⅰ、Ⅲ型胶原mRNA、TGF-β1蛋白高于对照组,PPARα蛋白低于对照组(P<0.01,P<0.05)。SHR组糜酶mRNA高于对照组(P<0.01)。结论:糜酶参与心肌纤维化,其机制与TGF-β1蛋白上调、PPARα蛋白下调有关。     

糜酶基因多态性与浙江地区汉族人群2型糖尿病肾病的相关性

目的探讨糜酶（CMA）基因多态性与浙江地区汉族2型糖尿病肾病（DN）患者的相关性。方法应用PCR—RFLP技术和遗传学方法,对145例汉族正常人群和130例汉族2型糖尿病患者进行CMA基因CMA／B多态性频率检测;同时根据尿白蛋白排泄率将糖尿病患者分为3组：正常蛋白尿组66例、微量白蛋白尿组40例和临床蛋白尿组24例,并分别检测各组CMA基因CMA／B多态性频率。结果CMA／B基因型频率分布均符合Hardy—Weinberg平衡;在汉族正常人群中,CMA基因AA、AG、GG型频率分别为0159、0497和0345。糖尿病组及其3个分组患者CMA／B基因型、等位基因频率与正常对照组的差异均无统计学意义（χ^2分别为1219、0．560和3．359、1．940,均P〉0.05）,而DN组患者与非DN组上述指标的差异均无统计学意义（χ^2分别为1403和1．062,均P〉0.05）。结论浙江地区汉族人群存在CMA基因CMA／B多态性,而CMA／B多态性可能与汉族人群糖尿病和DN的发病无相关性。     

慢性活动性乙肝患者胶原纤维、糜酶活性及羟脯氨酸含量表达研究

目的:研究慢性活动性乙型肝炎患者胶原纤维、糜酶活性及羟脯氨酸含量表达及相关性.方法:选取100例慢性活动性乙型肝炎患者,行肝穿刺活检染色确定肝纤维化程度分期及炎症活动度分级,以直接测定胶原纤维相对含量,酶联免疫吸附法测定肝组织中糜酶活性,胃酶酸解法测定胶原纤维含量,荧光定量PCR技术测定HBV-DNA含量.结果:100例慢性活动性乙型肝炎患者肝组织胶原纤维含量为(0.13±0.05)mmol/L,靡酶活性为(24.77±15.08)ng/mg,羟脯氨酸含量为(20.84±7.46)μmol/L,HBV-DNA水平为(9.04±1.44)10g10 IU/mL;胶原纤维、靡酶活性及羟脯氨酸含量S1+S2期、S3+S4期高于S0期患者(P＜0.05),S3+S4期高于S1+S2期(P＜0.05);炎症活动度分级G3+G4级患者胶原纤维、靡酶活性及羟脯氨酸含量明显高于G1+G2级(P＜0.05);胶原纤维、靡酶活性及羟脯氨酸含量与S分期、G分级间具有正相关性(P＜0.05),胶原纤维、靡酶活性及羟脯氨酸含量、S分期、G分级与HBV-DNA水平间具有正相关性(P＜0.05).结论:慢性活动性乙型肝炎患者胶原纤维、糜酶活性及羟脯氨酸含量随着纤维化程度分期、炎症活动度分级而逐渐升高,其含量与HBV-DNA水平关系密切.     

Chymase抑制对糖尿病大鼠心肌细胞凋亡的影响

目的探讨Chymase抑制对糖尿病大鼠心肌细胞凋亡的影响机制。方法 24只雄性SD大鼠随机分为正常对照组(NC组,n=7)、糖尿病组(DM组,n=7)、应用糜酶(Chymase)抑制剂的糖尿病组(DM+Chy-I组,n=10)。采用链脲佐菌素(streptozocin,STZ)单次腹腔注射方法建立糖尿病大鼠模型。其中DM+Chy-I组给予糜酶抑制剂[Suc-Val-Pro-Phe P-(OPh)2,1mg/(kg·d)],12周后处死,采用脱氧核糖核苷酸末端转移酶介导的缺口末端标记法(TUNEL)检测心肌细胞的凋亡指数(AI),Western blot法检测心肌caspase-3蛋白的表达,免疫组化检测心肌组织Chymase蛋白表达,采用黄嘌呤氧化酶法(羟胺法)测定心肌组织超氧化物歧化酶(SOD)活力,TBA(thiobarbituric acid)法测定丙二醛(MDA)含量。结果与NC组相比,DM组大鼠心肌细胞AI明显升高;caspase-3蛋白表达明显增加;MDA含量升高,SOD活力降低(P<0.05);与DM组相比,Chymase抑制剂使心肌细胞AI明显降低;caspase-3蛋白表达减少;MDA含量降低,SOD活力升高(P<0.05)。结论 Chymase抑制剂减少了糖尿病心肌病大鼠心肌细胞的凋亡可能是通过抑制caspase-3的表达和氧化应激损伤。