中国心脏外科医师奖(金刀奖)获得者 高秉仁 中国医师协会心血管外科医师奖(金刀奖)获得者高秉仁是新一代我国中青年心血管外科专家,在从事心血管外科工作的领域有着丰富的临床经验和极高的学术造诣。现任兰州大学第二医院心胸外科主任,主任医师,教授,博士后流动站导师,博士研究生导师,兼任中国医师协会心血管外科分会常务委员、中华医学会高原医学分会高原危重病急诊专业委员会常务委员、全国学位与研究生教育评估专家组成员、教材评审委员、甘肃省科学技术奖励评审委员、甘肃省高级职务评审委员会成员,甘肃省医学会和兰州医学会医疗事故鉴定委员会专家、甘肃省《司法鉴定人执业证》核准执业者。《科学中国人》专家委员会“生命科学与医药卫生”专业副主任委员、《中华实用医药杂志》常务编委、《现代心血管外科杂志》编委、《中华现代内科学杂志》常务编委、《中国临床医药实用杂志》特约编委。曾荣获“青年教师成才奖”、 1998年入选甘肃省跨世纪学术技术带头人“333科技人才工程”第一、二层次首批人选,2002年入选甘肃省“555”创新人才工程第一层次首批人选。2009年入选甘肃省领军人才第一层次人选。2009年获得第三届中国医师协会心血管外科医师奖(金刀奖),金刀奖是经中国医师协会批准中国心血管外科医师分会评选的心脏外科医师的最高奖项。高秉仁获第三届中国医师协会心血管外科医师奖“金刀奖” 在心血管外科领域耕耘了27个春秋的高秉仁教授在医、教、研方面均取得了累累硕果。高秉仁教授1982年本科毕业于兰州大学医学院医疗系,后获心胸外科医学硕士学位,一直从事心胸血管外科临床工作,在西北高原经济落后,医疗条件设备贫瘠的条件下,知难而进,艰苦创业,在临床实践工作中,他有着崇高的敬业风尚和吃苦耐劳的奉献精神,与同事们一道,团结一心、勤奋钻研,奋勇拼搏,对待每一位患者,他都像亲人般温暖,认真了解患者的病情,仔细检查,耐心解释,让每一位慕名而来的患者都能够得到满意的答复。在手术台上,精益求精,一丝不苟。为了救治危重患者,他经常吃住在科室里,有时在ICU几夜不能合眼,经常一两周才回家一次。即使身为科主任与教授,在节假日依然坚持查房,了解病人病情,风雨无阻。把“以病人为中心”的理念切实体现到医疗工作中,受到病人和同行一致的赞誉。同时非常注重专业领域科学研究工作,紧跟国际国内最新前沿,在微创心脏外科等多个研究领域均进行长期的连续性深入研究,开创了多项新业务。不论是在担任甘肃省心血管医院副院长还是在兰州大学第二医院心胸外科主任的工作中他都以科学发展观为主导,刻苦钻研,不断的提高医疗技术水平,成为西北高原甘肃省心血管外科专家。他在临床实践中不断注重学科建设,大力扶持心胸外科的亚专业,紧跟学科前沿,在科室管理方面努力达到战略管理层次,不断获得新的竞争优势,提高科室的核心竞争力,使兰大二院心胸外科在两年多的时间里达到了兄弟医院20余年走过的历程和发展速度,使科室获得了跨越式的发展。仅2008年共完成各类手术780例,其中心脏不停跳心内直视手术矫治先天性房间隔缺损和室间隔缺损200余例手术,右腋下微小切口心脏瓣膜置换手术69例,法洛四联症和复杂心脏畸形手术43例。疗效优良,各类手术总体治愈率达98%,取得了很好的社会效益和经济效益。 作为一名心脏外科专家,高秉仁教授博极医源,潜心学习专业知识,不断创新,钻研手术技巧,勇攀新高峰,积累了丰富的临床经验,掌握了高超的手术技能。他还敏锐地学习和采用国外先进的心胸血管外科技术,有力地提升了我国和我省心胸血管外科水平。早在1996年率先在甘肃省开展了微创心脏外科;次年率先在甘肃省进行了腋下微小切口心脏不停跳心内直视手术;率先在甘肃省进行了同种带瓣动脉移植治疗复杂先天性心脏病;率先在甘肃省完成了首例大动脉转位的根治手术。近几年在他的带领下,科室先后开展的瓣膜置换术中房颤的射频消融技术、冠状动脉肌桥切开成形术、复杂先天性心脏病矫治术、主动脉夹层象鼻支架手术及微创介入覆膜支架置入术及冠脉外科手术等新业务新技术18余项,均达到省内领先的地位或国内先进行列水平。部分项目填补了省内和西北地区的空白甚至国内空白。在临床工作中,他承担了科室大部分疑难重症病人的诊断和手术治疗,每年以精湛的技术亲自主刀完成心脏手术约130余例,其中多数为重症或疑难病例,如年龄2个月的主动脉弓-降主动脉广泛狭窄合并室间隔缺损在体外循环中停循环的同期手术治疗和78岁高龄的冠状动脉搭桥术。由他在早年完成甘肃省首例巨大室壁瘤切除同期乳内动脉-冠状动脉搭桥术,近年来此项业务已成为常规手术位于国内先进行列;右腋下小切口先心病修补、主动脉瓣替换、佛氏窦瘤破裂的修补等微创心脏外科已形成了自己的特色。在工作实践中认真总结经验,发表论文70余篇,其中SCI收录论文5篇,国外国际性杂志2篇,国家级和中文核心期刊62篇。参编专著4部,参编16余万字由人民卫生出版社出版的《遗传病学》获全国优秀专著。完成甘肃省级和教育卫生厅级科研基金课题15余项;获科技进步成果奖10余项,其中“微创体外循环下心脏不停跳心内直视手术临床研究”、 “抗钙化同种带瓣膜动脉液氮低温保存及其活性的研究”; “病残儿的病因学调查研究及其防治对策”; “甘肃省五地市先天性心脏病流行病学调查暨先天性心脏病遗传学研究”荣获甘肃省科学技术进步二等奖;全程参与完成的“高原危重病急症现场急救及综合诊治研究”荣获国务院颁发的国家科技进步二等奖。 作为医院心外科的领军人物,把人才梯队的建设放在重要位置。高秉仁教授把培养人才视为己任,言传身教,甘为人梯,以博大的情怀培养年轻技术骨干,结合每名医生的特点,制定了个性化的培养计划和主攻方向,为使大家尽快掌握复杂先心病技术,他常常手把手地教他们提高操作技能,无私的将个人经验与手术技巧传授给每一位医师,快速提高了心外科整体医疗水平。他着眼科室长远发展建设,注重挖掘、培养、贮备人才,先后选派多人次在国内、外研修。目前科室有博士3人,硕士以上学历9人,大家都以他的敬业精神和不懈的努力勇攀高峰的精神为楷模,共同打造一流的团队,使学科已具有一定的规模,并形成了一整套医疗、科研和教学体系。金刀奖是经中国医师协会批准中国心血管外科医师分会评选的心脏外科医师的最高奖项,2009年度共评选出5人。 右二为高秉仁教授 在教学工作中,不断提高教学艺术和教学效果,注重教书育人,因材施教。完成研究生的导师工作,已指导毕业博士研究生4人、硕士研究生17人,研究生毕业后均成为各单位的临床和学术骨干力量。再读博士研究生5人,再读硕士研究生8人。 在构建和谐社会方面也做出了突出贡献。1、民政部的“残疾孤儿手术康复明天计划”遴选心外科优秀专家,甘肃省民政厅选定高秉仁教授,于2006年9月与兰州大学第二医院签订协议,使兰州大学第二医院成为“残疾孤儿康复明天计划”先心病修补手术项目的定点医院。在一年半救助先心病儿童250余名。使先心病孤儿摆脱沉重的经济负担和巨大精神压力。高秉仁教授在该项目总结大会上获得全省先进工作者称号。2、积极响应卫生厅号召,在科室设立了济困病房,面向农村特困家庭和城市低保户,高秉仁教授带领他的团队利用这个平台救治了一大批瓣膜病、冠心病和先心病,为促进社会和谐、营造幸福家庭作出了重要贡献,使党和政府的形象在老百姓心目中更加高大。3、2008年5.12地震后,高秉仁教授积极地奔赴灾区,冒着极大的危险实施救援活动,受到了当地灾区人民和兰州军区的高度赞扬,受到了兰州大学校党委领导高度赞扬。
高秉仁 魏万胜 陈文胜 赵启明杨坤 苟永久 李斌 The Cardiothracic Surgery, The second Hospital of Lanzhou Univercity, Lanzhou 730000Abstract Objective: To carry out epidemiological investigation to children and adolescent with congenital heart diseases (CHD) and research of genetics. Methods: The CHD investigation team examined l15535 children and adolescent of 2~19 years old in 3 cities ( Lanzhou city, Baiyin city , Zhangye city )and in 3 region (Dingxi , Jiuquan, Jinchang ) in up-mid Yellow River drainage basin and Hexi-Corridor area. The rate of general investigation was 96.2%. Results: 660 patients with CHD were filtrated. The total sickening rate is 5.7126‰. The sickening rate of 2~5 years group is higher than the rate of 6~19 years group remarkably. 36 highly attacked families were fished out. The family sickening rate is 142.40‰. The sickening law of relattives of probed is that the closer the kin, the higher the incidence of the disease. Conclusions: The incidence and sickening rate of Hexi-Corridor area is higher than one of Yellow River field. The children with CHD in plateau region become severe pulmonary arteries pressure in preschool age and have high rate of die yonng. The weightiog average heritability of highly attacked families is (148.074 ±7.055 ) % .The inheritance pattern of CHD in highly attacked families is polygenic inheritance with one dominant major gene taking part in.Key words: Epidemiology; Sickening rate; highly attacked families; polygenic inheritance; Dominant major geneAn epidemiological investigation of congenital heart disease had been carried on in up-mid yellow river drainage basin and Hexi-corridor area in northwest tableland from 1994 to 1998, 115535 of 120349 children (96.2%) whose age ranged 2 and 19 years, now reports as follows.1 Materials and methods 1.1 General material There were males 57818 (50.02%) and females 57764 (49.98%) of 115535 children, their ages ranged 2 and 19 years. There were 54080 children came from Lanzhou city, 13959 children came from Silver city, 18755 children came from Jiuquan area, 11419 children came from Zhangye city, 8936 children came from Dingxi area, 8386 children came from Jingchang area.1.2 Methods First carry on training to investigator, to judge the cardiac murmur and sign [l,2]; thus achieved claim of concordance in filling in questionnaire and so on; lead to general mean rate to 96%. Special topic group carry on census according to plan, screen out children withcardiac murmur and suspect of heart disease; then further make special examination, such as ECG cordis x-ray and UCG. According to DC of "Practical cardiology" [l] and "PDC" as well "Guide of CAS" [2] to put Out the diagnosis with aggregate analysis, further confirm clinical diagnosis by radical correction and neoplasty in part cases, with coincidence as 1OO%. Draw a pedigree chart to the high density family.2 Results2.1 Attack rate and constituent of CHD 660 of 115535 cases with CHD had been found out, total attack rate were 5.7126‰; there were males 289 and females 371 of 660 cases. That disposition of entity as follows: there were 348 cases (52.72%) with VSD; 210 cases (31.82%) with ASD; 35 cases (5.30%) with PDA, in 6 of 35 cases with VSD, l of 35 cases with aorta; there were 10 cases (l.52%) with VSD combined with ASD and stenosis of right ventricular outfiow hract; 25 cases (3.79%) with tetralogy of Fallot; 7 cases (l.06%) with part ECD; 4 cases (0.61%) with congenital insufficiency of tricuspid valve; 5 cases (0.76%) with PS; every 3 cases (each 0.46%) with complete atrioventricular anomaly, sinus valsalva tumor breaken, aortic incompetence and arachnodactyly; furthermore every l case (0.15%) with single atrium, aortic valve stenosis, atresia of pulmonary artery, and transposition of conducting arteries.2.2 Attack rates of all age class and areaAccording to different attack and colonia, it is divided into three groups: the children group with ages between 2 and 5 years; primary pupil group with ages between 6 and 12 years; secondary students group with ages 13 and 19 years. Attack rates of all age class and area of Yellow River Valley aod River West Passage are seen the tab l. Tab 1 Attack rates of all age class and area of Yellow River Valley and River West Passage━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ Valley Area Age group(year) Number of people Number of cases Attack rate(‰) Casea rate of area (‰) ──────────────────────────────── 2~5 9280 52 5.6034Yellow Lanzhou city 6~12 22003 118 5.3629 4.863 黄 13~19 22797 93 4.0795River ───────────────────────────── 河 2~5 1490 15 10.0671Valley Baiyin city 6~12 3988 23 5.7673 5.230 流 13~19 8481 35 4.1269 ──────────────────────────── 域 2~5 1282 9 7.0203 Dingxi area 6~12 5354 24 4.4826 4.700 13~19 2300 9 3.9130──────────────────────────────── 2~5 2359 23 9.7499 He Jiuquan area 6~12 6951 64 9.2073 7.251 河 13~19 9445 49 5.1879 Xi ──────────────────────────── 西 2~5 915 19 20.7650 zou Zhangye city 6~12 5025 34 6.7662 7.356 走 13~19 5479 31 5.6580 lang ──────────────────────────── 廊 2~5 1342 14 10.4322 Jinchang area 6~12 3271 26 7.9486 7.393 13~19 3773 22 5.8309 ────────────────────────────────2.3 Attack rates of sex spaceThere were males 289 of 57715 cases with CHD, whose case rate were 5.0074‰; in 371 of 57820 females cases, whose case rate were 6.4165‰. It had no a significant different (x2=0.06, P> 0.05). Family proneness Total 36 high density family had been discovered in this investigation. There were 53 of 589 first and second degree relative in proband, whose case rate of CHD were 89.98‰; in 34 of 146 first degree retative whose case rate were 232.88‰; in 19 of 443 second degree relative whose case rate were 42.89‰ [see. Tab 2]. It is showed that had a very significant differencebetween first and second degree relatives, to make clearly that case of relatives had a close relationship with sibship.2.5 Invasion of CHD and height of elevation Lanzhou city locates on 1516m height above sea level, in 263 of 54080 cases with CHD, whose case rate were 4.863‰; Silver city locates on 1707m height above sea level, in 73 of 13959 cases with CHD, whose case rate were 5.230‰; Dingxi area locates on 1896m height above sea level, in 42 of 8936 cases with CHD, whose case rate were 4.700‰; Jiuquan area locates on 1477-2100m height above sea level, in 136 of 18755 cases with CHD, whose case rate were 7.251‰; Zhangye city locates on 1482-2000m height above sea level, in 84 of 11419 cases with CHD, whose case rate were 7.356‰; Gingchang area locates on 1496-2080m height above sea level, in 62 of 8386 cases with CHD, whose case rate were 7.393‰. Its case rate was higher in Jiuquan, Zhangye and Gingchang area than in Lanzhou and Dingxi area (x2=48.4, P
高秉仁 陈武林 陈文胜 赵启明 魏万胜 张建华 苟永久 杨坤【摘要】 目的 探讨先天性心脏病体外循环心脏停跳与不停跳手术围术期血清 TNF-α、 IL-6和IL-8 的动态变化及对比研究。方法 30例先天性心脏病病人分成两组( n = 15 ), A组在体外循环心脏停跳下行心脏畸形矫正手术, B组在体外循环心脏不停跳下施行心脏畸形矫正手术。每位病人分别在术前、体外循环开始30 min、体外循环结束2 h、术后6 h和24 h中心静脉采血,放免法测定TNF-α、IL-6和IL-8的血浆含量。结果 A、B两组病人术前血浆TNF-α、IL-6和IL-8平均水平无明显差异。体外循环开始30 min、体外循环结束2 h及术后6 h, 均比术前明显升高,并在体外循环结束2 h后达到高峰。术后24 h IL-8与TNF-α基本回落到术前水平,而IL-6仍保持较高水平。A组患者在体外循环结束2 h及术后6 h的 TNF-α、IL-6和IL-8水平显著高于B 组。结论 在心脏外科手术中,体外循环和心肌缺血-再灌注损伤会引起病人血浆致炎因子如TNF-α、IL-6 和IL-8 水平的升高。心脏停跳手术方法将加重这种改变,而心脏不停跳手术方法没有心肌缺血-再灌注的过程,减少了心肌损伤,具有良好的心肌保护作用。【关键词】 先天性心脏病;停跳与不停跳手术;TNF-α;IL-6;IL-8中图分类号:R654.1 文献标识吗: A Comparisons of changes in TNF-α,IL-6 and IL-8 levels in the Serum of patients with or without Cardiac Arrest Gao Bing-ren, Chen Wu-lin,et al.Cardiac Surgery Department, First Hospital Affiliated to Lanzhou University, Gansu, 730000 China 【Abstract】 Objective To compare the changes in TNF-α,IL-6 and IL-8 levels in the serum of patients with CHD before, after and during the operation with or without cardiac arrest. Methods Thirty patients were equally divided into two groups: Group A and Group B. During the operation, Group A experienced cardiac arrest, and Group B did not. Blood samples were collected from the central vein of each patient at five time points: before the operation, 30 mins after the beginning of CPB, 2 hrs after CPB, 6 and 24 hrs after the operation. The levels of TNF-α,IL-6 and IL-8 were measured. Results There was no significant difference between the two groups’ levels of TNF-α,IL-6 and IL-8 before the operation. 30 mins after the beginning of CPB, 2 hrs after CPB and 6 hrs after the operation, the levels of TNF-α,IL-6 and IL-8 increased significantly compared with preoperative level, and reached their peak at 2 hrs after CPB. The average level of TNF-αand IL-8 returned to normal 24 hrs after the operation, while IL-6 level remained high. Group A’s levels of IL-6,IL-8 and TNF-αat 2 hrs after CPB and 6 hrs after the operation were significantly higher than those in group B. Conclusion In heart operations, CPB and ischemia-reperfusion injury could increase the levels of IL-6 ,IL-8 and TNF-αin patients’serum. Operation technique with cardiac arrest aggravated these changes. Since the beating heart operation technique has no cardiac ischemia-reperfusion procedure, it reduces the injury to cardiac muscle and is good for cardiac protection. 【Key words】 CHD;Operation with or without cardiac arrest;TNF-α;IL-6;IL-8体外循环(Cardiopulmonary bypass,CPB)心脏手术能够引起全身性炎性反应, 肿瘤坏死因子、白细胞介素( IL-6、IL-8 等)、补体等在炎症反应过程中扮演了重要角色,它们有协同作用并通过增强血细胞之间相互作用来诱导和维持炎症反应[1],体外循环中的缺血-再灌注可增强炎症反应,加重组织损伤。为此, 笔者对体外循环手术患者围术期TNF-α、IL-6和IL-8进行检测,初步观测和探讨TNF-α、IL-6和IL-8在常规体外循环心脏停跳下和体外循环心脏不停跳下行心脏畸形矫正手术前后的动态变化和对比性研究。1 对象和资料1.1 实验对象 均为兰州大学附一院胸心外科2004年8月~2005年4月普通先天性心脏病病人,其中男17例,女13例,年龄3~28(12.3±7.1)岁。手术种类:室间隔缺损(VSD)14例,房间隔缺损(ASD)16例。 1.2 方法 手术均为择期手术,采用胸部正中切口。30例病人分成两组,体外循环心脏停跳手术15例(A组),体外循环心脏不停跳手术15例(B组)。 CBP降温范围(28±2)℃,时间35.2~128 min,平均69.6 min。主动脉阻断时间18~70.4 min,平均42.3 min。中度血液稀释,平衡液预充,全身肝素化(3mg·kg-1)。患者全部采用气管插管、静脉复合麻醉。术前诊断明确,排除先心病以外的其它疾病。手术过程顺利,术后患者恢复良好,术后1周余出院。1.3 设备 美国产Sarns7400型人工心肺机,美国产North-American容控型呼吸机,西安产87型鼓泡式氧合器,上海产SN-695B型智能放免r-测量仪。1.4 试剂盒 TNF-α、IL-6和IL-8药盒由北京华英生物技术研究所、北京美迪克生物技术有限公司提供。1.5 样品的收集和处理 在下列时间点1.全麻插管前;2. CPB开始后30 min;3. CPB结束后2 h; 4.术后6 h; 5.术后24 h计5次采集血标本。每次采静脉血2 ml,注入含有10%乙二胺四乙酸二钠30μl和抑肽酶40μl的聚丙烯试管内混匀,在4℃、3000 r/min离心15 min,分离血浆后于-20℃保存待测。1.6 测定方法 采用放射免疫分析法(RIA),在兰州大学附一院核医学科放免分析室进行。测定前,将样本置于室温中复融,混匀,再次4℃、3000 r/min离心10 min,取上清测定。1.7 统计学处理 测定结果以均值±标准差( )表示,用SPSS 12.0软件包,两组间比较用t检验,组内比较用方差分析进行数据的统计学处理,P <0.05为有显著性差异。 2 结 果2. 1 两组患者年龄、体重、体外循环时间均无显著性差异。2. 2 细胞因子 全麻插管前两组病人血浆TNF-α、IL-6和IL-8含量无显著性差异。在CPB开始30 min、CPB结束后2 h和术后6 h, 两组患者的TNF-α、IL-6和IL-8血浆水平均比术前明显升高(P <0.05),CPB结束后2 h达到高峰, 然后逐渐下降。两组患者的TNF-α和IL-8水平在手术后24 h后基本恢复正常,而IL-6术后24 h仍保持较高的水平。A组患者的TNF-α、IL-6和IL-8水平在体外循环结束后2 h和术后6 h都比B组的显著增高(P <0.05)。表 1 两组病人不同时间血浆TNF-α的变化(,ng/ml)组别T1 T2 T3T4T5停跳组 0.58±0.06 0.77±0.04** 1.07±0.08**▲▲ 1.01±0.05**▲▲ 0.68±0.07不停跳组 0.60±0.05 0.78±0.07* 0.84±0.06** 0.80±0.05* 0.65±0.07注: 与术前比较, *P < 0.05 , **P < 0.01 ;停跳组与不停跳组比较,▲▲P < 0.01表 2 两组病人不同时间血浆IL-6的变化(, pg/ml)组别 T1 T2 T3 T4 T5停跳组 91.87±8.18 116.86±8.30*1 37.02±5.27**▲ 130.88±5.15**106.30±7.23 不停跳组 87.94±12.10 113.46±7.68* 117.17±7.27* 115.44±7.06* 102.60±8.63注: 与术前比较,*P <0.05 , **P < 0.01;停跳组与不停跳组比较,▲P < 0.05表 3 两组病人不同时间血浆IL-8的变化(, ng/ml)组别 T1 T2 T3 T4 T5停跳组 0.59±0.05 0.75±0.04** 1.00±0.06**▲ 0.93±0.05**▲ 0.52±0.04不停跳组 0.51±0.04 0.73±0.13* 0.78±0.06* 0.73±0.05 0.47±0.04注: 与术前比较, *P <0.05 , **P <0.01;停跳组与不停跳组比较,▲P < 0.053 讨 论体外循环应用于心脏外科已经有50余年的历史了,它可以诱发全身炎症反应,导致包括心脏在内的全身各脏器功能障碍,甚至引起多器官功能衰竭,成为术后并发症和死亡率增高的主要原因。细胞因子是一类具有复杂生物学活性,且在机体免疫调节和炎症反应中起着重要作用的效应物质,与体外循环引起的全身炎症反应密切相关,目前检测到的有TNF-α、IL-6和IL-8等。这些情况在需心脏停跳的体外循环手术患者中表现得更为严重,这可能与心肌缺血-再灌注损伤有关[2]。TNF-α是一种重要的炎性细胞因子, CPB期间TNF-α血浆浓度的升高比其他细胞因子更快,在炎症反应中有启动、触发作用,可诱发其他炎性介质的释放。过量的TNF-α会导致心脏的损伤,引发低心排血量和低血压, TNF-α还能够刺激内皮细胞分泌IL-8,并促进中性粒细胞、巨噬细胞在损伤部位聚集[3]。血浆TNF-α在主动脉(或上下腔静脉)阻断后30 min 即明显升高,体外循环结束后2 h达到高峰,以后逐渐降低,术后24 h基本降到术前水平。我们推测早期血浆TNF-α的迅速升高是由于CPB引起的全身炎症反应和心肌急性缺血后由巨噬细胞、单核细胞和肥大细胞等大量释放造成的,而以后的升高(停跳组)是由于缺血-再灌注引起血管内皮细胞的损伤、激活中性粒细胞、心肌细胞死亡等所致。A,B两组相比,体外循环结束后2 h和术后6 h不停跳组血浆TNF-α含量增高低于停跳组( P < 0.05 )。因此,不停跳组具有良好的心肌保护作用可能是因为减少了损害性细胞因子如TNF-α的产生。IL-6 是一种主要的急性相反应促进因子,活化的单核细胞是血液中IL-6的主要来源,淋巴细胞、内皮细胞等都能在不同条件下产生IL-6,术后的炎症反应和组织损伤都与急性相反应有关[4]。血清IL-8水平与缺血-再灌注后细胞的实质性损伤紧密相关,是一种主要的中性粒细胞趋化因子,TNF-α能够刺激内皮细胞分泌IL-8并促进中性粒细胞、巨噬细胞在损伤部位聚集,激活的吞噬细胞能直接分泌IL-8,血清中增加的IL-8能进一步加重组织损伤。IL-8还能诱导多种白细胞趋化运动,激活中性粒细胞促进细胞脱颗粒,破坏内皮细胞基底膜的完整性和促进中性粒细胞跨内皮细胞游走[5]。笔者的研究显示,体外循环术后患者的IL-6和IL-8都不同程度的升高,特别是A组(停跳组)患者的IL-6和IL高程度更为显著,这种差异提示二者可能与心肌缺血-再灌注损伤有紧密的内在关系。研究显示CPB心脏手术后,血浆TNF-α升高常可引发低心排血量和低血压, 血浆IL-6浓度的升高可反映组织损伤的程度,与心肌损害具有很好的相关性,IL-6水平的异常增高对心脏功能也有抑制作用。 IL-8对中性粒细胞既具有活化作用又有选择性趋化作用,是典型的炎性介质,血浆IL-8水平升高与心血管系统功能恶化呈明显相关性,而且与CPB术后心脏功能不全有明显关系[6],这些情况在心脏停跳手术的病人表现得更为突出。而在体外循环心脏不停跳下手术的病人其心肌有持续的血液供应,没有缺血-再灌注损伤的过程,减少了心肌损害,减轻了术后全身炎症反应,有利于病人早日康复,缩短住院时间,减少花费。4 参考文献:[1] Ide H,Kakiuchi T, Furual N, et al. The effect of cardiopulmonary bypass on T cells and their subpopulation [ J ].Ann Thorac Surg, 1987, 44: 277-282.[2] 龙村. 体外循环学 [M]. 北京: 人民军医出版社,2004,193-197.[3] Levine B, Kalman J, Mayer L, et al. Elevated circulation levels of tumor necrosis factor in congestive heart failure [ J ]. NEng1 MED, 1990, 332: 236-241. [4] Kawamura T, Wakusawa R, Okada K. Evaluation of cytokines during open heart surgery with cardiopulmonary bypass: participation of IL-8 and IL reperfusion injury [ J ].Can J A naesth, 1993, 56(Suupl): 92-96.[5] 孙卫民,王慧琴. 细胞因子研究方法学[M].北京:人民卫生出版社,1999, 443- 453, 466- 485.[6] Joren PG, De Jongh R, DeBacker W, et al . Interleukin-8 production in patients undergoing cardiopulmonary bypass [ J ]. Am.Respir Dis, 1993, 148(4): 890-895.
高秉仁 李永顺 魏万胜 赵启明 陈文胜 李斌 苟永久 杨坤Keywords:transplantation;myocardial infarction;cell;regenerateBackground Most cardiac regenerative approaches restore injured heart muscle.In this study,we investigate if fibrin sealent can help neonatal cardiomyocytes restore myocardium infarction in rat.Methods The left anterior descending artery in adult female Sprague-Dawley (SD) rats was ligated to make myocardial infarct model.Neonatal ventricular cardiomyocytes from one day SD rats of both sexes were isolated, labeled and cultured.The cell were injected in the infarcted area three weeks later.The animals were randomized into four recipient groups:(1)received cardiomyocytes plus fibrin sealant(group CF,n=10), (2)received cardiomyocytes alone (group C,n=10), (3)fibrin sealant recipients alone (group F,n=10), (4)control group (n=10).Four weeks after transplantation, echocardiography and Langerdoff model were used to assess heart function. Immunohistochemical staining and polymerase chain reaction (PCR) were performed to track the implanted cardiomyocytes and detect the sex-determining region Y gene on Y chromosome. Results Echocardiography showed that the fraction shortening (FS) in each groups was as follows: group CF,27.80%±6.32%;group C, 22.29 %±4.54 %;group F,19.24% ±6.29%;control group, 20.36%±3.29%. The FS of group CF was significant difference compared with other groups ,respectively P=0.028,0.001,0.005. Langendoff model revealed significant differences of left ventricular development of higher pressures(LVDPmax,mmHg) among four groups.The LVDPmax were group CF, 104.81±17.05; group C, 80.97±21.60; group F, 72.07±26.17; control group, 71.42±17.55. The LVDPmax of group CF was significant difference compared with other groups ,respectively P=0.015,0.001,0.001. Pathological examination and PCR indicated that ransplanted cardiomyocytes survived better in group of cardiomyocytes plus fibrin sealant.Conclusion In a rat model, transplanted neonatal cardiomyocytes plus fibrin sealant can survive in myocardial infarct area and improve heart function more.The past 20 years have seen tremendous progress in the medical treatment of cardiac diseases,a progress that actually translated into decreased cardiovascular morbidity and mortality in clinical practice.1 Yet evidence accumulates that interventions intended to slow down that progression of chronic heart diseases have approached their limit.This may be one of reasons why the recent progress in stem cell biology has stirred so much attention.Human embryonic stem cell lines nowadays can be generated from human blastocysts at a high success rate and have generated human cardiac myocytes.2-4 Thus despite many open questions and some controversy in the field5-6. But the most straightforward strategy is to infuse or inject these cells into the injured heart and to develop methods to increase the fraction of cells that home and survive in the heart and differentiate to the desired functional elements.This strategy has already been clinically evaluated,but gave mixed resulted,7-10 potentially also because the homing rate after infusion and the survival rate after intramyocardial injections are very low.11 Given the creativity and technical expertise of invasive cardiology,significant progress in the efficacy of such delivery technique is to be expected.Several groups have demonstrated effectiveness in improving cardiac function though the injection of the cells in saline,cell culture medium,or bovine serum albumin into ischemic myocardium.12 However,the cells are not delivered derectly into the infarct. Another study demonstrated that fibrin glue was capable of preserving infarct wall thickness and cardiac function after myocardial infarction(MI) in rats and may be useful as a biomaterial scaffold for myocardial cell transplantation.13In the study we examined the neonatal cardiomyocytes plus fibrin sealant,as an injectable biopolymer, whether it could improve cardiac function after MI. METHODSAnimalsSixty adult female Sprague-Dawley (SD) rats weighing (210±10)g and neonatal SD rats (both genders) purchased from the Experimental Animal Center of Lanzhou University of China (Lanzhou,Certificate No.SCXK 2002-0010).All experimental procedures were performed according to the "Regulation to the Care and Use of Experimental Animals" (1996) of the Lanzhou Council on Animal Care.Rat model of myocardial infarctionFemale SD rats were anesthetized with 5% isoflurane by an airtight mask. The animal was intubated and ventilated at a rate of 60 cycles/min with a tidal volume of 2.5 mL under room air supplemented with oxygen (0.2 L/min) and 1% to 2.5% Isoflurane.Lead I of the ECG was monitored throughout the experiment.The chest was opened under sterile technique by left thoracotomy through the fourth intercostals space.The pericardium was removed.A single stitch of 6-0 prolene suture was placed through the myocardium at a depth slightly greater than the perceived level of the left anterior decending (LAD) portion of the left coronary artery while taking care not to enter the ventricular chamber.The suture was tightened to occlude the LAD for 30 minutes and then removed to allow for reperfusion.Myocardial ischemia was confirmed by typical ST-segment changes on the ECG, regional myocardial color changes and ventricular arrhythmias.After reperfusion air was expelled from the chest and the incision was closed.The rats recovered from anesthesia and were returned to their cages. Cell isolation and cultureVentricular cardiomyocytes of one day old SD neonatal rats (both sexes) were isolated and cultured according to the following described procedure. After the atria and great vessels were removed from the hearts, the ventricles were minced into small fragments (1 mm3). The minced tissue were incubated in digestion buffer (0.08% trypsin) and digested for 8-10 circles (8 minutes per circle). To reduce non-myocyte contamination, cells were preplated in cell culture flasks (Corning Life Science, NY, USA) for 30 minutes at 37℃. After preplating, to facillitate the identification of the transplanted cells after their implantation, the nonattached cells were resuspended in working dyeing solution with 2 g/ml of Chloromethylbenzamido (CellTrackerTM CM-DiI, C-7001, Molecular Probe, OR, USA) in phosphate buffer solution at 37℃ for 5 minutes and at 4℃ for 15 minutes. After labeling, the cells were plated in 150-mm culture flasks (Corning Life Science, NY, USA) and cultured in the presence of 5% CO2 and 95% air at 37℃ for 1 to 2 days. Fibrin sealantFibrin sealant (BD Biosciences,Bedford,MA.USA)was used in this study.This two component system remains liquid for several seconds before solidifying into a semirigid gel matrix.The first component consists of concentrated fibrinogen and aprotinin,a fibrinolysis inhibitor,whereas the second is a mixture of thrombin and CaCl2.It is delivered through the supplied Duploject applicator,which holds the two components in separate syringes and provides simultaneous mixing and delivery.The ratio of fibrinogen to thrombin components was 1:1.InjectionsThree weeks after myocardial infarction, animals were screened by echocardiograph for heart function as estimated by fraction shortening (FS) at the mid-papillary level of left ventricle(LV) in Short-axis 2-dimensional images. Animals with FS less than 25% were randomized into 4 groups and either 4×106 Ventricular cardiomyocytes in 100 uL of fibrin sealant, 4×106 Ventricular cardiomyocytes in 100 uL of 0.5% bovine serum albumin (BSA), 100 uL of fibrin sealant, or 0.5% BSA in 100 uL of phosphatebuffered saline (PBS) (control group) was injected into the ischemic LV.The first group was neonatal ventricular cardiomyocytes plus fibrin sealant injection (group CF,n=10). 4×106 neonatal ventricular cardiomyocytes were suspended in 50 uL of the thrombin component of the fibrin sealant. The thrombin–cell mixture was simultaneously injected into the myocardium with 50 uL of the fibrinogen component. The second group was cardiomyocytes injection alone (group C,n=10). .5×106 cardiomyocytes were suspended in 100 uL of 0.5% BSA and injected into the myocardium.The third group was fibrin sealant injection alone (group F,n=10).Thrombin (50 uL) and 50 uL of fibrinogen were simultaneously injected into ischemic myocardium .The fourth group was control group (n=10). 0.5% BSA in 100 uL of phosphatebuffered saline (PBS) was injected into the ischemic LV. Using a median sternotomy, one injection was made through a 29-guage needle into the area under epicardium of left ventricular scar. Approximately 100 μl liquid was injected per heart for 0ne minute injection time. Once injection was completed,the puncture holes were closed by sutures,which served as a marker for the area of transplantation at follow-up thoracotomy.The incision was closed by suturing sternum, muscle and skin continuously. Analgesics were given by oral injection of tramadol for three days after surgery. Cyclosporin A (Sandimmun, Novartis Pharma AG, Switzerland) was administed orally in both groups by injection in the mouth daily,starting on the day of transplantation at a dose of 50 mg/kg.EchocardiographyTransthoracic echocardiography was performed on all animals 3 weeks after myocardial infarction (baseline echocardiogram). A follow-up echocardiogram was performed 4 weeks after control or treatment injection. Rats were anesthetized as described aboved and transthoracic echocardiography was performed on all rats. The left parasternal images were taken in the right lateral decubitus position using Vivid 7 (GE Vingmed Ultrasound, Horten, Norway) with a 10-MHz multiphase array probe. After adequate Short-axis two-dimensional images at the mid-papillary level of left ventricle were obtained,the M-mode cursor was positioned perpendicular to the ventricular anteroseptal wall (at the site of infarct) and the posterior wall. Wall thickness and left ventricular internal dimensions were measured according to the leading edge method of the American Society of Echocardiography. Fractional shortening (FS) as a measure of systolic function was calculated as FS (%) =[(LVIDd - LVIDs) / LVIDd] ×100, where LVID was the left ventricular internal dimension, d was diastole, and s was systole. Two echocardiographer blinded to the experimental treatment acquired the images and performed the data analysis.Isolated Langendorff heart perfusionOne to three days after echocardiography performing, global heart function was evaluated using a non-working constant pressure Langendorff preparation. The rats were anesthetized, and sodium heparin (100 U) was administered intravenously. The heart was quickly isolated and perfused in a Langendorff apparatus with filtered Krebs-Henseleit buffer (mM: NaCl, 118; KCl, 4.7; KH2PO4, 1.2; CaCl2, 2.5; MgSO4, 1.2; NaHCO3, 25; and glucose, 11; pH 7.4) at a pressure of 65 mmHg equilibrated with 5% carbon dioxide and 95% oxygen. A latex balloon was passed into the left ventricle through the mitral valve and connected to pressure transducer (model ML110, AD Instruments, Australia) and differentiator amplifier (SP8520, Powerlab/8SP, AD Instruments Pty Ltd, Castle Hill, Australia). After 20 minutes of stabilization, the balloon size was increased by 0.02 mL increments from 0.04 mL by the stepwise addition of saline. LV peak systolic and end-diastolic pressures were measured at ventricular volumes from 0.04 mL and in 0.02-mL increments until the end-diastolic pressure was over 30 mm Hg. Developed pressure was calculated as the difference between the peak systolic and end-diastolic pressures at each ventricular volume. Passive diastolic pressures were recorded at each balloon volume in 0.04-mL increments until the diastolic pressure was over 30 mm Hg.Hearts were then arrested with 10 mL of KCl solution (20 mmol) for histological studies. Histolog and immunohistochemistryThe site of injected area was identified and dissected. Four to five specimens were used, and each of this was cut into 2 pieces. The first one was fixed in 10% phosphate-buffered formalin , embedded in paraffin, and sectioned to yield 10-m-thick slices. The second piece of each specimen was frozen in liquid nitrogen for 15 seconds and then transferred to -70℃ refrigerator to be stored until it was processed on a cryostat. Paraffin sections were stained with hematoxylin and eosin (H&E). Five-m-thick cryostat sections were used for immunohistochemistry. Primary antibodies included mouse Monoclonal Anti-α-Actinin (Sarcomeric) FITC-conjugated secondary antibodies were used against the primary antibodies. Stained tissue was examined with an inverted fluorescent microscope (IX70, Olympus Cooperation, Japan). H&E stains were used to confirm ensuing scarring and injection of cells. α-actinin accompanying CM-DiI was used to show implanted cells,Polymerase chain reaction for identifying Y chromosomeNeonatal cardiomyocytes from rats of both genders were implanted into female rats. The polymerase chain reaction (PCR) amplification was used to identify the Y chromosome to detect the surviving male cardiomyocytes. Left ventricular tissue from the injection site was isolated and refrigerated in liquid nitrogen. Genomic DNA for PCR template was extracted. Genomic DNA templates (100 ng) from different groups were used in PCR reactions (50 l) with rat SRY primers according to the protocols published by Muller-Ehmsen et al14. Forward primer sequence is as follows: 5’AGTGTTCAGCCCTACAGCCTGAGGAC—, and reverse sequence: —GCTGCAATGGGACAACAACCTACACAC 3’. PCR assays were performed in triplicate. Control reactions were performed using the reduced nicotinamide adenine dinucleotide phosphate primers and the same templates, PCR products were separated by electrophoresis on a 2% agarose gel with 1x tris-aminomethane tis-acetate-ethylenediaminetetraacetic acid buffer, stained with EtBr, and photographed in ultraviolet light.2.10. StatisticsAll data were expressed as the means±standard deviation. The software of SPSS 11.5 for windows (SPSS Inc., IL, USA) was used for analysis. Comparisons of continuous variables among the 4 groups were performed by a one-way analysis of variance (ANOVA). The LSD method was used to specify differences among groups. Probability values less than 0.05 were considered statistically significant.RESULTSThirteen of sixty rats died during ligation of left coronary artery. Three weeks after myocardial infarction, seven of fourty seven rats were excluded because the FS were more than 25%. The remaining fourty rats were randomized into 4 groups as described above. Echocardiographic evaluation of cardiac functionThe FS in each group was as follows (means±standard deviation): group CF, 27.80%±6.32%; group C, 22.29 %±4.54%; group F, 19.24%±6.29%; control group, 20.36 %±3.29%. The FS of group CF was significant difference compared with other groups ,respectively P=0.028,0.001,0.005( post hoc LSD test). The group treated by neonatal cardiomyocytes plus fibrin sealant showed significantly higher FS compared with other groups (Figure 1).Langendorff perfusion assessment of cardiac functionCardiac function assessed by Langendorff perfusion revealed significant differences among four groups when left ventricular balloon volume was at the level of 0.10mL. The left ventricular high developed pressure(LVDPmax, mmHg) in each group was as follows (means±standard deviation): group CF, 104.81±17.05; group C, 80.97±21.60; group F, 72.07±26.17; control group, 71.42±17.55. The LVDPmax of group CF was significant difference compared with other groups ,respectively P=0.015,0.001,0.001( post hoc LSD test). The group treated by neonatal cardiomyocytes plus fibrin sealant showed significantly higher LVDPmax compared with other groups (Figure 2).Histological studiesThe left coronary artery ligation led to a transmural infarction in the anterior wall of all examined rats. The anterior wall in group CF is the thickest among all groups (Figures 3). Results of immunohistochemical staining for α-actinin was showed in Figure 4. CM-DiI was used to trace implanted surviving cells. Survival of transplanted cells by PCR analysisLeft ventricular tissue from three rats in each group was used for detecting the SRY gene by PCR. All tissues from animals of group CF and group C, who received neonatal heart cells, were positive, confirming the presence and survival of transplanted cells, and all tissues from group F and control group which did not receive neonatal heart cells were negative (Figure 5). DISCUSSIONThe results of heart function assessment indicated transplantation of neonatal cardiomyocytes plus fibrin sealant preserved left ventricle fraction shortening and left ventricular high developed pressure most effectively.The present study indicate also that fibrin sealant may be used as a support and tissue-engineering scaffold to improve cardiac function after MI in rats.The optimal type of cells to restore cardiac function after myocardial infarction is still controversial. New hope focuses on embryonic stem cells (ESCs) and adult stem cells (ASCs), especially bone marrow stem cells (BMSCs). But for ESCs, transplantation of undifferentiated ESCs may lead to teratoma and it is difficult to obtain enough and purified cardiac-like cells from ESCs in vitro by now.15 However, only when 100% ESCs differentiated into cardiomyocytes, tumor formation can be excluded. For BMSCs, Orlic and colleagues reported that BMSCs could differentiate into cardiac-like cells,16 but the transdifferentiation ability of BMSCs was under suspicion recently.17 Jackson and associates indicated that BMSCs may have the ability to transdifferentiate into cardiac-like cells, but the transdifferentiation rate is very low (大鼠心功能的实验研究研究背景:目前有许多心肌再生技术来修复受损心肌,本实验研究移植乳鼠心肌细胞和纤维蛋白粘合剂,观察移植细胞能否存活及是否改善心肌梗塞大鼠的心功能. 方法: 结扎雌性Sprague-Dawley(SD)大鼠左冠状动脉前降支,制作大鼠心肌梗塞模型。培养雄性的新生1天SD乳鼠心肌细胞。雌性大鼠心梗3周后行细胞移植。实验动物随机分成4组:(1) CF组,即乳鼠心肌细胞加纤维蛋白粘合剂移植组(n=10),(2)C组,即乳鼠心肌细胞移植组(n=10),(3)F组,即纤维蛋白粘合剂移植组(n=10),(4)对照组(n=10)。移植术后4周,用超声心动图及离体心脏灌流模型评价心功能,心肌标本做石蜡切片观察梗塞心肌及免疫组化染色观察移植细胞,多聚酶链反应探测移植细胞中Y染色体SRY基因。结果: 超声心动图检测显示左心室短轴缩短率:CF组,27.80%±6.32%;C组, 22.29%±4.54%; F组,19.24%±6.29%;对照组,20.36%±3.29%。CF组明显高于其它三组,P值分别是0.028、0.001和0.005,有显著性差异,CF组最佳。离体心脏灌流模型显示左心室发展峰压(mmHg)CF组是104.81±17.05;C组是80.97±21.60;F组是72.07±26.17;对照组是71.42±17.55。CF组明显高于其它三组,P值分别是0.015,0.001和0.001,有显著性差异,CF组最佳。心肌标本石蜡切片显示CF组左室心肌生长优于其它各组。免疫组化染色显示心肌细胞生长CF组优于C组,多聚酶链反应显示移植细胞存活。结论:乳鼠心肌细胞和纤维蛋白粘合剂移植于心肌梗塞大鼠的心脏,移植细胞能够存活并提高了心肌梗塞大鼠的心功能。关键词:移植,心肌梗塞,细胞,再生