摘要
同种原位心脏移植是临床治疗终末期心脏病的有效方法。随着心脏移植的开展，移植心脏排斥反应的监测和预防已成为国内外学者广泛关注而又难以克服的问题。以往的研究中，多数学者将研究的重点集中在免疫学上，如抗原抗体及细胞因子的变化等，而-对T细胞本身的生物学特性的改变没有进行深入的研究。本课题应用大鼠腹腔异位心脏移植动物模型探索T细胞CD28分子的表达、CD8~+CD28~+T生物学特性的变化与急性排斥反应的关系，尝试开拓心脏移植急性排斥反应研究的新领域。
首先，我们制作了大鼠腹腔异位心脏移植动物模型。该模型制作的成败是本系列实验成败的关键。在制作模型过程中，我们对供心的切取、受体的肝素化、腹腔血管的准备、心肌的灌注和保护及血管的吻合等进行了一系列操作技术的改进，使得制作模型简便快捷。
在第二部分探索T细胞CD28分子的表达与心脏移植急性排斥反应的关系；T细胞是不均一的群体，根据其CD4和CD8分子的差别，可将成熟的T细胞分为CD4~+T和CD8~+T；CD4~+T也是不均一的群体，可以继续分为辅助性T细胞(Th)和迟发型超敏反应T细胞(T_(DTH))两个功能亚群；CD8~+T的功能亚群包括抑制性T细胞(Ts)和杀伤性T细胞(CTL)。静止的CD8~+T细胞以前体细胞的形势存在，在识别并结合APC表面抗原多肽-MHC-Ⅰ类分子复合物后，活化为抗原特异性的效应细胞。CD28分子主要表达在T细胞的共刺激分子，是T细胞激活的共刺激通路，激活的CD8~+T细胞主要是CD8~+CD28~+T细胞，起杀伤作用；CD8~+CD28~-T则是作为抑制性T细胞而起作用的。本实验期望阐明CD28分子与急性排斥反应的内在关系。
在第三部分探讨了起杀伤作用的CD8~+CD28~+T细胞生物学特性(包括钙离子浓度、膜电位、pH值)的变化与急性排斥反应的关系。实验是用荧光探针在T细胞内与钙离子、氢离子等结合，通过荧光强度的变化来证明细胞膜电位、钙离子浓度及pH值的变化。Fluo-3-AM产生的荧光强度与细胞内游离钙浓度成正比；BCECF-AM根据细胞内酸度不同发出
不同强度的荧光，根据荧光强度可知细胞内氢离子变化趋势;DIBAc4(3)
是膜电位敏感的亲脂性阴离子荧光染料，根据其在细胞内外的重新分布
可判断出细胞膜电位的变化，DIBAq(3)进入细胞多，荧光增强，表明细
胞膜电位出现去极化变化。根据三种荧光不同强度来检测细胞的膜电位、
钙离子浓度及pH值的变化。
第一部分大鼠异位心脏移植动物模型的制作
一、材料与方法
一)、材料SPF级雄性Wistard大鼠40只、SPF级雄性SD大鼠120只，
体质量250一3009(第一军医大学实验动物中心提供);显微外科手术器械
(上海医疗器械厂);手术显微镜(上海医用光学仪器厂);9/0无创伤缝线
(广州医疗器械厂)。
二)、手术方法供、受体动物均采用309/L戊巴比妥钠腹腔注射口om
g/kg)麻醉，整个手术由2人操作。制作心脏移植动物模型80例，40
例SD至SD(SD~SD)大鼠腹腔异位心脏移植，40例Wjstard至
SD(wistard一SD)大鼠腹腔异位心脏移植。
1、受体鼠的准备麻醉后大鼠仰卧位，固定，拔毛，消毒铺单，
腹部正中切口，长约3一4cm，暴露下腔静脉及腹主动脉，在肾血管平面
下选择适当长度的腹主动脉及下腔静脉作为吻合口部位，从下腔静脉注
入0.3mg的肝素。在血管上下两端分别用动脉夹阻断，用冠脉刀切开腹
主动脉及下腔静脉，长度为2一3mm。受体准备完毕。
2、供心准备切口同上，经下腔静脉注入肝素250u，腹腔冰水降
温，剪开两侧肋骨和隔肌，将胸壁向上翻，胸腔冰盐水降温，靠主动脉
弓剪断主动脉及肺动脉，集束结扎上腔静脉、下腔静脉及肺静脉;剪下
供心;在冰盐水中，用2m14℃St.Thomas液经主动脉灌注，灌注彻底。
3、心脏移植将供心主动脉与腹主动脉用9/0无创缝线作端侧连续
吻合;同法将下腔静脉与肺动脉吻合;排气后开下端动脉夹，下腔静脉
血逆灌，检查无漏血，开上端动脉夹，心脏复跳，全层缝合腹壁，术毕。
二、结果
制作模型80例(40例SD~SD;40例WIStar一SD)存活74例(SD~SD38
例;Wi Star~SD 36例)，存活率92.5%。供心总缺血时间30士6.smin。
手术总时间50士smin。成活大鼠无下肢瘫痪，无明显感染征象。
三、讨论
国内外在ono动物模型上做了各式各样的改进，动物模型的制作成
功率有所提高。XiuD等使用袖套技术能在smin准备好供心，3min将供
体的主动脉与颈总动脉、肺动脉与颈内静脉吻合完成，且吻合口不会出
血;供心缺血时间不会大于10min;但是需要特殊的器械，而且花费十分
昂贵。在国内目前的条件下，手工吻合仍然是心脏移植技术的主要手段。
我们在制作此经典模型上有如下体会和改进:
1.麻醉不能过深，麻醉后大鼠呼吸均匀、舌唇红润、安静，表明麻醉
效果好;如大鼠麻醉过深，影响手术存活率。
2.摘取供心时，应由横窦分离出主动脉及肺动脉，紧靠主动脉弓准确
剪断主动脉及肺动脉，切勿损伤心房组织及血管。为了暴露清楚，
首先剪断腹主动脉及下腔静脉，使血液流入腹腔。然后在左房下集
束结扎左右上腔静脉、下腔静脉及肺血管，减
To detect the guide line of supervise the acute rejection ,we made this kind of animal model. The success of this animal model determined the success of our experiment. In the process of making animal model we improved the the technique of donor cardiectomy , heparinization of recipient , preparative of celiac vessel , vascular anastomosis, myocardial perfusion and protection.,which made making heart transplantation animal model more convenient.
Heart transplantation is a efficiency method in treating terminal heart diseases therapy in clinic. With the improvement of heart transplantation, the prevention and monitoring of acute rejection become the most difficult problem in medical area. In the past time ,many medical researchers studied the acute rejection just in immunology including antibody antigen cell factors and so on. But they did not do a deep research in the biology character about T cell. Our research detected the relation between the acute rejection and expression of CD28 molecular of T cell in heteroheart transplantation. After that we explored the the relation between the acute rejection and biological character of CD8+CD28+T. We attempted to exploit a new world about the research of the acute rejection.
T cell is not a homogeneous group. Based on the expression CD4, CD8, we can divided T lymphocyte into CD4+T, CD8+T. CD4+T can be divided into Th and TDTH. CD8+T can be divided into Ts and CTL. Resting CTL presented in a preceding state. Recognized the antigen-MHC-I compound of the APC surface ,resting CTL was actived and became a effect cell against special antigen. CD28 molecule-a costimulated molecule was just expressed in surface of T cell.CD8+CD28+T was the main part of CTL.In part 3,our experiment detected the relation between the acute rejection and intracellular calcium, membrane potential and pH of the CD8+CD28+T.
Our experiment detected the intracellular calcium membrane potential and pH of the CD8+CD28+T by fluorescence probe,which can combined with intracellular calcium hydrogen ion etc. The concentration of Fluo-3-AM fluorescence was positive correlation with the intracellular calcium. Through the concentration of BCECF-3-AM fluorescence we can know the concentration of hydrogen ion. DiBAC4 (3) was a sensitive fluorescence to the membrane potential.When more DiBAC4(3) entering the cell , the fluorescence became stronger,which shew the membrance depolarized stronger.
Part 1 Animal Model of Heterotopic Heart Transplantation in rats
Abstract 1 Material and Method
Material 40 SPF grade male Wistar rats, 120 SPF grade male SD rats, weight 250-300g(offered by the experiment animal center of the first military medical university); operation appliance of microsurgery ( Shanghai medical appliance company); operation microscope(Shanghai medical optics appliance company);9/0 suture(Guangzhou medical appliance company)
Operation Method:
Anaesthesia by barbiturate(30mg/kg)
1) Preparing of Recipient After anaesthesia the rat was on its back,fixed,epilation and sterilized. Through the abdominal incision(3-4cm) we shew the descending vena cava and abdominal aorta. The anastomosing position was choosed under the kidney artery. Recipient heparinized by 0.3mg heperine. Blocked the blood vessel ,we made a 2-3mm length incision on the blood vessel.we finished the preparing recipient.
2) Donor Preparing Made the same abdominal incision , heparinized by 2mg heperine, droped the body temperature and then cut the two sides ribs and diaphragm, lifted the front chest wall, droped the heart temperature by ice water, cut the lung artery and arota and bundled the ascending and descending vena cava 1ung vein. Cut down the donor rat heart and perfused the donor heart by4CSt. Thomas saline water to make the heart muscle soft and pale.
3 Heart Transplantation Anastomosed the aorta of the donor heart and the aorta of the recipient abdominal aorta and Anastomosed the pulmonary artery of the donor heart and the abdominal descending vena cava of the recipient. Expeled the gas of the blood vessel,loosened the desce
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