【作者】 修宗昌；

【导师】 李德新；

【作者基本信息】 辽宁中医学院 ， 中医基础理论， 2001， 博士

【摘要】 脾为后天之本，气血生化之源，在生命活动过程中占有重要地位。长期以来，脾虚证一直是人们研究的热点。尤其近年来，中医证候动物模型研究的广泛开展，加之分子生物学的兴起，为更深入探讨“脾虚”的本质奠定了理论基础。所以，我们把“脾虚证Ca²⁺/CaM信号系统的实验研究”纳入研究范围，立其为题。 目 的 寻求脾虚失运，化源亏乏，同空肠平滑肌细胞内Ca²⁺/CaM信号系统的内在联系。继而从胃肠动力学角度，对“脾主运化”的实质以及“脾虚失运”的发生机制作出一个分子水平的诠释，并为阐明四君子汤的作用机理提供实验资料。 材料与方法 1、脾虚模型复制 将wistar大鼠按性别、体重随机分成3组，每组10只。①模型组：破气苦降加饥饱失常法，即每日灌饲小承气汤煎剂，隔日半量进食；②正常对照组：常规饲养，以等量生理盐水代替药物；③复健组：每日灌饲四君子汤煎剂，1～2h后，灌饲小承气汤煎剂，余同模型组。共15天。 2、空肠平滑肌细胞悬液制备 将大鼠断头处死，在十二指肠与空肠交界处下1cm处剪取4cm长的肠组织，剖开剥去浆膜与粘膜，洗净，剪碎，装入三角烧瓶内，加10ml消化液，通入100％氧气，30℃，消化10min；共2次。消化毕，1800r/min离心5min，弃上清，加HEPES-Ringer缓冲液洗涤2次，弃上清，再加入HEPES-Ringer缓冲液10ml，通入氧气，30℃，振荡20min；而后用尼龙网（500μm）过筛，充氧气。 3、空肠平滑肌细胞内[Ca²⁺]i测定 荧光法 一 取细胞悬液Zml，1800r／min离心 smin，用 Hanks液洗涤 2次，加 IMDM培养 液 Zml，振荡后加 10 ul含 Fura－2／AM（终浓度为 5 ＂in） 的 DMSO溶液；37’C水浴 摇床中经 Fura－二／AM负载 60min。弃上清，加 Hanks液洗 2次，1800r／min离心 smin。弃上清，加 IMDM培养液Zml，测定前将样品 37OC孵育2～3 min，在比色 杯内放入磁棒以防止细胞沉淀。最后用F3000荧光光度计测定荧光强度，其中激 发波长为 340urn，发射波长为 500urn，反应时间 2 min。结果以下式算出： ［Ca’”二＝224 X（F－F min）／（Fmax－F） 式中，F为静息荧光值，F min为加 EGTA后的荧光值，Fmax为加 tritonx．100 后的荧光值。 4、空肠平滑肌细胞内CaM活性测定 磷酸H酯酶（PDE）法 山制备PDE 新鲜小牛脑除去小脑、被膜、血管等，取150g于1000ml缓冲 液中匀浆，20000 X g4℃离心lh，上清过DEAE6ephadex－A50柱，NaCI线型梯度 洗脱，收集第二个活性峰处酶液，透析后，上DEAE一纤维素DE52柱，NaCI线 型梯度洗脱，收集活性峰处酶液，透析后分装，80 t保存。 （2）P＊E活性检测①酶促反应过程：②孔雀石绿定磷法测定无机磷：绘制 磷标准曲线，未知样品磷摩尔数可以从曲线上查得。按下式计算PDE 活性（V molpi．min）： 测定管磷含量（ug）一对照管磷含量（ug） — —XZ 30．97x30xPDE（mg） m标准 CaM的制备及其标准曲线制作①取新鲜小牛脑 1009，如＊)处理干 净，加入缓冲液，高速匀浆。浆液搅拌 lh后离心，保留上清。②将离心上清用 6mol／L 醋酸调PH为4．3，10min后加硫酸镣至饱和度为50％，PH不变。放置lh后离 心，弃去上清。加入 200ml缓冲液，用 lmol／L Tris调 PH至 7．5，离心，保留上－“清。③将离心上清过用缓冲液平衡的DEAE一纤维素DE52柱，并进行梯度洗脱， 测各管 OD。s沪合并第 2个蛋白洗脱峰，同上透析，浓缩便得纯化 CaM。将其以 0．l％ BSA配成浓度为0．05％，测其对PDE的激活作用。④绘制CaM激活PDE标准曲 线 （4）样品中 CaM含量测定 取 0．sml 细胞悬液在电磁搅拌器上磨 30s，4 oC 1300r／min 离心30nin，上清于100℃水中煮Zmin，冷却后11300r／min4℃离心 8 一 10min，上清用标难曲线制作法测定CaM 含量。绘出激活曲线，选择灵敏点 （OD660）查出待测样品中CaM活性。 5、统计学处理：采用t检验。 结果 1、脾虚动物模型塑造 模型组自第3天起出现泄泻体重下降。此后至造模结束，渐现便搪加重，倦 乏拱背，毛乱乏泽，纳少等脾虚症状，肛温下降不显（P＞0．05）；复健组偶见便 搪，毛略欠泽，体重缓慢增加(P更多还原

【Abstract】 The spleen provides the materiel basis of the acquired constitution, is the source of growth and development, and occupies an important position in the course of life activity. For a long time, spleen-deficiency syndrome has been a hot subject people have been studying. Especially in recent years, the extensive development of the studies for animal models showing syndrome of TCM and the upsurge of molecular biology have laid a theoretical foundation for further probe into the essence of nsufficiency of the spleen?.Therefore, the experimental research on the signal system, Ca2~+/Cam, of spleen-deficiency syndrome is brought into the range of research and has been established as research subject. Purposes The inner link of dysfunction of the spleen in transport and insufficient nutrient essence caused-by spleen-deficieney with the signal system, Ca>ICaM in jejuna snaoolh muscle cells is sought for. Then, from the angle of gastrointestinal dynamics, annotations at the molecular level are made both on the essence of that the spleen has the function to transport and transform nutrients and on the producing mechanism of dysfunction of the spleen due to its deficiency. And the experimental materials are provided in order to clarify the mechanism of action of Sijunzi Tang. Materials and methods 1. Building of Animal Model of Deficiency of Spleen-energy. 30 wistar rats, both male and female, were randomly denied into three groups. The rats in the control group were fed normally and pleased with normal saline every day. While the rats in the model group were fed with half full and perfuse with Xiaochengqi Tang every day. The rats in the third group were perfused with Sijunzi Tang first, and then were given the 2 Xiaochengqi Tang one or two hours later. The period of building animal model is about 15 days. 2. Preparation of Jejunal Smooth Muscle Cells The rats were killed with decapitation. The jejunal tissues lying 1cm distal to the duodero jejunalis junction were obtained. After the serosa and mucous layer were stripped, the tissues were cut into pieces and were kept in a container with I Oml of digestive liquid. They were digested for 10 minutes under the adequate oxygen and at the constant temperature of 3000 .After that, the tissues were recovered by centrifugation (1 800r/min, 5mm) and washed with buffer HEPES-Ringer twice. After agitation for 20minites at the constant temperature of 30C. The jejunal smooth muscle cells were obtained through a nylon Sieae (500~.m). 3. Assay of rCa2I in Jejunal Smooch Musele Cells Fluorescerce Method Take 2ml of cell suspension, centrifugate it for 5mm at the speed of 1 800r/min , add 2m1 of the culture solution , IMDM . After agitation, add 1 Oul of DMSD solution containing Fura-2/AM (the final concentration is 5uM). This solution is loaded for 60mm in water bath cradle at the temperature of 3700. Throw the upper supernatant liquid. Then wash it twice with Hanks solution, centrifugate it for 5mm at the speed of 1 800r/min, throw the upper supematant liquid again, add 2m1 of culture solution. IIvI7DM. Before the assay. incubate the sample for 2 to 3mm at the temperature oL370C. Then put a magnetic rod in a color comparison tube to avoid cell preipitation. At last assay the fluorescein intensity with F-3 000 fluorophotometer. In its assay, excitation wave length is 340nm, emission wave length is SOOnm. the time of更多还原