【作者】 朱华庆；

【导师】 汪渊； 袁凌云；

【作者基本信息】 安徽医科大学 ， 生物化学与分子生物学， 2002， 硕士

【摘要】 目的：兔MLCK cDNA表达载体的亚克隆，为探针的制备及原位杂交的研究奠定基础；探讨VitE对动脉粥样硬化模型兔动脉MLCK活性及与膜通透性变化的影响。方法：提取Pat21—CMV5（B-）质粒，经限制性内切酶Hind Ⅲ、XbaⅠ双酶切，插入质粒pBKcmv中，构建为重组质粒pBKcmv-MLCK，阳性克隆在大肠杆菌中扩增后提取质粒，经限制性内切酶Hind Ⅲ、EcoRI双酶切，插入质粒pET28b中，获重组质粒pET28b-MLCK；复制兔动脉粥样硬化模型，利用γ-³²P掺入法检测动脉MLCK活性；动脉经O.C.T包埋、冰冻切片，利用免疫荧光检测动脉内膜通透性的变化，同时用HE染色观察动脉壁的形态结构。结果：重组质粒pBKcmv-MLCK限制性内切酶谱分析表明其酶切位点与原设计相符。重组质粒pET28b-MLCK中插入片段大小为405 bp，与设计一致。动脉粥样硬化兔模型建立成功，大白兔在喂食高胆固醇膳食4周或12周后，与正常对照相比血液中胆固醇和低密度脂蛋白的含量显著增加（P＜0.05，P＜0.01），胆固醇含量增加约达18—30倍，低密度脂蛋白增加约达22—61倍，而甘油三酯和极低密度脂蛋白的含量没有明显变化（P＞0.05）。喂高胆固醇膳食4周组，高密度脂蛋白增加约为对照组的2倍（P＜0.05），喂高胆固醇12周组动物的高密度脂蛋白含量与对照比较没有统计学差异（P＞0.05），但在加喂维生素E之后，血浆中高密度脂蛋白含量增加，约为对照组的2倍（P＜0.05）。另外，喂食12周同时加维生素E组，甘油三酯和极低密度脂蛋白浓度均升高（P＜0.05）。在喂高胆固醇4周的动物组，动脉组织MLCK活性与正常对照比较显著增强（P＜0.05）。但是，在喂高胆固醇膳12周或同时添加维生素E后，动脉组织MLCK活性与正常对照组比较增加不明显（P＞0.05）。荧光显微镜下观察显示，兔主动脉内膜通透性在喂食高胆固醇膳食后4周有所增多，12周后显著增加，12周组的膳食中同时加维生素E后，主动脉的通透性有所下降。 安徽医科大学硕士学位论文 朱华庆·2002年 HE染色显示，喂高胆固醇4周组的兔主动脉内膜增厚，细胞间隙增大并有少量 泡沫细胞形成，喂高胆固醇12周形成明显的动脉粥样硬化，在加维生素E对兔 主动脉内膜病变似乎有一定改善作用。结论：成功获得兔 MLCK叨NA亚克隆， 在动脉粥样硬化早期，内膜屏障完整性的改变可能与MLCK活性增强相关。 Vi iE可能是降低动脉内膜 MLCK的活性因素。AS的形成是山内皮细胞的向心性 收缩所致，VitE对动脉粥样硬化模型兔动脉内膜的通透性有一定的抑制作用。更多还原

【Abstract】 Objective: To subclone of rabbit MLCK cDNA, To study the effect of VitE on the MLCK activity and the endothelial permeability of artery in atherosclerosis model rabbits. Methods: The pBKcmv-MLCK was constructed by insertting the cDNA fragment of Pal2]-CMV5 (B-) pi asm id cut by Hind III / Xba I. The pET28b-MLCK was subcloned by inserting MLCK cDNA of the pBKcmv-MLCK cut by Hind III / EcoRI .The MLCK activity of rabbit artery was measured by the method of Y -32P incorporated. The endothelial permeability was detected by immunofluorescent. Results: The restrictive endonuclease mapping showed that the restrictive endonuclease sites of pBKcmv-MLCK were identical with the original design, the insert fragment of pET28b-MLCK is 405bp which is identical with the design.the model of atherosclerosis was established, After being fed with cholesterol (Chol)and lard for four or twelve weeks, the Choi and low-density lipoprotein(LDL) increased obviously in the blood, there is significantly statistical difference when compared with the normal control (P < 0.05 or P < 0.01). No statistical difference is found in triglyceride (TG)and very low-density lipoprotein(VLDL) when compared with the control. In groups fed with Choi and lard for twelve weeks, the Choi and LDL increased markedly in the blood (P < 0.05), and there is significantly statistical difference when compared with the control, but no statistical difference is found in serum TO and high-density lipoprotein(HDL). In groups fed with Choi, lard and vitamin E, the TG, Choi, HDL, LDLand VLDL increased obviously and there is significant statistical difference when compared with the normal control (P < 0.05, P < 0.01. P> 0.05 )compared with the control. After rabbits were fed with Choi and lard for four weeks, the activity of MLCK increased markedly, and there is significantly statistical difference when compared with the normal control (P < 0.05). While therabbits were fed with Choi and lard for 12 weeks or with Choi and lard add vitamin E for 12 weeks, the activities of MLCK did not change markedly, there is no statistical difference when compared with the normal control respectively (P > 0.05). The permeability of arterial wall rised after the rabbits was fed with the feed of higher cholesterol for four weeks, the permeability increased more and more obviously after the rabbits was fed with the feed of higher cholesterol for a longer time. The permeability appears to be decreased when vitamin E was added into the high cholesterol fed groups for twelve weeks, the HE staining indicated that there are a lot of foam cell producted after the rabbits was fed with the feed of higher cholesterol for four weeks and the atherosclerosis was formated after the rabbits was fed with the feed of higher cholesterol for twelve weeks ,the endothelial symptom seem to be relifed when vitamin E was added into the high cholesterol fed groups for twelve weeks. Conclusions: the pBKcmv-MLCK recombinant plasmid was constructed and the subclone of MLCK was obtained. The change of integrity of arterial wall may be associate with the increase of activity of MLCK. VitE may be decrease the MLCK activity. Vitamin E may protect endothelial and arterial wall from injury. VitE may decrease the endothelial permeability of atherosclerosis rabbit.更多还原