The Study Of The Relation Between The Endothelial Permeability And The Variance Of Myosin Light Chain Kinase Activity Of The Artery In Atherosclerosis Model Rabbit

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.