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目的建立5-脂氧化酶(5-LO)转基因小鼠进行动脉粥样硬化的发病分子机制的研究。方法通过显微注射的方法,将5-脂氧化酶基因片段(6.8 kb)导入BDF1受精卵雄原核并移植到同期受孕的假孕母鼠输卵管中,对产出仔鼠的鼠尾组织DNA进行PCR、Southern blot检测,对9、20、24号转基因小鼠分别提取腹腔细胞、骨髓细胞及脾、肾组织总RNA和蛋白,并采用RT-PCR、Western blot方法进行转录水平检测和蛋白表达检测。结果共产生25只子代小鼠,经PCR和Southern检测获得7只阳性小鼠,经RT-PCR和Western blot检测结果表明,9、20、24号转基因小鼠腹腔细胞、骨髓细胞、脾、肾5-LO和5-脂氧化酶激活蛋白(FLAP)在RNA和蛋白水平表达均高于正常BDF1对照小鼠,且统计学分析腹腔细胞、骨髓细胞表达均具有显著差异(P<0.05)。结论成功建立5-LO转基因小鼠模型。
Objective To establish a molecular mechanism of atherosclerosis in 5-lipoxygenase (5-LO) transgenic mice. Methods The gene fragment of 5-lipoxygenase (6.8 kb) was introduced into the pronucleus of BDF1 fertilized ovum by microinjection and transplanted into the fallopian tube of pseudopregnant female rats that were conceived in the same period. PCR and Southern blot. Total RNA and protein of peritoneal cells, bone marrow cells, spleen and kidney were extracted from transgenic mice9,20,24 respectively. The transcriptional level and protein expression were detected by RT-PCR and Western blot . Results A total of 25 offspring mice were generated. Seven positive mice were obtained by PCR and Southern blot. The results of RT-PCR and Western blot showed that the number of peritoneal cells, bone marrow cells, spleen, The expression of renal 5-LO and 5-lipoxygenase activating protein (FLAP) in both RNA and protein levels were higher than those in normal BDF1 control mice, and statistically significant differences were observed in peritoneal cells and bone marrow cells (P <0.05). Conclusion A 5-LO transgenic mouse model was successfully established.