Objective： TO observe the effect of Huxin Formula （护心方, HXF） on expressions of the chief reverse cholesterol transport （RCT） associated genes, caveolin-1 and scavenger receptor-B I （SR-B I ） in ApoE-gene kno...Objective： TO observe the effect of Huxin Formula （护心方, HXF） on expressions of the chief reverse cholesterol transport （RCT） associated genes, caveolin-1 and scavenger receptor-B I （SR-B I ） in ApoE-gene knockout [ApoE （-/-）] mice. Methods： Thirty ApoE （-/-） mice of 4-6 weeks old were randomly divided into three groups （A-C）. After being fed with high-fat diet for 16 weeks, they were treated with HXF （1 mL/100 g）, pravachol （0.3 mg/100 g）, and saline in equal volume respectively for 16 weeks successively; in addition, a blank group was set up with 10 C57BL/6J mice of 6-week old received 16-week high-fat feeding and saline treatment. Animals were sacrificed at the termination of the experiment, their paraffin sections of aortic tissue were used to measure the size of plaque, expressions of cavolin-1 and SR-B I were detected by immunological histochemical method. Results： As compared with the blank group, levels of caveolin-1 and SR-B I were increased in Groups A and B （P〈0.01）; but the increase in Group A was more significant than that in Group B （P〈0.05）. The plaque/aorta area ratio decreased significantly in Groups A and B, but showed insignificant difference between the two groups. Conclusion： HXF could obviously increase the expressions of RCT associated genes, caveolin-1 and SR-B I, promote the RCT process, so as to reduce the formation of aorta atherosclerotic plaque in ApoE （-/-） mice.展开更多
目的:利用成簇规律性间隔短回文重复序列(clustered regularly interspaced short palindromic repeat,CRISPR)相关蛋白9(CRISP associated protein 9,Cas9)技术构建微小核糖核酸-551b(miR-551b)基因敲除小鼠模型。方法:选择健康C57BL/6...目的:利用成簇规律性间隔短回文重复序列(clustered regularly interspaced short palindromic repeat,CRISPR)相关蛋白9(CRISP associated protein 9,Cas9)技术构建微小核糖核酸-551b(miR-551b)基因敲除小鼠模型。方法:选择健康C57BL/6J小鼠,针对miR-551b外显子1区域,设计导向RNA(guide RNA,gRNA),构建Cas9载体质粒,将体外转录的Cas9 RNA及gRNA显微注射入小鼠的受精卵并体外培养。将培养合格的胚胎移植到代孕小鼠的输卵管中,待小鼠生育后得到F0代小鼠,使用基因测序确定基因敲除情况,与野生型小鼠繁育后,得到F1代杂合小鼠,F1代小鼠经自交繁育获得F2代小鼠,F3代小鼠由F2代纯合小鼠自交获得,采用电泳鉴定小鼠基因型,RT-PCR检测F3代小鼠组织miR-551b的表达。结果:利用CRISPR/Cas9技术构建模型小鼠得到F0代小鼠,通过测序筛选出缺失目标序列的F0代杂合子小鼠。与WT小鼠繁育后,琼脂糖凝胶电泳及测序筛选出F1代杂合小鼠,同样的方法鉴定并获得F2、F3代基因敲除小鼠,获取F3代纯合小鼠的心脏及下腔静脉样本,RT-PCR结果证实F3代纯合小鼠miR551b表达明显低于WT小鼠(P<0.05),成功敲除miR-551b基因。结论:通过CRISPR-Cas9技术成功构建miR⁃155基因敲除小鼠模型并稳定遗传,为进一步研究提供了有利条件。展开更多
基金Supported by the National Natural Science Foundation of China （No. 81102584）Guangdong Provincial Administration of Traditional Chinese Medicine （No. 2009386）
文摘Objective： TO observe the effect of Huxin Formula （护心方, HXF） on expressions of the chief reverse cholesterol transport （RCT） associated genes, caveolin-1 and scavenger receptor-B I （SR-B I ） in ApoE-gene knockout [ApoE （-/-）] mice. Methods： Thirty ApoE （-/-） mice of 4-6 weeks old were randomly divided into three groups （A-C）. After being fed with high-fat diet for 16 weeks, they were treated with HXF （1 mL/100 g）, pravachol （0.3 mg/100 g）, and saline in equal volume respectively for 16 weeks successively; in addition, a blank group was set up with 10 C57BL/6J mice of 6-week old received 16-week high-fat feeding and saline treatment. Animals were sacrificed at the termination of the experiment, their paraffin sections of aortic tissue were used to measure the size of plaque, expressions of cavolin-1 and SR-B I were detected by immunological histochemical method. Results： As compared with the blank group, levels of caveolin-1 and SR-B I were increased in Groups A and B （P〈0.01）; but the increase in Group A was more significant than that in Group B （P〈0.05）. The plaque/aorta area ratio decreased significantly in Groups A and B, but showed insignificant difference between the two groups. Conclusion： HXF could obviously increase the expressions of RCT associated genes, caveolin-1 and SR-B I, promote the RCT process, so as to reduce the formation of aorta atherosclerotic plaque in ApoE （-/-） mice.
