CAJ | 학술논문

目的：通过观察益心解毒方含药血清对Nox2、Nox4亚基过表达的H9 c2心肌细胞NADPH氧化酶活性的影响，揭示其作用环节是否与干预相应亚型的 NADPH 氧化酶调控环节有关。方法采用PCR方法扩增Nox2、Nox4基因全长序列，经双酶切、连接载体和转化后提取重组质粒，经酶切鉴定的阳性质粒进行DNA测序，测序正确后按照lipofectamine 2000试剂的说明书瞬时转染H9 c2细胞，转染后的心肌细胞分组给予不同的药物干预，24 h 后检测 NADPH 氧化酶活性。结果①含有Nox2／Nox4亚基的重组质粒载体通过测序鉴定，结果与GenBank报道的完全一致。②通过荧光显微镜下观察转染72 h后的H9 c2细胞，可见大量发绿色荧光的细胞，流式细胞术计数结果显示转染率均在60％左右，符合实验要求。③空质粒载体组、模型组、益心解毒方组的NAD－PH氧化酶活性表达明显高于正常组（ P＜0．01，P＜0．05）；模型组和益心解毒方组的NADPH氧化酶活性表达高于空载体组（ P＜0．01）；模型组NADPH氧化酶活性表达均明显高于其他各组，而益心解毒方各组的NADPH氧化酶活性表达均明显低于模型组（ P＜0．01， P＜0．05）。结论成功构建大鼠心肌细胞Nox2、Nox4亚基的重组质粒载体，该重组质粒载体可以在心肌细胞中过表达，益心解毒方可以有效地降低NADPH氧化酶活性的表达。提示此复方治疗心衰的机制可能是抑制了Nox2和Nox4型NADPH氧化酶的表达。
목적：통과관찰익심해독방함약혈청대Nox2、Nox4아기과표체적H9 c2심기세포NADPH양화매활성적영향，게시기작용배절시부여간예상응아형적 NADPH 양화매조공배절유관。방법채용PCR방법확증Nox2、Nox4기인전장서렬，경쌍매절、련접재체화전화후제취중조질립，경매절감정적양성질립진행DNA측서，측서정학후안조lipofectamine 2000시제적설명서순시전염H9 c2세포，전염후적심기세포분조급여불동적약물간예，24 h 후검측 NADPH 양화매활성。결과①함유Nox2／Nox4아기적중조질립재체통과측서감정，결과여GenBank보도적완전일치。②통과형광현미경하관찰전염72 h후적H9 c2세포，가견대량발록색형광적세포，류식세포술계수결과현시전염솔균재60％좌우，부합실험요구。③공질립재체조、모형조、익심해독방조적NAD－PH양화매활성표체명현고우정상조（ P＜0．01，P＜0．05）；모형조화익심해독방조적NADPH양화매활성표체고우공재체조（ P＜0．01）；모형조NADPH양화매활성표체균명현고우기타각조，이익심해독방각조적NADPH양화매활성표체균명현저우모형조（ P＜0．01， P＜0．05）。결론성공구건대서심기세포Nox2、Nox4아기적중조질립재체，해중조질립재체가이재심기세포중과표체，익심해독방가이유효지강저NADPH양화매활성적표체。제시차복방치료심쇠적궤제가능시억제료Nox2화Nox4형NADPH양화매적표체。
Objective To observe the effect of Yixin Jiedu Formula drug serum on NADPH oxidase activity in H9 c2 myocardial cells with overexpressed Nox2 and Nox4 subunits, and reveal whether it could influence the regulation and control process of corresponding subtypes ’ NADPH oxidase. Methods Nox2 and Nox4 genes’ total sequence were amplified by PCR, recombinant plasmid was extracted after cut of double enzyme, connection of carrier and conversion.DNA sequencing was carried out for positive plasmid after enzyme digestion identification. After that, H9c2 cells got transient transfection following the instruction of lipofectamine 2000 , and different drug intervention were given to the cells.After 24 h, NADPH oxidase activity was detected.Results ①Sequencing identification of recombinant plasmid carrier with Nox2/Nox4 subunits was completely accord with the reported results from GenBank.②Under fluorescence microscope, the transfected H9c2 cells were observed after 72 h, a great deal of green fluorescent cells were observed.And the transfected rate was about 60%according to flow cytometry count result, which met the requirements of the experiment.③Positive expression of NADPH oxidase in empty plasmid vector group, model group and Yixin Jiedu Formula group were significantly higher than the control group,the differences were significant ( P<0.01,P<0.05);Positive expression of NADPH oxidase in model group and Yixin Jiedu Formula group were higher than the empty plasmid vector group, the differences were significant ( P<0.01 );Positive expression of NADPH oxidase in model group was significantly higher than other groups ( P<0.01) , and positive expression of NADPH oxidase in different Yixin Jiedu Formula groups were significantly lower than the model group ( P <0.05).Conclusions Recombinant plasmid carrier of Nox2 and Nox4 subtypes in rats’ myocardial cells is constructed successfully, which has an overexpression in myocardial cells.Yixin Jiedu Formula could effectively decrease the positive expression of NADPH oxidase, which indicates the possible mechanism of this formula in treating heart fallure is restralning the expression of Nox2 and Nox4 NADPH oxidase.