体内抑制法尼基焦磷酸合成酶可以改善压力超负荷诱导的心脏重塑

发布时间：2018-01-05 06:38

本文关键词：体内抑制法尼基焦磷酸合成酶可以改善压力超负荷诱导的心脏重塑　出处：《浙江大学》2017年博士论文　论文类型：学位论文

【摘要】：第一部分体内抑制法尼基焦磷酸合成酶可以改善压力超负荷诱导的心脏重塑研究背景:法尼基焦磷酸合成酶(FPPS)是甲羟戊酸途径中一个重要的分支酶。作为胆固醇合成的中间步骤,FPPS催化C5单位的异戊烯焦磷酸(IPP)和其同分异构体二甲基烯丙基焦磷酸(DMAPP)连续缩合,生成10C单位的r{牛儿基焦磷酸(GPP)和15C单位的法尼基焦磷酸(FPP)。FPP不仅是合成胆固醇和辅酶Q的重要底物,也是众多小G蛋白法尼基化修饰的法尼基来源。已有研究表明抑制FPPS可以改善AngII诱导的心脏重塑,而压力超负荷诱导的高血压大鼠模型中FPPS和部分小G蛋白表达升高。我们通过基因干扰技术抑制小鼠FPPS表达,探索FPPS在压力超负荷诱导的心脏重塑中的效应以及作用机制。目的:通过基因干扰技术抑制小鼠FPPS表达,探索FPPS在压力超负荷诱导的心脏重塑中的效应以及作用机制。方法:(1)设计若干FPPS短发夹RNA序列,慢病毒包装感染小鼠原代心肌细胞。用免疫蛋白印迹法验证每一个序列的干扰效率。(2)筛选出FPPS基因干扰效率高的序列,将对应序列的慢病毒显微注射到小鼠胚胎细胞。(3)筛选含有干扰序列的阳性转基因小鼠,验证小鼠心脏组织FPPS干扰效率。同时鉴定子代阳性小鼠的FPPS干扰效率,确保其干扰效率能稳定遗传到子代。(4)将F3代雄性FPPS干扰转基因雄鼠和非转基因同窝对照雄鼠随机分为手术组和假手术组。用腹主动脉缩窄术建立压力超负荷模型。(5)术后12周,测量小鼠的心脏功能,尾动脉血压。(6)组织病理学分析心肌细胞大小和心肌纤维化水平。(7)免疫蛋白印迹法测量FPPS蛋白及其通路下游蛋白水平变化。(8)G-LISA法测量Ras,RhoA,Rac1和CDC42活性。(9)荧光定量PCR法测量心脏胚胎基因的表达。结果:(1)pLVT202序列可以抑制心肌细胞FPPS表达。(2)成功构建了 FPPS干扰的转基因小鼠,其心脏组织FPPS干扰效率约为50%。其子代转基因小鼠均能稳定干扰FPPS表达,转基因小鼠较同窝野生型小鼠表达量降低了 50%。(3)成功构建了腹主动脉缩窄术引起的小鼠压力超负荷模型。术后12周,小鼠心脏明显扩大,心肌细胞肥厚,心肌纤维化明显。(4)术后12周时,干扰FPPS表达可以降低压力超负荷引起的心力衰竭指标升高水平、减少心肌细胞肥厚水平和心肌纤维化水平,同时提高射血分数。(5)术后12周时,干扰FPPS可以降低压力超负荷引起的Ras蛋白的活化和ERK1/2磷酸化升高,但并不能抑制活化RhoA水平的升高。结论:干扰FPPS表达可以部分改善压力超负荷引起的心力衰竭、心肌细胞肥厚和心肌纤维化。其机制可能是降低Ras蛋白的活化水平,进而降低ERK1/2磷酸化水平,抑制心肌细胞增殖和纤维化。第二部分构建心肌细胞条件性法尼基焦磷酸合成酶基因敲除小鼠研究背景:法尼基焦磷酸合成酶(FPPS)是甲羟戊酸途径中一个重要的分支酶。作为胆固醇合成的中间步骤,FPPS催化C5单位的异戊烯焦磷酸(IPP)和其同分异构体二甲基烯丙基焦磷酸(DMAPP)连续缩合,生成10C单位的r{牛儿基焦磷酸(GPP)和15C单位的法尼基焦磷酸(FPP)。FPP不仅是合成胆固醇和辅酶Q的重要底物,也是众多小G蛋白法尼基化修饰的法尼基来源。基因敲除小鼠模型被认为是很好的研究基因功能的模型,而目前并没有FPPS基因敲除小鼠模型的参考文献。因此,为了更深入的研究FPPS基因在心脏重塑病理中的作用,我们构建心肌细胞条件性法尼基焦磷酸合成酶基因敲除小鼠模型。目的:构建心肌细胞条件性法尼基焦磷酸合成酶基因敲除小鼠模型。方法:(1)设计并构建FPPS条件性基因敲除打靶载体质粒。(2)ES细胞电穿孔转移线性化质粒,筛选序列正确的阳性克隆,移植胚胎。(3)鉴定嵌合体小鼠,繁殖获得FPPS-Flox纯合子小鼠。(4)繁殖获得与心肌特异性表达Cre重组酶的Myh6-MerCreMer的FPPSfl/flCre+/-小鼠。腹腔注射他莫昔芬后验证心肌组织特异性敲除FPPS的效率。结果:(1)成功获得了 FPPS-Flox纯合子小鼠和心肌特异性表达Cre重组酶的Myh6-MerCreMer 的 FPPSfl/flCre+/-小鼠。(2)腹腔注射他莫昔芬后8周,小鼠心肌组织FPPS表达降低了约80%。结论:我们成功构建了心肌细胞条件性法尼基焦磷酸合成酶基因敲除小鼠模型。
[Abstract]:The first part of inhibition of farnesyl pyrophosphate synthase can improve the pressure overload induced cardiac remodeling of farnesyl pyrophosphate synthase (FPPS) is an important branch of the enzyme in the mevalonate pathway. As an intermediate step in cholesterol synthesis catalyzed by FPPS C5, a isopentenyl pyrophosphate (IPP) and the same isomer two methyl allyl pyrophosphate (DMAPP) continuous condensation, r{geranylgeranyl pyrophosphate 10C generation unit (GPP) of farnesyl pyrophosphate and 15C units (FPP).FPP is an important substrate for synthesis of cholesterol and coenzyme Q, is also a large number of small G protein farnesylation farnesyl source. The existing research inhibition of FPPS can improve the cardiac remodeling induced by AngII, and the pressure increased the expression of FPPS and small G protein hypertensive rat model induced by load. Through RNA interference inhibited FPPS expression, exploration The effect of cable FPPS in cardiac remodeling induced by pressure overload and the mechanism. Objective: through the technique of gene interference inhibited FPPS expression, explore the effect of FPPS on cardiac remodeling induced by pressure overload in and mechanism. Methods: (1) the design of several FPPS short hairpin RNA sequences, lentiviral infection of primary packaging mouse cardiomyocytes. Verify the interference efficiency of every sequence by Western blot method. (2) screened FPPS gene interference and high efficiency, the corresponding sequence of lentivirus were microinjected into mouse embryonic cells. (3) positive transgenic mice containing siRNA screening, verification of mouse heart tissue FPPS interference efficiency. At the same time, the efficiency of FPPS interference identification of offspring positive mice, to ensure the efficiency of interference can be stably inherited by the offspring. (4) the F3 generation of male FPPS interference transgenic male rats and non transgenic littermates of male rats were randomly divided into control Operation group and sham operation group. The surgery to establish model of pressure overload by abdominal aorta. (5) 12 weeks after surgery, cardiac function measurement in mice, the blood pressure of tail artery. (6) analysis of myocardial cell size and the level of myocardial fibrosis pathological changes (7). Western blot was used to measure FPPS protein and the pathway downstream protein level. (8) measurement of Ras, G-LISA RhoA, Rac1 and CDC42 activity. (9) gene expression in embryonic heart measured by fluorescence quantitative PCR method. Results: (1) pLVT202 sequence can inhibit the expression of myocardial FPPS. (2) the successful construction of transgenic mice by FPPS interference, the heart tissue FPPS the interference efficiency is about 50%. of the offspring of transgenic mice expressed stable knockdown of FPPS transgenic mice, compared with wild type littermates decreased expression of 50%. (3) was constructed successfully pressure abdominal aorta constriction caused by overload model. After 12 weeks, the mouse heart obviously Expansion, myocardial hypertrophy, myocardial fibrosis significantly. (4) 12 weeks after the operation, the expression of FPPS can reduce the interference index of heart failure induced by pressure overload elevated levels, reduce myocardial cell hypertrophy and myocardial fibrosis, and improve LVEF. (5) 12 weeks after the operation, the interference of FPPS can reduce the pressure overload induced Ras protein activation and phosphorylation of ERK1/2 increased, but did not inhibit the activation level of RhoA increased. Conclusion: the expression of FPPS can improve the disturbance of heart failure induced by pressure overload, myocardial cell hypertrophy and cardiac fibrosis. The mechanism may be to reduce the activation level of Ras protein, thereby reducing the phosphorylation level of ERK1/2 and the inhibition of myocardial cell proliferation and fibrosis. The second part is the construction of farnesyl pyrophosphate synthase gene knockout mice myocardial cell background: farnesyl pyrophosphate synthase (FPPS) is An important branch of the enzyme in the mevalonate pathway. As an intermediate step in cholesterol synthesis, isopentenyl pyrophosphate catalyzed by FPPS C5 units (IPP) and its isomer two methyl allyl pyrophosphate (DMAPP) continuous condensation, r{geranylgeranyl pyrophosphate 10C generation unit (GPP) of farnesyl phosphate and 15C units (FPP).FPP is an important substrate for synthesis of cholesterol and coenzyme Q, is also a large number of small G protein farnesylation farnesyl. Gene knockout mice are considered to study gene function good model, and there is no FPPS gene knockout mice model reference. Therefore in order to study the role of FPPS, further gene in cardiac remodeling pathology, we construct farnesyl pyrophosphate synthase gene knockout mouse model of myocardial cells. Objective: to construct myocardial cell conditioned farnesyl pyrophosphate synthase base For the knockout mice. Methods: (1) the design and construction of knockout vector plasmid FPPS conditional gene. (2) ES cell electroporation transfer linearized plasmid, and positive clones were screened for the correct sequence of embryos. (3) Jian Dingqian chimeric mice to produce FPPS-Flox, homozygous mice. (4) to produce recombinant Cre myocardial enzymes and specific expression of Myh6-MerCreMer in FPPSfl/flCre+/- mice. After intraperitoneal injection of tamoxifen to verify myocardial tissue specificity of FPPS knockdown efficiency. Results: (1) successfully obtained FPPS-Flox homozygous mice and FPPSfl/flCre+/- mice cardiac specific table of Cre recombinase Myh6-MerCreMer. (2) 8 weeks after intraperitoneal injection of tamoxifen, expression mouse myocardial FPPS reduced about 80%. conclusion: we successfully constructed the farnesyl pyrophosphate synthase gene knockout mouse model of myocardial cells.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R54

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