FRET对活体细胞内APP裂解过程的动态研究

发布时间：2018-02-28 02:20

本文关键词： 融合基因 CFP YFP APP 突变 FRET 多光子共聚焦显微镜 C99　出处：《华中科技大学》2007年博士论文　论文类型：学位论文

【摘要】： 目的构建含有突变型APP的荧光真核表达系统以研究APP的酶解过程和Aβ产生的分子机制。方法以质粒pcDNA3.0-CFP-CaM-YFP的BglII酶切产物为模板,通过聚合酶链式反应(PCR)分别得到编码蓝色荧光蛋白(cyan fluorescence protein, CFP)和黄色荧光蛋白碱基序列(yellow fluorescence protein, YFP);以pcDNA3.0-APP为模板,通过聚合酶链式反应(PCR)得到含有APP717突变的最后300碱基片段(C99);生物合成含有Swedish突变的APP中间54个碱基片段(54bp)。利用基因工程技术将CFP、54bp、YFP、C99片段克隆至载体质粒pcDNA3.0中,得到重组质粒pcDNA3.0-CFP-54bp-YFP-C99和pcDNA3.0-CFP-54bp-YFP,酶切和测序鉴定。将构建的融合基因转染至SH-SY5Y细胞中,通过RT-PCR检测融合基因mRNA表达,利用多光子共聚焦显微镜观察不同时间点(12h、24h、48h、72h、96h)转染细胞内的荧光蛋白表达情况。细胞转染12h、18h、24h、36h、48h、72h后,检测FRET,予以波长为820nm的CFP激发光激发,采集发射波长为473nm的CFP图像,同时采集发射波长为525nm的YFP图像;计算CFP、YFP荧光强度(ICFP和IYFP)和两者的比率(Fret=IYFP/ICFP),绘制曲线。pcDNA3.0-CFP-54bp-YFP-C99转染细胞48h后,利用多光子共聚焦显微镜观察细胞内黄色荧光蛋白的表达、分布以及YFP标记的Aβ去向。免疫细胞化学鉴定Aβ的生成。pcDNA3.0-CFP-54bp-YFP-C99、pcDNA3.0-CFP-54bp-YFP转染细胞12h、24h、36h、48h、72h、96h后,MTT检测转染细胞活性,了解Aβ对细胞的影响。结果 1、CFP、YFP、C99的PCR产物大小分别为818bp、738bp、325bp,经过不同的双酶切得到的酶切产物大小分别为702bp、723bp和310bp,电泳证实与预期一致;重组质粒pcDNA3.0-CFP-54bp-YFP-C99、pcDNA3.0-CFP-54bp-YFP分别经过不同的组合酶切,得到的目的片段长度正确;测序鉴定证明融合基因CFP-54bp-YFP-C99、CFP-54bp-YFP的序列与“gene bank”的碱基信息相符。 2、将转染细胞进行RT-PCR显示有目的片段,分别对应于CFP-54bp-YFP-C99和YFP-C99,对照细胞内则无相应片段。 3、转染细胞内有蓝色(和)黄色荧光表达,在转染12h后已经可以观察到细胞内有荧光分布,24h荧光逐渐增多,至48h荧光进一步增多增强,72h荧光有所减少,96h荧光明显减少减弱。 4、pcDNA3.0-CFP-54bp-YFP细胞在转染后始终存在FRET,细胞轮廓光滑;pcDNA3.0-CFP-54bp-YFP-C99细胞在转染12h后有微弱FRET,以后无FRET,细胞内有颗粒广泛沉积。 5、CFP-54bp-YFP转染细胞Fret不变; CFP-54bp-YFP-C99转染细胞Fret下降。 6、CFP-54bp-YFP-C99在转染细胞48h后,能够被β、γ分泌酶裂解产生Aβ。 7、Aβ产生于首先细胞内,聚集成颗粒,广泛分布于细胞内和细胞膜上,细胞外间隙也有少量分布,但没有形成明显沉积。 8、Aβ形成后细胞形态异常。 9、免疫细胞化学进一步证实Aβ的生成。 10、MTT证实细胞内聚集的Aβ使得细胞活性下降,与对照组相比差异有统计学意义。结论 1、荧光标记并含有Swedish (和APP717 )突变的重组质粒pcDNA3.0-CFP-54bp-YFP-C99和pcDNA3.0-CFP-54bp-YFP构建成功。 2、融合基因在mRNA和蛋白水平均能够正确表达,为下一步研究APP裂解和Aβ产生提供了一种有力工具。 3、FRET能够敏感准确的检测APP有无发生β裂解。 4、CFP-54bp-YFP-C99能够被β分泌酶裂解而CFP-54bp-YFP不能被裂解,提示C99可能起到信号肽样的引导定位作用,对β分泌酶裂解APP具有重要意义;干扰C99可能会抑制Aβ的产生,本实验为探索AD的早期干预提供一种新思路。 5、融合基因能够正确表达并被裂解,生成YFP标记的Aβ。 6、Aβ产生于细胞内多个部位,并有少量被分泌至细胞外。 7、Aβ在细胞外形成沉积之前,首先在细胞内聚集并产生继发性细胞毒作用,造成细胞活性下降,形态异常。
[Abstract]:Purpose A fluorescent eukaryotic expression system containing mutant APP was constructed to study the enzymatic hydrolysis process of APP and the molecular mechanism produced by A尾. method The expression of yellow fluorescence protein ( C99 ) was detected by polymerase chain reaction ( PCR ) . The expression of yellow fluorescent protein was detected by polymerase chain reaction ( PCR ) . The expression of yellow fluorescent protein was detected by polymerase chain reaction ( PCR ) . Understand the effect of A.beta . on cells . Results 1 . The size of the PCR products of cfp , YFP and C99 was 818bp , 738bp and 325bp , respectively , and the size of the digested products was 702bp , 723bp and 310bp , respectively . 2 . The transfected cells were RT - PCR to show the target fragments , which corresponded to the results showed that there were no corresponding fragments in the control cells . 3 . There were blue ( and ) yellow fluorescent expression in transfected cells . After 12 hours of transfection , the fluorescence distribution was observed in the cells , the fluorescence intensity increased at 24 hours , the fluorescence intensity increased at 48 hours , the fluorescence in 72h decreased , and the fluorescence of 96h decreased obviously . 4 . After transfection , the pcDNA3 . 0 - cfp - 54bp - YFP cells were all the same after transfection , the cell profile was smooth ; the pcDNA3 . 0 - cfp - 54bp - YFP - C99 cells had a weak fluorescence after 12 hours of transfection . 5 . Fret was not the same as that of Fret transfected with YFP - 54bp - YFP , and Fret was decreased by Fret - 54bp - YFP - C99 transfected cells . 6 . After 48 hours of transfection , the 尾 - and 纬 - secreting enzymes could be cleaved by 尾 and 纬 - secreting enzymes to produce A尾. 7 . A尾is produced in the first cell , aggregates into particles , is widely distributed in the cells and the cell membrane , and the extracellular space of the cells also has a small amount of distribution , but no significant deposition is formed . 8 . After formation of A.beta . , the cell morphology was abnormal . 9 . Immunocytochemistry further confirmed the formation of A尾. 10 . MTT proved that the aggregation of A尾 in the cells decreased the activity of the cells , which was statistically significant as compared with the control group . Conclusion 1 . The recombinant plasmid pcDNA3 . 0 - cf4 - 54bp - YFP - C99 and pcDNA3 . 0 - cfp - 54bp - YFP were constructed successfully . 2 . The fusion gene can be expressed correctly at mRNA and protein levels , and provides a powerful tool for the next study of APP cleavage and A.beta . production . 3 . fret can sensitively and accurately detect whether or not the APP undergoes beta cracking . 4bp - 54bp - YFP - C99 can be cleaved by 尾 - secretory enzymes , and the Qp - 54bp - YFP can not be cleaved . It is suggested that C99 may play a role in signal peptide - like guidance and localization , and it has important significance for the cleavage of APP . The interference C99 may inhibit the production of A.beta . , and this experiment provides a new idea for exploring early intervention of AD . and 5 , the fusion gene can be correctly expressed and cleaved to generate a YFP labeled A beta . 6 . A . beta . is generated in a plurality of sites in the cell and a small amount is secreted out of the cell . 7 . A . beta . is first accumulated in the cells and secondary cytotoxic effects are generated before the formation of a deposit outside the cell , resulting in a decrease in cell activity and abnormal morphology .

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R346

【共引文献】