【作者】 李晓晴；

【导师】 张苏明；

【作者基本信息】 华中科技大学 ， 神经病学， 2007， 博士

【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。更多还原

【Abstract】 ObjectiveTo investigate the mechanism of the APP cleavage progress and Aβgeneration, the construction of recombinant eukaryotic expression plasmid was made encoding Swedish and APP717 mutations of amyloid precursor protein (APP) and fluorescent protein.MethodsThe cyan and yellow fluorescence protein sequences (which were named as CFP and YFP, respectively.) were obtained by polymerase chain reaction (PCR) with the template of BglII-digestion product of the plasmid pcDNA3.0-CFP-CaM-YFP. The last 300 bases of APP sequence (which was named as C99 containing APP717 mutation) were amplified by PCR with the template of the plasmid pcDNA3.0-APP harboring APP717 mutation. The 54 bases in the middle of APP sequence were synthesized (which was named as 54bp containing Swedish mutation) by biological company. The four fragments, mentioned above (which were CFP, YFP, C99 as well as 54bp.) were inserted into the vector pcDNA3.0. By genetic engineering the recombinant plasmid pcDNA3.0-CFP-54bp-YFP-C99 and pcDNA3.0-CFP-54bp-YFP were constructed and identified by enzyme digestion and sequencing assay. The recombinant plasmids pcDNA3.0-CFP-54bp-YFP-C99 and pcDNA3.0-CFP-54bp-YFP were transfected into SH-SY5Y cells respectively. The mRNA expression of the fusion gene was examined by reverse transcript polymerase chain reaction assay (RT-PCR). The protein expression of fusion gene and fluorescence change was detected by multi-photon con-focal microscopy at 12h, 24h, 48h, 72h and 96h after transfection. At 12h, 18h, 24h, 36h, 48h and 72h after transfection of pcDNA3.0-CFP-54bp-YFP-C99 or pcDNA3.0-CFP-54bp-YFP, as soon as the cells were excited at 820nm of CFP excitation wavelength, the CFP images were collected at the CFP emission wavelength of 473nm and YFP images were also collected at the YFP emission wavelength of 525nm. FRET phenomenon was observed to get an understanding ofβ-cleavage of APP. Calculate the intensities of CFP (ICFP) and YFP (IYFP) and then turn them into the ratio Fret ( Fret=IYFP : ICFP). At 48h after pcDNA3.0-CFP-54bp-YFP-C99 transfection, multi-photon con-focal microscope was used to detect the yellow fluorescence, to observe its distribution and trace Aβlabeled by YFP. Aβgeneration was confirmed by immunocytochemistry. The viability of transfection cells was measured via MTT assay to find the Aβeffect on the cells at specific time point.Results1. The length of PCR-product of CFP、YFP and C99 was 818bp、738bp and 325bp. The length of product of CFP、YFP and C99 via different double-digestion was 702bp、723bp and 310bp. The electrophoresis result is identical to our expectation. The different double-digestion of the plasmid pcDNA3.0-CFP-54bp-YFP-C99 and pcDNA3.0-CFP-54bp-YFP resulted in different fragments and confirmed the accomplishment of the two recombinant plasmids. Finally sequencing assay showed that the fusion genes of CFP-54bp-YFP-C99 and CFP-54bp-YFP were totally same to that base information in gene bank.2. The purpose fragment was amplified in transfected cells while it was absent in control cells.3. Cyan and yellow fluorescence could be detected in cells at 12h after transfection. At 24h the intensity of fluorescence strengthened. At 48h the fluorescence increased further. But at 72h the fluorescence became to decrease and by 96h it diminished.4. FRET occurred in pcDNA3.0-CFP-54bp-YFP transfection cells and the cell shape was clear. FRET was present in pcDNA3.0-CFP-54bp-YFP-C99 transfected-cells at 12h after transfection, but absent thereafter. Much intracellular fluorescence accumulation spread within the cells.5. In pcDNA3.0-CFP-54bp-YFP transfected-cells, Fret unchanged. In pcDNA3.0-CFP-54bp-YFP-C99 transfected-cells, Fret declined.6. At 48h in transfection cells, the fusion gene product of CFP-54bp-YFP-C99 could be cleaved byβ- andγ-secretase and liberate Aβ.7. Aβwas firstly generated within the cell. It accumulated and deposited widespread in the cell and membrane. Very little Aβwas secreted outside of the cell but did not form obvious deposition.8. The cell shape was abnormal due to Aβgeneration and deposition.9. Aβwas found by immunocytochemistry.10. Aβintracellular accumulation resulted in the decrease of transfected-cell viability examined by MTT assay and the difference was significant in comparison with the control group.Conclusion1. The construction of recombinant plasmid pcDNA3.0-CFP-54bp-YFP-C99 and pcDNA3.0-CFP-54bp-YFP containing Swedish (and APP717) mutation of APP and two types of fluorescent protein sequences was accomplished.2. The fusion gene could be expressed correctly at mRNA and protein levels and become to be a strong tool for investigating the mechanism of APP cleavage and Aβgeneration.3. FRET could be used to detect the occurrence ofβ-cleavage sensitively.4. CFP-54bp-YFP-C99 could be cleaved byβ-secretase and CFP-54bp-YFP could not which indicated that C99 would be significant forβ-cleavage of APP and might function as signal-peptide for directing the cleavage. If C99 function was disturbed, then the initiation of Aβmight be blocked. It may provide a new idea for the early therapy of AD.5. The fusion gene could be translated into correct protein. The expression product of fusion gene could be sequentially cleaved byβ- andγ-secretase and generate Aβlabeled by YFP.6. Aβwas produced mainly and firstly within the cell and little was liberated into the cell space.7. Before Aβdeposited outside of the cell, Aβaggregated within the cell and induced the secondary damage to the cell which leaded to the cell viability decrease and shape abnormality.更多还原