QDs-SA量子点荧光成像阿尔茨海默病细胞模型APP、Aβ蛋白的实验研究
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摘要
背景与目的
阿尔茨海默病是以进行性认知功能障碍和行为损害为主要特征的中枢神经系统退行性病变。β-淀粉样蛋白是阿尔茨海默病特征性的病理改变老年斑的主要成分,已有研究表明阿尔茨海默病发病早期即存在Aβ蛋白的沉积,其沉积部位、面积和量与AD认知功能障碍程度密切相关。量子点是一种新型的荧光分子探针,与传统的荧光染料相比,具有荧光强度高、光学稳定性好等优势,将量子点与APP、Aβ蛋白特异结合,发展AD分子光学成像技术,指导阿尔茨海默病的早期诊断具有广阔的发展前景。本项目拟构建pcDNA3.1/APP595/596并列突变质粒,稳定转染入人胚肾HEK293细胞,建立阿尔茨海默病转基因细胞模型;应用量子点对APP、Aβ蛋白靶向标记,并将其与传统荧光染料对比,在评估量已点生物相容性的同时,获取量子点荧光成像强度和荧光稳定性的相关资料,为量已点早期应扩于阿尔茨海默病的分子影像诊断打下基础。
方法
1)对pcDNA3.1/APP质粒(美国Pittsburgh大学的Perez博士惠赠)进行测序,证实除质粒本身第595和596位氨基酸密码子存在并列突变外,序列的第4位碱基有意外突变(C→G),并影响到正常氨基酸的表达,亮氨酸(Leu)变成了缬氨酸(Val)。因此实验拟行质粒APPcDNA第4位碱基的定点突变。从原始质粒克隆模板中,利用PCR方法扩增目的基因APP695cDNA,将目的基因和目的载体pcDNA3.1分别酶切;纯化酶切产物后定向连接,并将其转化细菌感受态细胞,对长出的克隆行PCR鉴定,PCR鉴定为阳性的克隆送测序并行比对分析。2)将点突变后pcDNA3.1/APP595/596质粒转染至HEK293细胞建立稳定表达株,细胞免疫荧光和Western-blot方法检测转染细胞APP的表达和Aβ蛋白生成情况。3)采用细胞形态学、MTT分析法结合流式细胞检测凋亡技术,系统考察QDs-SA量子点(浓度2.5～25nm)对稳定转染pcDNA3.1/APP595/596质粒的HEK293细胞的毒性作用。4)应用激光共聚焦荧光成像和流式细胞技术,对QDs-SA量子点靶向标记阿尔茨海默病转基因细胞模型中APP、Aβ蛋白的荧光进行检测,同时与基于传统荧光染料标记的免疫荧光分析方法进行比较。
结果
1)应用DNAStar软件对阳性克隆测序结果与GenBank中APP基因序列比对,证实原序列中的错义突变G已被突变回C,第4位点碱基定点突变完成,pcDNA3.1/APP595/596并列突变质粒构建成功。2)Western-blot在稳定转染了pcDNA3.1/APP595/596质粒的细胞中检测到目的条带,而转染空载体组细胞未见APP蛋白表达。细胞免疫荧光染色后,共聚焦荧光显微镜下观察APP基因转染后能够生成APP和Aβ蛋白,前者主要定位于细胞胞膜,Aβ蛋白主要表达于近膜胞浆,细胞膜上也有表达。3)在2.5～25nm浓度范围内,与QDs-SA量子点共育24小时后,转染质粒的HEK293细胞形态学无明显改变;MTT示量子点作用细胞吸光度值与对照组相比未见明显差异;不同浓度量子点组间吸光度值比较亦无统计学意义(P＞0.05)。流式细胞检测空白对照组与各浓度组间凋亡早期细胞百分率差异无统计学意义(P＞0.05)。4)在波长为388nm紫外光激发下,QDs-SA量子点的最大荧光发射峰波长约为605±10nm,半峰宽小于35nm。激光共聚焦显微镜荧光分析示QDs-SA量子点标记APP和Aβ蛋白相较于传统Cy3荧光染料的平均荧光密度值更大(P＜0.05);488nm激发光连续激发12分钟,QDs-SA量子点荧光标记的APP和Aβ蛋白仍发射较强的橙红色荧光,荧光强度分别下降了27.87%、29.25%,而传统荧光染料Cy3荧光强度下降幅度高达79.60%和76.82%。流式细胞仪检测QDs-SA量子点和Cy3活细胞标记膜蛋白APP阳性率差异无统计学意义(P＜0.05),QDs-SA量子点荧光标记APP蛋白的流式荧光图形呈宽端型,Cy3标记的APP流式荧光图形呈扁平形,平均荧光强度差异有统计学意义(P＜0.05)。
结论
1)成功构建了在真核细胞中能高效表达人APP和Aβ蛋白的真核表达载体,并稳定转染HEK293细胞建立阿尔茨海默病转基因细胞模型。2)在一定的浓度范围内,QDs-SA量子点对细胞形态学、细胞生长和增殖活性无明显影响,具有较好的生物相容性。3) QDs-SA量子点能有效识别阿尔茨海默病转基因细胞模型中APP和Aβ蛋白;QDs-SA量子点标记APP和Aβ蛋白荧光成像在光稳定性和荧光强度等方面均优于传统的Cy3荧光染料标记的免疫荧光成像。
Objectives:Alzheimer's disease is a degenerative disease of the nervous system which is characterized by deficits in memory and cognition.The clinical diagnosis of AD depends on the clinical findings, scale measuring and image examination.However,because of low sensitivity and specificity,the conventional structure or function imaging techniques have not been widespread used yet in the field of clinical diagnosis of AD.Senile plaques is the characteristic pathological features. Amyloidβ-peptide as the chief component of senile plaques,has currently been identified that it existed in the Pathogenesis of early acute AD and its deposit areas and quantity were closely related to cognitive-assessing.Therefore,detection of Amyloidβ-peptide is meaningful for diagnosis of AD.Quantum Dot is a new fluorescence probe that has significant advantages in fluorescent intensity and ray stability when compared to traditional fluorescent dye.Thus,Quantum Dot may be used as a powerful method for Aβprotein detection and early diagnosis of AD.This study was designed to evaluate the biocompatibility and get the preliminary data of Quantum Dot used as a molecular probe of APP、Aβproteins.The pcDNA3.1/APP595/596 plasmid with coordinate bas-mutation was constructed and stably transfected into HEK293 cells to set up the cell model of AD.Then,Quantum Dots were targeted into APP、Aβproteins respectively and the biocompatibility as well as fluorescence intensity and duration of Quantum Dot were compared with that of traditional fluorochromes.
Methods:1) The pcDNA3.1/APP plasmid was gifted by Pro.Perez, University of Pittsburgh,USA.The Coordinate bas-mutations(G→T,A→C) and a accidental mutation(C→G) had been confirmed by DNA sequencing.In this experiment,APP was PCR-amplified,digested by HindⅢand XbaⅠ,and ligated into purified pcDNA3.1 vector.This recombinant plasmid was transfected into E.colo SH5αby means of heat shock.The recombinant plasmid was identified by PCR,and the positive cloning was identified by DNA sequence analysis.2) The construct was transfected into HEK293 cell lines,screened by G418.The interest protein(including APP and Aβ) were identified by immunofluorescence and Western blot.3) The toxicity of QDs-SA to HEK293 cells stably transfected pcDNA3.1/APP595/596 plasmid was investigated by cell morphology,MTT analysis and flow cytometry technology.4) With the help of Laser Scanning Confocal Microscope and Flow Cytometry,this kind of flurescence probe based on the QDs-SA had been used to detect APP and Aβproteins in HEK293 cells stably transfected pcDNA3.1/APP595/596 plasmid,and compared with conventional fluroimmunoassay.
Results:1)The positive cloning gene sequence were aligned to the complete gene sequence of APP by DNAStar software.The result confirmed that the accidental mutation(C→G) had been mutated back(G→C).The recombinant pcDNA3.1/APP595/596 plasmid was constructed successfully.2)The detection verified that recombinant plasmid group expressed APP protein,the specific protein bands was only detected in HEK293 Cells stably transfected pcDNA3.1/APP595/596 plasmid,but no expression was found in control groups.The immunofluorescence staining detection indicated APP expression in the plasma membrane,but the Aβmainly located in the endochylema near the cell membrane,and slight Aβexpression also located in the plasma membrane.3) The HEK293 cells were incubated with QDs-SA on different concentrations between 2.5nm and 25nm and lasted for 24 hours, there was no evident morphological deviations.The QDs-SA group's cell activity assayed by MTT had no any apparente difference compared to control group.Meanwhile,the difference of absorbance intensity among the different concentration groups had no any statistical significance(P＞0.05 ).We used AnnexinV/FITC staining flow cytometry to observe the percentage of apoptosis.The percentage of apoptosis had no any significant difference among groups(P＞0.05 ).4) under the stimulation by 388nm excitation light,The maximal Emission of the QDs-SA was 605±10nm,and the full width at half maximum＜35nm.The mean fluorescence intensity of QDs-SA was greater than the Cy3's under confocal fluorescence microscopy(P＜0.05) There was no notable quench of exciting quantum dots for 12 minutes,and the APP and Aβproteins stained by QDs-SA showed intensive orange red fluorescence under confocal fluorescence microscopy.The fluorescence intensity decreased nearly 27.87%、29.25%respectively.The other stained by Cy3 decreased 79.60%and 76.82%.The positive rate of APP staining had no significant difference between the QDs-SA and Cy3 by flow cytometry, but the Mean fluorescence intensity had statistical significance(P＜0.05).
Conclusions:1) The recombinant plasmid containing the APP 595/596 Coordinate bas-mutations was successfully constructed.Then the recombinant plasmid was stably transfected into HEK293 cells to construct the transgenic cellular model of Alzheimer's disease.2) The QDs-SA were biocompatible with HEK293 cells in appropriate concentration rang,which provided basis for further biological applications of QDs.3) The QDs-SA fluorescence probes can effectively recognize APP and Aβproteins and exhibited good sensitivity and exceptional photostability,suggesting that QDs-SA fluorescence probes could be a potential method in Aβdetection and offer a novel way for the diagnosis of Alzheimer's disease.
阿尔茨海默病是以进行性认知功能障碍和行为损害为主要特征的中枢神经系统退行性病变。β-淀粉样蛋白是阿尔茨海默病特征性的病理改变老年斑的主要成分,已有研究表明阿尔茨海默病发病早期即存在Aβ蛋白的沉积,其沉积部位、面积和量与AD认知功能障碍程度密切相关。量子点是一种新型的荧光分子探针,与传统的荧光染料相比,具有荧光强度高、光学稳定性好等优势,将量子点与APP、Aβ蛋白特异结合,发展AD分子光学成像技术,指导阿尔茨海默病的早期诊断具有广阔的发展前景。本项目拟构建pcDNA3.1/APP595/596并列突变质粒,稳定转染入人胚肾HEK293细胞,建立阿尔茨海默病转基因细胞模型;应用量子点对APP、Aβ蛋白靶向标记,并将其与传统荧光染料对比,在评估量已点生物相容性的同时,获取量子点荧光成像强度和荧光稳定性的相关资料,为量已点早期应扩于阿尔茨海默病的分子影像诊断打下基础。
方法
1)对pcDNA3.1/APP质粒(美国Pittsburgh大学的Perez博士惠赠)进行测序,证实除质粒本身第595和596位氨基酸密码子存在并列突变外,序列的第4位碱基有意外突变(C→G),并影响到正常氨基酸的表达,亮氨酸(Leu)变成了缬氨酸(Val)。因此实验拟行质粒APPcDNA第4位碱基的定点突变。从原始质粒克隆模板中,利用PCR方法扩增目的基因APP695cDNA,将目的基因和目的载体pcDNA3.1分别酶切;纯化酶切产物后定向连接,并将其转化细菌感受态细胞,对长出的克隆行PCR鉴定,PCR鉴定为阳性的克隆送测序并行比对分析。2)将点突变后pcDNA3.1/APP595/596质粒转染至HEK293细胞建立稳定表达株,细胞免疫荧光和Western-blot方法检测转染细胞APP的表达和Aβ蛋白生成情况。3)采用细胞形态学、MTT分析法结合流式细胞检测凋亡技术,系统考察QDs-SA量子点(浓度2.5～25nm)对稳定转染pcDNA3.1/APP595/596质粒的HEK293细胞的毒性作用。4)应用激光共聚焦荧光成像和流式细胞技术,对QDs-SA量子点靶向标记阿尔茨海默病转基因细胞模型中APP、Aβ蛋白的荧光进行检测,同时与基于传统荧光染料标记的免疫荧光分析方法进行比较。
结果
1)应用DNAStar软件对阳性克隆测序结果与GenBank中APP基因序列比对,证实原序列中的错义突变G已被突变回C,第4位点碱基定点突变完成,pcDNA3.1/APP595/596并列突变质粒构建成功。2)Western-blot在稳定转染了pcDNA3.1/APP595/596质粒的细胞中检测到目的条带,而转染空载体组细胞未见APP蛋白表达。细胞免疫荧光染色后,共聚焦荧光显微镜下观察APP基因转染后能够生成APP和Aβ蛋白,前者主要定位于细胞胞膜,Aβ蛋白主要表达于近膜胞浆,细胞膜上也有表达。3)在2.5～25nm浓度范围内,与QDs-SA量子点共育24小时后,转染质粒的HEK293细胞形态学无明显改变;MTT示量子点作用细胞吸光度值与对照组相比未见明显差异;不同浓度量子点组间吸光度值比较亦无统计学意义(P＞0.05)。流式细胞检测空白对照组与各浓度组间凋亡早期细胞百分率差异无统计学意义(P＞0.05)。4)在波长为388nm紫外光激发下,QDs-SA量子点的最大荧光发射峰波长约为605±10nm,半峰宽小于35nm。激光共聚焦显微镜荧光分析示QDs-SA量子点标记APP和Aβ蛋白相较于传统Cy3荧光染料的平均荧光密度值更大(P＜0.05);488nm激发光连续激发12分钟,QDs-SA量子点荧光标记的APP和Aβ蛋白仍发射较强的橙红色荧光,荧光强度分别下降了27.87%、29.25%,而传统荧光染料Cy3荧光强度下降幅度高达79.60%和76.82%。流式细胞仪检测QDs-SA量子点和Cy3活细胞标记膜蛋白APP阳性率差异无统计学意义(P＜0.05),QDs-SA量子点荧光标记APP蛋白的流式荧光图形呈宽端型,Cy3标记的APP流式荧光图形呈扁平形,平均荧光强度差异有统计学意义(P＜0.05)。
结论
1)成功构建了在真核细胞中能高效表达人APP和Aβ蛋白的真核表达载体,并稳定转染HEK293细胞建立阿尔茨海默病转基因细胞模型。2)在一定的浓度范围内,QDs-SA量子点对细胞形态学、细胞生长和增殖活性无明显影响,具有较好的生物相容性。3) QDs-SA量子点能有效识别阿尔茨海默病转基因细胞模型中APP和Aβ蛋白;QDs-SA量子点标记APP和Aβ蛋白荧光成像在光稳定性和荧光强度等方面均优于传统的Cy3荧光染料标记的免疫荧光成像。
Objectives:Alzheimer's disease is a degenerative disease of the nervous system which is characterized by deficits in memory and cognition.The clinical diagnosis of AD depends on the clinical findings, scale measuring and image examination.However,because of low sensitivity and specificity,the conventional structure or function imaging techniques have not been widespread used yet in the field of clinical diagnosis of AD.Senile plaques is the characteristic pathological features. Amyloidβ-peptide as the chief component of senile plaques,has currently been identified that it existed in the Pathogenesis of early acute AD and its deposit areas and quantity were closely related to cognitive-assessing.Therefore,detection of Amyloidβ-peptide is meaningful for diagnosis of AD.Quantum Dot is a new fluorescence probe that has significant advantages in fluorescent intensity and ray stability when compared to traditional fluorescent dye.Thus,Quantum Dot may be used as a powerful method for Aβprotein detection and early diagnosis of AD.This study was designed to evaluate the biocompatibility and get the preliminary data of Quantum Dot used as a molecular probe of APP、Aβproteins.The pcDNA3.1/APP595/596 plasmid with coordinate bas-mutation was constructed and stably transfected into HEK293 cells to set up the cell model of AD.Then,Quantum Dots were targeted into APP、Aβproteins respectively and the biocompatibility as well as fluorescence intensity and duration of Quantum Dot were compared with that of traditional fluorochromes.
Methods:1) The pcDNA3.1/APP plasmid was gifted by Pro.Perez, University of Pittsburgh,USA.The Coordinate bas-mutations(G→T,A→C) and a accidental mutation(C→G) had been confirmed by DNA sequencing.In this experiment,APP was PCR-amplified,digested by HindⅢand XbaⅠ,and ligated into purified pcDNA3.1 vector.This recombinant plasmid was transfected into E.colo SH5αby means of heat shock.The recombinant plasmid was identified by PCR,and the positive cloning was identified by DNA sequence analysis.2) The construct was transfected into HEK293 cell lines,screened by G418.The interest protein(including APP and Aβ) were identified by immunofluorescence and Western blot.3) The toxicity of QDs-SA to HEK293 cells stably transfected pcDNA3.1/APP595/596 plasmid was investigated by cell morphology,MTT analysis and flow cytometry technology.4) With the help of Laser Scanning Confocal Microscope and Flow Cytometry,this kind of flurescence probe based on the QDs-SA had been used to detect APP and Aβproteins in HEK293 cells stably transfected pcDNA3.1/APP595/596 plasmid,and compared with conventional fluroimmunoassay.
Results:1)The positive cloning gene sequence were aligned to the complete gene sequence of APP by DNAStar software.The result confirmed that the accidental mutation(C→G) had been mutated back(G→C).The recombinant pcDNA3.1/APP595/596 plasmid was constructed successfully.2)The detection verified that recombinant plasmid group expressed APP protein,the specific protein bands was only detected in HEK293 Cells stably transfected pcDNA3.1/APP595/596 plasmid,but no expression was found in control groups.The immunofluorescence staining detection indicated APP expression in the plasma membrane,but the Aβmainly located in the endochylema near the cell membrane,and slight Aβexpression also located in the plasma membrane.3) The HEK293 cells were incubated with QDs-SA on different concentrations between 2.5nm and 25nm and lasted for 24 hours, there was no evident morphological deviations.The QDs-SA group's cell activity assayed by MTT had no any apparente difference compared to control group.Meanwhile,the difference of absorbance intensity among the different concentration groups had no any statistical significance(P＞0.05 ).We used AnnexinV/FITC staining flow cytometry to observe the percentage of apoptosis.The percentage of apoptosis had no any significant difference among groups(P＞0.05 ).4) under the stimulation by 388nm excitation light,The maximal Emission of the QDs-SA was 605±10nm,and the full width at half maximum＜35nm.The mean fluorescence intensity of QDs-SA was greater than the Cy3's under confocal fluorescence microscopy(P＜0.05) There was no notable quench of exciting quantum dots for 12 minutes,and the APP and Aβproteins stained by QDs-SA showed intensive orange red fluorescence under confocal fluorescence microscopy.The fluorescence intensity decreased nearly 27.87%、29.25%respectively.The other stained by Cy3 decreased 79.60%and 76.82%.The positive rate of APP staining had no significant difference between the QDs-SA and Cy3 by flow cytometry, but the Mean fluorescence intensity had statistical significance(P＜0.05).
Conclusions:1) The recombinant plasmid containing the APP 595/596 Coordinate bas-mutations was successfully constructed.Then the recombinant plasmid was stably transfected into HEK293 cells to construct the transgenic cellular model of Alzheimer's disease.2) The QDs-SA were biocompatible with HEK293 cells in appropriate concentration rang,which provided basis for further biological applications of QDs.3) The QDs-SA fluorescence probes can effectively recognize APP and Aβproteins and exhibited good sensitivity and exceptional photostability,suggesting that QDs-SA fluorescence probes could be a potential method in Aβdetection and offer a novel way for the diagnosis of Alzheimer's disease.
引文
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