半导体量子点单克隆抗体荧光探针的制备及对细胞内蛋白质分子的检测

发布时间：2018-01-21 15:53

本文关键词： 半导体量子点(半导体纳米微晶体) 大鼠牙乳头细胞生物相容性口腔活细胞免疫荧光分子探针　出处：《重庆医科大学》2008年硕士论文　论文类型：学位论文

【摘要】： 目的:用最大发射波长分别605nm、525nm的半导体量子点(semiconductor quantum dots QDs)分别与Smad4、Smad2单克隆抗体偶联后制备成水溶性的分子探针。并检测两种量子点在偶联前的光学性质、对大鼠牙乳头细胞(rat dental papillae cells,RDPCs)的生物相容性及偶联后分子探针的光学性质和生物识别特异性。方法:①选择刚出生3天的新生SD大鼠,取出磨牙牙胚,分离牙乳头,用酶消化法培养SD大鼠牙乳头细胞,用波形丝蛋白和细胞角蛋白免疫化学染色鉴定细胞来源,观察细胞的生长规律;②把605QDs、525QDs及两种量子点等浓度的混合物按3种不同浓度与RDPCs共同培养,观察QDs对RDPCs生长和形态的影响;③用MTT法检测不同浓度的605QDs、525QDs及两种量子等浓度的混合物对RDPCs的毒性作用。④用化学偶连法制备水溶性的605QDS-Smad4、525QDS-Smad2单抗荧光探针并纯化;⑤测定605QDS-Smad4、525QDS-Smad2单抗荧光探针的吸收光谱、发射光谱并用激光共聚焦显微镜对605QDS-Smad4、525QDS-Smad2单抗荧光探针的光学性质进行检测;⑥TGF-β1和大鼠牙乳头细胞共同孵育24h后,采用SP免疫细胞化学法和605QDS-Smad4、525QDS-Smad2单抗荧光探针直接免疫荧光成像法观察比较Smad4、Smad2在大鼠牙乳头细胞内的分布,并检测细胞内605QDS-Smad4、525QDS-Smad2单抗荧光探针的光学性质。结果:①:体外培养的RDPCs生长良好,波形丝蛋白染色阳性而细胞角蛋白染色阴性。②不同浓度的605QDs、525QDs及两种量子点的等浓度混合物对体外培养的RDPCs没有毒性作用,对其生长和形态也没有影响。③605QDs、525QDs分别与Smad4、Smad2单抗通过共价结合形成稳定的605QDs-Smad4、525QDs-Smad2单抗荧光探针,605QDs-Smad4、525QDs-Smad2单抗荧光探针对大鼠牙乳头细胞内Smad4分子仍具有特异性的识别能力;605QDs-Smad4、525QDs-Smad2单抗荧光探针仍具有QDS所具有的激发光谱宽,发射光谱窄,荧光度强,光化学稳定性好等光学特征。结论:①605QDs、525QDs及两种量子点等浓度的混合物在一定的浓度范围内对RPDCs具有良好的生物相容性,为605QDs、525QDs在活体生理条件下用于活细胞内的多通道信号研究提供了科学的证据。②生物修饰的半导体量子点和单抗共价结合形成分子探针后仍具有特异免疫识别能力和独特的光学性质,这为半导体量子点用于对活细胞内蛋白质等生物单分子进行实时、原位、长时间动态视踪检测提供了科学依据和基础。
[Abstract]:Objective: 605nm with the maximum emission wavelength of semiconductor quantum dots, 525nm (semiconductor quantum dots QDs) respectively with Smad4, Smad2 monoclonal antibody was prepared by coupling water soluble molecular probe. And the detection of two kinds of quantum dots in the optical properties of the coupling, of rat tooth milk (rat dental papillae cells cell RDPCs), compatible optical properties and biological identification of specific molecular probes and biological coupling.
Methods: Ninety SD rats born just 3 days, remove the molar, separation of dental papilla and dental papilla cells cultured in SD rats by enzymatic digestion with vimentin and cytokeratin immunohistochemical staining to identify the cell source, observation of cell growth; the mixture of 525QDs and 605QDs, two a quantum dot concentration by co culture of 3 different concentration and RDPCs, the effect of QDs on the growth and morphology of RDPCs; the detection of different concentrations of 605QDs by MTT method. The toxicity of mixture of 525QDs and two kinds of quantum concentration of RDPCs. The chemical coupling preparation of water soluble 605QDS-Smad4525QDS-Smad2 monoclonal antibody fluorescent probe the absorption spectra of the determination and purification; monoclonal antibody 605QDS-Smad4525QDS-Smad2 fluorescence probe, emission spectroscopy and laser confocal microscopy on optical properties of 605QDS-Smad4525QDS-Smad2 monoclonal antibody fluorescent probe for detection 6; TGF- beta 1 and rat dental papilla cells were incubated with 24h after SP by immunocytochemical staining and 605QDS-Smad4525QDS-Smad2 monoclonal antibody fluorescent probe direct immunofluorescence imaging method to observe and compare the distribution of Smad4, Smad2 in rat dental papilla cells, and the intracellular 605QDS-Smad4525QDS-Smad2 monoclonal antibody fluorescent probe optical properties.
Results: in vitro cultured RDPCs grew well, positive for vimentin staining and cytokeratin staining was negative. The different concentrations of 605QDs, 525QDs and two kinds of quantum dots concentration mixture has no toxic effect on the cultured RDPCs, there is no impact on its growth and morphology. The 605QDs, 525QDs and Smad4 respectively. Smad2, formed by covalent binding of monoclonal antibody 605QDs-Smad4525QDs-Smad2 monoclonal antibody fluorescent probe stable, 605QDs-Smad4525QDs-Smad2 monoclonal antibody fluorescent probe has specificity for dental papilla cell of rat Smad4 molecular recognition ability; 605QDs-Smad4525QDs-Smad2 monoclonal antibody fluorescent probe is QDS with the broad excitation spectrum, narrow emission spectrum, fluorescence intensity, good optical characteristics. The photochemical stability
Conclusion: 605QDs, a mixture of 525QDs and two kinds of quantum dots concentration has good biocompatibility, 605QDs to RPDCs in a certain concentration range, 525QDs in vivo physiological conditions for providing scientific evidence of multi-channel signal within a living cell. The semiconductor quantum dots and the covalent modification of biological monoclonal antibody with the formation of molecular probe has specific immune recognition ability and unique optical properties, such as semiconductor quantum dots for real time, on live cells such as protein molecules in situ, long time dynamic visual tracking detection provides a scientific basis.

【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R346

【引证文献】