本文關(guān)鍵詞： 半導(dǎo)體量子點(diǎn)(半導(dǎo)體納米微晶體) 大鼠牙乳頭細(xì)胞 生物相容性 口腔 活細(xì)胞 免疫熒光 分子探針　出處：《重慶醫(yī)科大學(xué)》2008年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】： 目的:用最大發(fā)射波長(zhǎng)分別605nm、525nm的半導(dǎo)體量子點(diǎn)(semiconductor quantum dots QDs)分別與Smad4、Smad2單克隆抗體偶聯(lián)后制備成水溶性的分子探針。并檢測(cè)兩種量子點(diǎn)在偶聯(lián)前的光學(xué)性質(zhì)、對(duì)大鼠牙乳頭細(xì)胞(rat dental papillae cells,RDPCs)的生物相容性及偶聯(lián)后分子探針的光學(xué)性質(zhì)和生物識(shí)別特異性。 方法:①選擇剛出生3天的新生SD大鼠,取出磨牙牙胚,分離牙乳頭,用酶消化法培養(yǎng)SD大鼠牙乳頭細(xì)胞,用波形絲蛋白和細(xì)胞角蛋白免疫化學(xué)染色鑒定細(xì)胞來(lái)源,觀察細(xì)胞的生長(zhǎng)規(guī)律;②把605QDs、525QDs及兩種量子點(diǎn)等濃度的混合物按3種不同濃度與RDPCs共同培養(yǎng),觀察QDs對(duì)RDPCs生長(zhǎng)和形態(tài)的影響;③用MTT法檢測(cè)不同濃度的605QDs、525QDs及兩種量子等濃度的混合物對(duì)RDPCs的毒性作用。④用化學(xué)偶連法制備水溶性的605QDS-Smad4、525QDS-Smad2單抗熒光探針并純化;⑤測(cè)定605QDS-Smad4、525QDS-Smad2單抗熒光探針的吸收光譜、發(fā)射光譜并用激光共聚焦顯微鏡對(duì)605QDS-Smad4、525QDS-Smad2單抗熒光探針的光學(xué)性質(zhì)進(jìn)行檢測(cè);⑥TGF-β1和大鼠牙乳頭細(xì)胞共同孵育24h后,采用SP免疫細(xì)胞化學(xué)法和605QDS-Smad4、525QDS-Smad2單抗熒光探針直接免疫熒光成像法觀察比較Smad4、Smad2在大鼠牙乳頭細(xì)胞內(nèi)的分布,并檢測(cè)細(xì)胞內(nèi)605QDS-Smad4、525QDS-Smad2單抗熒光探針的光學(xué)性質(zhì)。 結(jié)果:①:體外培養(yǎng)的RDPCs生長(zhǎng)良好,波形絲蛋白染色陽(yáng)性而細(xì)胞角蛋白染色陰性。②不同濃度的605QDs、525QDs及兩種量子點(diǎn)的等濃度混合物對(duì)體外培養(yǎng)的RDPCs沒(méi)有毒性作用,對(duì)其生長(zhǎng)和形態(tài)也沒(méi)有影響。③605QDs、525QDs分別與Smad4、Smad2單抗通過(guò)共價(jià)結(jié)合形成穩(wěn)定的605QDs-Smad4、525QDs-Smad2單抗熒光探針,605QDs-Smad4、525QDs-Smad2單抗熒光探針對(duì)大鼠牙乳頭細(xì)胞內(nèi)Smad4分子仍具有特異性的識(shí)別能力;605QDs-Smad4、525QDs-Smad2單抗熒光探針仍具有QDS所具有的激發(fā)光譜寬,發(fā)射光譜窄,熒光度強(qiáng),光化學(xué)穩(wěn)定性好等光學(xué)特征。 結(jié)論:①605QDs、525QDs及兩種量子點(diǎn)等濃度的混合物在一定的濃度范圍內(nèi)對(duì)RPDCs具有良好的生物相容性,為605QDs、525QDs在活體生理?xiàng)l件下用于活細(xì)胞內(nèi)的多通道信號(hào)研究提供了科學(xué)的證據(jù)。②生物修飾的半導(dǎo)體量子點(diǎn)和單抗共價(jià)結(jié)合形成分子探針后仍具有特異免疫識(shí)別能力和獨(dú)特的光學(xué)性質(zhì),這為半導(dǎo)體量子點(diǎn)用于對(duì)活細(xì)胞內(nèi)蛋白質(zhì)等生物單分子進(jìn)行實(shí)時(shí)、原位、長(zhǎng)時(shí)間動(dòng)態(tài)視蹤檢測(cè)提供了科學(xué)依據(jù)和基礎(chǔ)。
[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.

【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類(lèi)號(hào)】：R346

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 馬金杰;CdTe量子點(diǎn)與生物大分子相互作用研究[D];華中農(nóng)業(yè)大學(xué);2009年

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本文編號(hào)：1451904