碲化镉量子点对大鼠骨髓间充质干细胞增殖与向神经细胞分化的影响

发布时间：2018-05-20 01:46

本文选题：碲化镉量子点 + 骨髓间充质干细胞　；参考：《吉林大学》2013年硕士论文

【摘要】：碲化镉量子点（CdTe QDs）是一种纳米材料，具有独特光学特性，在活细胞标记方向具有广阔的应用前景。不同粒径的CdTe QDs可以在同一束激发光照射下发出不同颜色，，不同波长的发射光，而且发射光谱窄，易于区分辨认。而且CdTe合成较简便，产率高，在包被材料的保护下稳定性更强，是非常理想的活细胞标记物。然而，CdTe QDs中含有重金属Cd，可能对机体和细胞产生毒副作用，因此许多研究都探索了CdTe QDs对于机体和细胞的毒性，但已有的体外实验研究大多都是以非哺乳动物细胞系为实验对象，主要探讨其是对于细胞增殖的影响，对于细胞生理、生化及生物学特征研究甚少。BMSCs是一类具有多项分化潜能的细胞，能够在体外条件下诱导分化为神经细胞，成骨细胞，脂肪细胞，心肌细胞等，在疾病治疗方面有巨大潜力。但目前干细胞治疗技术大多处于试验阶段，活细胞荧光标记示踪技术对于追踪干细胞在动物体内的移行和作用机制非常重要。因此，鉴于CdTe QDs作为活细胞标记物的优势，如果能将其用于BMSCs活细胞标记是非常有意义的，但是标记活细胞用于动物体内的示踪，仅仅通过细胞毒作用确定标记剂量是不完善的，还应该观察对于干细胞分化的影响。本研究以大鼠骨髓间充质干细胞（BMSCs）为对象，利用BMSCs具有多向分化潜能的特点，检测CdTe QDs对于大鼠BMSCs的增殖以及神经分化的影响，为寻找CdTeQDs标记活细胞的安全、合理的应用剂量提供重要依据。本实验首先通过荧光光谱结果和透射电镜观察CdTe QDs在DMEM/F-12溶液中的分散和聚合情况。采用贴壁法分离并扩增培养大鼠BMSCs，并进行传代扩增。使用地塞米松，β甘油磷酸钠，维生素C诱导其分化为成骨细胞，验证其多向分化潜能。将BMSCs暴露于不同浓度的CdTe QDs下（0.00pM、3.05pM、6.10pM、12.21pM、24.41pM、48.83pM、97.66pM、195.31pM、390.63pM和781.25pM），用MTT法检测细胞活力，观察CdTe QDs对BMSCs增殖能力的影响。BMSCs暴露于不同浓度的（3.05pM、6.10pM、12.21pM、24.41pM和48.83pM）CdTe QDs48h，然后再对其进行向神经细胞分化诱导（bFGF和EGF预诱导3d，维甲酸诱导7d），用免疫细胞化学法和免疫细胞荧光法检测神经元的特异标志蛋白MAP-2、NeuN、NSE和星形胶质特异标志蛋白GFAP的表达情况，用RT-PCR法检测NSE，GFAP基因的转录水平，从而评价CdTe QDs对BMSCs向神经细胞分化能力的影响。结果表明荧光光谱结果和透射电镜结果显示，CdTe QDs在DMEM/F-12溶液中分散良好，未发生聚合或降解。经过7d的原代培养，大鼠BMSCs生长状态良好，增殖旺盛。诱导向成骨细胞分化以后，茜素红染色结果显示出明显的钙化结节，表明分化为成骨细胞。MTT结果显示，在CdTe QDs暴露24h的情况下，大于48.83pM的暴露浓度能够对BMSCs的增殖产生明显抑制（P＜0.05），而在暴露48h的情况下，24.41pM的暴露浓度就能抑制BMSCs的增殖，暴露时间越长，暴露剂量越大，CdTe QDs的毒性就越大。CdTe QDs暴露48h，诱导BMSCs分化成神经细胞7d后，免疫细胞化学和免疫细胞荧光结果显示，MAP-2，NeuN，NSE，GFAP四种蛋白在12.21pM暴露浓度下均发生表达水平显著下降（P＜0.05）。RT-PCR结果显示，NSE和GFAP两种基因的转录水平同样在12.21pM浓度下发生显著下降（P＜0.05）。结论：在一定剂量范围内，CdTe QDs对大鼠BMSCs细胞活力具有抑制作用，表明其具有细胞毒性作用。在一定剂量范围内，CdTe QDs影响大鼠BMSCs分化的神经细胞的特异标志蛋白MAP-2、NeuN、NSE和GFAP表达减少。在一定剂量范围内，CdTe QDs引起大鼠BMSCs分化的神经细胞的神经特异标志NSE和GFAP mRNA表达量减少。CdTe QDs在未影响细胞活力的剂量下，可对BMSCs的分化产生影响。
[Abstract]:Cadmium telluride quantum dot (CdTe QDs) is a kind of nanomaterial. It has unique optical properties and has a broad application prospect in the direction of living cell marking. The CdTe QDs with different particle sizes can emit different colors and wavelengths under the same beam illuminating light, and the emission spectrum is narrow, and it is easy to distinguish and identify. And the CdTe synthesis is simple and simple. However, CdTe QDs contains heavy metal Cd, which may have toxic side effects on the body and cells, so many studies have explored the toxicity of CdTe QDs to the body and cells, but most of the existing experimental studies in vitro are non mammals. The cell line is the experimental object. It mainly discusses its effect on cell proliferation. The study of cell physiology, biochemistry and biological characteristics is very small..BMSCs is a kind of cells with multiple differentiation potential. It can be induced to differentiate into nerve cells, osteoblasts, fat cells and cardiac myocytes in vitro. But at present, most of the stem cell therapies are in the experimental stage, and the fluorescence labeling technique of living cells is very important for tracking the migration and action of stem cells in animals. Therefore, in view of the advantages of CdTe QDs as a living cell marker, it is very meaningful if it can be used to mark BMSCs living cell markers, but the markers are marked. The use of living cells to trace in animals, it is not perfect to determine the labeling dose only by cytotoxic effect. It should also be observed for the effect of stem cell differentiation. In this study, rat bone marrow mesenchymal stem cells (BMSCs) were used to detect the proliferation of CdTe QDs to BMSCs in rats and to detect the proliferation of BMSCs in rats. The effect of differentiation provides important basis for finding the safety and reasonable dosage of CdTeQDs labelled living cells.
In this experiment, the dispersion and polymerization of CdTe QDs in DMEM/F-12 solution were observed by fluorescence spectrum and transmission electron microscopy. The rat BMSCs was isolated and amplified by adherent method and amplified. Using dexamethasone, sodium beta glycerphosphate and vitamin C to induce its differentiation into osteoblasts to verify its multidirectional differentiation potential. BMSCs Exposure to different concentrations of CdTe QDs (0.00pM, 3.05pM, 6.10pM, 12.21pM, 24.41pM, 48.83pM, 97.66pM, 195.31pM, 390.63pM and 781.25pM). Nerve cell differentiation induced (bFGF and EGF preinduced 3D, retinoic acid induced 7D). The expression of specific marker protein MAP-2, NeuN, NSE and astrocyte specific marker protein GFAP of neurons was detected by immunocytochemistry and immunocytofluorescence, and the transcriptional level of NSE and GFAP genes was detected by RT-PCR method. The effect of cell differentiation ability.
The results showed that the results of fluorescence and transmission electron microscopy showed that CdTe QDs was well dispersed in DMEM/F-12 solution and did not polymerize or degrade. After the primary culture of 7D, the growth state of BMSCs was good and the proliferation was strong. After the differentiation of the osteoblasts, the alizarin red stained fruit showed obvious calcified nodules, indicating that the differentiation was osteogenesis. The results of cell.MTT showed that the exposure concentration greater than 48.83pM could significantly inhibit the proliferation of BMSCs (P < 0.05) when CdTe QDs was exposed to 24h (P < 0.05). The exposure concentration of 24.41pM could inhibit the proliferation of BMSCs in the case of 48h, and the longer the exposure time, the greater the exposure dose, the greater the toxicity of CdTe QDs. The results of immunocytochemistry and immunofluorescence showed that the expression level of four proteins of MAP-2, NeuN, NSE and GFAP decreased significantly (P < 0.05) at 12.21pM exposure (P < 0.05). The results of MAP-2, NeuN, NSE, and GFAP showed that the transcriptional level of NSE and GFAP two genes decreased significantly at the 12.21pM concentration (0.05).
Conclusion: in a certain dose range, CdTe QDs has a inhibitory effect on the viability of rat BMSCs cells, indicating that it has a cytotoxic effect. In a certain dose range, CdTe QDs affects the specific marker protein MAP-2, NeuN, NSE and GFAP expression of BMSCs differentiated neurons in rat BMSCs. In a certain dose range, CdTe QDs induces rats. The neuronal specific markers of differentiated nerve cells, NSE and GFAP mRNA, decrease.CdTe QDs at the dose of cell viability, which can affect the differentiation of BMSCs.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R114

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