發(fā)布時(shí)間：2018-05-20 01:46

  本文選題：碲化鎘量子點(diǎn) + 骨髓間充質(zhì)干細(xì)胞�。� 參考：《吉林大學(xué)》2013年碩士論文

【摘要】：碲化鎘量子點(diǎn)（CdTe QDs）是一種納米材料，具有獨(dú)特光學(xué)特性，在活細(xì)胞標(biāo)記方向具有廣闊的應(yīng)用前景。不同粒徑的CdTe QDs可以在同一束激發(fā)光照射下發(fā)出不同顏色，，不同波長的發(fā)射光，而且發(fā)射光譜窄，易于區(qū)分辨認(rèn)。而且CdTe合成較簡(jiǎn)便，產(chǎn)率高，在包被材料的保護(hù)下穩(wěn)定性更強(qiáng)，是非常理想的活細(xì)胞標(biāo)記物。然而，CdTe QDs中含有重金屬Cd，可能對(duì)機(jī)體和細(xì)胞產(chǎn)生毒副作用，因此許多研究都探索了CdTe QDs對(duì)于機(jī)體和細(xì)胞的毒性，但已有的體外實(shí)驗(yàn)研究大多都是以非哺乳動(dòng)物細(xì)胞系為實(shí)驗(yàn)對(duì)象，主要探討其是對(duì)于細(xì)胞增殖的影響，對(duì)于細(xì)胞生理、生化及生物學(xué)特征研究甚少。BMSCs是一類具有多項(xiàng)分化潛能的細(xì)胞，能夠在體外條件下誘導(dǎo)分化為神經(jīng)細(xì)胞，成骨細(xì)胞，脂肪細(xì)胞，心肌細(xì)胞等，在疾病治療方面有巨大潛力。但目前干細(xì)胞治療技術(shù)大多處于試驗(yàn)階段，活細(xì)胞熒光標(biāo)記示蹤技術(shù)對(duì)于追蹤干細(xì)胞在動(dòng)物體內(nèi)的移行和作用機(jī)制非常重要。因此，鑒于CdTe QDs作為活細(xì)胞標(biāo)記物的優(yōu)勢(shì)，如果能將其用于BMSCs活細(xì)胞標(biāo)記是非常有意義的，但是標(biāo)記活細(xì)胞用于動(dòng)物體內(nèi)的示蹤，僅僅通過細(xì)胞毒作用確定標(biāo)記劑量是不完善的，還應(yīng)該觀察對(duì)于干細(xì)胞分化的影響。本研究以大鼠骨髓間充質(zhì)干細(xì)胞（BMSCs）為對(duì)象，利用BMSCs具有多向分化潛能的特點(diǎn)，檢測(cè)CdTe QDs對(duì)于大鼠BMSCs的增殖以及神經(jīng)分化的影響，為尋找CdTeQDs標(biāo)記活細(xì)胞的安全、合理的應(yīng)用劑量提供重要依據(jù)。 本實(shí)驗(yàn)首先通過熒光光譜結(jié)果和透射電鏡觀察CdTe QDs在DMEM/F-12溶液中的分散和聚合情況。采用貼壁法分離并擴(kuò)增培養(yǎng)大鼠BMSCs，并進(jìn)行傳代擴(kuò)增。使用地塞米松，β甘油磷酸鈉，維生素C誘導(dǎo)其分化為成骨細(xì)胞，驗(yàn)證其多向分化潛能。將BMSCs暴露于不同濃度的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法檢測(cè)細(xì)胞活力，觀察CdTe QDs對(duì)BMSCs增殖能力的影響。BMSCs暴露于不同濃度的（3.05pM、6.10pM、12.21pM、24.41pM和48.83pM）CdTe QDs48h，然后再對(duì)其進(jìn)行向神經(jīng)細(xì)胞分化誘導(dǎo)（bFGF和EGF預(yù)誘導(dǎo)3d，維甲酸誘導(dǎo)7d），用免疫細(xì)胞化學(xué)法和免疫細(xì)胞熒光法檢測(cè)神經(jīng)元的特異標(biāo)志蛋白MAP-2、NeuN、NSE和星形膠質(zhì)特異標(biāo)志蛋白GFAP的表達(dá)情況，用RT-PCR法檢測(cè)NSE，GFAP基因的轉(zhuǎn)錄水平，從而評(píng)價(jià)CdTe QDs對(duì)BMSCs向神經(jīng)細(xì)胞分化能力的影響。 結(jié)果表明熒光光譜結(jié)果和透射電鏡結(jié)果顯示，CdTe QDs在DMEM/F-12溶液中分散良好，未發(fā)生聚合或降解。經(jīng)過7d的原代培養(yǎng)，大鼠BMSCs生長狀態(tài)良好，增殖旺盛。誘導(dǎo)向成骨細(xì)胞分化以后，茜素紅染色結(jié)果顯示出明顯的鈣化結(jié)節(jié)，表明分化為成骨細(xì)胞。MTT結(jié)果顯示，在CdTe QDs暴露24h的情況下，大于48.83pM的暴露濃度能夠?qū)MSCs的增殖產(chǎn)生明顯抑制（P＜0.05），而在暴露48h的情況下，24.41pM的暴露濃度就能抑制BMSCs的增殖，暴露時(shí)間越長，暴露劑量越大，CdTe QDs的毒性就越大。CdTe QDs暴露48h，誘導(dǎo)BMSCs分化成神經(jīng)細(xì)胞7d后，免疫細(xì)胞化學(xué)和免疫細(xì)胞熒光結(jié)果顯示，MAP-2，NeuN，NSE，GFAP四種蛋白在12.21pM暴露濃度下均發(fā)生表達(dá)水平顯著下降（P＜0.05）。RT-PCR結(jié)果顯示，NSE和GFAP兩種基因的轉(zhuǎn)錄水平同樣在12.21pM濃度下發(fā)生顯著下降（P＜0.05）。 結(jié)論：在一定劑量范圍內(nèi)，CdTe QDs對(duì)大鼠BMSCs細(xì)胞活力具有抑制作用，表明其具有細(xì)胞毒性作用。在一定劑量范圍內(nèi)，CdTe QDs影響大鼠BMSCs分化的神經(jīng)細(xì)胞的特異標(biāo)志蛋白MAP-2、NeuN、NSE和GFAP表達(dá)減少。在一定劑量范圍內(nèi)，CdTe QDs引起大鼠BMSCs分化的神經(jīng)細(xì)胞的神經(jīng)特異標(biāo)志NSE和GFAP mRNA表達(dá)量減少。CdTe QDs在未影響細(xì)胞活力的劑量下，可對(duì)BMSCs的分化產(chǎn)生影響。
[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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R114

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