本文關(guān)鍵詞： 骨髓間充質(zhì)干細(xì)胞 超順磁性氧化鐵 DAPI CM-DiI 標(biāo)記　出處：《山西醫(yī)科大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

【摘要】：目的：骨髓間充質(zhì)干細(xì)胞(Bone marrow mesenchymal stem cells,BMSCs)已經(jīng)成為組織工程中重要的的種子細(xì)胞,在多種疾病的臨床治療中具有廣闊的應(yīng)用前景。而若想深入研究BMSCs的存活、增殖、分化及在體內(nèi)的遷移、歸巢情況,首先需要解決如何高效、安全地標(biāo)記BMSCs的問題。本文通過觀察SPIO、DAPI和CM-DiI對家兔BMSCs的聯(lián)合標(biāo)記效果及其對細(xì)胞存活、增殖以及分化的影響,為BMSCs的活體示蹤研究奠定理論及實(shí)驗(yàn)基礎(chǔ)。 方法：分離培養(yǎng)家兔骨髓間充質(zhì)干細(xì)胞,用SPIO、DAPI和CM-DiI聯(lián)合標(biāo)記后,采用普魯士藍(lán)染色法檢測SPIO標(biāo)記率,熒光顯微鏡檢測熒光標(biāo)記率,透射電子顯微鏡觀察細(xì)胞的超微結(jié)構(gòu)及SPIO在細(xì)胞內(nèi)的分布,臺盼藍(lán)拒染法檢測細(xì)胞活力,MTT法檢測細(xì)胞增殖力,并使用地塞米松、胰島素、3-異丁基-1-甲基黃嘌呤、吲哚美辛體外誘導(dǎo)標(biāo)記細(xì)胞向脂肪細(xì)胞分化并用透射電子顯微鏡鑒定誘導(dǎo)后細(xì)胞,使用5-氮雜胞苷體外誘導(dǎo)標(biāo)記細(xì)胞向心肌細(xì)胞分化并用免疫熒光鑒定誘導(dǎo)后細(xì)胞。 結(jié)果：SPIO、DAPI和CM-DiI在體外對家兔BMSCs的聯(lián)合標(biāo)記率幾乎達(dá)100%；透射電鏡下可見電子密度高的SPIO顆粒主要分布于溶酶體、線粒體內(nèi)部及胞內(nèi)其他膜性結(jié)構(gòu)上,細(xì)胞核內(nèi)未見分布,且聯(lián)合標(biāo)記BMSCs的超微結(jié)構(gòu)顯示清晰、形態(tài)保存良好；臺盼藍(lán)拒染法顯示聯(lián)合標(biāo)記BMSCs組的細(xì)胞活力與未標(biāo)記BMSCs組相比差異無統(tǒng)計學(xué)意義(P0.05)；MTT法檢測發(fā)現(xiàn)聯(lián)合標(biāo)記BMSCs組的細(xì)胞增殖力與未標(biāo)記BMSCs組相比差異無統(tǒng)計學(xué)意義(P0.05)；聯(lián)合標(biāo)記BMSCs經(jīng)地塞米松、胰島素、3-異丁基-1-甲基黃嘌呤、吲哚美辛體外誘導(dǎo)后,透射電鏡下可見胞漿內(nèi)出現(xiàn)脂滴；聯(lián)合標(biāo)記BMSCs經(jīng)5-氮雜胞苷體外誘導(dǎo)后,免疫熒光鑒定心肌特異性肌鈣蛋白T(cTnT)陽性。 結(jié)論：SPIO、DAPI和CM-DiI對家兔骨髓間充質(zhì)干細(xì)胞的聯(lián)合標(biāo)記率高,并對其存活、增殖以及分化無影響。
[Abstract]:Objective: bone marrow mesenchymal stem cells (BMSCs) have become important seed cells in tissue engineering and have wide application prospects in clinical treatment of various diseases. In the process of differentiation, migration in vivo and homing, we need to solve the problem of how to label BMSCs efficiently and safely. In this paper, we observed the co-labeling effect of SPION DAPI and CM-DiI on rabbit BMSCs and its effects on cell survival, proliferation and differentiation. It lays a theoretical and experimental foundation for the study of BMSCs in vivo. Methods: rabbit bone marrow mesenchymal stem cells (BMSCs) were isolated and cultured. After labeled with SPIO-DAPI and CM-DiI, the labeling rate of SPIO was detected by Prussian blue staining and the fluorescence labeling rate was detected by fluorescence microscope. Transmission electron microscopy (TEM) was used to observe the ultrastructure of cells and the distribution of SPIO in the cells. The viability of cells was detected by trypan blue exclusion assay. The proliferation of cells was detected by using dexamethasone, insulin 3-isobutyl-1-methylxanthine, dexamethasone, insulin 3-isobutyl-1-methylxanthine. Indomethacin induced the differentiation of labeled cells into adipocytes and identified them by transmission electron microscopy. 5-azacytidine was used to induce the differentiation of labeled cells into cardiomyocytes in vitro and immunofluorescence was used to identify the induced cells. Results the combined labeling rate of BMSCs in rabbit with CM-DiI was almost 100% in vitro, and the SPIO particles with high electron density were mainly distributed in lysosome, mitochondria and other membranous structures in mitochondria, but not in nucleus under transmission electron microscope. The ultrastructure of BMSCs was clear and the morphology was well preserved. Trypan blue exclusion assay showed that there was no significant difference in cell viability between the combined labeled BMSCs group and the unlabeled BMSCs group. It was found that there was no significant difference in cell proliferation between the combined labeled BMSCs group and the unlabeled BMSCs group. Combined labeling of BMSCs by dexamethasone, After induction of insulin 3-isobutyl -1-methylxanthine and indomethacin in vitro, lipid droplets in the cytoplasm were observed under transmission electron microscope, and BMSCs labeled by 5-azacytidine was induced in vitro. The positive expression of cardiac troponin TcTnT was identified by immunofluorescence. Conclusion the combined labeling rate of bone marrow mesenchymal stem cells (BMSCs) in rabbits was higher than that of CM-DiI, and had no effect on survival, proliferation and differentiation.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R329

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