發(fā)布時(shí)間：2018-01-13 22:00

  本文關(guān)鍵詞：熒光磁性納米粒子標(biāo)記骨髓間充質(zhì)干細(xì)胞靶向胃癌的研究　出處：《上海交通大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 骨髓間充質(zhì)干細(xì)胞 熒光磁性納米粒子 胃癌 靶向遷移

【摘要】：目的:(l)建立一種高效、簡(jiǎn)便的分離純化骨髓間充質(zhì)干細(xì)胞(bone mesenchymal stem cells,BMSCs)的方法;(2)明確熒光磁性納米粒子(fluorescent magnetic nanoparticles,FMNPs)標(biāo)記骨髓間充質(zhì)干細(xì)胞的適當(dāng)濃度;(3)檢驗(yàn)FMNPs體外標(biāo)記BMSCs的效果;(4)探討B(tài)MSCs靶向胃癌的遷移的能力。 方法:(1)利用貼壁培養(yǎng)法分離、純化大鼠的骨髓間充質(zhì)干細(xì)胞,流式細(xì)胞儀檢測(cè)骨髓間充質(zhì)干細(xì)胞CD29、CD90和CD45的表達(dá)情況,分別用成骨細(xì)胞和脂肪細(xì)胞誘導(dǎo)劑誘導(dǎo)干細(xì)胞檢測(cè)骨髓間充質(zhì)干細(xì)胞的分化能力;(2)體外用不同濃度的FMNPs標(biāo)記BMSCs,觀察不同濃度的FMNPs對(duì)細(xì)胞活力的影響,進(jìn)而選取最佳標(biāo)記的FMNPs濃度量;利用普魯士藍(lán)染色,激光共聚焦顯微鏡,核磁共振成像儀觀察熒光磁性納米粒子標(biāo)記的干細(xì)胞體外成像效果;(3)建立裸鼠的胃癌模型,應(yīng)用小動(dòng)物分子影像系統(tǒng)和核磁共振成像儀驗(yàn)證靜脈注射被標(biāo)記的BMSCs靶向胃癌的能力。 結(jié)果:(1)利用貼壁培養(yǎng)法分離、純化大鼠的骨髓間充質(zhì)干細(xì)胞,流式細(xì)胞儀檢測(cè)第三代BMSCs的CD29,CD90和CD45的陽(yáng)性表達(dá)率分別為84.69%,90.28%,0.72%,堿性磷酸酶染色和油紅O染色結(jié)果均表明骨髓間充質(zhì)干細(xì)胞成功分化為成骨細(xì)胞和脂肪細(xì)胞;(2)熒光磁性納米粒子與骨髓間充質(zhì)干細(xì)胞共同培養(yǎng),不同時(shí)間點(diǎn)檢測(cè)干細(xì)胞存活率,進(jìn)而選取熒光磁性納米粒子的最佳標(biāo)記濃度為50?g/mL。普魯士藍(lán)染色,激光共聚焦成像和核磁共振成像表明熒光磁性納米粒子體外成功標(biāo)記骨髓間充質(zhì)干細(xì)胞并且不影響細(xì)胞活性;(3)建立裸鼠的胃癌模型,胃癌細(xì)胞MGC803皮下注射入裸鼠體內(nèi),四周后篩選成瘤效果好的裸鼠胃癌模型,并將標(biāo)記后的干細(xì)胞尾靜脈注射入裸鼠體內(nèi),14天后用小動(dòng)物分子成像系統(tǒng)觀察到BMSCs遷移靶向到胃癌部位。隨后,處死裸鼠,取出心、肺、肝、脾、腎和腫瘤塊進(jìn)行器官離體熒光成像,結(jié)果顯示被標(biāo)記組分的腫瘤呈現(xiàn)出顯著的熒光信號(hào),除了肝(具有代謝異物的能力),其他臟器如心、脾、肺、腎都沒(méi)有熒光信號(hào)。對(duì)另一組FMNPs標(biāo)記的BMSCs移植后的胃癌裸鼠模型進(jìn)行MRI掃描,在腫瘤部位3.0 T的磁場(chǎng)強(qiáng)度下有清晰的磁共振信號(hào)。 結(jié)論:(l)貼壁篩選法操作簡(jiǎn)單,篩選出的細(xì)胞易于成活,是一種簡(jiǎn)便有效的方法;(2)適當(dāng)濃度FMNPs能夠高效標(biāo)記BMSCs; (3) BMSCs具有高度特異性地靶向胃癌的能力。 意義:本實(shí)驗(yàn)證明BMSCs具有靶向遷移到胃癌的能力。此外,BMSCs具有取材便捷、體外擴(kuò)增能力強(qiáng)、基因轉(zhuǎn)染效率高、轉(zhuǎn)染后穩(wěn)定表達(dá)、可自體回輸從而避免了免疫排斥反應(yīng)等特點(diǎn),被認(rèn)為是基因工程的理想種子細(xì)胞,這為以BMSCs為載體的胃癌靶向基因治療提供了理論依據(jù)。然而,目前關(guān)于BMSCs向胃癌細(xì)胞靶向遷移的分子機(jī)制尚未明確,有待于深入研究。
[Abstract]:Objective to establish an efficient and simple method for the isolation and purification of bone mesenchymal stem cells from bone marrow mesenchymal stem cells (BMSCs). (2) the fluorescent magnetic nanoparticles were identified as fluorescent magnetic nanoparticles. Proper concentration of bone marrow mesenchymal stem cells labeled with NPFM; (3) to test the effect of FMNPs labeling BMSCs in vitro; To explore the migration ability of BMSCs targeted gastric cancer. Methods Bone marrow mesenchymal stem cells (BMSCs) were isolated and purified by adherent culture. The expression of CD29 CD90 and CD45 in bone marrow mesenchymal stem cells (BMSCs) was detected by flow cytometry. The differentiation ability of bone marrow mesenchymal stem cells was detected by osteoblast and adipocyte inducer induced stem cells. (2) BMSCs were labeled with different concentrations of FMNPs in vitro. The effects of different concentrations of FMNPs on cell viability were observed, and the best FMNPs concentration was selected. Prussian blue staining, laser confocal microscopy and nuclear magnetic resonance imager were used to observe the imaging effect of stem cells labeled with fluorescent magnetic nanoparticles in vitro. The gastric cancer model of nude mice was established and the ability of intravenous injection of labeled BMSCs to target gastric cancer was verified by small animal molecular imaging system and nuclear magnetic resonance imager. Results the bone marrow mesenchymal stem cells of rats were purified by adherent culture and the CD29 of the third generation of BMSCs was detected by flow cytometry. The positive expression rates of CD90 and CD45 were 84.69% and 90.28%, respectively. Alkaline phosphatase staining and oil red O staining showed that bone marrow mesenchymal stem cells were successfully differentiated into osteoblasts and adipocytes. (2) fluorescence magnetic nanoparticles and bone marrow mesenchymal stem cells were co-cultured. The survival rate of stem cells was measured at different time points, and the optimal labeling concentration of fluorescent magnetic nanoparticles was 50? G / mL.Prussian blue staining laser confocal imaging and magnetic resonance imaging showed that fluorescent magnetic nanoparticles were successfully labeled with BMSCs in vitro without affecting cell viability. Gastric cancer model was established in nude mice. Gastric cancer cell MGC803 was injected subcutaneously into nude mice. The labeled stem cells were injected into the tail vein of nude mice for 14 days. The migration of BMSCs was observed to gastric carcinoma by small animal molecular imaging system. Then, the nude mice were killed and heart, lung, liver and spleen were removed. In vitro fluorescence imaging of the kidney and tumor mass showed that the labeled tumor showed significant fluorescence signals except the liver (with the ability to metabolize foreign bodies) and other organs such as heart spleen and lung. No fluorescence signal was found in the kidneys. MRI scanning was performed on another group of nude mice models of gastric cancer after BMSCs transplantation labeled with FMNPs. There were clear magnetic resonance signals at 3. 0 T magnetic field. Conclusion the adherent screening method is simple and easy to survive, so it is a simple and effective method. 2) appropriate concentration of FMNPs could effectively label BMSCs; BMSCs has a highly specific ability to target gastric cancer. Significance: this experiment proved that BMSCs has the ability of targeting to gastric cancer. In addition, BMSCs has the advantages of convenient material collection, strong ability of amplification in vitro, high efficiency of gene transfection, and stable expression after transfection. Autotransfusion can avoid the characteristics of immune rejection, so it is considered to be the ideal seed cells for genetic engineering, which provides a theoretical basis for targeted gene therapy of gastric cancer based on BMSCs. At present, the molecular mechanism of BMSCs targeted migration to gastric cancer cells has not been clarified, which needs further study.
【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R329

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本文編號(hào)：1420698