荧光磁性纳米粒子标记骨髓间充质干细胞靶向胃癌的研究

发布时间：2018-01-13 22:00

本文关键词：荧光磁性纳米粒子标记骨髓间充质干细胞靶向胃癌的研究　出处：《上海交通大学》2011年硕士论文　论文类型：学位论文

【摘要】：目的:(l)建立一种高效、简便的分离纯化骨髓间充质干细胞(bone mesenchymal stem cells,BMSCs)的方法;(2)明确荧光磁性纳米粒子(fluorescent magnetic nanoparticles,FMNPs)标记骨髓间充质干细胞的适当浓度;(3)检验FMNPs体外标记BMSCs的效果;(4)探讨BMSCs靶向胃癌的迁移的能力。方法:(1)利用贴壁培养法分离、纯化大鼠的骨髓间充质干细胞,流式细胞仪检测骨髓间充质干细胞CD29、CD90和CD45的表达情况,分别用成骨细胞和脂肪细胞诱导剂诱导干细胞检测骨髓间充质干细胞的分化能力;(2)体外用不同浓度的FMNPs标记BMSCs,观察不同浓度的FMNPs对细胞活力的影响,进而选取最佳标记的FMNPs浓度量;利用普鲁士蓝染色,激光共聚焦显微镜,核磁共振成像仪观察荧光磁性纳米粒子标记的干细胞体外成像效果;(3)建立裸鼠的胃癌模型,应用小动物分子影像系统和核磁共振成像仪验证静脉注射被标记的BMSCs靶向胃癌的能力。结果:(1)利用贴壁培养法分离、纯化大鼠的骨髓间充质干细胞,流式细胞仪检测第三代BMSCs的CD29,CD90和CD45的阳性表达率分别为84.69%,90.28%,0.72%,碱性磷酸酶染色和油红O染色结果均表明骨髓间充质干细胞成功分化为成骨细胞和脂肪细胞;(2)荧光磁性纳米粒子与骨髓间充质干细胞共同培养,不同时间点检测干细胞存活率,进而选取荧光磁性纳米粒子的最佳标记浓度为50?g/mL。普鲁士蓝染色,激光共聚焦成像和核磁共振成像表明荧光磁性纳米粒子体外成功标记骨髓间充质干细胞并且不影响细胞活性;(3)建立裸鼠的胃癌模型,胃癌细胞MGC803皮下注射入裸鼠体内,四周后筛选成瘤效果好的裸鼠胃癌模型,并将标记后的干细胞尾静脉注射入裸鼠体内,14天后用小动物分子成像系统观察到BMSCs迁移靶向到胃癌部位。随后,处死裸鼠,取出心、肺、肝、脾、肾和肿瘤块进行器官离体荧光成像,结果显示被标记组分的肿瘤呈现出显著的荧光信号,除了肝(具有代谢异物的能力),其他脏器如心、脾、肺、肾都没有荧光信号。对另一组FMNPs标记的BMSCs移植后的胃癌裸鼠模型进行MRI扫描,在肿瘤部位3.0 T的磁场强度下有清晰的磁共振信号。结论:(l)贴壁筛选法操作简单,筛选出的细胞易于成活,是一种简便有效的方法;(2)适当浓度FMNPs能够高效标记BMSCs; (3) BMSCs具有高度特异性地靶向胃癌的能力。意义:本实验证明BMSCs具有靶向迁移到胃癌的能力。此外,BMSCs具有取材便捷、体外扩增能力强、基因转染效率高、转染后稳定表达、可自体回输从而避免了免疫排斥反应等特点,被认为是基因工程的理想种子细胞,这为以BMSCs为载体的胃癌靶向基因治疗提供了理论依据。然而,目前关于BMSCs向胃癌细胞靶向迁移的分子机制尚未明确,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.
【学位授予单位】：上海交通大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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