阳离子脂质体和慢病毒载体转染MSCs的比较研究
发布时间：2018-10-29 13:24
【摘要】：干细胞疗法为组织修复和再生医学开辟了新的道路。骨髓间充质干细胞(Mesenchymal stem cells, MSCs)是一种重要的成体干细胞,具有自我更新的特性,并且具有向多种细胞分化的能力,例如脂肪细胞、软骨细胞和成骨细胞。由于这些内在的特性,MSCs被认为再生医学的理想细胞来源。但是,体外的培养条件以及细胞培养周期的延长会影响MSCs的多潜能性和表型。为了解决这些问题并增加治疗的成功率,MSCs的基因修饰开始成为新的研究途径。各种各样的病毒以及非病毒转染方法不断进行优化,目的都是使目的基因理想表达。病毒载体的转染方法具有较高的转染率和较长时间的基因表达,但是缺点也比较明显,例如细胞毒性、免疫原性、致癌性、低细胞特异性、价格昂贵以及无法转染较长基因序列等。非病毒载体的转染方法虽然转染率较低,基因表达时间较短,然而,具有安全、可转染长基因序列、低毒性、易操作、可通过组织或细胞特异性配体的修饰来操控靶基因的运输等优点。基因修饰的MSCs使干细胞治疗的优势进一步提升。通过转染相应的基因,可以诱导MSCs向特定细胞系分化。在基因修饰后,MSCs同样可以成为基因或药物的载体。而且经过基因修饰的MSCs无论在体内还是体外都可以使用荧光蛋白来定位。所以,基因修饰成为了研究分子生物学机制的有力工具,从而促进了MSCs在临床中的应用。 目的： 1.通过阳离子脂质体载体介导GFP基因感染大鼠骨髓间充质干细胞,探索非病毒转染技术,获得最佳阳离子脂质体/DNA比。 2.通过慢病毒载体介导GFP基因感染大鼠骨髓间充质干细胞,探索慢病毒转染技术,获得最佳MOI值。 方法： 1.本实验使用全骨髓贴壁法分离提取骨髓间充质干细胞,使用流式细胞仪检测细胞表面抗原CD29. CD45和CD90的表达。使用骨诱导培养液培养骨髓间充质干细胞,培养7d后进行碱性磷酸酶成骨鉴定。 2.设置不同阳离子脂质体含量(0.2μl、0.3μl、0.4μl)及不同质粒DNA含量(60ng、70ng.80ng)共9组,进行非病毒转染MSCs,使用荧光显微镜观察目的基因表达情况和细胞状态筛选最佳阳离子脂质体/DNA比。 3.构建慢病毒载体,使用不同MOI值,设置MOI=0,10,20,50,100,200,400共7组,对MSCs进行转染,使用荧光显微镜观察目的基因表达情况和细胞状态筛选最佳MOI值。 结果： 1.使用全骨髓法成功提取MSCs,倒置显微镜下观察MSCs贴壁生长,流式细胞仪检测第三代MSCs稳定均一表达CD29. CD90,几乎不表达CD45. MSCs经成骨诱导液诱导后7d,碱性磷酸酶检测显示促分化组ALP分泌明显高于未分化组,继续培养至10d后可见钙结节形成。 2.使用不同组别阳离子脂质体转染MSCs72h后,荧光显微镜下可见阳离子脂质体含量0.3μl与质粒DNA含量80ng组转染效率相对较高,细胞状态良好。 3.使用不同MOI组别慢病毒转染MSCs,分别于3d、7d和10d荧光显微镜下观察,可见MOI=20时转染效率相对较高,细胞状态较好。 结论： 阳离子脂质体作为载体转染骨髓间充质干细胞,较低的转染率仍然是非病毒转染的主要缺点,但是较低的细胞毒性和损伤有利于对转染细胞的进一步利用。病毒转染具有较高的转染率,但是其细胞毒性不容忽视。通过对骨髓间充质干细胞的基因修饰,为实现干细胞治疗的临床应用提供了可能性。
[Abstract]:Stem cell therapy has opened up new roads for tissue repair and regenerative medicine. Bone marrow mesenchymal stem cells (MSCs) are important adult stem cells, have self-renewing characteristics, and have the ability to differentiate into a variety of cells, such as fat cells, chondrocytes and osteoblasts. Due to these inherent characteristics MSCs are believed to be the ideal cell source for regenerative medicine. However, the culture conditions in vitro and the extension of cell culture cycles may affect the multipotential and phenotype of MSCs. In order to solve these problems and increase the success rate of treatment, the gene modification of MSCs became a new approach. All kinds of viruses, as well as non-viral transfection methods, are constantly optimized for the purpose of making the target gene ideal. The transfection methods of viral vectors have higher transfection efficiency and longer gene expression, but the disadvantages are also obvious, such as cytotoxicity, immunogenicity, carcinogenicity, low cell specificity, high price, and inability to transfect longer gene sequences and the like. Although the transfection rate of the non-viral vector is low, the gene expression time is short, however, the transfection method has the advantages of being safe, can transfect long gene sequences, is easy to operate, can control the transportation of the target gene by modifying the tissue or the cell specific ligand, and the like. The genetically modified MSCs further enhance the advantages of stem cell therapy. MSCs can be induced to differentiate into specific cell lines by transfection of corresponding genes. After gene modification, MSCs can also be vectors of genes or drugs. but also genetically modified MSCs can be positioned in vivo or in vitro using fluorescent proteins. Therefore, gene modification has become a powerful tool to study molecular biology mechanism, thus promoting the application of MSCs in clinic. Objective: 1. Using cationic liposome carrier to mediate GFP gene infection in rat bone marrow mesenchymal stem cells, explore the non-virus transfection technology and obtain the best cationic liposome./ DNA ratio. 2. Through lentiviral vector-mediated GFP gene infection in rat bone marrow mesenchymal stem cells, explore lentivirus transfection technology, get the most Methods: 1. Bone marrow mesenchymal stem cells were isolated and extracted by bone marrow adherent method in this experiment. Detection of surface antigen of surface antigen by cytograph 9. Expression of CD45 and CD90. Bone marrow mesenchymal stem cells were cultured using bone induction culture medium. After 7d, alkaline phosphatase was identified as bone identification. 2. Different cationic liposome contents (0.2. l, 0.3. l, 0.4. l) and different plasmid DNA contents (60ng, 70ng. 80ng) were set in 9 groups. MSCs were transfected with non-virus, and the expression of target genes and cells were observed using fluorescence microscopy. The best cationic liposome/ DNA ratio was selected. 3. Construction lentiviral vector, using different MOI values, set MOI = 0, 10, 20, 50, 100, 200, 400 in 7 groups, transfected with MSCs, and observed with fluorescence microscope. Genes Results: 1. MSCs were extracted from MSCs and MSCs were observed under inverted microscope. Detection of third generation MSC by flow cytometry The expression of CD29. CD90 was expressed almost without CD45. MSCs were induced by bone inducing fluid 7d, and alkaline phosphatase was used to detect ALP in differentiation group. After the MSCs72h was transfected with different groups of cationic liposomes, the content of cationic liposomes was 0. 3. m Compared with the 80ng group of plasmid DNA, the transfection efficiency was relatively high, and the cell status was good. light microscopy It was observed that the transfection efficiency was relatively high when MOI = 20, and the cell status was good. Conclusion: Cationic liposome is used as vector to transfect bone marrow mesenchymal stem cells, and the lower transfection efficiency is still low. No major disadvantages of non-viral transfection, but lower cytotoxicity and damage It is beneficial to further use of transfected cells. Virus transfection has a higher transfection rate, but its cytotoxicity cannot be ignored.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R457.7
本文编号：2297810
[Abstract]:Stem cell therapy has opened up new roads for tissue repair and regenerative medicine. Bone marrow mesenchymal stem cells (MSCs) are important adult stem cells, have self-renewing characteristics, and have the ability to differentiate into a variety of cells, such as fat cells, chondrocytes and osteoblasts. Due to these inherent characteristics MSCs are believed to be the ideal cell source for regenerative medicine. However, the culture conditions in vitro and the extension of cell culture cycles may affect the multipotential and phenotype of MSCs. In order to solve these problems and increase the success rate of treatment, the gene modification of MSCs became a new approach. All kinds of viruses, as well as non-viral transfection methods, are constantly optimized for the purpose of making the target gene ideal. The transfection methods of viral vectors have higher transfection efficiency and longer gene expression, but the disadvantages are also obvious, such as cytotoxicity, immunogenicity, carcinogenicity, low cell specificity, high price, and inability to transfect longer gene sequences and the like. Although the transfection rate of the non-viral vector is low, the gene expression time is short, however, the transfection method has the advantages of being safe, can transfect long gene sequences, is easy to operate, can control the transportation of the target gene by modifying the tissue or the cell specific ligand, and the like. The genetically modified MSCs further enhance the advantages of stem cell therapy. MSCs can be induced to differentiate into specific cell lines by transfection of corresponding genes. After gene modification, MSCs can also be vectors of genes or drugs. but also genetically modified MSCs can be positioned in vivo or in vitro using fluorescent proteins. Therefore, gene modification has become a powerful tool to study molecular biology mechanism, thus promoting the application of MSCs in clinic. Objective: 1. Using cationic liposome carrier to mediate GFP gene infection in rat bone marrow mesenchymal stem cells, explore the non-virus transfection technology and obtain the best cationic liposome./ DNA ratio. 2. Through lentiviral vector-mediated GFP gene infection in rat bone marrow mesenchymal stem cells, explore lentivirus transfection technology, get the most Methods: 1. Bone marrow mesenchymal stem cells were isolated and extracted by bone marrow adherent method in this experiment. Detection of surface antigen of surface antigen by cytograph 9. Expression of CD45 and CD90. Bone marrow mesenchymal stem cells were cultured using bone induction culture medium. After 7d, alkaline phosphatase was identified as bone identification. 2. Different cationic liposome contents (0.2. l, 0.3. l, 0.4. l) and different plasmid DNA contents (60ng, 70ng. 80ng) were set in 9 groups. MSCs were transfected with non-virus, and the expression of target genes and cells were observed using fluorescence microscopy. The best cationic liposome/ DNA ratio was selected. 3. Construction lentiviral vector, using different MOI values, set MOI = 0, 10, 20, 50, 100, 200, 400 in 7 groups, transfected with MSCs, and observed with fluorescence microscope. Genes Results: 1. MSCs were extracted from MSCs and MSCs were observed under inverted microscope. Detection of third generation MSC by flow cytometry The expression of CD29. CD90 was expressed almost without CD45. MSCs were induced by bone inducing fluid 7d, and alkaline phosphatase was used to detect ALP in differentiation group. After the MSCs72h was transfected with different groups of cationic liposomes, the content of cationic liposomes was 0. 3. m Compared with the 80ng group of plasmid DNA, the transfection efficiency was relatively high, and the cell status was good. light microscopy It was observed that the transfection efficiency was relatively high when MOI = 20, and the cell status was good. Conclusion: Cationic liposome is used as vector to transfect bone marrow mesenchymal stem cells, and the lower transfection efficiency is still low. No major disadvantages of non-viral transfection, but lower cytotoxicity and damage It is beneficial to further use of transfected cells. Virus transfection has a higher transfection rate, but its cytotoxicity cannot be ignored.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R457.7
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相关期刊论文 前2条
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