陽離子脂質(zhì)體和慢病毒載體轉(zhuǎn)染MSCs的比較研究
發(fā)布時間：2018-10-29 13:24
【摘要】：干細(xì)胞療法為組織修復(fù)和再生醫(yī)學(xué)開辟了新的道路。骨髓間充質(zhì)干細(xì)胞(Mesenchymal stem cells, MSCs)是一種重要的成體干細(xì)胞,具有自我更新的特性,并且具有向多種細(xì)胞分化的能力,例如脂肪細(xì)胞、軟骨細(xì)胞和成骨細(xì)胞。由于這些內(nèi)在的特性,MSCs被認(rèn)為再生醫(yī)學(xué)的理想細(xì)胞來源。但是,體外的培養(yǎng)條件以及細(xì)胞培養(yǎng)周期的延長會影響MSCs的多潛能性和表型。為了解決這些問題并增加治療的成功率,MSCs的基因修飾開始成為新的研究途徑。各種各樣的病毒以及非病毒轉(zhuǎn)染方法不斷進(jìn)行優(yōu)化,目的都是使目的基因理想表達(dá)。病毒載體的轉(zhuǎn)染方法具有較高的轉(zhuǎn)染率和較長時間的基因表達(dá),但是缺點(diǎn)也比較明顯,例如細(xì)胞毒性、免疫原性、致癌性、低細(xì)胞特異性、價格昂貴以及無法轉(zhuǎn)染較長基因序列等。非病毒載體的轉(zhuǎn)染方法雖然轉(zhuǎn)染率較低,基因表達(dá)時間較短,然而,具有安全、可轉(zhuǎn)染長基因序列、低毒性、易操作、可通過組織或細(xì)胞特異性配體的修飾來操控靶基因的運(yùn)輸?shù)葍?yōu)點(diǎn)。基因修飾的MSCs使干細(xì)胞治療的優(yōu)勢進(jìn)一步提升。通過轉(zhuǎn)染相應(yīng)的基因,可以誘導(dǎo)MSCs向特定細(xì)胞系分化。在基因修飾后,MSCs同樣可以成為基因或藥物的載體。而且經(jīng)過基因修飾的MSCs無論在體內(nèi)還是體外都可以使用熒光蛋白來定位。所以,基因修飾成為了研究分子生物學(xué)機(jī)制的有力工具,從而促進(jìn)了MSCs在臨床中的應(yīng)用。 目的： 1.通過陽離子脂質(zhì)體載體介導(dǎo)GFP基因感染大鼠骨髓間充質(zhì)干細(xì)胞,探索非病毒轉(zhuǎn)染技術(shù),獲得最佳陽離子脂質(zhì)體/DNA比。 2.通過慢病毒載體介導(dǎo)GFP基因感染大鼠骨髓間充質(zhì)干細(xì)胞,探索慢病毒轉(zhuǎn)染技術(shù),獲得最佳MOI值。 方法： 1.本實(shí)驗(yàn)使用全骨髓貼壁法分離提取骨髓間充質(zhì)干細(xì)胞,使用流式細(xì)胞儀檢測細(xì)胞表面抗原CD29. CD45和CD90的表達(dá)。使用骨誘導(dǎo)培養(yǎng)液培養(yǎng)骨髓間充質(zhì)干細(xì)胞,培養(yǎng)7d后進(jìn)行堿性磷酸酶成骨鑒定。 2.設(shè)置不同陽離子脂質(zhì)體含量(0.2μl、0.3μl、0.4μl)及不同質(zhì)粒DNA含量(60ng、70ng.80ng)共9組,進(jìn)行非病毒轉(zhuǎn)染MSCs,使用熒光顯微鏡觀察目的基因表達(dá)情況和細(xì)胞狀態(tài)篩選最佳陽離子脂質(zhì)體/DNA比。 3.構(gòu)建慢病毒載體,使用不同MOI值,設(shè)置MOI=0,10,20,50,100,200,400共7組,對MSCs進(jìn)行轉(zhuǎn)染,使用熒光顯微鏡觀察目的基因表達(dá)情況和細(xì)胞狀態(tài)篩選最佳MOI值。 結(jié)果： 1.使用全骨髓法成功提取MSCs,倒置顯微鏡下觀察MSCs貼壁生長,流式細(xì)胞儀檢測第三代MSCs穩(wěn)定均一表達(dá)CD29. CD90,幾乎不表達(dá)CD45. MSCs經(jīng)成骨誘導(dǎo)液誘導(dǎo)后7d,堿性磷酸酶檢測顯示促分化組ALP分泌明顯高于未分化組,繼續(xù)培養(yǎng)至10d后可見鈣結(jié)節(jié)形成。 2.使用不同組別陽離子脂質(zhì)體轉(zhuǎn)染MSCs72h后,熒光顯微鏡下可見陽離子脂質(zhì)體含量0.3μl與質(zhì)粒DNA含量80ng組轉(zhuǎn)染效率相對較高,細(xì)胞狀態(tài)良好。 3.使用不同MOI組別慢病毒轉(zhuǎn)染MSCs,分別于3d、7d和10d熒光顯微鏡下觀察,可見MOI=20時轉(zhuǎn)染效率相對較高,細(xì)胞狀態(tài)較好。 結(jié)論： 陽離子脂質(zhì)體作為載體轉(zhuǎn)染骨髓間充質(zhì)干細(xì)胞,較低的轉(zhuǎn)染率仍然是非病毒轉(zhuǎn)染的主要缺點(diǎn),但是較低的細(xì)胞毒性和損傷有利于對轉(zhuǎn)染細(xì)胞的進(jìn)一步利用。病毒轉(zhuǎn)染具有較高的轉(zhuǎn)染率,但是其細(xì)胞毒性不容忽視。通過對骨髓間充質(zhì)干細(xì)胞的基因修飾,為實(shí)現(xiàn)干細(xì)胞治療的臨床應(yīng)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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R457.7
本文編號：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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R457.7
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相關(guān)期刊論文 前2條
1 唐莉;常靜;;攜帶GFP的慢病毒感染人臍帶間充質(zhì)干細(xì)胞及其對Oct4表達(dá)的影響[J];細(xì)胞與分子免疫學(xué)雜志;2013年03期
2 Tao Wang;Hua-shan Zhao;Qiu-ling Zhang;Chang-lin Xu;Chang-bai Liu;;Generation of Transgene-free Induced Pluripotent Stem Cells with Non-viral Methods[J];Chinese Medical Sciences Journal;2013年01期
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