新型非病毒基因转染体系的构建及其在骨髓间充质干细胞基因重组中的应用
发布时间：2019-01-16 06:49
【摘要】：目的：为提高非病毒载体转染细胞后的基因表达水平并延长表达时间,并为骨髓间充质干细胞(MSC)生长创造更好的环境,以期用于软骨损伤的体内再生治疗。 方法：1、合成普鲁兰糖-精胺,与DNA孵育制备普鲁兰糖-精胺/DNA复合物,考察该复合物的粒径与电位。2、考察基因复合物在不同细胞上的转染效果,利用阴离子化明胶构建反向转染体系,比较反向转染方法与常规转染方法的差异3、在体外选择PET无纺纤维与胶原海绵作为支架,结合反向转染构建了反向三维转染体系,在MSC上考察报告基因转染效果与转染编码TGFβ-1的DNA后诱导MSC向软骨方向分化的能力。4、以可降解的壳聚糖温敏凝胶与明胶海绵作为三维支架,携载以普鲁兰糖-精胺转染TGFβ-1基因的MSC,考察其软骨修复的能力。 结果：普鲁兰糖-精胺/DNA复合物可以成功转染MSC。在血清存在情况下,反向转染方法相比常规方法可以产生更高的基因表达。阴离子化明胶的带电性与反向转染体系的转染效率密切相关,所带负电荷的多少可以影响到基因复合物的释放过程,以及血清蛋白在表面上的吸附量。不同的转染方法可能影响到细胞对基因复合物的摄取途径,摄取的改变与载体类别和细胞类型都有关系。培养在三维支架上的MSC转染后相比二维环境下可以保持更长期的基因表达,在PET无纺纤维上的基因表达水平也更高。以壳聚糖温敏凝胶为支架携载转染pTGFβ-1的MSC治疗组与未处理损伤组修复效果相似；以含有TGFβ-1基因的明胶海绵或经过TGFβ-1基因重组的MSC进行修复,具有一定的软骨修复效果。 结论：血清的加入对反向转染体系的基因转染效果不产生明显影响,克服了常规转染时血清对转染效率的负面影响。将反向转染与三维支架结合后更有利于细胞的生长,使MSC在体外保持长期的基因表达。以明胶海绵携载转染pTGFβ-1后的MSC体内移植后可促进大鼠膝关节软骨损伤修复。
[Abstract]:Aim: to improve the gene expression level and prolong the expression time after transfection of non-viral vector and to create a better environment for the growth of bone marrow mesenchymal stem cells (BMSCs) in vivo for regeneration of cartilage injury. Methods: 1. Plulanose-spermidine was synthesized and incubated with DNA to prepare Prulanose-spermidine / DNA complex. The particle size and potential of the complex were investigated, and the transfection effect of the gene complex on different cells was investigated. The reverse transfection system was constructed with anionic gelatin, and the difference between the reverse transfection method and the conventional transfection method was compared. In vitro, the PET non-woven fiber and collagen sponge were selected as scaffolds, and the reverse three-dimensional transfection system was constructed in combination with reverse transfection. The effect of gene transfection and the ability of transfection of DNA encoding TGF 尾-1 to induce MSC to differentiate into cartilage were investigated on MSC. 4. The degradable chitosan thermo-sensitive gel and gelatin sponge were used as three-dimensional scaffolds. The ability of cartilage repair was investigated by MSC, carrying TGF 尾-1 gene transfected with Pructose-spermine. Results: Prulanose-spermidine / DNA complex could transfect MSC. successfully. In the presence of serum, reverse transfection can produce higher gene expression than conventional methods. The charge of anionic gelatin is closely related to the transfection efficiency of the reverse transfection system. The negative charge can affect the release process of the gene complex and the amount of serum protein adsorbed on the surface. Different transfection methods may affect the uptake pathway of gene complex, and the changes of uptake may be related to the type of vector and cell type. The MSC transfected on three-dimensional scaffold could maintain longer gene expression than that in two-dimensional environment, and the gene expression level on PET non-woven fiber was higher than that on PET non-woven fiber. The repair effect of pTGF 尾-1 transfected with chitosan thermosensitive gel was similar to that of untreated MSC group. A gelfoam containing TGF 尾 -1 gene or a recombinant MSC containing TGF 尾 -1 gene were used to repair the cartilage. Conclusion: the addition of serum has no significant effect on the transfection efficiency of the reverse transfection system, and it overcomes the negative effect of the serum on the transfection efficiency during conventional transfection. The combination of reverse transfection and three-dimensional scaffold is more favorable to cell growth and maintains long-term gene expression of MSC in vitro. In vivo transplantation of MSC transfected with pTGF 尾-1 with gelatin sponge could promote the repair of articular cartilage injury in rats.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R346
本文编号：2409565
[Abstract]:Aim: to improve the gene expression level and prolong the expression time after transfection of non-viral vector and to create a better environment for the growth of bone marrow mesenchymal stem cells (BMSCs) in vivo for regeneration of cartilage injury. Methods: 1. Plulanose-spermidine was synthesized and incubated with DNA to prepare Prulanose-spermidine / DNA complex. The particle size and potential of the complex were investigated, and the transfection effect of the gene complex on different cells was investigated. The reverse transfection system was constructed with anionic gelatin, and the difference between the reverse transfection method and the conventional transfection method was compared. In vitro, the PET non-woven fiber and collagen sponge were selected as scaffolds, and the reverse three-dimensional transfection system was constructed in combination with reverse transfection. The effect of gene transfection and the ability of transfection of DNA encoding TGF 尾-1 to induce MSC to differentiate into cartilage were investigated on MSC. 4. The degradable chitosan thermo-sensitive gel and gelatin sponge were used as three-dimensional scaffolds. The ability of cartilage repair was investigated by MSC, carrying TGF 尾-1 gene transfected with Pructose-spermine. Results: Prulanose-spermidine / DNA complex could transfect MSC. successfully. In the presence of serum, reverse transfection can produce higher gene expression than conventional methods. The charge of anionic gelatin is closely related to the transfection efficiency of the reverse transfection system. The negative charge can affect the release process of the gene complex and the amount of serum protein adsorbed on the surface. Different transfection methods may affect the uptake pathway of gene complex, and the changes of uptake may be related to the type of vector and cell type. The MSC transfected on three-dimensional scaffold could maintain longer gene expression than that in two-dimensional environment, and the gene expression level on PET non-woven fiber was higher than that on PET non-woven fiber. The repair effect of pTGF 尾-1 transfected with chitosan thermosensitive gel was similar to that of untreated MSC group. A gelfoam containing TGF 尾 -1 gene or a recombinant MSC containing TGF 尾 -1 gene were used to repair the cartilage. Conclusion: the addition of serum has no significant effect on the transfection efficiency of the reverse transfection system, and it overcomes the negative effect of the serum on the transfection efficiency during conventional transfection. The combination of reverse transfection and three-dimensional scaffold is more favorable to cell growth and maintains long-term gene expression of MSC in vitro. In vivo transplantation of MSC transfected with pTGF 尾-1 with gelatin sponge could promote the repair of articular cartilage injury in rats.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R346
【参考文献】
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1 章永望;赵会英;张娜;;新型可注射温敏水凝胶的制备及其释药性能[J];北京化工大学学报(自然科学版);2007年05期
，本文编号：2409565
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