新型非病毒基因轉(zhuǎn)染體系的構(gòu)建及其在骨髓間充質(zhì)干細(xì)胞基因重組中的應(yīng)用
發(fā)布時(shí)間：2019-01-16 06:49
【摘要】：目的：為提高非病毒載體轉(zhuǎn)染細(xì)胞后的基因表達(dá)水平并延長(zhǎng)表達(dá)時(shí)間,并為骨髓間充質(zhì)干細(xì)胞(MSC)生長(zhǎng)創(chuàng)造更好的環(huán)境,以期用于軟骨損傷的體內(nèi)再生治療。 方法：1、合成普魯蘭糖-精胺,與DNA孵育制備普魯蘭糖-精胺/DNA復(fù)合物,考察該復(fù)合物的粒徑與電位。2、考察基因復(fù)合物在不同細(xì)胞上的轉(zhuǎn)染效果,利用陰離子化明膠構(gòu)建反向轉(zhuǎn)染體系,比較反向轉(zhuǎn)染方法與常規(guī)轉(zhuǎn)染方法的差異3、在體外選擇PET無(wú)紡纖維與膠原海綿作為支架,結(jié)合反向轉(zhuǎn)染構(gòu)建了反向三維轉(zhuǎn)染體系,在MSC上考察報(bào)告基因轉(zhuǎn)染效果與轉(zhuǎn)染編碼TGFβ-1的DNA后誘導(dǎo)MSC向軟骨方向分化的能力。4、以可降解的殼聚糖溫敏凝膠與明膠海綿作為三維支架,攜載以普魯蘭糖-精胺轉(zhuǎn)染TGFβ-1基因的MSC,考察其軟骨修復(fù)的能力。 結(jié)果：普魯蘭糖-精胺/DNA復(fù)合物可以成功轉(zhuǎn)染MSC。在血清存在情況下,反向轉(zhuǎn)染方法相比常規(guī)方法可以產(chǎn)生更高的基因表達(dá)。陰離子化明膠的帶電性與反向轉(zhuǎn)染體系的轉(zhuǎn)染效率密切相關(guān),所帶負(fù)電荷的多少可以影響到基因復(fù)合物的釋放過(guò)程,以及血清蛋白在表面上的吸附量。不同的轉(zhuǎn)染方法可能影響到細(xì)胞對(duì)基因復(fù)合物的攝取途徑,攝取的改變與載體類(lèi)別和細(xì)胞類(lèi)型都有關(guān)系。培養(yǎng)在三維支架上的MSC轉(zhuǎn)染后相比二維環(huán)境下可以保持更長(zhǎng)期的基因表達(dá),在PET無(wú)紡纖維上的基因表達(dá)水平也更高。以殼聚糖溫敏凝膠為支架攜載轉(zhuǎn)染pTGFβ-1的MSC治療組與未處理?yè)p傷組修復(fù)效果相似；以含有TGFβ-1基因的明膠海綿或經(jīng)過(guò)TGFβ-1基因重組的MSC進(jìn)行修復(fù),具有一定的軟骨修復(fù)效果。 結(jié)論：血清的加入對(duì)反向轉(zhuǎn)染體系的基因轉(zhuǎn)染效果不產(chǎn)生明顯影響,克服了常規(guī)轉(zhuǎn)染時(shí)血清對(duì)轉(zhuǎn)染效率的負(fù)面影響。將反向轉(zhuǎn)染與三維支架結(jié)合后更有利于細(xì)胞的生長(zhǎng),使MSC在體外保持長(zhǎng)期的基因表達(dá)。以明膠海綿攜載轉(zhuǎn)染pTGFβ-1后的MSC體內(nèi)移植后可促進(jìn)大鼠膝關(guān)節(jié)軟骨損傷修復(fù)。
[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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R346
本文編號(hào)：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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R346
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 章永望;趙會(huì)英;張娜;;新型可注射溫敏水凝膠的制備及其釋藥性能[J];北京化工大學(xué)學(xué)報(bào)(自然科學(xué)版);2007年05期
，本文編號(hào)：2409565
本文鏈接：http://sikaile.net/xiyixuelunwen/2409565.html
最近更新
教材專著
