攜載BMP4基因的核殼結(jié)構(gòu)納米粒子修復(fù)兔橈骨臨界骨缺損的研究
發(fā)布時間：2018-08-13 16:02
【摘要】：背景及目的:骨缺損常因創(chuàng)傷、感染、腫瘤等所致,傳統(tǒng)的治療方法如自體骨移植、異體骨移植或非骨性材料等并發(fā)癥多且效果不理想。因此,研究者們將骨組織工程與基因治療技術(shù)結(jié)合,探討能夠在體外安全有效緩釋并表達(dá)目的基因的載體。最近研究表明,殼聚糖(CS)也可以作為基因載體,其具有獨特的生物安全性和生物相容性,可與基因形成穩(wěn)定的納米粒子,在體內(nèi)緩慢降解而緩釋基因。骨形成蛋白(BMP)在骨修復(fù)及骨再生中起著非常重要的作用,BMP4蛋白是BMP家族中重要的蛋白之一,具有誘導(dǎo)骨細(xì)胞分化和促進(jìn)新生骨的形成作用。本研究旨在探究修飾后的殼聚糖包裹BMP4基因形成的核結(jié)構(gòu)及核殼結(jié)構(gòu)在體外轉(zhuǎn)染效率及體內(nèi)對兔橈骨臨界骨缺損的修復(fù)作用。方法:巰基烷基化殼聚糖(TACS)與BMP4質(zhì)粒通過正負(fù)電作用形成核結(jié)構(gòu),外包裹聚乙二醇修飾羥丁基殼聚糖(EG-HBC)形成殼結(jié)構(gòu),對HEK293T細(xì)胞進(jìn)行體外轉(zhuǎn)染,Western blot,免疫熒光實驗探索核及核殼結(jié)構(gòu)的轉(zhuǎn)染效率。構(gòu)建兔子雙前肢18mm完全性臨界骨缺損模型,分別植入含有TACS@EG-HBC/pBMP4或TACS/pBMP4的明膠海綿,對照組僅植入單純的明膠海綿。術(shù)后2、4、8、12周取標(biāo)本做分別處理,檢測缺損部位的大體標(biāo)本、骨密度及骨礦物質(zhì)含量、X線、HE染色、免疫組化法、生物力學(xué)。結(jié)果:Western blot顯示TACS@EG-HBC/pBMP4蛋白表達(dá)最高,免疫熒光結(jié)果示TACS@EG-HBC/pBMP4轉(zhuǎn)染效率高于TACS/pBMP4組和對照組。大體標(biāo)本示:TACS@EG-HBC/pBMP4/G組2周可見新生骨痂,4周缺損處骨痂接近橋接,中間有纖維組織,8周骨痂骨化并橋接,12周骨缺損完全骨化修復(fù);骨密度及骨礦物質(zhì)含量顯著高于TACS/pBMP4/G組和G組;X線示:術(shù)后12周骨缺損處已完全修復(fù),骨髓腔已再通;HE染色:4周時可見大量軟骨細(xì)胞,后形成骨小梁相互連接成片,到12周時新生骨小梁相互連接成板層骨;免疫組化:BMP4棕色蛋白染色明顯;生物力學(xué)測定:所形成的新生骨與正常骨組織生物力學(xué)無統(tǒng)計學(xué)差異。結(jié)論:TACS@EG-HBC/pBMP4/G具有良好的緩釋基因和成骨能力。
[Abstract]:Background and objective: bone defect is often caused by trauma, infection, tumor and so on. The traditional treatment methods such as autogenous bone transplantation, allograft bone graft or non-bone material have many complications and the effect is not satisfactory. Therefore, the researchers combine bone tissue engineering with gene therapy to explore vectors that can effectively and safely release and express the target gene in vitro. Recent studies have shown that chitosan (CS) can also be used as gene carrier, which has unique biological safety and biocompatibility, and can form stable nanoparticles with gene, and slow degradation and slow release of genes in vivo. Bone morphogenetic protein (BMP) plays a very important role in bone repair and bone regeneration. BMP4 protein is one of the most important proteins in the BMP family, which can induce bone cell differentiation and promote the formation of new bone. The purpose of this study was to investigate the transfection efficiency of the modified chitosan coated BMP4 gene and its core-shell structure in vitro and the repair effect of the modified chitosan on the critical bone defect of rabbit radius in vivo. Methods: mercaptoalkylated chitosan (TACS) and BMP4 plasmids were used to form nuclear structure by positive and negative electric interaction, and EG-HBC was coated with polyethylene glycol to form shell structure. HEK293T cells were transfected with Western blot in vitro. The transfection efficiency of nuclear and core-shell structures was investigated by immunofluorescence assay. A complete critical bone defect model of 18mm in rabbit forelimbs was established and gelfoam containing TACS@EG-HBC/pBMP4 or TACS/pBMP4 was implanted into the control group. The specimens were collected for 12 weeks after operation, and the gross specimens were detected, bone mineral density (BMD) and bone mineral content (BMD), bone mineral content (BMD) and bone mineral content (BMD) were stained with HE, immunohistochemistry and biomechanics. Results the highest expression of TACS@EG-HBC/pBMP4 protein was detected by Western blot, and the transfection efficiency of TACS@EG-HBC/pBMP4 was higher than that of TACS/pBMP4 group and control group. Gross specimens showed that the callus at the defect of 4 weeks was close to the bridge in the group of EG-HBC / pBMP4 / G with fibrous tissue at 8 weeks and complete ossification of bone defect at 12 weeks. Bone mineral density (BMD) and bone mineral content (BMD) in TACS/pBMP4/G group and G group were significantly higher than those in TACS/pBMP4/G group and G group. At 12 weeks after operation, bone defects had been repaired completely, and a large number of chondrocytes could be seen in the medullary cavity at 4 weeks after HE staining, and trabeculae were connected to each other. At the 12th week, trabeculae were connected to each other to form lamellar bone; immunohistochemical staining of brown protein of BMP4 was obvious; biomechanical measurement: there was no significant difference in biomechanics between the formed new bone and normal bone tissue. Conclusion the EG-HBC / pBMP4 / G has good ability of sustained release gene and osteogenesis.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R687
本文編號：2181482
[Abstract]:Background and objective: bone defect is often caused by trauma, infection, tumor and so on. The traditional treatment methods such as autogenous bone transplantation, allograft bone graft or non-bone material have many complications and the effect is not satisfactory. Therefore, the researchers combine bone tissue engineering with gene therapy to explore vectors that can effectively and safely release and express the target gene in vitro. Recent studies have shown that chitosan (CS) can also be used as gene carrier, which has unique biological safety and biocompatibility, and can form stable nanoparticles with gene, and slow degradation and slow release of genes in vivo. Bone morphogenetic protein (BMP) plays a very important role in bone repair and bone regeneration. BMP4 protein is one of the most important proteins in the BMP family, which can induce bone cell differentiation and promote the formation of new bone. The purpose of this study was to investigate the transfection efficiency of the modified chitosan coated BMP4 gene and its core-shell structure in vitro and the repair effect of the modified chitosan on the critical bone defect of rabbit radius in vivo. Methods: mercaptoalkylated chitosan (TACS) and BMP4 plasmids were used to form nuclear structure by positive and negative electric interaction, and EG-HBC was coated with polyethylene glycol to form shell structure. HEK293T cells were transfected with Western blot in vitro. The transfection efficiency of nuclear and core-shell structures was investigated by immunofluorescence assay. A complete critical bone defect model of 18mm in rabbit forelimbs was established and gelfoam containing TACS@EG-HBC/pBMP4 or TACS/pBMP4 was implanted into the control group. The specimens were collected for 12 weeks after operation, and the gross specimens were detected, bone mineral density (BMD) and bone mineral content (BMD), bone mineral content (BMD) and bone mineral content (BMD) were stained with HE, immunohistochemistry and biomechanics. Results the highest expression of TACS@EG-HBC/pBMP4 protein was detected by Western blot, and the transfection efficiency of TACS@EG-HBC/pBMP4 was higher than that of TACS/pBMP4 group and control group. Gross specimens showed that the callus at the defect of 4 weeks was close to the bridge in the group of EG-HBC / pBMP4 / G with fibrous tissue at 8 weeks and complete ossification of bone defect at 12 weeks. Bone mineral density (BMD) and bone mineral content (BMD) in TACS/pBMP4/G group and G group were significantly higher than those in TACS/pBMP4/G group and G group. At 12 weeks after operation, bone defects had been repaired completely, and a large number of chondrocytes could be seen in the medullary cavity at 4 weeks after HE staining, and trabeculae were connected to each other. At the 12th week, trabeculae were connected to each other to form lamellar bone; immunohistochemical staining of brown protein of BMP4 was obvious; biomechanical measurement: there was no significant difference in biomechanics between the formed new bone and normal bone tissue. Conclusion the EG-HBC / pBMP4 / G has good ability of sustained release gene and osteogenesis.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R687
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 Sébastien Gittens;The ectopic study of tissue-engineered bone with hBMP-4 gene modified bone marrow stromal cells in rabbits[J];Chinese Medical Journal;2005年04期
，本文編號：2181482
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