【作者】 蒋佳；

【导师】 曾炳芳；

【作者基本信息】 上海交通大学 ， 骨外科学， 2008， 博士

【摘要】 背景:大段骨缺损的修复是骨科临床治疗的难题。近年来,骨组织工程发展迅速,利用骨组织工程治疗骨缺损成为研究的热点。组织工程骨的血管化问题是影响其应用于临床的重要原因之一。骨形态发生蛋白2(BMP2)的成骨作用已获得公认,内皮细胞生长因子(VEGF)是血管内皮细胞的特异性有丝分裂原,可促进新生血管形成。研究表明,在骨形成和骨折愈合过程中,VEGF和BMP2的表达相互促进,并在骨再生过程中产生协同效应。联合应用BMP2和VEGF双基因,可实现骨与血供的联合重建,成为促进骨缺损修复的一种有效方法。利用重组病毒载体转染外源性基因到组织工程骨的种子细胞并与支架材料复合形成基因修饰组织工程骨是当今骨缺损的基因治疗研究的热点。慢病毒载体能够感染大多数非分裂细胞,并具有高效、稳定地表达目的基因的能力,已被广泛应用于基因治疗实验研究。目的:(1)利用BMP2和VEGF165两种基因间的协同作用,促进组织工程骨的成骨及血管化。(2)掌握重组慢病毒的生产技术,以便应用于今后其它研究中,从而使相应的外源基因高效稳定地表达。材料和方法:(1)从人骨肉瘤成骨样细胞(MG-63细胞)中调取人BMP2和VEGF165基因;分别构建携带BMP2、VEGF165基因的重组慢病毒表达载体;分别进行携带BMP2、VEGF165基因的重组慢病毒(LV-BMP、LV-VEGF)的包装和生产。(2)Wistar大鼠的骨髓间充质干细胞(MSCs)的分离培养、体外扩增。(3)按照未转染细胞组、VEGF165单转染细胞组、BMP2单转染细胞组、BMP2及VEGF165共转染细胞组分别转染MSCs;(4)转染后7天,通过实时荧光定量检测各组人BMP2和VEGF165基因的表达状况;转染后1、4、8周后,通过ELISA检测BMP2和VEGF165蛋白表达水平变化(5)转染后14天,按上述分组对各组细胞行成骨能力的检测(碱性磷酸酶染色及定量检测)。(6)按分组将细胞与支架材料复合并行细胞材料复合物的检测:细胞接种后14天行电镜观察、接种后14天行碱性磷酸酶定量检测、接种后2周观察细胞生长曲线。(7)按分组将组织工程骨回植到裸鼠皮下试验,回植后4、8周组织学观察体内成骨情况,回植后4周CD31免疫组化染色观察血管生成情况。结果:(1)分别成功构建携带BMP2、VEGF165的重组慢病毒。(2)仅在BMP+VEGF组,人BMP2和VEGF165基因在mRNA和蛋白水平高效共表达,且其表达水平与相应单转组无明显差异(P>0.05)。ELISA检测显示人BMP2和VEGF165基因在蛋白水平高效共表达可长达8周。(3)双转组中MSCs的ALP活性明显高于各单转组(P<0.01)。(4)细胞接种到支架材料后,扫描电镜观察细胞在接种后14天在支架材料上生长增殖良好。HOECHST DNA检测表明接种后14天内各组细胞生长曲线无明显异常(P>0.05)。但双转组中MSCs的ALP活性明显高于各单转组(P<0.01)。(5)裸鼠实验中,联合应用BMP2与VEGF165组和单用BMP2组有异位骨形成,其他各组均无成骨。植骨后4、8周,HE染色显示联合应用BMP2组与VEGF165组成骨面积多于单用BMP组(P<0.01)。植骨后4周,双转组CD31染色阳性细胞明显多于单转BMP组。结论:通过慢病毒载体系统进行双基因转染的大鼠骨髓间充质干细胞(MSCs)复合磷酸三钙(TCP)体外构建组织工程骨及体内成骨的实验研究,证实(1)利用较新的慢病毒载体系统共转染的方法可使种子细胞高效稳定地共表达人BMP2及VEGF165基因(2)利用两种基因间的协同作用,联合应用BMP2和VEGF可明显促进组织工程骨的成骨及血管化,其成骨及血管化效果均优于单用一种生长因子。更多还原

【Abstract】 Background: It still remains as a clinical problem for orthopaedic surgeons to repair large bone defect. Tissue engineered bone has been advancing rapidly in recent years and has become a research focus in the treatment of bone defect. But the vascularization process is a major bottleneck for its clinical application. The osteogenic ability of bone morphogenetic protein 2 (BMP2) has been widely admitted and vascular endothelial growth factors (VEGF) is special mitogen in vascular endothelial cell, accelerating the growth of new vessels. Researches show that the expression of BMP and VEGF exists mutual influence relations, leading to a synergetic effect on bone regeneration. United reconstruction of bone formation and bone supply can be realized by combined application of both genes, becoming a novel method to repair bone defect. It has become a research focus to transfer endogenous gene to seed cell of tissue-engineered bone through recombinant virus vector system and then combined with scaffolds to form gene-modified tissue engineered bone. Lentiviral vector can infect most none-dividing cells and owns the capacity to express exogenous genes efficiently and stably. It has been widely applied in researches on gene therapy.Objectives: (1) Promote the angiogenesis and osteogenesis processes of tissue-engineered bone by virtue of the synergetic effect of BMP2 and VEGF genes. (2) Master the technique of recombinant lentivirus production to apply in future researches, leading to a stable and high expression of relating exogenous genes.Materials and methods: (1) Human VEGF165 and BMP2 cDNAs were obtained from human osteosarcoma cell line MG63. The expression lentivirus vectors carrying VEGF165 or BMP2 were constructed respectively. Recombinant lentivirus carrying VEGF165 (Lv-VEGF) or BMP2 (Lv-BMP) were packaged and produced respectively. (2) Rat bone marrow mesenchymal stem cells (MSCs) were isolated and expanded in vitro. (3) MSCs were co-transfected with Lv-VEGF and Lv-BMP (BMP+VEGF group), or each alone (BMP group and VEGF group), or with no virus (control group). (4) The mRNA expression of human VEGF165 and BMP2 genes in each group was detected by real-time PCR at 7 days after transfection. And the protein expression of human VEGF165 and BMP2 genes in each group was detected by enzyme linked immunosorbent assay (ELISA) at 1,4,8 weeks after transfection.(5) Alkaline phosphatase activity (ALP) staining as well as ALP activity was performed in each group at 14 days after transfection. (6) MSCs of each group were collected and seeded on theβ-tricalcium phosphate (β-TCP) scaffolds. Theβ-TCP scaffolds combining MSCs of BMP+VEGF group were randomly chosen for scanning electron microscopy (SEM) observation at 14 days after cell seeding. Cell proliferation of each group was determined every day by a hoechst assay for up to 2 weeks after cell seeding. The ALP activity assay of cell-seeded scaffolds in each group were analyzed 14 days after cell seeding.(7)Engineered constructs of each group were implanted subcutaneously to nude mice. The implants were harvested and used for histological analysis at 4 and 8 weeks after implantation. Angiogenesis was also observed at 4 and 8 weeks after implantation by CD31 staining.Results: (1) Lentiviral expression vectors carrying hVEGF165 or hBMP2 were correctly constructed. (2) VEGF165 and BMP2 genes were co-expressed in BMP+VEGF group. No significant difference of BMP2 expression was detected between BMP+VEGF and BMP groups (P>0.05), similarly no significant difference of VEGF165 expression between BMP+VEGF and VEGF groups (P>0.05). And both genes were successfully co-expressed in the co-transfection group for up to 8 weeks confirmed by ELISA. (3) The ALP activity of MSCs was significantly augmented by the co-transfection with both genes than any single gene transfection (p<0.01).(4) After seeding MSCs onto the scaffolds, SEM observation showed that MSCs grew and proliferated well in co-transfection group at 14 days. There was no significant difference among all the groups in hoechst DNA assay for cell proliferation for 14 days after cell seeding (P>0.05), but the highest ALP activity was observed in the co-transfection group at 14 days after cell seeding (p<0.01). In nude mice, ectopic osteogenesis was observed in BMP group and BMP+VEGF group, and no ectopic osteogenesis was found in the other groups. At 4 and 8 weeks post-operation, BMP+VEGF group has better bone formation than BMP group in the HE staining sections (p<0.01). At 4 weeks post-operation, more CD31 positive cells were observed in BMP+VEGF group than in BMP group.Conclusions:In our study, we constructed tissue-engineered bone usingβ-TCP combined with MSCs lentivirally co-transfected with BMP2 and VEGF165 and implant these engineered constructs subcutaneously to nude mice to observe the bone formation in vivo. We can conclude: (1) A stable and relatively long term co-expression of both genes can be achieved through lentivirus mediated co-transfection of both genes. (2) Combined use of BMP2 and VEGF gene can produce better osteogenesis and angiogenesis than each single gene alone in virtue of the synergistic effect of both genes.更多还原