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LMP-1基因转染的MC3T3-E1细胞修饰材料促进骨再生
马俊轩
2016-04-16
导师肖德明
专业外科学
授予单位北京大学
授予地点北京大学深圳医院
学位博士
关键词生物材料 生物加工 细胞外基质 骨再生 基因治疗
其他题名LMP-1 Gene Transfected MC3T3-E1 Cells Modified Biomaterials to Promote Bone Regeneration
分类号R687.3
摘要

目的:为了更好地促进骨组织再生,需要研发具有骨诱导活性的生物材料。现有生物材料多由人工方法合成,难以模仿及控制骨组织再生修复过程中复杂、精细的调节机制,由此导致了诸多问题。本研究通过将治疗性基因LMP-1导入细胞,利用细胞对骨天然支架材料进行生物加工,使此材料具有与体内微环境相近的调节方式,并对此生物加工材料的疗效进行体内实验验证。

方法:首先通过慢病毒载体转染,将LMP-1基因导入小鼠前成骨MC3T3-E1细胞系,在基因及蛋白质水平分析LMP-1的过表达是否促进了细胞分泌成骨诱导活性物质。其次用骨组织有机、无机天然成分构建了两种支架材料,即糖胺聚糖水凝胶及煅烧牛骨碳羟基磷灰石,并通过基因分析评价这两种材料对干细胞成骨分化的影响。最后将稳定表达LMP-1基因的MC3T3-E1细胞植入骨天然支架材料培养7天、14天及21天,对支架材料进行生物加工后植入家兔股骨远端显著骨缺损,评价其促进骨再生的治疗效果。

结果:稳定转染LMP-1基因的MC3T3-E1细胞相对于对照Ⅰ型胶原蛋白、BMP-4及BMP-7表达显著增高(P < 0.05)。煅烧牛骨碳羟磷灰石材料中的干细胞在成骨诱导剂的作用下,成骨相关基因Col1A1、RUNX2及OPN的转录水平均显著增高(P<0.05)。而糖胺聚糖水凝胶中的干细胞仅Col1A1升高(P<0.05)。低氧环境下,煅烧牛骨材料内培养的干细胞血管化相关基因VEGF转录水平升高(P<0.05)。而糖胺聚糖水凝胶中的干细胞VEGF转录水平无显著变化(P>0.05)。水凝胶内干细胞VEGF转录水平从7天到21天表达随时间逐渐降低(P<0.05)。用LMP-1的稳定表达细胞生物改造煅烧牛骨碳羟基磷灰石材料后,植入家兔股骨远端显著骨缺损,不同时间取材进行MicroCT评价,发现生物加工7天的材料植入后12周,缺损处的新骨量平均达到79.3%,明显高于单纯煅烧牛骨材料植入的对照(33.3%新骨)(P<0.05)。对标本进行硬组织切片染色分析发现,构建7天的生物加工材料植入家兔骨缺损后12周,骨缺损内的骨量显著高于对照单纯煅烧骨材料植入(P<0.05)。骨与材料界面观察提示此生物加工材料可具有良好的骨组织整合作用及生物相容性。

结论:LMP-1基因的过表达可使细胞Ⅰ型胶原蛋白及多种骨形态蛋白的的分泌增加,煅烧牛骨碳羟基磷灰石材料经过稳定表达LMP-1基因的MC3T3-E1细胞系生物加工后,可有效促进骨组织再生修复。

英文摘要

Object: Biomaterials are needed for bone tissue regeneration. Most of current biomaterials which are produced using artificial methods have many problems. This can be attributed to the fact that artificial methods can hardly duplicate and control the multiple complex and sophisticated mechanisms of regulation involved in the process of normal bone formation. This study used cells that transfected with therapeutic gene of LMP-1 to modify natural bone derived scaffolds. The biosynthesized and biomodified biomaterials may provide a microenvironment which regulate regeneration very close to physiological process. The therapeutic effect of this biomaterial was then tested in vivo.

Methods: Lentivirus was used to transfect LMP-1 gene into MC3T3-E1 cells for a sustained expression. RNA and protein analysis were performed to identify whether the introduction of LMP-1 could promote cells to secrete osteogenic inducing growth factors.  Then organic and inorganic components of natural bone were used to produce two scaffold materials: chemically crosslinked glycosaminoglycan hydrogels and calcined bovine carbonated hydroxyapatite materials. The influence of the two materials on stem cells’ functions were evaluated in vitro by RNA analysis. Finally, the LMP-1 over-expressing cells were seeded into the scaffolds and cultured for 7 days, 14 days and 21 day of modifications. The materials were then implanted into the critical defects performed in the distal femora of rabbits to evaluate its effect on the amount of new bone formation.

Results: Protein and RNA analysis indicated that over expression of LMP-1 can significantly upregulate type Ⅰ collage, BMP-4 and BMP-7 expression (P<0.05). Stem cells in calcined bovine carbonated hydroxyapatite materials had a higher tranion of osteogenesis related genes which include Col1A1, RUNX2 and OPN (P<0.05). Cells in glycosaminoglycan hydrogels otherwise only showed an increased expression of Col1A1 (P<0.05). Under hypoxia, vascularization related gene VEGF was upregulated by cells in calcined bone (P<0.05), but not those in hydrogels (P>0.05). Cells in hydrogels showed a decreased Expression of VEGF over time from 7 days to 21 days (P<0.05). After the biosynthesized materials were implanted into the bone defects, micro CT indicated that after 12 weeks of implantation, the materials biosynthesized by cells for 7 days obtained 79.3% of new bone formation which was significantly higher than the scaffold control (33.3% of new bone formation) (P<0.05). Hard tissue sections also showed that after the implantation of 7-day-biosythesized biomaterials for 12 weeks, significantly larger amounts of bone were found in the defects compared with those implanted with only calcined bovine scaffolds (P<0.05). The interface of bone and materials indicated satisfactory bone integration and biocompatibility.

Conclusion: Over expression of LMP-1 gene by cells could result in an increased expression of type Ⅰ collagen and multiple bone morphogenic proteins. Biosynthesized materials by LMP-1 over expressing cells on calcined bovine bone carbonated hydroxyapatite could significantly promote bone regeneration.

语种中文
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文献类型学位论文
条目标识符http://ir.bjmu.edu.cn/handle/400002259/125373
专题北京大学深圳医院
作者单位北京大学深圳医院
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马俊轩. LMP-1基因转染的MC3T3-E1细胞修饰材料促进骨再生[D]. 北京大学深圳医院. 北京大学,2016.
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