microRNA纳米微囊功能化促进钛合金表面骨愈合研究

发布时间：2018-11-09 15:57

【摘要】：钛合金具有良好的生物相容性,出色的抗腐蚀性能和机械性能,在骨损伤治疗中得到了广泛应用。但其表面惰性诱发多种临床问题,需要对其表面进行功能化改性。microRNAs参与调控骨修复的各个过程,投递成骨相关microRNAs已经被证明能够有效促进骨愈合。因此,本文目的是设计一种microRNAs投递系统,实现体内稳定有效的microRNA转导,并对钛合金表面进行修饰,实现体内促进骨再生的功能。本文首先以商用基因转染剂分别将miR-21和反义miR-21(as-miR-21)转染到脐带血间充质干细胞(hUMSCs)中,研究miR-21促进hUMSCs成骨分化的能力及其机制。结果表明,hUMSCs内投递miR-21可显著提高成骨标志物碱性磷酸酶(ALP)、RUNT相关基因2(RUNX-2)和骨钙素(OCN)的表达,茜素红染色证明miR-21可促进hUMSCs成骨分化。本文同时证明,随着miR-21的上游调控作用,PI3K-AKT信号通路得以激活,进而促进GSK-3β的磷酸化,使得β-catenin得以稳定并入核后增加RUNX-2基因的表达,最终促进hUMSCs成骨分化。其次本文以原位聚合法制备了包载miRNA的纳米微囊,进一步通过静电作用与O-羧甲基壳聚糖(CMCS)复合,形成miRNA活化基质,避免了miRNA的漏出并且为细胞转染提供了更好的稳定性,同时活化基质具有良好的生物相容性。以此基质投递miR-21至hUMSCs,ALP及RUNX-2的表达均呈上升趋势,证明miR-21活化基质具有诱导干细胞成骨分化的能力。SD大鼠体内胫骨平台骨损伤部位注射基质后,分别于2周和4周考察活化基质促进骨修复能力,结果表明:注射基质后,miR-21相关靶点蛋白PTEN下调,β-catenin上调。并且与对照组相比具有更好的促进骨再生能力第三,本文以miR-29b活化基质修饰钛合金表面。该涂层不仅有利于细胞黏附以及增值,而且能够提供较高的细胞转染效率。此外,涂层可以调控miR-29b的靶点HDAC-4,并可体外诱导hUMSCs成骨分化。将miR-29b活化基质修饰的钛合金植入大鼠胫骨平台,分别在2周、4周、6周和8周考察钛合金表面骨的修复能力,结果表明:钛合金表面miR-29b涂层修饰后,能够极大提高其表面骨缺损愈合能力。此外,成骨相关标志物RUNX-2和OCN的表达水平提高且miR-29b靶点HDAC-4的表达下调,进一步证明miR-29b功能化涂层修饰后,能够极大提高钛合金表面骨再生能力。
[Abstract]:Titanium alloys have been widely used in the treatment of bone injury because of their good biocompatibility, excellent corrosion resistance and mechanical properties. However, the surface inertia induces a variety of clinical problems and needs to be functionalized. MicroRNAs is involved in various processes of bone repair, and the delivery of osteoblast related microRNAs has been proved to be effective in promoting bone healing. Therefore, the aim of this paper is to design a microRNAs delivery system, to realize stable and effective microRNA transduction in vivo, and to modify the surface of titanium alloy to promote bone regeneration in vivo. In this paper, miR-21 and antisense miR-21 (as-miR-21) were transfected into umbilical cord blood mesenchymal stem cells (hUMSCs) by commercial gene transfer agent, respectively, to study the ability and mechanism of miR-21 to promote osteogenic differentiation of hUMSCs. The results showed that the expression of (ALP), RUNT related gene 2 (RUNX-2) and osteocalcin (OCN) was significantly increased by miR-21 in hUMSCs, and miR-21 could promote the differentiation of hUMSCs by alizarin red staining. We also demonstrated that with the upstream regulation of miR-21, the PI3K-AKT signaling pathway was activated, which promoted the phosphorylation of GSK-3 尾, and increased the expression of RUNX-2 gene after the stable incorporation of 尾-catenin into the nucleus. Finally, hUMSCs osteogenic differentiation was promoted. Secondly, nano-microcapsules coated with miRNA were prepared by in-situ polymerization. Furthermore, by electrostatic interaction with O-carboxymethyl chitosan (CMCS), the activated miRNA matrix was formed. It can avoid the leakage of miRNA and provide better stability for cell transfection, and the activated matrix has good biocompatibility. The expression of miR-21, hUMSCs,ALP and RUNX-2 in this medium was on the rise, which proved that miR-21 activated matrix had the ability to induce osteogenic differentiation of stem cells. After SD rats were injected with matrix at the site of tibial plateau bone injury in vivo, the expression of miR-21 activated matrix was found to be able to induce osteogenic differentiation of stem cells. The ability of activated matrix to promote bone repair was investigated at 2 and 4 weeks, respectively. The results showed that miR-21 related target protein PTEN was down-regulated and 尾-catenin upregulated after injection of matrix. Compared with the control group, the surface of titanium alloy was modified with miR-29b activated matrix. The coating can not only facilitate cell adhesion and proliferation, but also provide higher cell transfection efficiency. In addition, the coating can regulate the target HDAC-4, of miR-29b and induce hUMSCs osteogenic differentiation in vitro. Titanium alloy modified with miR-29b activated matrix was implanted into tibial plateau of rats. The repair ability of titanium alloy surface was investigated at 2 weeks, 4 weeks, 6 weeks and 8 weeks, respectively. The results showed that the surface of titanium alloy was modified by miR-29b coating. It can greatly improve the healing ability of the surface bone defect. In addition, the increased expression of RUNX-2 and OCN and the down-regulation of HDAC-4, the target of miR-29b, further proved that the ability of bone regeneration on the surface of titanium alloy could be greatly improved by the modification of miR-29b functionalized coating.
【学位授予单位】：天津大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：TG178

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