CRISPR靶向导入SDF-1调控BMSCs促进眼眶骨再生及其机制探讨

The orbit, adjacent to the paranasal sinus, is an osteal cavity with thin orbital walls. Trauma can easily cause large bone defects in the orbit and make it hard for osteogenesis repair after injury. Nonabsorbable materials were used to repair the orbital defect, but might cause several complications and unsatisfied effects. Previous researches have pointed out that gene-modified BMSCs tissue engineering bone aimed at improving orbital microenvironment can effectively promote bone regeneration. However, due to the blindness and randomness in the process of gene integration, carcinogenic risk greatly improved as a result of the activation of “cancer genes”, which leads to poor clinical prospect. In order to solve this problem and shortage of endogenous osteogenic and angiogenic cells in the orbit, CRISPR technique will be used to specifically edit SDF-1 gene-modified BMSCs combined with tissue engineering bone to repair orbital critical-sized bone defect. SDF-1 has the ability to recruit hematopoietic and osteogenic stem cells, promote the osteogenic capability of BMP2, optimize the orbital microenvironment and regenerate and reconstruct the orbit anatomically and physiologically. This study applies CRISPR technique to orbital tissue engineering for the first time nationwide, and will throw light on the clinical treatment of large bone defect.

眼眶为骨性空腔，骨质菲薄，毗邻副鼻窦腔，外伤及肿瘤易导致大片骨缺损，可导致面部畸形和视功能障碍。由于传统材料的局限性，临床修复效果不尽如人意。我们前期研究表明基因修饰BMSCs组织工程骨，可以有效促进骨再生，但由于基因整合的盲目性及随机性，可导致“原癌基因”的激活，具有致癌风险，成为临床应用的瓶颈。针对这一问题及眼眶骨缺损所特有内源性成骨成血管细胞匮乏的环境因素，本项目采用CRISPR靶向犬BMSC基因组“安全港”导入外源目的基因SDF-1，在高表达SDF-1的同时，很好解决了临床应用的安全性问题。SDF-1可以招募成骨/成血管干细胞至损伤区域，活化破骨细胞分泌BMP，优化眼眶成骨微环境，促进眼眶骨再生。本实验属国内外首次将CRISPR应用于骨再生医学的研究，有望为临床上治疗以大型骨缺损为主要病变的疾病提供新思路、新方法。

眼眶是构成面中部轮廓的重要骨组织结构，对眼球正常位置，面部外形、及视功能的维持至关重要。头面部外伤容易累及眼眶，引起骨缺损,导致严重后果。用组织工程技术真正一体化“修复”眼眶骨缺损，是目前的研究热点。但由于传统材料的局限性，临床修复效果不尽如人意。我们前期研究表明基因修饰BMSCs组织工程骨，可以有效促进骨再生，但由于基因整合的盲目性及随机性，可导致“原癌基因”的激活，具有致癌风险，成为临床应用的瓶颈。针对这一问题及眼眶骨缺损所特有内源性成骨成血管细胞匮乏的环境因素，本项目采用CRISPR靶向犬BMSC基因组“全港”导入外源目的基因SDF-1，在高表达SDF-1的同时，很好解决了临床应用的安全性问题。SDF-1可以招募成骨/成血管干细胞至损伤区域，活化破骨细胞分泌BMP，优化眼眶成骨微环境，促进眼眶骨再生。本课题在已建立的眼眶功能性重建手术和组织工程技术修复眼眶骨缺损的研究基础上，将BMSC细胞分离培养，CRISPR对种子细胞进行安全又有效的基因编辑，使其高高表达SDF-1，并对其增殖分化过程中促进血管形成的能力、招募成骨细胞能力及促成骨能力进行论证，以弥补内源性成骨细胞的缺乏，长效稳定地提供促成骨及促血管生长因子，以改善眼眶局部成骨微环境 ，为眶骨缺损修复提供全新的视角和坚实的实验基础。