利用miRNA基因改質人類脂肪幹細胞於頭蓋骨典型缺損修復之應用

Repair of Critical Size Calvarial Defects Using Adipose-Derived Stem Cell Modulated with miRNA

廖亞信(Ya-Hsin Liao)

指導教授 ： 胡育誠

國立清華大學/工學院/化學工程學系/碩士(2014年)

摘要

大範圍的頭蓋骨缺損修復在臨床上是骨科與整形外科醫師的重大挑戰。近年來，幹細胞結合基因治療之新穎技術，已可加速骨修復，而脂肪幹細胞(adipose derived stem cell, ASC)比骨髓間葉幹細胞更容易分離與培養，深具臨床應用潛力。我們已證實使用桿狀病毒轉導ASC使其表現人類骨型態蛋白第二型(bone morphogenetic protein 2, BMP-2)，可以修復兔子股骨的大範圍缺損，但是同樣的轉導策略並無法應用於兔子頭蓋骨大範圍缺損之修復。最近發現某些微小片段RNA (miRNA)具有調控幹細胞分化的能力，因此本研究希望利用miRNA的調控，結合BMP-2的表現，使人類脂肪幹細胞(hASC)被誘導往硬骨路徑分化，並應用於修復頭蓋骨缺損。我們建構了帶有促骨分化miRNA (miR-148b)的桿狀病毒，與FLPo/Frt長效表現BMP-2的桿狀病毒共同轉導hASC後，我們發現細胞內骨分化指標基因表現量有著大幅的提升。之後我們以電腦斷層掃描(CT)分析了長效表現BMP-2結合短效或長效表現miR-148b在裸鼠頭蓋骨缺陷修復的效率，在術後第12周可以修復約九成的缺陷面積與體積，且組織切片染色結果顯示新生骨修復近似於天然的平板骨結構。後續我們證實miR-148b可能藉由調控BMP pathway的抑制劑Noggin (NOG)，以提升hASC的骨分化能力。本研究結合了miR-148b與生長因子BMP-2的共同表現，提升了人類脂肪幹細胞的硬骨分化能力，並成功地將hASC應用於頭蓋骨缺陷之修復，本研究成果對於未來在臨床上的應用應會有相當大的助益。

關鍵字

脂肪幹細胞 ； 桿狀病毒 ； 頭蓋骨缺陷 ； miR-148b ； BMP-2

並列摘要

Bony defects in the craniofacial skeleton remain a major and challenging health concern. Recent studies have shown that baculovirus-mediated gene therapy combined with bone marrow-derived mesenchymal stem cell (BMSC) therapy can improve the calvarial bone healing. Aside from BMSC, adipose-derived stem cells (ASC) have proven to be an abundant source of multipotent stem cells. However, ASC are less superior to BMSC in their osteogenesis potential. Therefore, hereby we aimed to combine baculovirus-mediated microRNA (miRNA) expression with ASC therapy, hoping that the miRNA can assist the regulation of osteogenesis and improve bone healing. We constructed multiple baculoviruses harboring miRNAs putatively associated with osteogenesis, and discovered that miR-148b can up-regulate osteogenic marker gene expression in osteogenic medium. Transduction of human ASC (hASC) with miR-148b and BMP-2, which not only extended the BMP2 expression beyond 14 days but augmented the ASC osteogenesis. Furthermore, the gelatin-coated poly(lactic-co-glycolic acid) (PLGA) scaffold seeded with engineered hASC was transplanted into mice critical size calvarial defect. After transplantation, μCT and histological analyses demonstrated that miR-148b remarkably ameliorated the regeneration of critical size calvarial defect, when compared with the group implanted with hASC only expressing BMP2. The hASC modulated with miR-148b accelerated the bone remodeling and regenerated the bone through the intramembranous pathway, filling ≈90% of the area and volume in 12 weeks. Furthermore, target prediction analysis tools and experimental validation by luciferase 3’ UTR reporter assay identified NOG (Noggin, a BMP antagonist) as a direct target of miR-148b. It was seen that over-expression of miR-148b and BMP-2 leads promotion of BMP-2/p-Smad1/5 signaling than that only expression of BMP-2. This strongly suggests that miR-148b promotes osteogenic differentiation by repressing NOG and its downstream signaling. Our study provides evidence that miRNA-based therapy can be a valuable tool to promote bone regeneration.

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