- 學位論文
膠原蛋白接受器Discoidin Domain Receptor 1對次發性股骨骨折癒合有正面效果
Discoidin domain receptor 1 is required for femur fracture healing
摘要
介紹: 骨折(fracture)是一種綜合性疾病，骨折癒合主要的生物學階段包括發炎，軟骨內骨化和重塑過程。Discoidin domain receptor 1 (Ddr1)是一種具有酪胺酸激酶活性的膠原蛋白接受體。在我們實驗室先前研究結果證實，在小鼠軟骨中特異性剔除Ddr1會延遲軟骨細胞之軟骨內骨化、終端分化及凋亡的現象，而導致長骨發育不全。顯示Ddr1於骨骼發育扮演重要角色，而軟骨細胞之軟骨內骨化乃骨折癒合重要機制之一，因此DDR1的表現與否對於骨折癒合可能具有重要意義。 假設： 在軟骨細胞特異性剔除Ddr1會減少軟骨內骨化過程並延遲骨折癒合。 方法: 實驗組別分別為Ddr1f/f -4OHT mice及CKOΔDdr1 mice，在公鼠第七週齡時連續五天注射4-Hydroxytamoxifen (4-OHT)誘導Cre-Loxp系統活化，使Ddr1在軟骨細胞中被剔除，於第八週齡進行股骨骨折手術，術後以每週兩次注射4-OHT維持Ddr1剔除狀態。術後利用X-ray觀察癒合組織之變化，再藉由Micro-CT system分析礦化癒合組織之相關數據及評估骨折部位的力學強度。藉由免疫組織化學染色法針對癒合組織中glycosaminoglycans含量、Ki67細胞繁殖標的物及TUNEL細胞凋亡標的物進行分析，為了瞭解骨頭癒合相關調控機制，以Real-time PCR及免疫組織化學染色法針對在骨折癒合期間可能受DDR1影響的每個階段的標的物等分析其蛋白質及mRNA表現量。 結果: X-ray影像顯示CKOΔDdr1 小鼠癒合組織面積於術後第3週明顯小於Ddr1f/f -4OHT 小鼠。Micro-CT system中的3D影像、組織體積（TV）及骨量（BV），CKOΔDdr1 小鼠於術後第5週明顯少於Ddr1f/f -4OHT小鼠。在三點彎曲試驗結果顯示CKOΔDdr1 小鼠有著較差的力學強度。Safranin-O-Fast Green 染色結果顯示CKOΔDdr1 小鼠癒合組織中sGAG含量於術後第3週明顯少於Ddr1f/f -4OHT小鼠。軟骨細胞中Ki67染色結果顯示CKOΔDdr1 小鼠的於術後第1週明顯少於Ddr1f/f -4OHT小鼠。TUNEL染色結果顯示CKOΔDdr1 小鼠的於術後第5週明顯少於Ddr1f/f -4OHT小鼠。在軟骨中特異性剃除Ddr1會顯著性的降低骨折癒合過程中涉及的基因表達。免疫組織化學染色結果顯示TGFß1、TGFß3及VWF的表現量具有顯著性的差異。 結論 : 結果表明，在軟骨細胞中特異性剔除Ddr1，CKOΔDdr1小鼠表現出延遲的股骨骨折癒合之特徵。證明Ddr1在早期骨骼癒合的過程中有著重要的角色，DDR1有潛力成為骨折癒合過程中的生物標的物。
並列摘要
Background The bone fracture is a comprehensive disease, the major biological stages of bone fracture healing including inflammatory, endochondral ossification and coupled remodeling. Discoidin domain receptor 1 (DDR1) is a receptor tyrosine kinase, and is activated by various collagens. Our previous results suggest that chondrocytes specific knockout Ddr1 may delay chondrocytes proliferation, differentiation, and apoptosis in long bone development. The Ddr1 has important role in skeletal development. Endochondral ossification is one of the important stage in the early fracture healing process. Thus, this study aimed to investigate the impact of DDR1 in fracture healing process. Hypothesis Ddr1 knockout in chondrocytes may reduce endochondral ossification and impair bone fracture healing Methods: The experimental animals are the Ddr1f/f -4OHT and CKOΔDdr1 mice, the CKOΔDdr1 mice was produced by using Cre-Lox recombination system and induction of 4-OHT, using the intramedullary fixation to create the fracture model. The monitored of callus and hard callus by using radiographic analysis and Micro-CT analysis. The mechanical strength of the femoral fracture mice was evaluated by a three-point bending test. To investigate may be affected markers of DDR1 the relate mechanism by IHC staining and mRNA expression. Results: The X-ray images and Micro-CT analysis showed that the callus area of CKOΔDdr1 mice were smaller than that of Ddr1f/f -4OHT and the mechanical strength exhibited weaker than that of Ddr1f/f -4OHT. The Safranin-O-Fast green stain, proliferation Ki67 and apoptosis TUNEL in CKO∆Ddr1 mice were less than those in Ddr1f/f -4OHT. Moreover, we found that Ddr1-specific knockout significant reduced the gene expression involved in chondrocytes during fracture healing. The IHC results showed that tgfß1, ß3, VWF of callus in CKO∆Ddr1 mice were significantly reduced compared with control Ddr1f/f -4OHT. conclusion: These results suggest that chondrocytes specifically knockout Ddr1 was exhibit a delay fracture healing features. To prove the important role of Ddr1 in early bone healing stage. The DDR1 has potential become biological marker in bone fracture healing.