CAJ | 학술논문

目的研究PTHrP、Notch双信号系统对骨骺干细胞增殖的调控作用.方法体内实验:取24 h内新生大鼠股骨体外器官培养,分别用PTHrP信号系统激活剂PTHrP(1～34)和PTHrP受体竞争抑制物PTHrP(7～34),及Notch信号系统激活剂Jagged1/Fc和抑制剂DAPT处理,空白对照加PBS缓冲液,培养72 h后收集标本行HE、Brdu及免疫组化染色方法检测.体外实验:构建PTHrP重组质粒和RNAi慢病毒表达载体转染体外培养的骨骺干细胞,收集转染后细胞用免疫印迹检测.结果与对照组相比,PTHrP(1～34)、Jagged1/Fc处理组静止区骨骺干细胞层长度在生长板全长中比值及Brdu阳性细胞率明显增高,促细胞增殖作用明显,而PTHrP(7～34)、DAPT处理组则抑制骨骺干细胞增殖.且PTHrP明显促进Notch信号通路配体Jagged1与受体NICD蛋白表达.结论 PTHrP、Notch信号通路均可促进骨骺干细胞增殖,且PTHrP可上调Notch表达进而促进骨骺干细胞的增殖.
목적 연구PTHrP、Notch쌍신호계통대골후간세포증식적조공작용.방법 체내실험:취24 h내신생대서고골체외기관배양,분별용PTHrP신호계통격활제PTHrP(1～34)화PTHrP수체경쟁억제물PTHrP(7～34),급Notch신호계통격활제Jagged1/Fc화억제제DAPT처리,공백대조가PBS완충액,배양72 h후수집표본행HE、Brdu급면역조화염색방법검측.체외실험:구건PTHrP중조질립화RNAi만병독표체재체전염체외배양적골후간세포,수집전염후세포용면역인적검측.결과 여대조조상비,PTHrP(1～34)、Jagged1/Fc처리조정지구골후간세포층장도재생장판전장중비치급Brdu양성세포솔명현증고,촉세포증식작용명현,이PTHrP(7～34)、DAPT처리조칙억제골후간세포증식.차PTHrP명현촉진Notch신호통로배체Jagged1여수체NICD단백표체.결론 PTHrP、Notch신호통로균가촉진골후간세포증식,차PTHrP가상조Notch표체진이촉진골후간세포적증식.
Objective To study the regulation of the proliferation of epiphysis stem cells by the PTHrP (parathyroidhormone related peptide) and Notch signaling systems. Methods An organ culture system of femurs of SD rat in 24 h after birth was employed. PTHrP ( 1 - 34) was used as the activator of the PTHrP signaling pathway and PTHrP (7 - 34) as the antagonist of PTH (parathyroidhormone) -receptor. For Notch signaling system, Jaggedl/Fc was used as the activator and DAPT as its inhibitor. The femurs were cultured in DMEM ( Dulbecco's modified Eagle's medium)/F12 medium while phosphate buffered saline was used for the control groups. Hhematoxylin and eosin staining and bromodeoxyuridine analysis were used to analyze the length of the epiphysis stem cells zone and the proliferation of epiphysis stem cells. The expression of NICD (Notch intra-cellular domain) and Jaggedl were analyzed by immunohistochemistry. The epiphysis stem cells were transfected with the lentiviral vectors with rat PTHrP gene overexpression or inhibition properties, the cells transfected with the PGC-GFP-lentivirus or NC-GFP-lentivirus were used as control. Western blot was employed to detect the expression of NICD and Jaggedl genes. Results PTHrP (1 -34) and Jagged1/Fc could dramatically elevate the rate of epiphysis stem cells zone by the whole growth plate length measurement while PTHrP (7 - 34 ) and DAPT could decrease the rate. Brdu analysis also showed that the number of proliferative epiphysis stem cells could be up-regulated by the PTHrP (1 -34 ) or Jagged1/Fc signaling. By contrast, the treatment with PTHrP (7 -34) or DAPT reduced the number of proliferative epiphysis stem cells. Immunohistochemistry and Western blot showed a significantly elevated expression of NICD and Jagged1 when PTHrP signaling was activated while a reductive expression of NICD and Jagged1 when PTHrP signaling was inactivated. Conclusion Both of PTHrP and Notch signaling system could promote the proliferation of epiphysis stem cells. And the PTHrP signaling can stimulate Notch signaling to promote the proliferation of epiphysis stem cells.