PTHrP和Notch双信号通路调控骨骺干细胞的增殖

Effects of PTHrP and Notch signaling on the proliferation of epiphysis stem cells

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24h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Jaggedl／Fc和抑制剂DAPT处理，空白对照加PBS缓冲液，培养72h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）、Jaggedl／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Jaggedl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。

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 Jaggedl/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 Jaggedl/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 Jaggedl when PTHrP signaling was activated while a reductive expression of NICD and Jaggedl 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.