克罗米酚对骨髓间质干细胞增殖和向成骨细胞分化的影响

目的　在离体条件下研究克罗米酚(CLO)对小鼠骨髓间质干细胞(BMSCs)增殖和向成骨细胞分化的影响。方法　在含经活性炭吸附的 10%血清的无酚红α MEM中加入β 磷酸甘油和维生素C诱导小鼠BMSCs向成骨细胞分化,同时加入 0 1nmol·L-1至 10nmol·L-1CLO处理细胞。用细胞计数来反映细胞增殖情况;测定碱性磷酸酶 (ALP)活性与钙的沉积量反映细胞向成骨细胞分化状态;用试剂盒来检测一氧化氮 (NO)的产物。结果　CLO ( 0 1 ~10nmol·L-1 )呈剂量依赖性地增加小鼠BMSCs的数目,ALP活性和钙的沉积量,同时培养基中NO代谢产物明显增加。分别加入雌激素受体 (ER)拮抗剂ICI182, 780 (0 1μmol·L-1 )及一氧化氮合酶抑制剂L NAME( 6mmol·L-1 )均可阻止CLO促进BMSCs的增殖及向成骨细胞分化的作用,并取消NO代谢产物的增加。结论　CLO在 0 1 ~10nmol·L-1剂量范围内具有雌激素样受体激活效应,可通过ER/NO途径促进小鼠骨髓间质干细胞的增殖及向成骨细胞分化。

The effects of Clomiphene on the proliferation and osteoblastic differentiation of mouse bone marrow-derived mesenchymal stem cells

Aim To investigate the effects of Clomiphene (CLO) o n proliferation and osteoblastic differentiation of mouse bone marrow-derived mes enchymal stem cells (BMSCs) in vitro.Methods Mouse BMSCs, cultured in phenol red-free, α-MEM supplemented with 10% charcoal-stripped fetal bovine serum, were added β-glycerolphosphate and Vitamin C for inducing osteoblastic differentiation, and treated with 0.1 nmol·L -1 to 10 nmol· L -1 CLO. The cell proliferation was measured by cell number count. The tem poral sequence of osteoblastic differentiation in BMSCs was assessed by measurin g alkaline phosphatase (ALP) activity and calcium deposition. Nitric oxide (NO) production was determined by measuring the stable end product of NO, nitrite, in the culture medium using commercial NO kit.Results CLO (0.1～10 nmol·L -1) res ulted in a dose-dependent increase in cell number, ALP activity and calcium dep osition in the BMSCs cultures. CLO treatment was associated with elevated nitrit e formation in conditioned media of BMCSs cultures. Concurrent treatment with IC I 182,780 (0.1 μmol·L -1), an estrogen receptor antagonist, or Nω-ni tro-L-arginine methylester (L-NAME, 6 mmol·L -1), a NOS inhibitor e liminated the stimulation of CLO on the proliferation and osteoblastic different iation of BMSCs cultures, and abolished the CLO (1 nmol·L -1)-induced inc rease in NO production.Conclusion Our results indicated that CL O at the range of 0.1～10 nmol·L -1 might be a estrogen-like receptor (E R) agonist, and stimulate the proliferation and osteoblastic differentiation of mouse BMSCs through ER/NO pathway.