重组人甲状旁腺素(1-34)影响兔骨髓间充质干细胞增殖及向成骨细胞分化的量效关系

Effects of recombinant human parathyroid hormone(1-34) on the proliferation of rabbit bone marrow-derived mesenchymal stem cells and the dose-effect relationship with differentiation to osteoblast

目的:分析重组人甲状旁腺素(1-34)影响兔骨髓间充质干细胞增殖、向成骨细胞分化与其作用时间及剂量的关系,认识重组人甲状旁腺素(1-34)作为骨形成促进剂的部分作用方式。方法:实验于2004-11/2005-04在云南省天然药物药理重点实验室完成。选用日本大耳兔,无菌取出双侧股骨、胫骨,分离骨髓间充质干细胞并进行原代培养、传代及诱导培养。将骨髓间充质干细胞分为8组:空白对照组(完全培养基)、阳性对照组(地塞米松10-11mol/L,β-甘油磷酸10mmol/L,维生素C50mg/L)及重组人甲状旁腺素(1-34)4000,400,40μg/L组,3个剂量组又分两种给药方式,即以48h为1个循环,药物作用时间分别为6h(间歇给药,6h后换为不含药培养基)和48h(连续给药,48h换液),共作用14d。于给药后第1～7天,用改良MTT法测各组细胞吸光度值(570nm),并绘制细胞生长曲线。分别于给药后第4,7,14天用超声粉碎仪粉碎细胞,取上清液用全自动生化分析仪进行碱性磷酸酶活性测定。结果:①各组骨髓间充质干细胞的生长增殖情况比较:重组人甲状旁腺素(1-34)4000,400,40μg/L6h组间充质干细胞在给药6d后,生长速度明显高于空白对照组、阳性对照组及重组人甲状旁腺素(1-34)4000,400,40μg/L48h组(P<0.05)。②各组骨髓间充质干细胞的碱性磷酸酶活性比较:给药第14天,重组人甲状旁腺素(1-34)4000,400μg/L48h组碱性磷酸酶活性显著低于空白对照组(P<0.05)。结论:重组人甲状旁腺素(1-34)促进骨形成的作用与剂量及作用时间有关。间歇性给药有利于骨髓间充质干细胞的增殖,而连续大剂量给药则抑制骨髓间充质干细胞向成骨细胞方向分化。

AIM： To analyze the effect in vitro of recombinant human parathyroid hormone （rh-PTH） （1-34） on the proliferation and osteoblastic differentiation of rabbit bone marrow derived mesenchymal stem cells （BMSCs） as well as the correlation between the action time and dose, so as to explore the action mechanism of rh-PTH （1-34） as the accelerator of bone formation. METHODS： The experiment was conducted in the Key Pharmacological Laboratory of Yunnan Natural Products between November 2004 and April 2005. Bilateral femur and tibia were obtained from Japan rabbits under sterile condition to separate the BMSCs for primary culture, passage and induced culture, The BMSCs were divided into 8 groups： blank control group （complete medium）, positive control group （dexamethasone 10^-11 mol/L, β-glycerophosphoric acid 10 mmol/L,Vitamin C 50 mg/L） and 4 000, 400,40 μg /L rh-PTH groups, moreover, three dose groups were administrated by two ways： 48 hours was taken as one cycle with the action time of 6 hours （intermittent administration and the medicine was taken place by the medium without medicine after 6 hours） and 48 hours respectively （continuous administration and the medicine was replaced at 48 hours later） for totally 14 days. The absorbance of cells （570 nm） in each group was detected by modified MTr method and the cell growth curve was drawn from the 1^st to the 7^th day after administration. Cells were smashed by ultrasonication respectively on the 4^th, 7^th and 14^th day after administration, and the alkaline phosphatase activities of supernatant was determined by automatic biochemistry analyzer. RESULTS： （1） Comparison in BMSCs growth and proliferation： The growth velocities of BMSCs in 4 000,400,40 μg /L rh-PTH groups at 6 hours after administration were significantly higher than that in the blank control group, positive control group and 4 000,400,40 μg/L rh-PTH groups at 48 hours after administration （P 〈 0.05）. （2） Comparison in the alkaline phosphatase activities of BMSCs： On the 14^th day of administration, it was remarkably lower in the 4 000,400,40 μg/L rh-PTH groups at 48 hours after administration than that in the blank control group （P 〈 0.05）. CONCLUSION： The promotion of rh-PTH （1-34） on bone formation is related with the exposing time and dose. Intermittent administration facilitates the proliferation of BMSCs, while continuous large-amount of administration inhibits the differentiation of BMSCs to osteoblast.