牵张应力对Hedgehog通路相关基因和细胞增殖的影响研究

The Influence of Hedgehog Pathway and Proliferation of Osteoblasts Upon Stretch Forces

目的:观察牵张力下Hh通路相关基因的表达以及牵张力对成骨细胞增殖的影响,探索机械应力对成骨细胞的调控机制。方法:应用Flexcell 4000TM加力系统对大鼠成骨细胞施加0.5Hz,3%、6%和9%的张应变,作用时间4h、12h和24h。用机械通道抑制剂三氯化钆(GdCl3)对成骨细胞进行干预。采用流式细胞技术测定细胞周期分布与细胞增殖变化,定量PCR检测Hedgehog通路相关基因Ihh、Shh、Ptch和Smo的表达。采用SAS8.0软件包对结果进行统计分析。结果:不同强度、不同作用时间的张应变对Ihh表达量的上调作用亦不相同,6%张应变作用24h的对Ihh表达量上调最为明显,其下游基因Ptch和Smo的表达变化与Ihh类似;上述上调Ihh表达的作用均可被GdCl3抑制。;6%同3%牵张力相比,更能促进成骨细胞的增殖。结论:Ihh基因对力学刺激敏感;Ihh基因表达量的变化与成骨细胞的增殖相关。

Objective： To observe the expression of Hh genes and the proliferation of osteoblasts upon mechanical tensile strains and to clarify their effects on the osteoblasts. Methods： Tensile stretches were applied to the cells for periods of 4,12 and 24 hours using Flexcell--4000TM strain unit. The amplitude of tensile stretches applied to the ceils were 3 %, 6% and 9% respectively at a frequency of 0.5Hz. The cultures were also treated with gadolinium （GdCla）, an inhibitor of stretch--activated channels. The temporary expression of Ihh, Shh, Ptch, Smo were quantified by Realtime PCR. Statistical analysis was performed using SAS8.0 software package. Results： The up-- regulation of Ihh is higher at 6% tensile stretch than 3% tensile stretch（P〈0.05）after 4h, 12h and 24h of strain application, while the expression of Shh was not altered. Pcth and Smo showed a similar expression pattern with Ihh. The up--regulation of Ihh, Ptch and Smo by tensile strain was inhibited by GdC13 strain. Conclusion： Ihh signaling is sensitive to stretch forces. The changes of expression of Ihh closely relate to the changes of the proliferation of the osteoblasts. The amplitude of 6% is an optimal amplitude as stimulus for up-- regulation of expression of Ihh in osteoblasts and it increase the proliferation activity.