外源性1-磷酸神经鞘氨醇(S1P)对大鼠骨骼肌成肌细胞DNA合成的影响

Effects of sphingosine 1-phosphate(S1P) on DNA synthesis of skeletal myoblast cells in rats

目的观察外源性1-磷酸神经鞘氨醇(sphingosine 1-phosphate,S1P)对大鼠骨骼肌成肌细胞增殖的影响。方法分离和培养大鼠骨骼肌成肌细胞,通过制备S1P脂质体使S1P转运到骨骼肌成肌细胞内或直接加入外源性S1P,采用3 H-TdR渗入法检测S1P进入细胞内和在细胞外对骨骼肌成肌细胞DNA的合成;进一步使用磷酸神经鞘氨醇激酶(sphingosine kinase,SphK)活性抑制剂N,N-二甲基鞘氨醇(N,N-dimethyl sphingosine,DMS)和HACPT[2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole],观察其对S1P促进DNA合成作用的影响。结果 S1P脂质体转入可明显促进骨骼肌成肌细胞DNA合成。100～1 000nmol/L剂量范围内S1P脂质体使细胞胸腺嘧啶核苷(3 H-TdR)的摄入量较对照组增加12.3%～50.7%(P<0.01),并与剂量呈正相关。该促进作用不会被SphK活性抑制剂DMS和HACPT所阻断。S1P直接加入培养液同样能促进细胞DNA合成(P<0.01),500和1 000nmol/L剂量组3 H-TdR的摄入量较对照组分别增加18.8%(P<0.05)和26.5%(P<0.05),增幅低于同剂量的S1P脂质体。该促进作用可被DMS和HACPT所阻断。结论外源性S1P可促进体外培养的大鼠骨骼肌成肌细胞增殖,其作用可能与细胞内SphK活性增加引起细胞内S1P生成增多。

Objective To explore the effects of sphingosine 1-phosphate （S1P） on proliferation of skeletal myoblast cells in rats. Methods Primary skeletal myoblast cells in rats were separated and cultured, then roles of SiP in the regulation of DNA synthesis were described by dissecting them into intracellular and extracellular actions. Liposomal S1P transfer technique was used to make SIP- containing liposomes and control liposomes to transfer S1P into cells. Then incorporation of ~H-TdR method was applied to deteet the proliferation of skeletal myoblast cells. Meanwhile,sphingosine kinase （SphK） inhibitors N, N-dimethyl sphingosine （DMS） and 2-（p-hydroxynilino）-4-（p-chlorophenyl） thiazole （HACPT） were used to observe the effect on DNA synthesis promoted by extracellular and intracellular S1P in skeletal myoblast cells. Results Intraeellular S1P markedly promoted DNA synthesis in skeletal myoblast cells. Compared with the control,liposomal S1P of 100- 1 000 nmol/L increased 3 H-TdR incorporation of 12.3 %- 50.7% in a dose-dependent pattern with no relationship to Sphk inhibitors. Extracellular S1P also promoted DNA synthesis, which was strongly inhibited by SphK inhibitors. Extracellular S1P of 500 and 1 000 nmol/L increased 3 H-TdR incorporation of 18.5 and 26.5% ,respectively （all P〈0. 05） ,and the growth rates were less than that induced by the same dose of liposomal SIP. Conclusions myoblast ceils in rats,and SIP generation Extraceltular S1P promotes the proliferation of skeletal in skeletal myoblast cells may by promoted through SphK.