西罗莫司体外诱导Colo-16细胞凋亡的研究

In vitro induction of apoptosis of a cutaneous squamous cell carcinoma cell line, Colo-16 cells, by sirolimus

目的研究西罗莫司对体外培养的皮肤鳞状细胞癌细胞株Colo-16细胞凋亡的影响。方法用不同浓度的西罗莫司处理Colo-16细胞，以四甲基偶氮唑蓝（MTT）法检测西罗莫司对Colo-16细胞体外增殖的影响；AnnexinV-FITC和PI双染检测Colo-16细胞凋亡率；Hoechst33258荧光染色法观察凋亡细胞的形态学变化；半定量逆转录聚合酶链反应法（RT-PCR）测定Bcl-2、Bax mRNA的表达。结果不同浓度的西罗莫司对Colo-16细胞体外增殖均具有抑制作用，这种抑制作用与西罗莫司的作用时间和剂量呈明显的依赖关系（P〈0．05）。西罗莫司显著增加Colo-16细胞的早期凋亡，西罗莫司（50，100，150，200nmol/L）作用48h后诱导Colo-16细胞的早期凋亡率分别为7．26％±0．26％、8．34％±0．19％、9．86％±0．14％、11．92％±0．15％，与对照组1．53％±0．09％比较，差异有统计学意义（P〈0．05），且Colo-16细胞早期凋亡率与西罗莫司浓度呈剂量依赖关系（r=0．955，P=0．000）。荧光染色可见细胞核染色质凝聚、边集、核裂解的形态学改变；Bcl-2 mRNA表达显著下调而Bax mRNA的表达显著增强。结论西罗莫司在体外能诱导Colo-16细胞凋亡，其发生机制可能与其调节Bcl-2、Bax等凋亡调控基因的表达有关。

Objective To investigate the in vitro effect of sirolimus on the apoptosis of a cutaneous squamous cell carcinoma cell line, Colo-16 cells. Methods Cultured Colo-16 cells were treated with different concentrations （50, 100, 150, 200 nmol/L） of sirolimus for various durations （12, 24, 48, 72 hours）. Subsequently, cell proliferation was detected by MTT assay, and cell apoptosis by Annexin V-FITC and PI double staining. Morphological changes of the cells were observed with Hoechst 33258 fluorescent staining. Total RNA was extracted from Colo-16 cells treated with sirolimus for 48 hours, and subjected to reverse transcription （RT）-PCR for the detection of mRNA expression of B cell lymphoma/leukmia-2 （Bcl-2） and Bcl-2-associated X Protein （Bax）. Results Sirolimus inhibited the proliferation of Colo-16 cells in a time- and dose-dependent fashion. The early apoptosis rate was 7.26% ± 0.26%, 8.34% ±0.19%, 9.86% ±0.14%, 11.92%± 0.15% in Colo-16 cells treated with sirolimus of 50, 100, 150, and 200 nmol/L, respectively, significantly higher than that in untreated cells （ 1.53% ± 0.09%, P 〈 0.05 ）; a positive correlation was observed between the apoptosis rate and concentrations of sirolimus （r = 0.955, P = 0.000）. Typical morphological changes of apoptosis, such as chromatin condensation and margination as well as nuclear fragmentation were observed by fluorescence staining. After treatment with sirolimus for 48 hours, a significant decrease was observed in the mRNA expression of Bcl-2, while an increase in that of Bax was noticed. Conclusion Sirolimus could induce Colo-16 cells apoptosis in vitro, which may be associated with the modulation of expression of apoptosis-regulating genes, such as Bcl-2 and Bax.