γ干扰素与阿霉素联用对成神经细胞瘤细胞生长及凋亡的影响

Study of Anti-proliferation and Apoptosis after Combined Treatment of IFN-γ with Doxorubicin in Caspase-8 Sile-nced Neuroblastoma Cell Line

目的探讨γ干扰素(IFN-γ)对阿霉素抗人成神经细胞瘤细胞作用的影响,并探讨其影响机制。方法应用四甲基偶氮唑蓝(MTT)分析和流式细胞仪,研究IFN-γ与化疗药阿霉素联合应用前后人成神经细胞瘤细胞SH-SY5Y的存活率及凋亡率;免疫组化方法检测IFN-γ作用前后半胱氨酸蛋白酶-8(caspase-8)蛋白的表达。结果阿霉素(0.03,0.10,0.30μg/ml)单独作用24h后,SY5Y细胞的存活率分别为(95.62±13.03)%,(82.62±7.94)%和(64.84±9.19)%,各组间差异显著。IFN-γ(10,100,1000U/ml)单独作用48h后,SY5Y细胞的存活率分别为(98.37±11.25)%,(97.15±5.36)%和(98.84±7.41)%,各组间差异无显著性。IFN-γ(1000U/ml)与不同浓度的阿霉素(0.03,0.10,0.30μg/ml)联合作用后,SY5Y细胞的存活率同单独应用阿霉素组比较明显下降,差异有显著性;阿霉素(0.30μg/ml)单独作用24h,SH-SY5Y的凋亡率为(22.00±6.55)%,IFN-γ(1000U/ml)单独作用48h,凋亡率为(8.22.±4.00)%。二者联合作用后,凋亡率较单用阿霉素组比较明显升高,差异有显著性;免疫组化方法发现SH-SY5Y细胞株不表达caspase-8,IFN-γ作用48h后caspase-8表达阳性。结论阿霉素对成神经细胞瘤细胞SH-SY5Y具有抑制增殖和诱导凋亡的作用,IFN-γ可以使其作用明显增强。其发生机制可能是通过IFN-γ上调caspase-8蛋白的表达而实现的。

Objective To explore the influence of IFN-γ on anti-proliferation and apoptosis of the Caspase-8 silenced neuroblastoma cell line induced by doxorubiein and the influence mechanism.Methods MTT and flow eytometrie analysis were used to detect the survival and apoptesis rates before and after the combined treatment of IFN-γ and doxorubicin.Immunohistochemical staining was used to detect the expression of caspase-8 protein before and after the treatment of IFN-γ. Results The survival rates of doxorubiein（0.03,0.10,0.30 μg/ml） used alone for 24 h were（95.62 ± 13.03）%,（82.62± 7.94）%,（64.84±9.19）%,IFN-γ（10, 100, 1000U/ml）used alone for 48h were（98.37% ± 11.25）%, （ 97.15 ± 5.36） %, （98.84 ± 7.41 ） %. The survival rates of IFN-γ （ 1000 U/ml ） combined with different concentration of doxorubicin （ 0.03,0.10,0.30 μg/ml ） were significantly lower than that of doxorubiein （ 0.03,0.10,0.30μg/ml ） done. The apoptosis rate of doxorubiein（0.30 μg/ml）done was（22.00 ±6.55）%, IFN-γ（ 1 000U/ml） done was （8.22 ±4.00）%, whereas the apoptesis rate of the combined treatment of IFN-γ and doxorubiein wassigniticantly higher than that of doxorubiein done; Immunohistochemical staining showed that caspase-8 protein was negative in SH-SYSY cell line, it become positiveafter the treatment of IFN-γ（ 1000 U/ml）for 48 h. Conclusion Treating human neuroblastoma cell line SH-SYSY cell with doxorubiein may induce anti-proliferation and apoptesis. Combined treatment with IFN-γ may significantly increase this effect. The mechanism may be realized by upregulating the expression of caspase-8 protein.