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目的瘦素(Leptin)作为一种脂肪细胞分泌的激素,在低氧条件下对肿瘤细胞具有促进生长、血管生成、侵袭转移等作用。本研究体外观察Leptin对低氧状态下人骨肉瘤MG-63细胞增殖和凋亡的影响,探讨其可能的相关机制。方法将骨肉瘤MG-63细胞分为4组,分别在常氧、常氧联合100μg/L Leptin、低氧、低氧联合100μg/L Leptin下培养。蛋白质印迹法检测MG-63细胞中低氧诱导因子HIF-1α和凋亡相关蛋白Bax、Bcl-2及Caspase-3的表达;流式细胞技术检测各组细胞凋亡;采用MTT比色法测定细胞增殖率。结果低氧联合Leptin组细胞增殖活性明显强于常氧组、低氧组和低氧联合Leptin组。低氧联合Leptin组的HIF-1α和抗凋亡蛋白Bcl-2的表达水平明显高于其他组,促凋亡蛋白Bax、caspase-3表达明显低于其他组,差异有统计学意义(P<0.001)。析因分析结果显示,低氧(P<0.001)和Leptin(P<0.001)的主效应均可以使HIF-1α表达增加,但其交互作用对其无明显意义,P=0.109;低氧(P=0.001)和Leptin(P<0.001)的主效应同样可以使Bcl-2表达增加,但其交互作用对其无明显意义(P=0.1472);低氧(P<0.001)和Leptin(P<0.001)的主效应及交互作用效应(P=0.002)对于凋亡相关蛋白Bax均有显著影响;低氧(P<0.001)及Leptin(P<0.001)的主效应及交互作用效应(P<0.001)对于凋亡相关蛋白caspase-3均有显著影响。流式细胞仪检测结果显示,常氧组凋亡率为(6.50±0.35)%,常氧联合Leptin组凋亡率为(2.50±0.46)%,低氧组为(3.10±0.31)%,低氧联合Leptin组为(1.60±0.15)%,组间比较差异有统计学意义,P<0.001。MTT法检测常氧组、常氧联合Leptin组、低氧组、低氧联合Leptin组MG-63细胞增殖率分别为0.35±0.02、0.59±0.03、0.58±0.02和0.65±0.04。低氧联合Leptin组增殖率显著高于其他3组,P<0.001。结论低氧状态下Leptin能促进骨肉瘤MG-63细胞的增殖,促进抗凋亡蛋白表达,并抑制促凋亡蛋白表达。
Objective Leptin, a hormone secreted by adipocytes, can promote growth, angiogenesis, invasion and metastasis of tumor cells under hypoxic conditions. This study was designed to observe the effect of Leptin on the proliferation and apoptosis of human osteosarcoma MG-63 cells in vitro and to explore its possible mechanism. Methods Osteosarcoma MG-63 cells were divided into 4 groups and cultured under normoxic and normoxia combined with 100μg / L Leptin, hypoxia and hypoxia combined with 100μg / L Leptin. The expression of HIF-1αand Bax, Bcl-2 and Caspase-3 in MG-63 cells were detected by Western blotting. Apoptosis was detected by flow cytometry (FCM) Cell proliferation rate. Results The proliferation of cells in hypoxia combined with Leptin group was significantly stronger than that in normoxia group, hypoxia group and hypoxia group. The expression of HIF-1α and anti-apoptotic protein Bcl-2 in hypoxia combined with Leptin group was significantly higher than that in other groups, and the expressions of Bax and caspase-3 were significantly lower than those in other groups (P < 0.001). The analysis of factorial analysis showed that the main effect of hypoxia (P <0.001) and Leptin (P <0.001) could increase the expression of HIF-1α, but its interaction had no significant effect, P = 0.109; (P = 0.001) and Leptin (P <0.001) also increased the expression of Bcl-2, but the interaction had no significant effect on it (P = 0.1472) ) (P = 0.002) had a significant effect on apoptosis-related protein Bax. The main effect and interaction effect of hypoxia (P <0.001) and Leptin (P <0.001) Apoptosis-related protein caspase-3 have a significant impact. The results of flow cytometry showed that the apoptosis rate was (6.50 ± 0.35)% in normoxia group and (2.50 ± 0.46)% in normoxia combined with Leptin group and (3.10 ± 0.31)% in hypoxia group Oxygen combined Leptin group was (1.60 ± 0.15)%, the difference between the two groups was statistically significant, P <0.001. MTT assay showed that the proliferation rates of MG-63 cells in normoxic group, normoxia combined with Leptin group, hypoxia group and hypoxia combined with Leptin group were 0.35 ± 0.02, 0.59 ± 0.03, 0.58 ± 0.02 and 0.65 ± 0.04, respectively. The proliferation rate of hypoxia combined with Leptin group was significantly higher than the other three groups, P <0.001. Conclusion Leptin can promote the proliferation of osteosarcoma MG-63 cells under hypoxic condition, promote the expression of anti-apoptotic proteins and inhibit the expression of pro-apoptotic proteins.