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目的探讨瘦素对子宫内膜癌细胞增殖的影响。方法荧光倒置显微镜下观测Ishikawa细胞瘦素相关受体(OBRb)的定位。分别使用0、10、50、100、150 ng/ml浓度梯度的瘦素分别在6、12、24 h 3个时间点对Ishikawa细胞进行干预处理,然后采用MTT比色法测定不同处理组细胞的增殖水平。Ishikawa细胞在使用瘦素进行干预后,同时分别加用STAT3通路阻断剂AG490和ERK1/2通路阻断剂PD98059,采用MTT比色法测定两种通路阻断剂对增殖水平的影响。采用蛋白免疫印迹法(Western blot)检测100 ng/ml浓度瘦素处理Ishikawa细胞不同时间点(0、20、40、60 min)STAT3和ERK1/2的蛋白磷酸化水平。结果倒置显微镜在Ishikawa细胞中能够观察到瘦素受体表达。Ishikawa细胞在体外经过瘦素干预后,其增殖水平明显增强,并随着药物剂量升高和处理时间延长而增强。在24 h时间点,Ishikawa细胞在100ng/ml浓度的瘦素刺激下其增殖能力达到最高水平,而100 ng/ml组与150 ng/ml组比较,差异无统计学意义(P>0.05);不同浓度梯度的STAT3通路阻断剂AG490与ERK1/2通路阻断剂PD98059都能明显下调细胞的增殖水平,并且其抑制作用与药物浓度呈正相关;采用100ng/ml浓度的瘦素处Ishikawa细胞后,STAT3和ERK1/2蛋白磷酸化升高水平与处理时间延长呈正相关。结论瘦素可能通过上调激活STAT3和ERK信号传导通路,进而增强子宫内膜癌Ishikawa细胞的增殖能力。
Objective To investigate the effect of leptin on the proliferation of endometrial carcinoma cells. Methods The localization of leptin related receptor (OBRb) in Ishikawa cells was observed under a fluorescence inverted microscope. Ishikawa cells were treated with leptin at concentrations of 0, 10, 50, 100 and 150 ng / ml for 3, 6, 12 and 24 h, respectively. Then MTT assay was used to determine the expression of Proliferation level. After Ishikawa cells were treated with leptin, STAT3 pathway inhibitor AG490 and ERK1 / 2 pathway inhibitor PD98059 were added respectively. The effects of two blockers on the proliferation were determined by MTT assay. The protein phosphorylation levels of STAT3 and ERK1 / 2 at different time points (0, 20, 40 and 60 min) in Ishikawa cells treated with 100 ng / ml leptin were detected by Western blot. Results Inverted microscope Leptin receptor expression was observed in Ishikawa cells. Ishikawa cells in vitro leptin intervention, the proliferation was significantly enhanced, and with the drug dose increases and the treatment time increases. At 24 h, Ishikawa cells proliferated to the highest level under the stimulation of leptin at a concentration of 100ng / ml, while there was no significant difference between the 100ng / ml and 150ng / ml groups (P> 0.05). Different concentrations of STAT3 pathway inhibitor AG490 and ERK1 / 2 pathway inhibitor PD98059 can significantly reduce cell proliferation, and its inhibitory effect and drug concentration was positively correlated; with 100ng / ml concentration of leptin Ishikawa cells The level of phosphorylation of STAT3 and ERK1 / 2 was positively correlated with the prolongation of treatment time. Conclusion Leptin may enhance the proliferation of Ishikawa cells of endometrial carcinoma by upregulating the STAT3 and ERK signal transduction pathways.