【摘要】 多项研究发现，趋化素样因子1(chemokine-like factor 1,CKLF1)是人类多种肿瘤发生和进展的重要调节因子。然而，关于CKLF1对肾癌细胞生物活性的影响知之甚少。通过质粒转染实现CKLF1在肾癌细胞ACHN中过表达，探究CKLF1对肾癌细胞ACHN细胞增殖和凋亡的影响及其作用机制。将实验组pEGFP-N1-CKLF1和对照组pEGFP-N1真核细胞表达质粒分别转染肾癌ACHN细胞，实现趋化素样因子1在ACHN细胞中的过表达。利用荧光显微镜，镜下观察质粒转染效率。利用细胞计数试剂盒8(cell counting Kit-8,CCK8)观察肾癌细胞ACHN转染24、48、72 h后增殖情况；利用流式细胞术检测ACHN细胞转染24、48、72 h后细胞周期的变化情况；利用Hoechst33258染色法观察转染48 h后，ACHN细胞形态变化，利用流式细胞术检测各组细胞凋亡率。采用Western blot法检测CKLF1过表达后，细胞周期蛋白D1(Cyclin D1)、Bcl-2样蛋白1(Bcl-2 like 1,Bcl-xL)、信号传导及转录激活因子3(signal transducers and activators of transcription 3,STAT3)、磷酸化信号传导及转录激活因子3(phosphorylation signal transducers and activators of transcription 3,P-STAT3)表达情况。研究结果显示：荧光显微镜下90%以上的细胞有绿色荧光蛋白表达，证明转染效率良好。CCK8检测结果显示：转染48、72 h后，实验组肾癌细胞ACHN的增殖情况均明显高于对照组，差异有显著性意义48 h(P<0.05),72 h(P<0.05);流式细胞术检测结果显示：实验组增值指数PI明显高于对照组，差异有显著性意义24 h(P<0.01),48 h(P<0.01),72 h(P<0.01)。荧光显微镜下Hoechst染色结果显示：与对照组相比较，实验组凋亡小体数量较少。流式细胞术检测结果显示，与对照组相比较，转染后48 h后实验组细胞凋亡率明显降低(P<0.05)。Western blot结果：与对照组相比较，周期相关蛋白Cyclin D1表达明显高于对照组，差异有显著性意义(P<0.05);实验组凋亡相关蛋白Bcl-xL表达量明显高于对照组(P<0.05);实验组JAK激酶(Janus kinase, JAK/STAT3,JAK-STAT3)信号通路蛋白P-STAT3明显高于对照组(P<0.05)。趋化素样因子1过表达可促进肾癌细胞ACHN的增殖、抑制其凋亡，其发生机制可能与JAK-STAT3信号通路有关。CKLF1可能是肾透明细胞癌的新的治疗靶点。更多还原

【Abstract】 Chemokine-like factor 1(CKLF1) has been found by many studies to be an important regulator of the occurrence and progression of various human tumors. However, little is known about the effect of CKLF1 on the biological activity of renal cancer cells. CKLF1 was overexpressed in renal cancer cell ACHN by plasmid transfection, and the effect of CKLF1 on the proliferation and apoptosis of renal cancer cell ACHN cells and its mechanism were explored.The eukaryotic expression plasmids of the experimental group pEGFP-N1-CKLF1 and the control group pEGFP-N1 were respectively transfected into renal cancer ACHN cells to achieve the overexpression of chemokine-like factor 1(CKLF1) in ACHN cells. Plasmid transfection efficiency was observed under a fluorescence microscope. Cell Counting Kit-8(CCK8) was used to observe renal cancer cells Proliferation of ACHN cells 24, 48, and 72 h after transfection. Flow cytometry was used to detect cell cycle changes of ACHN cells 24, 48, and 72 h after transfection. The Hoechst33258 staining method was used to observe the morphological changes of ACHN cells 48 hours after transfection, and the apoptosis rate of each group was detected by flow cytometry. Western blot was used to detect the expression of Cyclin D1、BCL-2 like 1(Bcl-xL)、Signal Transducers and Activators of Transcription 3(STAT3) and Phosphorylation Signal Transducers and Activators of Transcription 3(P-STAT3)after overexpression of CKLF1. The results show as follows. More than 90% of the cells under the fluorescence microscope had green fluorescent protein expression, and the transfection efficiency is good. CCK8 test results show: 48 h and 72 h after transfection, the proliferation of renal cancer cell ACHN in the experimental group is significantly higher than that of the control group 48 h(P <0.01),72 h(P <0.01). low cytometry test results showed that the proliferation index PI of the experimental group was significantly higher than that of the control Group 24 h(P<0.01),48 h(P<0.01),72 h(P<0.01). Compared with the control group, the experimental group had fewer apoptotic bodies. The results of flow cytometry showed that compared with the control group, the apoptosis rate of the experimental group is significantly reduced 48 hours after transfection(P<0.05). Western blot results show: compared with the control group, the expression of cycle-related protein Cyclin D1 is significantly higher than that in the control group, and the difference is significant(P<0.05) The expression of apoptosis-related protein Bcl-xL in the experimental group is significantly higher than that in the control group(P<0.05). The Janus kinase, JAK/STAT3(JAK-STAT3) signaling pathway protein P-STAT3 in the experimental group was significantly higher than that in the control group(P<0.05). The overexpression of chemokine-like factor 1 can promote the proliferation and inhibit the apoptosis of renal cancer cell ACHN, and its mechanism may be related to the JAK-STAT3 signaling pathway. CKLF1 may be a new therapeutic target for renal clear cell carcinoma.更多还原