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目的目前临床应用血必净注射液治疗急性放射性炎症反应取得了良好的疗效,但血必净注射液是否会影响肿瘤的生长和发展,此问题目前罕见报道。本实验探讨了血必净注射液对人鼻咽癌HNE-2细胞生物学行为的影响,为临床应用奠定基础。方法应用不同浓度(0、0.25、0.50、0.75、1.00、1.25 mg/mL)血必净注射液刺激鼻咽癌细胞HNE-2,四甲基偶氮唑蓝〔3-(4,5-dimethylthiazol-yl)-2,5-diphenyl tetrazolium bromide,MTT〕实验检测鼻咽癌细胞的生长增殖;应用流式细胞仪检测细胞凋亡及细胞周期;应用Transwell实验及蛋白质印迹法检测检测血必净注射液对鼻咽癌细胞侵袭、转移的影响及上皮间质转化(epithelial-mesenchymal transition,EMT)相关蛋白的表达。在裸鼠移植瘤模型中观察对照组、血必净组、放疗组、放疗+血必净组荷瘤鼠肿瘤生长曲线。结果 MTT实验显示,小剂量血必净刺激鼻咽癌细胞生长,但随浓度的增加,血必净注射液对鼻咽癌细胞增殖的抑制作用逐渐增强。空白对照组吸光度值为1.012±0.069,血必净0.25mg/mL组为1.034±0.061,0.50 mg/mL组为0.974±0.077,0.75 mg/mL组为0.806±0.103,1.00mg/mL组为0.656±0.098,1.25mg/mL组为0.626±0.017,F=28.353,P<0.001。凋亡实验显示,血必净处理20h,空白对照组细胞凋亡率为(3.97±0.13)%,血必净0.25mg/mL组为(8.79±0.36)%,0.50mg/mL组为(16.07±0.52)%,0.75mg/mL组为(25.60±0.57)%,1.0mg/mL组为(26.93±0.74)%,1.25mg/mL组为(30.84±0.69)%,F=1 167.35,P<0.001;血必净处理40h,空白对照组细胞凋亡率为(5.58±0.31)%,血必净0.25mg/mL组为(7.42±0.23)%,0.50mg/mL组为(9.30±0.08)%,0.75mg/mL组为(10.02±0.39)%,1.00mg/mL组为(12.6±0.31)%,1.25mg/mL组为(13.65±0.35)%,F=311.28,P<0.001。随着血必净浓度增大,细胞凋亡率增加。鼻咽癌荷瘤动物模型实验显示,血必净注射液可以抑制鼻咽癌移植瘤的生长,与放疗有协同作用。单独应用血必净注射液对鼻咽癌细胞的细胞周期无明显影响,可抑制EMT相关蛋白的表达和细胞的运动。Transwell实验结果显示,空白对照组细胞穿孔数为120.50±8.35,血必净0.25mg/mL组为94.5±2.08,0.50mg/mL组为85.00±2.58,0.75mg/mL组为77.25±2.21,1.00mg/mL组为74.00±4.83,1.25mg/mL组为61.75±1.71,F=88.873,P<0.001。结论血必净注射液对人鼻咽癌细胞的细胞周期无明显影响,但对细胞增殖和移植瘤生长,以及细胞运动有一定抑制作用,具有潜在的放疗增敏作用。
Objective The present clinical application of Xuebijing injection in the treatment of acute radiation inflammatory response has achieved good results, but Xuebijing injection will affect the growth and development of tumors, this issue is rarely reported. This experiment explored Xuebijing injection of human nasopharyngeal carcinoma cell line HNE-2 biological behavior, and lay the foundation for clinical application. Methods Xuebijing Injection at different concentrations (0,0.25,0.50,0.75,1.00,1.25 mg / mL) was used to stimulate nasopharyngeal carcinoma cells HNE-2, 4- (4,5-dimethylthiazol -yl) -2,5-diphenyl tetrazolium bromide (MTT) assay was used to detect the proliferation and proliferation of nasopharyngeal carcinoma cells. Flow cytometry was used to detect apoptosis and cell cycle. Transwell assay and Western blotting Fluid on the invasion and metastasis of nasopharyngeal carcinoma cells and the expression of epithelial-mesenchymal transition (EMT) -related proteins. The tumor growth curve of tumor-bearing mice in the control group, Xuebijing group, radiotherapy group, radiotherapy + Xuebijing group was observed in the nude mouse xenograft model. Results MTT experiments showed that a small dose of Xuebijing stimulate nasopharyngeal carcinoma cell growth, but with the concentration increased, Xuebijing injection on nasopharyngeal carcinoma cell proliferation was gradually enhanced. The absorbance value of blank control group was 1.012 ± 0.069, that of Xuebijing group was 1.034 ± 0.061, 0.974 ± 0.077 of 0.50 mg / mL group, 0.806 ± 0.103 of 0.75 mg / mL group and 0.656 of 1.00 mg / mL group ± 0.098 in the 1.25 mg / mL group and 0.626 ± 0.017 in the 1.25 mg / mL group, F = 28.353, P <0.001. Apoptosis experiments showed that the blood was treated with hematoporphyrin for 20h, the apoptotic rate in the blank control group was (3.97 ± 0.13)%, that in the Xuebijing group was (8.79 ± 0.36)% and that in the 0.50mg / mL group was (16.07 (25.60 ± 0.57)% in the 0.75 mg / mL group and (26.93 ± 0.74)% in the 1.0 mg / mL group and (30.84 ± 0.69)% and 1.257 <0.001; the blood was treated with BSA for 40h, the apoptotic rate in blank control group was (5.58 ± 0.31)%, that in Xuebijing group was (7.42 ± 0.23)% and that in 0.50mg / mL group was ), (10.02 ± 0.39)% in the 0.75 mg / mL group, (12.6 ± 0.31)% in the 1.00 mg / mL group, and 13.65 ± 0.35% for the 1.25 mg / mL group, F = 311.28, P <0.001. With the increase of blood concentration, apoptosis rate increased. The experiment of nasopharyngeal carcinoma tumor-bearing animal model shows that Xuebijing Injection can inhibit the growth of nasopharyngeal carcinoma and has a synergistic effect with radiotherapy. Xuebijing injection alone had no significant effect on the cell cycle of NPC cells and inhibited the expression of EMT-related proteins and cell motility. Transwell experimental results showed that the number of cells in the blank control group was 120.50 ± 8.35, blood chlorhexidine 0.25mg / mL group was 94.5 ± 2.08,0.50mg / mL group was 85.00 ± 2.58,0.75mg / mL group was 77.25 ± 2.21, 1.00 74.00 ± 4.83 for the mg / mL group, 61.75 ± 1.71 for the 1.25 mg / mL group, and F = 88.873, P <0.001. Conclusion Xuebijing Injection has no significant effect on the cell cycle of human nasopharyngeal carcinoma cells, but it has some inhibitory effects on cell proliferation, xenograft tumor growth and cell motility, and has the potential of radiosensitization.