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目的筛选中药成分干预人口腔鳞癌耐药细胞株KBV200前后差异性表达蛋白质。方法采用四甲基偶氮噻唑蓝(MTT)法检测5种中药对KBV200的细胞毒性,选择对细胞抑制率低于10%(IC10),即无细胞毒作用的浓度,联合200 ng/mL长春新碱(VCR)对KBV200细胞株进行干预;取干预前后的细胞总蛋白提取液,用表面加强激光解析电离-飞行时间质谱(SELD I-TOF-MS)和CM-10蛋白质芯片技术筛选差异性蛋白。结果质荷比2 000～50 000Da范围内,表没食子儿茶素没食子酸酯(EGCG)干预KBV200前后共捕获13个差异蛋白质峰(均P<0.05),对于3997、4941、4968、5971、6084 Da 5个蛋白质峰,人口腔鳞癌细胞株KB表达较低,而KBV200表达明显升高(P<0.05),EGCG干预后表达明显降低(P<0.05);大黄素干预KBV200前后共捕获到6个差异蛋白质峰(P<0.05),对于4968,5971,6084 Da 3个蛋白质峰,KB表达较低,KBV200表达明显升高(P<0.05),大黄素干预后表达明显降低(P<0.05);未检测到两面针碱、板蓝根粗提物和岩黄连粗提物干预KBV200前后细胞和KB细胞间的差异性蛋白峰。结论 EGCG和大黄素干预KBV200前后均捕获到差异性蛋白质峰,这些差异性表达蛋白质可能与逆转KBV200多药耐药性有关。
Objective To screen the differentially expressed proteins of human oral squamous cell carcinoma KBV200 cells before and after treatment with traditional Chinese medicine. Methods The cytotoxicity of five kinds of Chinese herbs on KBV200 was detected by MTT assay. The cytotoxicity of KBV200 was determined by MTT assay. Neutral alkali (VCR) was used to intervene in KBV200 cell line; Total cellular protein extract before and after intervention was screened by surface enhanced laser desorption ionization-time of flight mass spectrometry (SELD I-TOF-MS) and CM-10 protein chip protein. Results There were 13 differentially expressed protein peaks (both P <0.05) before and after intervention of epigallocatechin gallate (EGCG) in the range of 2 000 ~ 50 000 Da. For 3997, 4941, 4968, 5971, 6084 Da 5 protein peak in human oral squamous cell carcinoma cell line was lower than that in KBV200 group, while the expression of KBV200 was significantly increased (P <0.05), while the expression of EGCG was significantly decreased (P <0.05) (P <0.05). The protein peak of 4968, 5971 and 6084 Da were lower than that of the other three protein peaks (P <0.05), and the expression of KBV200 was significantly lower (P <0.05) ; No detectable peaks of differential protein between KBV200 cells and KB cells after the intervention of crude extract of Rhizoma coptidis, crude extract of Radix Isatidis and crude extract of Rhizoma Coptidis. Conclusions Both EGCG and emodin can capture differential protein peaks before and after KBV200. These differentially expressed proteins may be related to reversing KBV200 multidrug resistance.