苦参素通过抑制P-糖蛋白表达逆转结肠癌细胞的多药耐药性
发布时间：2019-01-05 21:39
【摘要】：目的:探讨苦参素(Oxymatrine,OMT)对结肠癌细胞HCT-8及其对长春新碱耐药的HCT-8/VCR细胞的增殖抑制作用及对化疗药的多药耐药逆转作用及可能分子机制。方法:(1)采用四甲基偶氮唑蓝法(Methyl Thiazolyl Tetrazolium,MTT)检测HCT-8/VCR细胞对化疗药长春新碱(Vincristine,VCR)、顺铂(Cisplatin,CDDP)及5-氟尿嘧啶(5-Fluorouracil,5-Fu)的多药耐药性。(2)采用MTT法检测苦参素对结肠癌细胞HCT-8及耐药细胞HCT-8/VCR的增殖抑制作用及1mg/ml苦参素作用后对各化疗药耐药性的变化。(3)实验分为HCT-8组、HCT-8/VCR组及HCT-8/VCR+苦参素组,苦参素组经1mg/ml苦参素作用48小时后,于倒置显微镜下观察各组细胞的形态,流式细胞术检测各组细胞内Rho123的荧光强度,qPCR检测ABCB1基因表达,Western blot法检测ABCB1基因编码的P-gp蛋白表达。结果:(1)MTT结果显示,HCT-8细胞对化疗药VCR,CDDP及5-Fu的IC50分别为25.63?3.13,12.33?2.01,2.89?0.62,耐药细胞HCT-8/VCR为320.95?6.44,45.95?4.76,18.32?3.91,两组相比差异均有统计学意义(P0.001),HCT-8/VCR细胞对VCR,CDDP及5-Fu的耐药倍数分别为12.52,3.73,6.34,提示该细胞为多药耐药细胞,可用于耐药相关实验。(2)苦参素可抑制耐药细胞HCT-8/VCR及其亲本细胞HCT-8的增殖,呈浓度依赖性(P0.05),选择抑制率低于10%的1mg/ml苦参素浓度作用48h作为后续逆转耐药相关实验。(3)经1mg/ml苦参素作用后,HCT-8/VCR细胞对化疗药VCR,CDDP及5-Fu的敏感性增加,各化疗药IC50均降低(P0.01),各化疗药耐药性逆转倍数分别为3.44,2.15,2.04。倒置显微镜下显示苦参素作用组细胞生长速度变慢,细胞漂浮死亡增加,可见空泡,贴壁性差。qPCR结果显示,HCT-8组及苦参素作用组ABCB1 mRNA表达均低于HCT-8-VCR组(P0.01);Western blot结果显示,HCT-8组及苦参素作用组中P-gp蛋白表达也明显低于HCT-8-VCR组(P0.05)。流式细胞术显示,苦参素作用组平均荧光强度为(23.45±0.64)%,高于HCT-8/VCR组(8.3±2.12)%,差异有统计学意义(P0.05).结论:苦参素可抑制结肠癌细胞增殖并逆转结肠癌细胞的多药耐药性,其机制可能与抑制ABCB1基因及其编码的P-gp的表达,提高细胞内药物浓度有关。
[Abstract]:Aim: to investigate the inhibitory effect of matrine (Oxymatrine,OMT) on the proliferation of colon cancer cell line HCT-8 and vincristine resistant HCT-8/VCR cell line and the reversal of multidrug resistance to chemotherapeutic agents and its possible molecular mechanism. Methods: (1) (Methyl Thiazolyl Tetrazolium,MTT was used to detect the chemotherapeutic agents of HCT-8/VCR cells, including vincristine (Vincristine,VCR), cisplatin (Cisplatin,CDDP) and 5-Fluorouracil (5-Fluorouracil). (2) MTT assay was used to detect the inhibitory effect of matrine on the proliferation of colon cancer cell line HCT-8 and drug resistant cell line HCT-8/VCR and the change of drug resistance of 1mg/ml matrine to various chemotherapeutic agents. (3) the experiment was divided into HCT-8 group, HCT-8/VCR group and HCT-8/VCR matrine group. After treated with 1mg/ml matrine for 48 hours, the morphology of cells in each group was observed under inverted microscope, and the fluorescence intensity of Rho123 in each group was detected by flow cytometry. QPCR was used to detect the expression of ABCB1 gene., Western blot method was used to detect the expression of P-gp protein encoded by ABCB1 gene. Results: (1) MTT results showed that the IC50 of HCT-8 cells to the chemotherapeutic drugs VCR,CDDP and 5-Fu were 25.63 ~ 3.1312.332.01and 2.890.62, respectively. The HCT-8/VCR of drug-resistant cells was 320.95 / 6.44 (45.95 / 4.76) and 18.32 / 3.91, respectively. There was significant difference between the two groups (P0.001), and there was a significant difference between the two groups (P0.001), and the difference between HCT-8/VCR cells and VCR, was significant (P0.001). The multiples of drug resistance of CDDP and 5-Fu were 12.52 ~ 3.73 ~ 6.34, respectively, which suggested that the cells were multidrug resistant and could be used in drug-resistance related experiments. (2) Matrine could inhibit the proliferation of HCT-8/VCR and its parent cells HCT-8. In a concentration-dependent manner (P0.05), the concentration of 1mg/ml, whose inhibitory rate was less than 10%, was selected for 48h as a subsequent reversal of drug-resistance related experiments. (3) after treated with 1mg/ml matrine, HCT-8/VCR cells were treated with the chemotherapeutic drug VCR,. The sensitivity of CDDP and 5-Fu was increased, the IC50 of each chemotherapeutic drug was decreased (P0.01), and the reversal times of drug resistance of each chemotherapeutic drug was 3.44 ~ 2.15 ~ 2.04respectively. The inverted microscope showed that the cell growth rate was slower, the cell floating death increased, the vacuole was visible, and the adhesiveness was poor in the matrine group. QPCR results showed that the growth rate of the cells in the matrine group was lower than that in the control group. The expression of ABCB1 mRNA in HCT-8 group and matrine treated group was lower than that in HCT-8-VCR group (P0.01). Western blot results showed that the expression of P-gp protein in HCT-8 group and matrine treated group was significantly lower than that in HCT-8-VCR group (P0.05). Flow cytometry showed that the average fluorescence intensity of the matrine treated group was (23.45 卤0.64)%, higher than that of the HCT-8/VCR group (8.3 卤2.12)%, the difference was statistically significant (P0.05). Conclusion: matrine can inhibit the proliferation of colon cancer cells and reverse the multidrug resistance of colon cancer cells. The mechanism may be related to the inhibition of the expression of ABCB1 gene and its encoded P-gp and the increase of intracellular drug concentration.
【学位授予单位】：右江民族医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.3
本文编号：2402342
[Abstract]:Aim: to investigate the inhibitory effect of matrine (Oxymatrine,OMT) on the proliferation of colon cancer cell line HCT-8 and vincristine resistant HCT-8/VCR cell line and the reversal of multidrug resistance to chemotherapeutic agents and its possible molecular mechanism. Methods: (1) (Methyl Thiazolyl Tetrazolium,MTT was used to detect the chemotherapeutic agents of HCT-8/VCR cells, including vincristine (Vincristine,VCR), cisplatin (Cisplatin,CDDP) and 5-Fluorouracil (5-Fluorouracil). (2) MTT assay was used to detect the inhibitory effect of matrine on the proliferation of colon cancer cell line HCT-8 and drug resistant cell line HCT-8/VCR and the change of drug resistance of 1mg/ml matrine to various chemotherapeutic agents. (3) the experiment was divided into HCT-8 group, HCT-8/VCR group and HCT-8/VCR matrine group. After treated with 1mg/ml matrine for 48 hours, the morphology of cells in each group was observed under inverted microscope, and the fluorescence intensity of Rho123 in each group was detected by flow cytometry. QPCR was used to detect the expression of ABCB1 gene., Western blot method was used to detect the expression of P-gp protein encoded by ABCB1 gene. Results: (1) MTT results showed that the IC50 of HCT-8 cells to the chemotherapeutic drugs VCR,CDDP and 5-Fu were 25.63 ~ 3.1312.332.01and 2.890.62, respectively. The HCT-8/VCR of drug-resistant cells was 320.95 / 6.44 (45.95 / 4.76) and 18.32 / 3.91, respectively. There was significant difference between the two groups (P0.001), and there was a significant difference between the two groups (P0.001), and the difference between HCT-8/VCR cells and VCR, was significant (P0.001). The multiples of drug resistance of CDDP and 5-Fu were 12.52 ~ 3.73 ~ 6.34, respectively, which suggested that the cells were multidrug resistant and could be used in drug-resistance related experiments. (2) Matrine could inhibit the proliferation of HCT-8/VCR and its parent cells HCT-8. In a concentration-dependent manner (P0.05), the concentration of 1mg/ml, whose inhibitory rate was less than 10%, was selected for 48h as a subsequent reversal of drug-resistance related experiments. (3) after treated with 1mg/ml matrine, HCT-8/VCR cells were treated with the chemotherapeutic drug VCR,. The sensitivity of CDDP and 5-Fu was increased, the IC50 of each chemotherapeutic drug was decreased (P0.01), and the reversal times of drug resistance of each chemotherapeutic drug was 3.44 ~ 2.15 ~ 2.04respectively. The inverted microscope showed that the cell growth rate was slower, the cell floating death increased, the vacuole was visible, and the adhesiveness was poor in the matrine group. QPCR results showed that the growth rate of the cells in the matrine group was lower than that in the control group. The expression of ABCB1 mRNA in HCT-8 group and matrine treated group was lower than that in HCT-8-VCR group (P0.01). Western blot results showed that the expression of P-gp protein in HCT-8 group and matrine treated group was significantly lower than that in HCT-8-VCR group (P0.05). Flow cytometry showed that the average fluorescence intensity of the matrine treated group was (23.45 卤0.64)%, higher than that of the HCT-8/VCR group (8.3 卤2.12)%, the difference was statistically significant (P0.05). Conclusion: matrine can inhibit the proliferation of colon cancer cells and reverse the multidrug resistance of colon cancer cells. The mechanism may be related to the inhibition of the expression of ABCB1 gene and its encoded P-gp and the increase of intracellular drug concentration.
【学位授予单位】：右江民族医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.3
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