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妇科肿瘤中卵巢癌的恶性程度较高,易复发转移,且发现时通常是晚期从而难治。目前卵巢癌在临床上因其首诊时通常病期较晚,通常需要新辅助化疗[1]后再评估手术机会,但在治疗上仍然缺少切实有效的靶点。
学者在果蝇中最早发现了Hedgehog通路[2],因其可致使果蝇的胚胎表面形成刺凸,外形酷似刺猬得名,在细胞增殖及分化中扮演着重要角色[3],其绝大多数时间处于静息状态,主要起修复及维持作用[4]。近年来的研究[5]认为该途径为一条促进恶性肿瘤发生发展的通路。
在卵巢癌的临床治疗中,奈达铂作为第二代铂类药物,因消化系统毒性及神经毒性均低于顺铂,从而被应用于临床[6]。同时在寻求中西医结合抗肿瘤的过程中,近些年祖国传统医学在抗肿瘤的治疗中发挥了重要的作用。人参皂苷Rg1主要存在于人参属药材中,被视作是人参的活性成分之一,也有研究者表明其具备明确的抗癌作用[7]。
本文基于奈达铂对卵巢癌的作用,同时联合人参皂苷Rg1作用于卵巢癌OVCAR-3细胞,检测对细胞增殖、周期以及Hedgehog通路蛋白的作用,进而探讨奈达铂通过该通路影响卵巢癌发生发展的机制以及人参皂苷Rg1对奈达铂作用是否具有协同增效作用。
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4组在同浓度药物干预48h后,与Contral组相比药物干预后的卵巢癌细胞OVCAR-3增殖能力明显有减弱,并且随着药物浓度的增加,增殖抑制作用越明显(P < 0.01)。在最高浓度位置G(100μg/mL)、N(100μg/mL)、G(6.25μg/mL)+N(100μg/mL)组的增殖抑制率分别为16.50%、91.62%、95.70%;IC50分别为未达到、4.607μg/mL、3.387μg/mL(见表 1)。
浓度/
(μg/mL)OD(x±s) 抑制率/% G组 N组 G+N组* G组 N组 G组+N组 0 2.656±0.064 2.849±0.010 2.772±0.018 — — — 0.39 2.647±0.051 2.724±0.020 2.514±0.017 0.34 4.40 9.30 0.78 2.612±0.035 2.535±0.017 2.332±0.017 1.65 11.04 15.88 1.56 2.559±0.029 2.177±0.025 1.916±0.089 3.64 23.61 30.88 3.12 2.492±0.017 1.657±0.153 1.479±0.059 6.18 41.86 46.64 6.25 2.414±0.031 0.970±0.073 0.845±0.047 9.11 65.95 69.52 12.50 2.378±0.019 0.650±0.047 0.452±0.021 10.47 77.20 83.71 25.00 2.333±0.032 0.403±0.021 0.333±0.007 12.14 85.87 87.98 50.00 2.235±0.031 0.313±0.007 0.230±0.014 15.85 89.02 91.71 100.00 2.217±0.047 0.239±0.020 0.119±0.020 16.50 91.62 95.70 F 81.5 1 466.60 3 389.81 — — — P < 0.01 < 0.01 < 0.01 — — — MS组内 0.001 0.004 0.002 — — — *:G+N组浓度为固定人参皂苷Rg1的终浓度为6.25 μg/mL,依次增加奈达铂药物浓度形成梯度 表 1 不同浓度各组药物干预48h后的细胞增殖能力(ni=5)
选取Control组(不含药完全培养基)、G组(6.25 μg/mL,依据增殖抑制率 < 10%的最大无毒剂量)、N组(4.607 μg/mL,依据IC50)、G(6.25 μg/mL)+N(4.607 μg/mL)组,CCK-8法绘制药物干预7d细胞增殖曲线,与对照组比较,各组药物干预后均有明显抑制细胞增殖的现象(P < 0.05),且实验组间两两比较的差异在大多数时间点上有统计学意义(P < 0.05)(见图 1)。
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依据“2.1”实验结果,设定分组为对照组及实验组同“2.1”。PTCH1、SMO、Gli1蛋白表达(相对表达量,目的蛋白/内参)在经过G组、N组、G+N组干预后相比较对照组均有明显调低(P < 0.05),趋势按次序依次降低(P < 0.05)(见表 2、图 2)。
分组 n SMO/GAPDH PTCH1/GAPDH Gil1/GAPDH Control组 3 0.523±0.021 0.777±0.038 0.790±0.030 G组 3 0.38±0.017* 0.627±0.042* 0.583±0.049* N组 3 0.210±0.020*# 0.467±0.029*# 0.310±0.030*# G+N组 3 0.077±0.012*#△ 0.310±0.035*#△ 0.087±0.021*#△ F — 357.72 94.42 245.10 P — < 0.01 < 0.01 < 0.01 MS组内 — 0.000 0.001 0.001 注:与Control组比较*P < 0.05;与G组比较#P < 0.05;与N组比较△P < 0.05 表 2 不同分组药物干预后OVCAR-3细胞中PTCH1、SMO、Gli1的相对表达量(x±s)
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分组同“2.1”,不同分组药物干预后,组内比较药物干预后的细胞周期变化显示,在3组实验组中的表现均为G1、S、G2期细胞所占比例呈逐期下降趋势(P < 0.01);组间比较显示,与Control组相比,G+N组中G2期细胞比例差异明显低于其他组(P < 0.05)(见表 3、图 3)。
分组 n G1 S G2 F P MS组内 Control组 3 56.12±4.37 30.16±3.32 13.72±2.33 115.70 < 0.01 11.849 G组 3 58.07±6.18 30.06±7.91 11.87±1.78 46.91 < 0.01 34.634 N组 3 60.17±5.73 29.77±6.52 10.06±0.79 75.50 < 0.01 25.323 G+N组 3 67.89±4.78 26.23±4.73 5.88±0.07*#△ 198.83 < 0.01 15.075 F — 2.83 0.31 14.62 — — — P — > 0.05 > 0.05 < 0.01 — — — MS组内 — 28.243 34.619 2.307 — — — 注:与Control组比较*P < 0.05;与G组比较#P < 0.05;与N组比较△P < 0.05 表 3 不同分组药物干预后OVCAR-3细胞周期比例(x±s;%)
人参皂苷Rg1联合奈达铂通过Hedgehog通路抑制卵巢癌细胞OVCAR-3的增殖及机制探究
Study on the inhibitory effects of ginsenoside Rg1 combined with nedaplatin on the proliferation of ovarian cancer cells OVCAR-3 through Hedgehog pathway and its mechanism
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摘要:
目的 探究人参皂苷Rg1联合奈达铂对卵巢癌细胞OVCAR-3增殖的影响、可能的机制以及二者的协同作用。 方法 CCK-8法检测人参皂苷Rg1、奈达铂以及两者联合对OVCAR-3细胞增殖的影响;Western blotting检测3组药物干预后Hedgehog通路关键蛋白PTCH1、SMO、Gli1表达的改变;流式细胞仪检测3组药物干预后OVCAR-3细胞周期的变化。 结果 与未进行药物干预的对照组相比,人参皂苷Rg1组、奈达铂组、联合药物组均对OVCAR-3细胞的增殖产生了抑制作用,差异有统计学意义(P < 0.05),人参皂苷Rg1与奈达铂表现出协同增效作用;与对照组相比,奈达铂组、人参皂苷Rg1组、联合药物组降低了PTCH1、SMO、Gli1蛋白的表达,差异有统计学意义(P < 0.05),人参皂苷Rg1与奈达铂表现出协同增效作用;与对照组相比,仅有联合药物组将细胞周期阻滞在G2期(P < 0.05)。 结论 人参皂苷Rg1联合奈达铂可能通过调低Hedgehog通路相关蛋白将OVCAR-3细胞的周期阻滞在G2期从而抑制其增殖,人参皂苷Rg1对奈达铂表现出协同增效作用。 -
关键词:
- 卵巢肿瘤 /
- Hedgehog通路 /
- 奈达铂 /
- 人参皂苷Rg1
Abstract:Objective To investigate the effects of nedaplatin combined with ginsenoside Rg1 on the proliferation of ovarian cancer cells OVCAR-3, and its possible mechanism. Methods The CCK-8 assay was used to detect the effects of ginsenoside Rg1, nedaplatin and their combination on the proliferation of OVCAR-3 cells.Western-blot was used to detect the expression levels of key proteins of Hedgehog pathway(PTCH1, SMO and Gli1).The flow cytometry was used to detect the changes of cell cycle of OVCAR-3 cells after drug intervention. Results Compared with the control group, the proliferation of OVCAR-3 cells in ginsenoside Rg1 group, nedaplatin group and combined drug group were inhibited, and the difference of which among three groups were statistically significant(P < 0.05).At the same time, ginsenoside Rg1 and nedaplatin showed synergistic effects.Compared with the control group, the expression levels of PTCH1, SMO and Gli1 proteins in nedaplatin group, ginsenoside Rg1 group and combined drug group decreased significantly, the difference of which among three groups were statistically significant(P < 0.05), and the synergistic effect between ginsenoside Rg1 and nedaplatin was found.Compared with the control group, only the combined drug group, the cell cycle was arrested at G2 phase(P < 0.05). Conclusions Ginsenoside Rg1 combined with nedaplatin may inhibit the proliferation of OVCAR-3 cells by modulating Hedgehog pathway related proteins to arrest the cell cycle at G2 phase, and ginsenoside Rg1 shows the synergistic effect on nedaplatin. -
Key words:
- ovarian neoplasms /
- Hedgehog pathway /
- nedaplatin /
- ginsenoside Rg1
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表 1 不同浓度各组药物干预48h后的细胞增殖能力(ni=5)
浓度/
(μg/mL)OD(x±s) 抑制率/% G组 N组 G+N组* G组 N组 G组+N组 0 2.656±0.064 2.849±0.010 2.772±0.018 — — — 0.39 2.647±0.051 2.724±0.020 2.514±0.017 0.34 4.40 9.30 0.78 2.612±0.035 2.535±0.017 2.332±0.017 1.65 11.04 15.88 1.56 2.559±0.029 2.177±0.025 1.916±0.089 3.64 23.61 30.88 3.12 2.492±0.017 1.657±0.153 1.479±0.059 6.18 41.86 46.64 6.25 2.414±0.031 0.970±0.073 0.845±0.047 9.11 65.95 69.52 12.50 2.378±0.019 0.650±0.047 0.452±0.021 10.47 77.20 83.71 25.00 2.333±0.032 0.403±0.021 0.333±0.007 12.14 85.87 87.98 50.00 2.235±0.031 0.313±0.007 0.230±0.014 15.85 89.02 91.71 100.00 2.217±0.047 0.239±0.020 0.119±0.020 16.50 91.62 95.70 F 81.5 1 466.60 3 389.81 — — — P < 0.01 < 0.01 < 0.01 — — — MS组内 0.001 0.004 0.002 — — — *:G+N组浓度为固定人参皂苷Rg1的终浓度为6.25 μg/mL,依次增加奈达铂药物浓度形成梯度 表 2 不同分组药物干预后OVCAR-3细胞中PTCH1、SMO、Gli1的相对表达量(x±s)
分组 n SMO/GAPDH PTCH1/GAPDH Gil1/GAPDH Control组 3 0.523±0.021 0.777±0.038 0.790±0.030 G组 3 0.38±0.017* 0.627±0.042* 0.583±0.049* N组 3 0.210±0.020*# 0.467±0.029*# 0.310±0.030*# G+N组 3 0.077±0.012*#△ 0.310±0.035*#△ 0.087±0.021*#△ F — 357.72 94.42 245.10 P — < 0.01 < 0.01 < 0.01 MS组内 — 0.000 0.001 0.001 注:与Control组比较*P < 0.05;与G组比较#P < 0.05;与N组比较△P < 0.05 表 3 不同分组药物干预后OVCAR-3细胞周期比例(x±s;%)
分组 n G1 S G2 F P MS组内 Control组 3 56.12±4.37 30.16±3.32 13.72±2.33 115.70 < 0.01 11.849 G组 3 58.07±6.18 30.06±7.91 11.87±1.78 46.91 < 0.01 34.634 N组 3 60.17±5.73 29.77±6.52 10.06±0.79 75.50 < 0.01 25.323 G+N组 3 67.89±4.78 26.23±4.73 5.88±0.07*#△ 198.83 < 0.01 15.075 F — 2.83 0.31 14.62 — — — P — > 0.05 > 0.05 < 0.01 — — — MS组内 — 28.243 34.619 2.307 — — — 注:与Control组比较*P < 0.05;与G组比较#P < 0.05;与N组比较△P < 0.05 -
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