扁蒴藤素对肝细胞癌细胞生物学行为及吉西他滨化疗敏感性的影响
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1.河北省沧州市中心医院,肝胆胰外二科,河北 沧州 061000;2.河北省沧州市中心医院,肝胆胰外一科,河北 沧州 061000;3.河北省东光县中医医院 急诊科,河北 东光 061600

作者简介:

于彬,河北省沧州市中心医院主治医师,主要从事肝胆胰疾病相关研究。

基金项目:

河北省医学科学研究课题基金资助项目(20211343)。


Effects of pristimerin on biological behavior and the sensitivity to gemcitabine chemotherapy in hepatocellular carcinoma cells
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1.The Second Department of Hepatobiliary and Pancreatic Surgery, Cangzhou Central Hospital, Cangzhou, Hebei 061000, China;2.the First Department of Hepatobiliary and Pancreatic Surgery, Cangzhou Central Hospital, Cangzhou, Hebei 061000, China;3.Emergency Department, Dongguang County Hospital of Traditional Chinese Medicine, Dongguang, Hebei 061600, China

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    摘要:

    背景与目的 研究显示,扁蒴藤素(PT)可通过多种机制调控癌细胞增殖、凋亡、迁移及血管生成,然而其对肝细胞癌(HCC)的作用研究较少。因此,本研究探讨PT对体外培养的HCC细胞生物学行为及其对HCC细胞吉西他滨(GEM)化疗敏感性的影响,并分析机制。方法 CCK-8法检测PT、GEM对HCC细胞株Huh7、SMMC-7721及HepG2细胞增殖抑制作用;根据CCK-8实验结果,选择合适的HCC细胞株与药物浓度用于后续实验;分别用GEM(GEM组)、PT(PT组)、GEM联合PT(GEM+PT组)、Wnt/β-catenin信号通路抑制剂FH535(FH535组)处理HCC细胞,以单纯培养的细胞为对照组,然后分别用流式细胞术、平板克隆形成实验、细胞划痕、Transwell实验及qRT-PCR技术检测各组细胞凋亡、集落形成、迁移、侵袭及细胞c-myc、cyclin D1及survivin mRNA表达情况;同时用Western blot法检测β-catenin、GSK-3β、p-GSK-3β、波形蛋白(Vim)、E-钙黏蛋白(E-cad)及cleaved caspase 3(C-caspase-3)蛋白的表达。结果 PT与GEM均能明显抑制Huh7、SMMC-7721及HepG2细胞的增殖,且呈浓度依赖性(均P<0.05)。选择Huh7细胞为研究对象,PT、GEM、FH535的作用浓度分别为2.0 μmol/L、20 μmol/L、20 μmol/L。与对照组比较,GEM组、PT组、GEM+PT组及FH535组细胞凋亡率、E-cad及C-caspase-3蛋白相对表达量升高,集落形成数、划痕愈合率、侵袭细胞数、c-myc、cyclin D1和survivin mRNA及β-catenin、p-GSK-3β和Vim蛋白相对表达量降低(P<0.05);且GEM组、PT组、GEM+PT组、FH535组对Huh7细胞的上述变化依次逐渐增强(均P<0.05)。结论 PT对HCC细胞的恶性生物学行为有明显的抑制作用,且可增强HCC细胞的GEM化疗敏感性,其作用机制可能与抑制Wnt/β-catenin通路激活有关。

    Abstract:

    Background and Aims Studies have demonstrated that pristimerin (PT) can regulate the proliferation, apoptosis, migration and angiogenesis of cancer cells through multiple mechanisms. However, its role in hepatocellular carcinoma (HCC) has been less studied. Therefore, this study was conducted to investigate the effect of PT on the biological behavior of HCC cultured in vitro and its impact on the sensitivity of HCC cells to gemcitabine (GEM) chemotherapy, as well as the mechanism.Methods The inhibitory effects of PT and GEM on the proliferation of HCC cell lines (Huh7, SMMC-7721 and HepG2) were detected by CCK-8 assay. The suitable HCC cell line and drug concentrations were selected for subsequent experiments according to the results of CCK-8 assay. HCC cells were exposed to GEM (GEM group), PT (PT group), GEM plus PT (GEM+PT group) or Wnt/β-catenin signaling pathway inhibitor FH535 (FH535 group), respectively, using the purely cultured cells as the control group. Then, the apoptosis, colony formation, migration, invasion and the mRNA expressions of c-myc, cyclin D1 and surviving were detected by flow cytometry, plate cloning test, cell scratch assay, Transwell test and qRT-PCR, respectively. At the same time, Western blot technology was used to detect the protein expressions of β-catenin, GSK-3β, p-GSK-3β, vimentin (Vim), E-cadherin (E-cad) and cleaved caspase 3 (C-caspase-3).Results The proliferation rates of Huh7, SMMC-7721 and HepG2 cells were significantly decreased after exposure to both PT and GEM, with a concentration-dependent manner (all P<0.05). The Huh7 cells were chosen as the study object, and the treatment concentrations of PT, GEM and FH535 used were 2.0 μmol/L, 20 μmol/L and 20 μmol/L, respectively. Compared with control group, the apoptosis rate, the relative protein expressions of E-cad and C-caspase-3 were increased, the number of colony formation, scratch healing rate, the number of invasive cells, the relative mRNA expressions of c-myc, cyclin D1, survivin and protein expressions of β-catenin, p-GSK-3β and Vim were decreased in GEM group, PT group, GEM+PT group (all P<0.05). The above changes in Huh7 cells were increased successively in order of GEM group, PT group, GEM+PT group and FH535 groups (all P<0.05).Conclusion PT has significant inhibitory effect on the malignant biological behavior of HCC cells, and can enhance the sensitivity of HCC cells to GEM. The mechanism may be related to its inhibition of the activation of Wnt/β-catenin pathway.

    表 5 各组Huh7细胞划痕愈合率比较(%,x¯±sTable 5 Comparison of scratch healing rate of Huh7 cells in each group (%, x¯±s)
    表 1 不同浓度PT培养下各细胞存活率(%,x¯±sTable 1 Cell survival rates under different concentrations of PT (%, x¯±s)
    表 7 各组Huh7细胞相关基因相对表达量比较(x¯±sTable 7 Comparison of relative expression levels of related genes in Huh7 cells in each group (x¯±s)
    表 3 各组Huh7细胞凋亡率比较(%,x¯±sTable 3 Comparison of apoptosis rate of Huh7 cells in each group (%, x¯±s)
    表 2 不同浓度GEM培养下各细胞存活率比较(%,x¯±sTable 2 Cell survival rates under different concentrations of GEM (%, x¯±s)
    图1 流式细胞仪检测各组Huh7细胞凋亡情况Fig.1 The apoptosis of Huh7 cells in each group detected by flow cytometry
    图2 各组Huh7细胞集落形成情况Fig.2 Colony formation of Huh7 cells in each group
    图3 各组Huh7细胞迁移情况Fig.3 Migration of Huh7 cell in each group
    图4 各组Huh7细胞侵袭情况(×200)Fig.4 Invasion of Huh7 cells in each group (×200)
    图5 Western blot检测各组Huh7细胞相关蛋白表达Fig.5 Detection of the expressions of related proteins in Huh7 cells in each group by Western blot
    表 8 各组Huh7细胞相关蛋白相对表达量比较(x¯±sTable 8 Comparison of relative expression levels of related proteins in Huh7 cells in each group (x¯±s)
    表 6 各组Huh7细胞侵袭数比较(个,x¯±sTable 6 Comparison of invasion number of Huh7 cells in each group (x¯±s)
    表 4 各组Huh7细胞集落形成数比较(个,x¯±sTable 4 Comparison of colony formation number of Huh7 cells in each group (x¯±s)
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于彬,于勇,时磊,袁俊建.扁蒴藤素对肝细胞癌细胞生物学行为及吉西他滨化疗敏感性的影响[J].中国普通外科杂志,2023,32(2):221-230.
DOI:10.7659/j. issn.1005-6947.2023.02.007

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  • 收稿日期:2022-08-18
  • 最后修改日期:2023-01-20
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  • 在线发布日期: 2023-03-02