纳米羟基磷灰石对肝癌细胞蛋白质合成的抑制作用
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摘要
纳米羟基磷灰石nHAP)具有显著的体外抗肝癌细胞作用,而对肝细胞的抑制作用不明显。纳米羟基磷灰石的这种抗肝癌作用,为抗肝癌医学研究提供了一条新的途径。目前已知其主要作用机理是通过调节c-myc、p53的基因表达,使肝癌细胞阻止于G1期,激活caspase-9诱导肝癌细胞非凋亡性程序性死亡。为了进一步探究纳米羟基磷灰石抑癌作用的机理,本文主要通过研究纳米羟基磷灰石对肝癌细胞蛋白合成的影响来探究其抑制肝癌细胞增殖的机理。
选用人肝癌细胞系Bel-7402和人肝细胞系L02,用不同浓度的同种纳米羟基磷灰石进行不同时间处理,然后通过MTT比色法测定其对两种细胞的增殖抑制率。结果表明,在一定处理浓度和时间范围内,纳米羟基磷灰石对L02细胞影响很小的情况下,对Bel-7402细胞的增殖产生明显抑制作用,起到抑制肝癌细胞增长的作用。
通过用Eu标记的纳米羟基磷灰石Eu-nHAP处理Bel-7402细胞和L02细胞,然后用DAPI作为细胞核特异荧光探针对两种细胞的细胞核染色,用DiOC6(3)作为内质网特异荧光探针对两种细胞的内质网染色,最后再在激光共聚焦显微镜下观察到经荧光探针标记的Bel-7402细胞和L02细胞,发现Eu-nHAP处于内质网。说明纳米羟基磷灰石进入细胞后的作用靶点为内质网,并可能通过内质网,影响细胞蛋白的合成、分泌、空间折叠、蛋白质糖基化修饰等功能。
用含有3H-Leucine的1640培养基培养经纳米羟基磷灰石处理过的细胞,然后经液体闪烁计数器检测细胞合成蛋白质的速率。结果表明,经纳米羟基磷灰石处理后的细胞,总蛋白质的合成速率均有不同程度减缓。在小于0.7mg／ml的浓度范围内,处理3天的纳米羟基磷灰石明显抑制了Bel-7402细胞蛋白质的合成,而L02细胞蛋白质的合成受到的影响却不大,L02细胞仍然维持着正常的合成蛋白的功能。这说明纳米羟基磷灰石通过破坏Bel-7402细胞的蛋白质合成功能,抑制肝癌细胞增长。
用异硫氰酸荧光素(FITC)标记转铁蛋白制成Fe-TfFITC,并与纳米羟基磷灰石处理过的Bel-7402细胞,L02细胞共孵育,利用荧光光度计检测两种细胞转铁蛋白受体的含量。结果表明,经纳米羟基磷灰石处理的Bel-7402细胞转铁蛋白受体的含量明显减少,L02细胞转铁蛋白受体含量几乎没有影响。这表明纳米羟基磷灰石进入肝癌细胞后通过抑制转铁蛋白受体的表达,降低铁进入细胞的数量,来抑制核糖核苷酸还原酶的活性,阻碍DNA的合成,将无限增殖的肝癌细胞阻滞于G1期,起到抑制肝癌细胞生长和增殖的作用。
综上所述,纳米羟基磷灰石在进入肝癌细胞后作用于内质网,影响细胞总蛋白的生成,并通过对细胞转铁蛋白受体表达的抑制,使肝癌细胞阻滞于G1期,起到抑制肝癌细胞增殖的作用。
Previous studies show that nano-hydroxyapatite (nHAP) significantly inhibits the proliferation of hepatocellular carcinoma cells, but not that of hepatocytes in vitro. The inhibition of nHAP on carcinoma cells may provide a new way for medical research. The known main mechanism is that nHAP inhibits carcinoma cell proliferation by regulating the mRNA expression of c-myc and p53, arresting the hepatocellular carcinoma cells at G1 phase, and activating caspase-9 which ultimately leads to paraptosis. In order to further explore the mechanisms, this paper studied the influnence of nHAP on cell protein synthesis.
The inhibition rate of cells was measured using MTT method. After treating human hepatocellular carcinoma Bel-7402 cells and human liver L02 cells with nHAP of different concentrations for different time periods, the cell activity was measusred. It was found that, in a certain rang of concentration and time, nHAP inhibited the growth of Bel-7402 cells while had little effect on L02 cells.
The location of nHAP in cells is observed with confocal laser scanning microscopy. After treating Bel-7402 cells and L02 cells with Eu-nHAP, the nucleus and endoplasmic reticulum were stained with DAPI and DiOC6(3) separately. Eu-nHAP was found locating in the endoplasmic reticulum. The results showed that nHAP may effect the protein synthesis, secreting, space folding and glycosylation of cells.
The protein synthesis of nHAP was measured by H3-Leucine incorporation. It was found that the total protein synthesis rate of Bel-7402 cells slowed in varying degrees after treated with nHAP of less than 0.7mg/ml for 3days. In contrast, nHAP had less effect on L02 cells. It showed that nHAP inhibited hepatocellular carcinoma cells growth and proliferation by disrupting protein synthesis.
The expression of transferrin receptor in cells was measured by fluorescence intensity using FITC-labelled transrerrin. It was found that nHAP significantly decreased the expression of transferrin receptor in Bel-7402 cells, in contrast, nHAP had little effect on the expression of transferrin receptor in L02 cells. It showed that nHAP inhibited hepatocellular carcinoma cells growth and proliferation by decreasing the expression of transferrin receptor and arresting human hepatocellular carcinoma at G1 phase.
In summary, nHAP enters hepatocellular carcinoma cells, locates in endoplasmic reticulum, and inhibits human hepatocellular carcinoma growth and proliferation by disrupting protein synthesis, decreaseing the expression of transferrin receptor and arresting human hepatocellular carcinoma at G1 phase.
选用人肝癌细胞系Bel-7402和人肝细胞系L02,用不同浓度的同种纳米羟基磷灰石进行不同时间处理,然后通过MTT比色法测定其对两种细胞的增殖抑制率。结果表明,在一定处理浓度和时间范围内,纳米羟基磷灰石对L02细胞影响很小的情况下,对Bel-7402细胞的增殖产生明显抑制作用,起到抑制肝癌细胞增长的作用。
通过用Eu标记的纳米羟基磷灰石Eu-nHAP处理Bel-7402细胞和L02细胞,然后用DAPI作为细胞核特异荧光探针对两种细胞的细胞核染色,用DiOC6(3)作为内质网特异荧光探针对两种细胞的内质网染色,最后再在激光共聚焦显微镜下观察到经荧光探针标记的Bel-7402细胞和L02细胞,发现Eu-nHAP处于内质网。说明纳米羟基磷灰石进入细胞后的作用靶点为内质网,并可能通过内质网,影响细胞蛋白的合成、分泌、空间折叠、蛋白质糖基化修饰等功能。
用含有3H-Leucine的1640培养基培养经纳米羟基磷灰石处理过的细胞,然后经液体闪烁计数器检测细胞合成蛋白质的速率。结果表明,经纳米羟基磷灰石处理后的细胞,总蛋白质的合成速率均有不同程度减缓。在小于0.7mg／ml的浓度范围内,处理3天的纳米羟基磷灰石明显抑制了Bel-7402细胞蛋白质的合成,而L02细胞蛋白质的合成受到的影响却不大,L02细胞仍然维持着正常的合成蛋白的功能。这说明纳米羟基磷灰石通过破坏Bel-7402细胞的蛋白质合成功能,抑制肝癌细胞增长。
用异硫氰酸荧光素(FITC)标记转铁蛋白制成Fe-TfFITC,并与纳米羟基磷灰石处理过的Bel-7402细胞,L02细胞共孵育,利用荧光光度计检测两种细胞转铁蛋白受体的含量。结果表明,经纳米羟基磷灰石处理的Bel-7402细胞转铁蛋白受体的含量明显减少,L02细胞转铁蛋白受体含量几乎没有影响。这表明纳米羟基磷灰石进入肝癌细胞后通过抑制转铁蛋白受体的表达,降低铁进入细胞的数量,来抑制核糖核苷酸还原酶的活性,阻碍DNA的合成,将无限增殖的肝癌细胞阻滞于G1期,起到抑制肝癌细胞生长和增殖的作用。
综上所述,纳米羟基磷灰石在进入肝癌细胞后作用于内质网,影响细胞总蛋白的生成,并通过对细胞转铁蛋白受体表达的抑制,使肝癌细胞阻滞于G1期,起到抑制肝癌细胞增殖的作用。
Previous studies show that nano-hydroxyapatite (nHAP) significantly inhibits the proliferation of hepatocellular carcinoma cells, but not that of hepatocytes in vitro. The inhibition of nHAP on carcinoma cells may provide a new way for medical research. The known main mechanism is that nHAP inhibits carcinoma cell proliferation by regulating the mRNA expression of c-myc and p53, arresting the hepatocellular carcinoma cells at G1 phase, and activating caspase-9 which ultimately leads to paraptosis. In order to further explore the mechanisms, this paper studied the influnence of nHAP on cell protein synthesis.
The inhibition rate of cells was measured using MTT method. After treating human hepatocellular carcinoma Bel-7402 cells and human liver L02 cells with nHAP of different concentrations for different time periods, the cell activity was measusred. It was found that, in a certain rang of concentration and time, nHAP inhibited the growth of Bel-7402 cells while had little effect on L02 cells.
The location of nHAP in cells is observed with confocal laser scanning microscopy. After treating Bel-7402 cells and L02 cells with Eu-nHAP, the nucleus and endoplasmic reticulum were stained with DAPI and DiOC6(3) separately. Eu-nHAP was found locating in the endoplasmic reticulum. The results showed that nHAP may effect the protein synthesis, secreting, space folding and glycosylation of cells.
The protein synthesis of nHAP was measured by H3-Leucine incorporation. It was found that the total protein synthesis rate of Bel-7402 cells slowed in varying degrees after treated with nHAP of less than 0.7mg/ml for 3days. In contrast, nHAP had less effect on L02 cells. It showed that nHAP inhibited hepatocellular carcinoma cells growth and proliferation by disrupting protein synthesis.
The expression of transferrin receptor in cells was measured by fluorescence intensity using FITC-labelled transrerrin. It was found that nHAP significantly decreased the expression of transferrin receptor in Bel-7402 cells, in contrast, nHAP had little effect on the expression of transferrin receptor in L02 cells. It showed that nHAP inhibited hepatocellular carcinoma cells growth and proliferation by decreasing the expression of transferrin receptor and arresting human hepatocellular carcinoma at G1 phase.
In summary, nHAP enters hepatocellular carcinoma cells, locates in endoplasmic reticulum, and inhibits human hepatocellular carcinoma growth and proliferation by disrupting protein synthesis, decreaseing the expression of transferrin receptor and arresting human hepatocellular carcinoma at G1 phase.
引文
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