甲基丙烯酸环氧丙酯致人支气管上皮细胞恶性转化基因表达谱改变的研究
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摘要
甲基丙烯酸环氧丙酯(Glycidyl Methacrylate,简称GMA)作为一种聚合用单体,具有良好的防紫外、耐水和耐热等特性,在树脂、涂料、粘合剂、塑料工业中得到广泛应用。GMA分子含有碳碳双键和环氧基团,可参与离子型和自由基型反应,具有很高的活性,其合成的产品兼具丙烯酸的耐候性和环氧的耐化学性。
已有研究显示,GMA在动物体内是由还氧化物水化酶和非特异性羧酸酯酶这两个相互竞争的酶系统催化而代谢,属低毒类、具有明显的刺激性、引起速发型和迟发型变态反应、多项致突变试验结果阳性、存在生殖发育毒性,并可诱导多种哺乳类细胞发生恶性转化,具有高度可疑的致癌性。
细胞体外转化试验是研究癌变机制的重要方法,16HBE细胞(human bronchialepithelial cells)是SV40 large T抗原永生化的人支气管上皮细胞株,保留着正常人支气管上皮细胞的特定形态和功能,在体外易于培养,裸鼠体内无致瘤性,在种属和组织差异方面更具优势,可以作为一种较理想的靶细胞用于潜在化学致癌物检测及细胞恶性转化机制的研究。
本研究采用刀豆凝集素A凝集实验、锚着非依赖性实验和染色体畸变实验动态观察GMA诱导的转化前期细胞和转化细胞生物学特性的改变和染色体的损伤。采用Agilent公司生产的“人类全基因组表达谱芯片”对转化前期、转化细胞基因表达进行动态分析,绘制转化前期细胞和转化细胞的同/共基因表达谱,分析GMA诱导16HBE细胞恶性转化过程中相关基因表达及相关基因通路的变化,探讨GMA诱导16HBE细胞恶性转化的分子机制。
1.GMA诱导人支气管上皮细胞发生恶性转化
染毒细胞的生长曲线显示,GMA转化组细胞生长速度明显高于DMSO对照组,提示转化组细胞呈增殖性改变。转化前期细胞可在conA溶液中形成少量细胞团,不能在软琼脂中形成集落;而转化细胞对conA的凝集敏感性增加,发生细胞凝集现象,且可在软琼脂中形成细胞集落,提示转化细胞获得恶性细胞的生物学特性。转化前期细胞染色体结构畸变率明显高于对照组,未观察到染色体数目畸变,转化细胞中可见明显的染色体数目畸变。
2.GMA诱导16HBE细胞恶性转化过程中基因表达谱改变的研究
2.1GMA诱导16HBE细胞恶性转化过程中基因表达谱的改变
以基因表达数值标准值比值两倍以上作为差异表达基因的筛选标准,GMA诱导的转化前期细胞与同代龄的DMSO对照组细胞相比,3667个基因的表达显示出明显差异,其中包括2975个上调基因和692个下调基因;转化细胞与同代龄的DMSO对照组细胞相比,有1789个基因的表达显示出明显差异,其中包括719个上调基因和1070个下调基因。分析上述两不同时点差异基因表达谱,绘制转化前期细胞、转化细胞同/共表达基因谱,结果显示,有283个基因在转化前期细胞和转化细胞中均与对照细胞相比呈差异表达;231个基因在转化前期细胞和转化细胞中表达差异一致,其中88个基因在转化前期细胞和转化细胞中均呈上调表达,143个基因在转化前期细胞和转化细胞中均呈下调表达;48个基因在转化前期细胞中表达上调而在转化细胞中表达下调,4个基因在转化前期细胞中表达下调而在转化细胞中表达上调。
2.2 GMA诱导人支气管上皮细胞恶性转化过程中相关功能基因表达的改变
采用实时定量PCR技术分析差异基因表达谱中表达差异较大的基因,结果显示,C19orf20基因、EPB49基因、前列腺干细胞抗原基因、RGS14基因在转化前期细胞中表达上调;着丝粒蛋白F、结缔组织生长因子基因在转化细胞中表达下调;RASD1基因在转化前期细胞中表达上调而在转化细胞中表达下调。
2.3 GMA诱导16HBE细胞恶性转化过程中相关基因通路表达的改变
按照KEGG通路分类方法,转化前期细胞与同代龄对照组细胞相比有28个基因通路的基因表达存在统计学差异;而转化细胞与同代龄对照组细胞相比有7个基因通路的基因表达存在统计学差异。其中粘附因子通路、白细胞内皮迁移通路、致病性大肠杆菌感染通路在转化前期细胞和转化细胞中表达均与同代龄细胞存在统计学差异。
综上所述,本研究探讨了转化前期细胞和转化细胞基因表达谱的改变,研究了表达差异较大的基因,并对相关基因通路表达的改变进行了分析。结果提示,GMA诱导16HBE细胞恶性转化过程是涉及信号转导、细胞骨架、感染相关等多个通路C19orf20基因、EPB49基因、前列腺干细胞抗原基因、GS14基因、着丝粒蛋白F、结缔组织生长因子基因、RASD1基因等众多基因参与的复杂过程,这些研究结果为探讨GMA致癌机制及早期分子标志物的筛选提供了理论基础。
Glycidyl methacrylate (GMA), as a comonomer in resin, is made for a wide application, inchluding coating, adhesive and plastic industries. There are two reactive functional groups: a very reactive epoxy group and an acrylic group. The product of GMA is brought together the chemical resistance of an epoxy with the weatherability of an acrylic due to its dual functionality.
The metabolism of glycidyl methacrylate in mammals will likely proceed by at least two different and competing enzyme systems, epoxide hydratase and non-specific carboxylesterases. It has been previously reported that GMA is classified into the slight toxic category, with high irritation and immediate hypersensitivity and delayed hypersensitivity. The reproductive and development toxicity to rats has been also reported. Positive results are reported in several mutation assay. And it is capable of inducing malignant transformation in several types mammalia cells. Many results have demonstrated its carcinogenic potential.
In vitro cell transformation test is an important way to study on carcinogenesis mechanism. 16HBE (human bronchial epithelial cells), an SV40 large T antigen-immortalized human airway epithelial cell line, retains the phenotype of the differentiated lineage of the parent. This cell line retains contact inhibition and rarely produces tumors when injected into nude mice, which provides us an ideal model for detecting carcinogenic potential of chemicals and for studying on the mechanisms of malignant transformation.
In the study, biological characteristics of malignant transformation were dynamicly identified by the tests of conA and colony forming frequency on soft agar. The damage of chromosome in malignant transformation protophase cells and malignant transformation cells were observed dynamicly. The Agilent whole genome oligo microarray was used to detect the change of the expression of genes in malignant transformation protophase cells and malignant transformation cells induced by GMA, in order to discuss the mechanism of its potential carcinogenicity.
1 .Malignant transformation of humanbronchial epithelial cells induced by glycidylmethacrylate.
It can be observed that the growth of cells induced by GMA is faster than the growth ofthe normal 16HBE cells through the growth curvature. The cells of 14~(th) generation intest group could be agglutinated in the conA solution. The transformed cells beinganchorage independence could grow in semi-solid agar. It is indicated that the rate ofchromosome structure aberration in malignant transformation protophase cells issignificative increased compared with the solvent control group (P<0.05); while themalignant transformation cells lack the normal nuclear style(P<0.05).
2. Change of expression of genes in malignant transformation of 16HBE cells inducedby GMA.
2.1 The list of changed expression genes in malignant transformation of 16HBE cellsinduced by GMA.
There are 3667 genes showing different expression, including 2975 up-regulated genesand 692 down-regulated genes, in malignant transformation protophase cells. And 1789different expression genes was screened in the array, including 719 up-regulated genesand 1070 down-regulated genes, in malignant transformation cells. It was observed that283 genes expressed differently in both malignant transformation protophase cells andmalignant transformation cells. 88 of them showed up-regulation and 143 of themshowed down-regulation unanimously. 48 up-regulated genes and 4 down-regulatedgenes in malignant transformation protophase cells showed reverse trendecy inmalignant transformation cells.
2.2Change of expression of several relative genes in malignant transformation of16HBE cells induced by GMA.
The C19orf20, EPB49, PSCA, and RGS14 were observed up-regulated expression inthe malignant transformation protophase cells. The CENPF and CTGF were observeddown-regulated expression in the malignant transformation cells. The RASD1 wasobserved up-regulated expression in the malignant transformation protophase cellswhile down-regulated expression in the malignant transformation cells.
2.3 Relative pathways with genes differently expressing in malignant transformation of 16HBE cells induced by GMA.
There are 28 pathways in the malignant transformation protophase cells and 7 pathwaysin the malignant transformation cells expressing differently, with significative differencein statistics. And 3 pathways were observed significatively expressing differently inboth malignant transformation protophase cells and malignant transformation cells.
As stated above, the malignant transformation of human bronchial epithelial cells is acomplicated process involved many genes,such as C19orf20, EPB49, PSCA, RGS1,CENPF , CTGF, and RASD1, in many pathways, such as signal transduction,cystoskeleton, infection, and so on.
已有研究显示,GMA在动物体内是由还氧化物水化酶和非特异性羧酸酯酶这两个相互竞争的酶系统催化而代谢,属低毒类、具有明显的刺激性、引起速发型和迟发型变态反应、多项致突变试验结果阳性、存在生殖发育毒性,并可诱导多种哺乳类细胞发生恶性转化,具有高度可疑的致癌性。
细胞体外转化试验是研究癌变机制的重要方法,16HBE细胞(human bronchialepithelial cells)是SV40 large T抗原永生化的人支气管上皮细胞株,保留着正常人支气管上皮细胞的特定形态和功能,在体外易于培养,裸鼠体内无致瘤性,在种属和组织差异方面更具优势,可以作为一种较理想的靶细胞用于潜在化学致癌物检测及细胞恶性转化机制的研究。
本研究采用刀豆凝集素A凝集实验、锚着非依赖性实验和染色体畸变实验动态观察GMA诱导的转化前期细胞和转化细胞生物学特性的改变和染色体的损伤。采用Agilent公司生产的“人类全基因组表达谱芯片”对转化前期、转化细胞基因表达进行动态分析,绘制转化前期细胞和转化细胞的同/共基因表达谱,分析GMA诱导16HBE细胞恶性转化过程中相关基因表达及相关基因通路的变化,探讨GMA诱导16HBE细胞恶性转化的分子机制。
1.GMA诱导人支气管上皮细胞发生恶性转化
染毒细胞的生长曲线显示,GMA转化组细胞生长速度明显高于DMSO对照组,提示转化组细胞呈增殖性改变。转化前期细胞可在conA溶液中形成少量细胞团,不能在软琼脂中形成集落;而转化细胞对conA的凝集敏感性增加,发生细胞凝集现象,且可在软琼脂中形成细胞集落,提示转化细胞获得恶性细胞的生物学特性。转化前期细胞染色体结构畸变率明显高于对照组,未观察到染色体数目畸变,转化细胞中可见明显的染色体数目畸变。
2.GMA诱导16HBE细胞恶性转化过程中基因表达谱改变的研究
2.1GMA诱导16HBE细胞恶性转化过程中基因表达谱的改变
以基因表达数值标准值比值两倍以上作为差异表达基因的筛选标准,GMA诱导的转化前期细胞与同代龄的DMSO对照组细胞相比,3667个基因的表达显示出明显差异,其中包括2975个上调基因和692个下调基因;转化细胞与同代龄的DMSO对照组细胞相比,有1789个基因的表达显示出明显差异,其中包括719个上调基因和1070个下调基因。分析上述两不同时点差异基因表达谱,绘制转化前期细胞、转化细胞同/共表达基因谱,结果显示,有283个基因在转化前期细胞和转化细胞中均与对照细胞相比呈差异表达;231个基因在转化前期细胞和转化细胞中表达差异一致,其中88个基因在转化前期细胞和转化细胞中均呈上调表达,143个基因在转化前期细胞和转化细胞中均呈下调表达;48个基因在转化前期细胞中表达上调而在转化细胞中表达下调,4个基因在转化前期细胞中表达下调而在转化细胞中表达上调。
2.2 GMA诱导人支气管上皮细胞恶性转化过程中相关功能基因表达的改变
采用实时定量PCR技术分析差异基因表达谱中表达差异较大的基因,结果显示,C19orf20基因、EPB49基因、前列腺干细胞抗原基因、RGS14基因在转化前期细胞中表达上调;着丝粒蛋白F、结缔组织生长因子基因在转化细胞中表达下调;RASD1基因在转化前期细胞中表达上调而在转化细胞中表达下调。
2.3 GMA诱导16HBE细胞恶性转化过程中相关基因通路表达的改变
按照KEGG通路分类方法,转化前期细胞与同代龄对照组细胞相比有28个基因通路的基因表达存在统计学差异;而转化细胞与同代龄对照组细胞相比有7个基因通路的基因表达存在统计学差异。其中粘附因子通路、白细胞内皮迁移通路、致病性大肠杆菌感染通路在转化前期细胞和转化细胞中表达均与同代龄细胞存在统计学差异。
综上所述,本研究探讨了转化前期细胞和转化细胞基因表达谱的改变,研究了表达差异较大的基因,并对相关基因通路表达的改变进行了分析。结果提示,GMA诱导16HBE细胞恶性转化过程是涉及信号转导、细胞骨架、感染相关等多个通路C19orf20基因、EPB49基因、前列腺干细胞抗原基因、GS14基因、着丝粒蛋白F、结缔组织生长因子基因、RASD1基因等众多基因参与的复杂过程,这些研究结果为探讨GMA致癌机制及早期分子标志物的筛选提供了理论基础。
Glycidyl methacrylate (GMA), as a comonomer in resin, is made for a wide application, inchluding coating, adhesive and plastic industries. There are two reactive functional groups: a very reactive epoxy group and an acrylic group. The product of GMA is brought together the chemical resistance of an epoxy with the weatherability of an acrylic due to its dual functionality.
The metabolism of glycidyl methacrylate in mammals will likely proceed by at least two different and competing enzyme systems, epoxide hydratase and non-specific carboxylesterases. It has been previously reported that GMA is classified into the slight toxic category, with high irritation and immediate hypersensitivity and delayed hypersensitivity. The reproductive and development toxicity to rats has been also reported. Positive results are reported in several mutation assay. And it is capable of inducing malignant transformation in several types mammalia cells. Many results have demonstrated its carcinogenic potential.
In vitro cell transformation test is an important way to study on carcinogenesis mechanism. 16HBE (human bronchial epithelial cells), an SV40 large T antigen-immortalized human airway epithelial cell line, retains the phenotype of the differentiated lineage of the parent. This cell line retains contact inhibition and rarely produces tumors when injected into nude mice, which provides us an ideal model for detecting carcinogenic potential of chemicals and for studying on the mechanisms of malignant transformation.
In the study, biological characteristics of malignant transformation were dynamicly identified by the tests of conA and colony forming frequency on soft agar. The damage of chromosome in malignant transformation protophase cells and malignant transformation cells were observed dynamicly. The Agilent whole genome oligo microarray was used to detect the change of the expression of genes in malignant transformation protophase cells and malignant transformation cells induced by GMA, in order to discuss the mechanism of its potential carcinogenicity.
1 .Malignant transformation of humanbronchial epithelial cells induced by glycidylmethacrylate.
It can be observed that the growth of cells induced by GMA is faster than the growth ofthe normal 16HBE cells through the growth curvature. The cells of 14~(th) generation intest group could be agglutinated in the conA solution. The transformed cells beinganchorage independence could grow in semi-solid agar. It is indicated that the rate ofchromosome structure aberration in malignant transformation protophase cells issignificative increased compared with the solvent control group (P<0.05); while themalignant transformation cells lack the normal nuclear style(P<0.05).
2. Change of expression of genes in malignant transformation of 16HBE cells inducedby GMA.
2.1 The list of changed expression genes in malignant transformation of 16HBE cellsinduced by GMA.
There are 3667 genes showing different expression, including 2975 up-regulated genesand 692 down-regulated genes, in malignant transformation protophase cells. And 1789different expression genes was screened in the array, including 719 up-regulated genesand 1070 down-regulated genes, in malignant transformation cells. It was observed that283 genes expressed differently in both malignant transformation protophase cells andmalignant transformation cells. 88 of them showed up-regulation and 143 of themshowed down-regulation unanimously. 48 up-regulated genes and 4 down-regulatedgenes in malignant transformation protophase cells showed reverse trendecy inmalignant transformation cells.
2.2Change of expression of several relative genes in malignant transformation of16HBE cells induced by GMA.
The C19orf20, EPB49, PSCA, and RGS14 were observed up-regulated expression inthe malignant transformation protophase cells. The CENPF and CTGF were observeddown-regulated expression in the malignant transformation cells. The RASD1 wasobserved up-regulated expression in the malignant transformation protophase cellswhile down-regulated expression in the malignant transformation cells.
2.3 Relative pathways with genes differently expressing in malignant transformation of 16HBE cells induced by GMA.
There are 28 pathways in the malignant transformation protophase cells and 7 pathwaysin the malignant transformation cells expressing differently, with significative differencein statistics. And 3 pathways were observed significatively expressing differently inboth malignant transformation protophase cells and malignant transformation cells.
As stated above, the malignant transformation of human bronchial epithelial cells is acomplicated process involved many genes,such as C19orf20, EPB49, PSCA, RGS1,CENPF , CTGF, and RASD1, in many pathways, such as signal transduction,cystoskeleton, infection, and so on.
引文
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