煤焦沥青烟提取物诱导BEAS-2B细胞恶性转化及其机制
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摘要
煤焦沥青(coal tar pitch, CTP)是煤炭在炼焦过程中的副产物,成分极其复杂,流行病学调查显示,煤焦沥青接触者肺癌的发病率明显高于非接触者,现已将煤焦沥青引发的肿瘤划定为职业肿瘤。在以往的研究中,多是以煤焦沥青的某单一成分为研究对象来探讨煤焦沥青对接触人群的危害和致癌机制的,而煤焦沥青这一混合物的致癌机制如何,鲜见报道,需要进一步研究。
该研究利用中温煤焦沥青烟提取物(coal tar pitch smoke extract)作为诱导剂,建立永生化人支气管上皮细胞BEAS-2B的恶性转化模型,在确定细胞发生恶性转化的前提下,对细胞整个转化过程进行分期,观察不同时期细胞的染色体不稳性,通过研究染毒细胞染色体损伤及恶性转化早期纺锤体检测点相关基因及蛋白的改变,探讨煤焦沥青烟提取物致细胞染色体不稳定性及其可能的致肺癌机制,为进一步研究煤焦沥青接触者罹患职业性肺癌发病机制提供一定的理论基础。
方法
1细胞恶性转化模型的建立
制备煤焦沥青烟提取物,通过细胞活力技术的方法测定合适的染毒剂量诱导细胞恶性转化,并观察BEAS-2B细胞转化过程中细胞表型的改变情况。
2染色体不稳定性观察
观察细胞受煤焦沥青烟提取物作用后传代过程中染色体的动态变化,观察染色体不稳定性。
3煤焦沥青烟提取物遗传毒性及纺锤体检测点功能检测
用彗星实验分析观察煤焦沥青烟提取物染毒对BEAS-2B细胞造成的遗传毒性进行观察RealTime PCR、免疫细胞化学技术检测纺锤体检测点相关基因Mad2、Bub1及其蛋白的表达情况。
4统计学分析
采用SPSS12.0对数据进行统计学分析,对符合正态分布的数据,以x±s表示。两组数据比较采用t检验,构成比比较采用χ2检验,多组数据比较采用单因素方差分析,两两比较采用LSD法,以α=0.05为检验水准。
结果
1细胞恶性转化试验
染毒组20代细胞形态发生改变,细胞体积增大,圆形类圆形细胞比例增加,部分细胞呈现成纤维细胞样改变,胞浆丰富,锚定独立生长实验结果显示细胞恶性程度不高,仅有极少数细胞3周时可以长成大的集落；30代,细胞大小差异显著,形态大部分变为圆形类圆形,面积增大,细胞内颗粒物增加,部分边缘呈膜状向外扩展,软琼脂克隆形成细胞经体外扩增培养后生长紊乱,无接触抑制,细胞已经发生恶性转化。流式细胞仪测定30代细胞的细胞周期显示G1期细胞比例明显减少,G2/M期细胞比例明显增加。
2染色体不稳定性的观察
恶性转化早期染毒组细胞已表现出明显的染色体不稳定性,差异较对照组有统计学意义(P<0.05),主要表现为细胞染色体数目和结构异常,且异常细胞数目随着细胞代数的增加有逐渐增多的趋势。
3煤焦沥青烟提取物遗传毒性及纺锤体检测点功能检测
煤焦油烟提取物可对受试细胞的DNA造成损伤,染毒组细胞拖尾长度较对照组均明显增高(P<0.05)。染毒组30代细胞Mad2、Bubl基因的表达均较对照组基因表达减低(P<0.05),Mad2、Bub1蛋白的平均光密度值较对照组都有所降低(P<0.05)。对照组之间各个基因与蛋白的表达差异无统计学意义(P>0.05)。结论
染色体不稳定性是煤焦沥青烟提取物染毒BEAS-2B细胞发生恶性转化的一种重要机制。在细胞染色体不稳定性及恶性转化过程中,DNA损伤可能是其始动因素,纺锤体检测点功能异常可能是其一个重要原因。
Coal tar pitch (CTP) is by-product made from coal duing the process of coking is mainly used in paint, rubber production. Epidemiological studies showed that there was a high incidence rate of lung cancer among the workors exposed to CTP defined as occupational tumor. In previous studies, single component of CTP was used to study the hazards and carcinogenic mechanisms of people exposed to coal tar pitch h, but as mixture there is rare reports about the carcinogenic mechanism of coal tar pitch.
To study the malignant transformation effects of coal tar pitch smoke extract on BEAS-2B in vitro and establish malignant transformation of BEAS-2B model induced by coal tar pitch extract, then further investigate the genotoxicity of coal tar pitch smoke extract by single cell gel electrophoresis (SCGE). Using RealTime PCR and cellular immune staining method to analyze the differences in the gene and proteins expression profiles between the early transformation cells and normal BEAS-2B cells for revealing the carcinogenesis and mechanism of lung cancer in coal tar pitch smoke extracts.
Methods
1 Malignant transformation model of BEAS-2B induced by coal tar pitch smoke extract.
BEAS-2B cells were reacted with coal tar pitch smoke, including a DMSO control group and a blank control grounp then with conventional culture. The characteristics of the cellular biology were observed and the malignant transformation of cells were identified through observing, anchorage independent growth and soon. Then the malignant transformation model of cells at different phases were established.
2 The CIN of BEAS-2B induced by coal tar pitch smoke extracts.
The chromosome aberration analyses showed that the CIN of BEAS-2B cells reacted by coal tar pitch smoke and the chromosome changes of BEAS-2B with cell transformation experiment.
3 The genotoxicity of coal tar pitch smoke extract and the function test of spindle checkpoint.
Investigating the genotoxicity of coal tar pitch smoke extract by SCGE. Using RealTime PCR and cellular immune staining method to analyze the differences of the spindle checkpoint-related genes and proteins expression profiles between the different groups of 30th generation cells.
4 Statistical analysis
Statistical software SPSS 12.0 was used for data analysis.χ2 test; one-way ANOVA, LSD were used to analyze (α=0.05)
Results
1 Malignant transformation experiment
The 30th generation cell of experiment group showed malignant transformation phenotype:different sizes; loss of the original spindle appearance, most of the changes for the class circle shape; cytoplasm increased; nuclear atypia; compound layer growth.Flow cytometry showed that the proportion of G1 phase cells was more decreased and G2/M phase cells was more increased of the 30th generations cell in experiment group than those of the 30th generations cell in control group, and there were significantly statistical difference between these two groups (P<0.05) indicating that cells altered growth kineties. The experimental group cells were formed small cell colonies in soft agar in 20th colonies The anchorage independent growth was appeared in 30th generation, after three weeks, there was few number of cells can grow into large experimental group cells.
2 Chromosome instability
In earlier period of malignant transformation, the cell included by coal tar pitch smoke extract had shown chromosome instability and had statistical difference with control group cells significantly (P<0.05), which mainly showed that the abnormal number and structure of chromosome.
3 Test of the genetoxic of coal tar pitch smoke extract and the function of spindle checkpoint
In the comet assay analyses in the BEAS-2B cells was included by coal tar pitch smoke extract, the results are positive. It means that coal tar pitch smoke extract may cause DNA damage. The cells of experiment group have longer tail length and had statistical difference with control group cells significantly (P<0.05). Coal tar pitch smoke extract has genetoxic for cell. The mRNA expression of spindle checkpoint-related genes were detected by RealTime PCR in the control group (P<0.05). Gene expression of Mad2, Bub1 in exposure group was lower than that of control group significantly. The protein expression of Mad2,Bub1 mainly located in the cytoplasm in coal tar pitch smoke extract cells, and the expression of Mad2, Bub1 were lower in the group of cells included by coal tar pitch smoke extract and had statistical difference with control group cells significantly (P<0.05)
Conclusion
Coal tar pitch smoke extract can induce malignant transformation of BEAS-2B cells in vitro.Coal tar pitch smoke extracts exposured can lead to chromosomal instability. DNA damage may be the initiating agent of the CIN and malignant transformation to the cell included with coal tar pitch smoke extract. The disfunction of spindle checkpoint may be an important agent of the CIN and malignant transformation to the cell included with coal tar pitch smoke extract.
该研究利用中温煤焦沥青烟提取物(coal tar pitch smoke extract)作为诱导剂,建立永生化人支气管上皮细胞BEAS-2B的恶性转化模型,在确定细胞发生恶性转化的前提下,对细胞整个转化过程进行分期,观察不同时期细胞的染色体不稳性,通过研究染毒细胞染色体损伤及恶性转化早期纺锤体检测点相关基因及蛋白的改变,探讨煤焦沥青烟提取物致细胞染色体不稳定性及其可能的致肺癌机制,为进一步研究煤焦沥青接触者罹患职业性肺癌发病机制提供一定的理论基础。
方法
1细胞恶性转化模型的建立
制备煤焦沥青烟提取物,通过细胞活力技术的方法测定合适的染毒剂量诱导细胞恶性转化,并观察BEAS-2B细胞转化过程中细胞表型的改变情况。
2染色体不稳定性观察
观察细胞受煤焦沥青烟提取物作用后传代过程中染色体的动态变化,观察染色体不稳定性。
3煤焦沥青烟提取物遗传毒性及纺锤体检测点功能检测
用彗星实验分析观察煤焦沥青烟提取物染毒对BEAS-2B细胞造成的遗传毒性进行观察RealTime PCR、免疫细胞化学技术检测纺锤体检测点相关基因Mad2、Bub1及其蛋白的表达情况。
4统计学分析
采用SPSS12.0对数据进行统计学分析,对符合正态分布的数据,以x±s表示。两组数据比较采用t检验,构成比比较采用χ2检验,多组数据比较采用单因素方差分析,两两比较采用LSD法,以α=0.05为检验水准。
结果
1细胞恶性转化试验
染毒组20代细胞形态发生改变,细胞体积增大,圆形类圆形细胞比例增加,部分细胞呈现成纤维细胞样改变,胞浆丰富,锚定独立生长实验结果显示细胞恶性程度不高,仅有极少数细胞3周时可以长成大的集落；30代,细胞大小差异显著,形态大部分变为圆形类圆形,面积增大,细胞内颗粒物增加,部分边缘呈膜状向外扩展,软琼脂克隆形成细胞经体外扩增培养后生长紊乱,无接触抑制,细胞已经发生恶性转化。流式细胞仪测定30代细胞的细胞周期显示G1期细胞比例明显减少,G2/M期细胞比例明显增加。
2染色体不稳定性的观察
恶性转化早期染毒组细胞已表现出明显的染色体不稳定性,差异较对照组有统计学意义(P<0.05),主要表现为细胞染色体数目和结构异常,且异常细胞数目随着细胞代数的增加有逐渐增多的趋势。
3煤焦沥青烟提取物遗传毒性及纺锤体检测点功能检测
煤焦油烟提取物可对受试细胞的DNA造成损伤,染毒组细胞拖尾长度较对照组均明显增高(P<0.05)。染毒组30代细胞Mad2、Bubl基因的表达均较对照组基因表达减低(P<0.05),Mad2、Bub1蛋白的平均光密度值较对照组都有所降低(P<0.05)。对照组之间各个基因与蛋白的表达差异无统计学意义(P>0.05)。结论
染色体不稳定性是煤焦沥青烟提取物染毒BEAS-2B细胞发生恶性转化的一种重要机制。在细胞染色体不稳定性及恶性转化过程中,DNA损伤可能是其始动因素,纺锤体检测点功能异常可能是其一个重要原因。
Coal tar pitch (CTP) is by-product made from coal duing the process of coking is mainly used in paint, rubber production. Epidemiological studies showed that there was a high incidence rate of lung cancer among the workors exposed to CTP defined as occupational tumor. In previous studies, single component of CTP was used to study the hazards and carcinogenic mechanisms of people exposed to coal tar pitch h, but as mixture there is rare reports about the carcinogenic mechanism of coal tar pitch.
To study the malignant transformation effects of coal tar pitch smoke extract on BEAS-2B in vitro and establish malignant transformation of BEAS-2B model induced by coal tar pitch extract, then further investigate the genotoxicity of coal tar pitch smoke extract by single cell gel electrophoresis (SCGE). Using RealTime PCR and cellular immune staining method to analyze the differences in the gene and proteins expression profiles between the early transformation cells and normal BEAS-2B cells for revealing the carcinogenesis and mechanism of lung cancer in coal tar pitch smoke extracts.
Methods
1 Malignant transformation model of BEAS-2B induced by coal tar pitch smoke extract.
BEAS-2B cells were reacted with coal tar pitch smoke, including a DMSO control group and a blank control grounp then with conventional culture. The characteristics of the cellular biology were observed and the malignant transformation of cells were identified through observing, anchorage independent growth and soon. Then the malignant transformation model of cells at different phases were established.
2 The CIN of BEAS-2B induced by coal tar pitch smoke extracts.
The chromosome aberration analyses showed that the CIN of BEAS-2B cells reacted by coal tar pitch smoke and the chromosome changes of BEAS-2B with cell transformation experiment.
3 The genotoxicity of coal tar pitch smoke extract and the function test of spindle checkpoint.
Investigating the genotoxicity of coal tar pitch smoke extract by SCGE. Using RealTime PCR and cellular immune staining method to analyze the differences of the spindle checkpoint-related genes and proteins expression profiles between the different groups of 30th generation cells.
4 Statistical analysis
Statistical software SPSS 12.0 was used for data analysis.χ2 test; one-way ANOVA, LSD were used to analyze (α=0.05)
Results
1 Malignant transformation experiment
The 30th generation cell of experiment group showed malignant transformation phenotype:different sizes; loss of the original spindle appearance, most of the changes for the class circle shape; cytoplasm increased; nuclear atypia; compound layer growth.Flow cytometry showed that the proportion of G1 phase cells was more decreased and G2/M phase cells was more increased of the 30th generations cell in experiment group than those of the 30th generations cell in control group, and there were significantly statistical difference between these two groups (P<0.05) indicating that cells altered growth kineties. The experimental group cells were formed small cell colonies in soft agar in 20th colonies The anchorage independent growth was appeared in 30th generation, after three weeks, there was few number of cells can grow into large experimental group cells.
2 Chromosome instability
In earlier period of malignant transformation, the cell included by coal tar pitch smoke extract had shown chromosome instability and had statistical difference with control group cells significantly (P<0.05), which mainly showed that the abnormal number and structure of chromosome.
3 Test of the genetoxic of coal tar pitch smoke extract and the function of spindle checkpoint
In the comet assay analyses in the BEAS-2B cells was included by coal tar pitch smoke extract, the results are positive. It means that coal tar pitch smoke extract may cause DNA damage. The cells of experiment group have longer tail length and had statistical difference with control group cells significantly (P<0.05). Coal tar pitch smoke extract has genetoxic for cell. The mRNA expression of spindle checkpoint-related genes were detected by RealTime PCR in the control group (P<0.05). Gene expression of Mad2, Bub1 in exposure group was lower than that of control group significantly. The protein expression of Mad2,Bub1 mainly located in the cytoplasm in coal tar pitch smoke extract cells, and the expression of Mad2, Bub1 were lower in the group of cells included by coal tar pitch smoke extract and had statistical difference with control group cells significantly (P<0.05)
Conclusion
Coal tar pitch smoke extract can induce malignant transformation of BEAS-2B cells in vitro.Coal tar pitch smoke extracts exposured can lead to chromosomal instability. DNA damage may be the initiating agent of the CIN and malignant transformation to the cell included with coal tar pitch smoke extract. The disfunction of spindle checkpoint may be an important agent of the CIN and malignant transformation to the cell included with coal tar pitch smoke extract.
引文
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