HSP70在煤焦沥青烟提取物致BEAS-2B细胞恶变过程中的表达变化

发布时间：2018-05-17 21:41

本文选题：永生化人支气管上皮细胞 + 煤焦沥青烟提取物　；参考：《郑州大学》2012年硕士论文

【摘要】：背景热休克蛋白70(Heat shock protein70, HSP70)是热休克蛋白家族中被研究最多的成员,是一类与肿瘤密切相关的具有分子伴侣和免疫调节作用的蛋白。HSP70能帮助细胞适应各种应激反应,减轻环境有害因子对细胞的损害作用,从而增强机体细胞的生存能力。国内外许多研究已经表明,HSP70在许多肿瘤中高表达,并在其发生、发展、治疗及预后的整个疾病过程中都扮演着重要的角色。煤焦沥青(Coal tar pitch, CTP)是煤炭在炼焦过程中产生的副产物,成分复杂,其所致肺癌是严重危害职业人群健康的恶性肿瘤之一。关于煤焦沥青致肿癌的研究很多,但其致癌机制尚未阐释清楚,有待进一步研究。该研究采用中温煤焦沥青烟提取物(Coal tar pitch smoke extract)作为诱导剂,建立永生化的人支气管上皮细胞BEAS-2B的恶性转化模型,检测该细胞系在细胞发生恶性转化的过程中HSP70与核苷酸切除修复因子的表达情况,探索HSP70及核苷酸切除修复因子在细胞恶性转化过程中的作用及其可能的机制,为进一步研究HSP70的生物学作用和煤焦沥青的致癌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实验分三组：即空白对照组、DMSO溶剂对照组、煤焦沥青组(煤焦沥青烟提取物溶液)。制备煤焦沥青烟提取物作为染毒剂,通过细胞活力计数法确定合适的染毒剂量,并以此剂量来诱导BEAS-2B细胞恶性转化,然后观察细胞转化过程中细胞表型的改变情况。 2.细胞恶性转化过程的检测用彗星实验检测染毒后BEAS-2B细胞损伤情况,并观察各代细胞形态的变化；用染色体核型分析和软琼脂集落形成实验检测细胞是否发生恶性转变。 3.HSP70蛋白表达水平与核苷酸切除修复因子表达水平的检测 Western blot法检测HSP70及核苷酸切除修复因子着色性干皮病互补基因蛋白质C(Xeroderma pigmentosum group, XPC)、切除修复交叉互补蛋白1(Excision repair cross complementing1, ERCC1)的表达,观察细胞受煤焦沥青烟提取物作用后,HSP70、XPC及ERCC1在传代过程中表达水平的动态变化。 4.统计学分析采用SPSS12.0对数据进行统计学分析,符合正态分布的数据以x±s表示；多组数据比较采用单因素方差分析；两组数据比较采用t检验,构成比采用卡方检验：两两比较采用LSD法,检验水准a=0.05。结果 1.细胞恶性转化试验彗星实验结果显示,染毒组细胞出现DNA损伤,与对照组比较,染毒组细胞拖尾长度较对照组均显著增高,差异有统计学意义(P0.05)；表明煤焦沥青烟提取物对BEAS-2B细胞具有遗传毒性。染毒组20代细胞发生形态学改变,细胞胞浆丰富,体积增大,类圆形、圆形比例增加,甚至部分细胞出现纤维细胞样改变；此代细胞核型出现明显异常,亚二倍体和超二倍体出现较多；但锚着力生长实验显示细胞仅有少数细胞能形成细胞集落,恶性程度不高。染毒组30代,胞体大小差异大,细胞内颗粒物增加,面积增大,大部分细胞外形变为类圆形、圆形,部分细胞膜边缘呈膜状向往扩展；细胞出现大量亚二倍体和超二倍体,畸变比率高；锚着力生长实验发现,此代细胞可以形成大细胞集落,无接触抑制,细胞发生了恶性转化。 2.BEAS-2B细胞中,蛋白质HSP70、XPC、ERCC1的表达染毒组在0代和30代细胞HSP70的表达与对照组比较,表达水平均显著升高(P0.05)；0代细胞XPC及ERCC1蛋白表达显著高于对照组(P0.05)；30代细胞中XPC蛋白显著低于2个对照组(P0.05), ERCC1蛋白表达显著高于对照组(P0.05)；对照组各组之间各个蛋白的表达差异均无统计学意义(P0.05)。结论 HSP70参与煤焦沥青烟提取物所致的BEAS-2B细胞恶性转化过程；细胞恶变后,HSP70能抑制XPC的表达；在细胞染色体突变及恶性转化过程中,DNA损伤可能是其始动因素,HSP70蛋白和XPC及ERCC1蛋白功能异常可能是其重要原因。
[Abstract]:background
Heat shock protein 70 (Heat shock protein70, HSP70) is the most studied member of the heat shock protein family. It is a kind of protein.HSP70, which is closely related to the tumor, with molecular chaperone and immunomodulatory function, can help cells adapt to various stress responses, reduce the damage of environmental harmful factors to cells, and thus enhance the cell of the body. Many studies at home and abroad have shown that HSP70 is highly expressed in many tumors and plays an important role in the whole process of its occurrence, development, treatment and prognosis.
Coal tar pitch (Coal tar pitch, CTP) is a by-product of coal in the process of coking, and its composition is complex. The cause of lung cancer is one of the malignant tumors that seriously harm the health of the occupational population. There are many researches on the swell cancer of coal tar pitch, but its carcinogenic mechanism has not been explained clearly and needs further study.
In this study, the medium temperature coal coke tar extract (Coal tar pitch smoke extract) was used as an inducer to establish an immortalized malignant transformation model of BEAS-2B in human bronchial epithelial cells, and to detect the expression of HSP70 and nucleotide excision factors during the malignant transformation of the cell line, and to explore the removal of HSP70 and nucleotides. The role of the repair factor in the process of cell malignant transformation and its possible mechanism provide a theoretical basis for further study of the biological role of HSP70 and the carcinogenic mechanism of coal tar pitch.
Method
Establishment of 1. cell malignant transformation model
The experiment was divided into three groups: blank control group, DMSO solvent control group, coal tar pitch group (coal tar leached tobacco smoke extract solution).
The extract of coal tar leachate was used as a dye agent to determine the appropriate dose by counting the cell vitality count, and to induce the malignant transformation of BEAS-2B cells by this dose, and then observe the change of cell phenotype in the process of cell transformation.
Detection of malignant transformation of 2. cells
The comet assay was used to detect the damage of BEAS-2B cells after exposure, and the changes of cell morphology were observed. The malignant transformation of the cells was detected by karyotype analysis and soft agar colony formation.
Expression level of 3.HSP70 protein and expression level of nucleotide excision repair factor
Western blot method was used to detect the expression of complementary gene protein C (Xeroderma pigmentosum group, XPC) of HSP70 and nucleotide excision repair factor coloring dry skin disease (Xeroderma pigmentosum group, XPC), and the expression of cross complementary protein 1 (Excision repair cross complementing1) was excised. Dynamic changes in the level of expression in the medium.
4. statistical analysis
The data were statistically analyzed with SPSS12.0, and the data conformed to normal distribution were expressed in X + s; the multiple groups of data were compared with single factor analysis of variance; the two groups of data were compared with t test, and the composition was compared to the chi square test: 22 compared with LSD, and the test level was a=0.05.
Result
1. cell malignant transformation test
The comet experiment showed that the cells of the infected group had DNA damage. Compared with the control group, the tail length of the cells in the infected group was significantly higher than that of the control group, and the difference was statistically significant (P0.05). It showed that the extract of coal tar leachate had a genetic toxicity to BEAS-2B cells. The 20 generation cells in the infected group had morphological changes, the cytoplasm was rich and the volume increased. Large, round, round, round, and even part of the cells appeared fibrous change. The karyotype of this generation was obviously abnormal, the diploid and the hyper diploid appeared more. But the anchorage growth experiment showed that only a few cells could form cell colonies, and the malignant range was not high. The size of the cells in the 30 generation of the infected group was large, and the cell size difference was large. The size of the inner particles increased and the area increased. Most of the cells changed into round and round shape, and the edge of the membrane of some cells was membranous. The cells appeared a large number of subdiploid and hyperdiploid, and the ratio of aberrations was high.
Expression of protein HSP70, XPC and ERCC1 in 2.BEAS-2B cells
The expression level of HSP70 in the 0 and 30 generation cells was significantly higher than that in the control group (P0.05), and the expression of XPC and ERCC1 protein in the 0 generation cells was significantly higher than that in the control group (P0.05), and the XPC protein in the 30 generation cells was significantly lower than that of the control group (P0.05), and the expression of ERCC1 protein was significantly higher than that of the control group (P0.05); the eggs of the control group were all eggs. There was no significant difference in the difference of white expression (P0.05).
conclusion
HSP70 participates in the malignant transformation of BEAS-2B cells caused by coal tar pitch smoke extract, and HSP70 can inhibit the expression of XPC after the malignant transformation of the cells. In the process of chromosome mutation and malignant transformation, the DNA damage may be its initiating factor, and the abnormal function of HSP70 protein and XPC and ERCC1 protein may be the important reason.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R114

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