【作者】 陆皓泉；

【导师】 周航； 吴逸明；

【作者基本信息】 郑州大学 ， 劳动卫生与环境卫生学， 2012， 硕士

【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(P<0.05)；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(P<0.05)；0代细胞XPC及ERCC1蛋白表达显著高于对照组(P<0.05)；30代细胞中XPC蛋白显著低于2个对照组(P<0.05), ERCC1蛋白表达显著高于对照组(P<0.05)；对照组各组之间各个蛋白的表达差异均无统计学意义(P>0.05)。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。更多还原

【Abstract】 Background Heat shock protein70(HSP70) is a class of protein with the role of molecular chaperone and immune regulatory, which is closely related with tumor and was studied most in recent years. HSP70can help cells adapt to all kinds of stress responses and reduce the cell damage by the harmful factors of environment, which contributes to the improved the survival ability of body’s cells. Many researches had reported overexpressed HSP70in many tumors and sueggested that it was involved in tumor occurrence, development, treatment and prognosis at home and abroad.Coal tar pitch(CTP), with complicated compositions, is the by-products of coal during the process of coking coal. CTP induced lung cancer is one of the malignant tumors that severely affect professional workers. Many Studies showed that CTP can cause lung cancer, but the mechanism was yet unknown,and the real mechanism needs further research.This study tries to uncover the malignant transformation effects of coal tar pitch smoke extract on BEAS-2B in vitro and establish malignant transformation model of BEAS-2B induced by coal tar pitch extract, and then detect the expression of HSP70and nucleotide resection repair factor in BEAS-2B cell. To study the possible mechanism of HSP70and nucleotide resection repair factor during the process of malignant transformation, which will provide a basic tool for further studies on the biological function of HSP70and the mechanism of lung cancer induced by coal tar pitch.Methods1Establish malignant transformation model of BEAS-2B BEAS-2B cell were divided into three groups:a blank control group, DMSO solvent treated group, coal tar piteh smoke treated group (coal tar pitch smoke extract solution). Preparation of coal tar pitch smoke extract as dyeing agents and the appropriate dose was determined by the cell vitality count method; the change of cellular phenotype and the characteristics of the cellular biology were observed in the malignant transforxnation of BEAS-2B cells.2Detection of the process of malignant transformationThe damage of BEAS-2B cells were detected by single cell gel electrophoresis and observed the change of cell forms in differents generation. Conventional chromosomal analysis and the soft agar colony formation test were used to detect whether cells have been induced to malignant transformation.3Detect the express level of HSP70and nucleotide resection repair factorObserve the dynamic expression of xeroderma pigmentosum group C and excision repair cross complementing1in differents generation of BEAS-2B cells induced by coal tar pitch smoke extract.4Statistic analysisSPSS12.0was used for data analysis. Normal distribution data was expressed by x±s,and one-way ANOVA, t-test,χ2test,LSD were used to analyze the data(a=0.05).Results1Malignant transformation experimentComet assay results showed that coal tar pitch smoke extract may has genetoxic in BEAS-2B cell, and DNA damage of experiment group have longer tail length and showed significant statistical difference compared with control group(P<0.05). The20th generation cell of experiment group showed that cell had morphological changes, cell cytoplasm rich, cells became larger, the proportion of circular or similar to round shape cells rised, some cells even showed fibroblast-like changes. This generation cell in experiment group have significantly differents on cell’s karyotype,appeared more sub-lipliod or hyperdiploid cell, but the soft agar colony formation test showed that only a few cells can form macroscopic colonies, degree of malignancy is not high.The30th generation cell of experiment group have signficant different on the cell body size, Cells particles increased, area increased, most of cell were circular or similar to round shape cells, part of cell membrane edge is membrane yearning extension; appeared a amount of sub-lipliod or hyperdiploid cell; high distortion rate, the soft agar colony formation test showed that cell can form macroscopic colonies, degree of malignancy is high. BEAS-2B cell have completed malignant transformation.2The expression of HSP70、XPC and ERCC1.In the0th and30th generation cell, the expression of HSP70in the experiment group,were notable higher than the contronl group(P<0.05); at the0th generation cell, the protein expression of XPC and ERCC1significant higher than contronl group(P <0.05); at the30th generation cell,the protein expression of XPC significantly lower than the two control group(P<0.05); the protein expression of ERCC1significantly higher than the two control group(P<0.05); there were no signifieant difference between Solvent control group and blank control group(P<0.05). ConclusionHSP70take participate in the process of malignant trans formation induced by coal tar pitch smoke extract in BEAS-2B cell; HSP70can restrain the expression of XPC after malignant transformation of cells; The DNA damage may be the originating factor and the function abnormality of HSP70、XPC and ERCC1may be the important reasons in the process of chromosome mutation and malignant transformation in BEAS-2B cell.更多还原