The Expression Changes Of HSP70in The Process Of Malignant Transformation Of BEAS-2B Cells Induced By Coal Tar Pitch Fume Extract

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.