本文選題：永生化人支氣管上皮細胞 + 煤焦瀝青煙提取物�。� 參考：《鄭州大學(xué)》2012年碩士論文

【摘要】：背景 熱休克蛋白70(Heat shock protein70, HSP70)是熱休克蛋白家族中被研究最多的成員,是一類與腫瘤密切相關(guān)的具有分子伴侶和免疫調(diào)節(jié)作用的蛋白。HSP70能幫助細胞適應(yīng)各種應(yīng)激反應(yīng),減輕環(huán)境有害因子對細胞的損害作用,從而增強機體細胞的生存能力。國內(nèi)外許多研究已經(jīng)表明,HSP70在許多腫瘤中高表達,并在其發(fā)生、發(fā)展、治療及預(yù)后的整個疾病過程中都扮演著重要的角色。 煤焦瀝青(Coal tar pitch, CTP)是煤炭在煉焦過程中產(chǎn)生的副產(chǎn)物,成分復(fù)雜,其所致肺癌是嚴重危害職業(yè)人群健康的惡性腫瘤之一。關(guān)于煤焦瀝青致腫癌的研究很多,但其致癌機制尚未闡釋清楚,有待進一步研究。 該研究采用中溫煤焦瀝青煙提取物(Coal tar pitch smoke extract)作為誘導(dǎo)劑,建立永生化的人支氣管上皮細胞BEAS-2B的惡性轉(zhuǎn)化模型,檢測該細胞系在細胞發(fā)生惡性轉(zhuǎn)化的過程中HSP70與核苷酸切除修復(fù)因子的表達情況,探索HSP70及核苷酸切除修復(fù)因子在細胞惡性轉(zhuǎn)化過程中的作用及其可能的機制,為進一步研究HSP70的生物學(xué)作用和煤焦瀝青的致癌機制提供一定的理論基礎(chǔ)。 方法 1.細胞惡性轉(zhuǎn)化模型的建立 實驗分三組：即空白對照組、DMSO溶劑對照組、煤焦瀝青組(煤焦瀝青煙提取物溶液)。 制備煤焦瀝青煙提取物作為染毒劑,通過細胞活力計數(shù)法確定合適的染毒劑量,并以此劑量來誘導(dǎo)BEAS-2B細胞惡性轉(zhuǎn)化,然后觀察細胞轉(zhuǎn)化過程中細胞表型的改變情況。 2.細胞惡性轉(zhuǎn)化過程的檢測 用彗星實驗檢測染毒后BEAS-2B細胞損傷情況,并觀察各代細胞形態(tài)的變化；用染色體核型分析和軟瓊脂集落形成實驗檢測細胞是否發(fā)生惡性轉(zhuǎn)變。 3.HSP70蛋白表達水平與核苷酸切除修復(fù)因子表達水平的檢測 Western blot法檢測HSP70及核苷酸切除修復(fù)因子著色性干皮病互補基因蛋白質(zhì)C(Xeroderma pigmentosum group, XPC)、切除修復(fù)交叉互補蛋白1(Excision repair cross complementing1, ERCC1)的表達,觀察細胞受煤焦瀝青煙提取物作用后,HSP70、XPC及ERCC1在傳代過程中表達水平的動態(tài)變化。 4.統(tǒng)計學(xué)分析 采用SPSS12.0對數(shù)據(jù)進行統(tǒng)計學(xué)分析,符合正態(tài)分布的數(shù)據(jù)以x±s表示；多組數(shù)據(jù)比較采用單因素方差分析；兩組數(shù)據(jù)比較采用t檢驗,構(gòu)成比采用卡方檢驗：兩兩比較采用LSD法,檢驗水準a=0.05。 結(jié)果 1.細胞惡性轉(zhuǎn)化試驗 彗星實驗結(jié)果顯示,染毒組細胞出現(xiàn)DNA損傷,與對照組比較,染毒組細胞拖尾長度較對照組均顯著增高,差異有統(tǒng)計學(xué)意義(P0.05)；表明煤焦瀝青煙提取物對BEAS-2B細胞具有遺傳毒性。染毒組20代細胞發(fā)生形態(tài)學(xué)改變,細胞胞漿豐富,體積增大,類圓形、圓形比例增加,甚至部分細胞出現(xiàn)纖維細胞樣改變；此代細胞核型出現(xiàn)明顯異常,亞二倍體和超二倍體出現(xiàn)較多；但錨著力生長實驗顯示細胞僅有少數(shù)細胞能形成細胞集落,惡性程度不高。染毒組30代,胞體大小差異大,細胞內(nèi)顆粒物增加,面積增大,大部分細胞外形變?yōu)轭悎A形、圓形,部分細胞膜邊緣呈膜狀向往擴展；細胞出現(xiàn)大量亞二倍體和超二倍體,畸變比率高；錨著力生長實驗發(fā)現(xiàn),此代細胞可以形成大細胞集落,無接觸抑制,細胞發(fā)生了惡性轉(zhuǎn)化。 2.BEAS-2B細胞中,蛋白質(zhì)HSP70、XPC、ERCC1的表達 染毒組在0代和30代細胞HSP70的表達與對照組比較,表達水平均顯著升高(P0.05)；0代細胞XPC及ERCC1蛋白表達顯著高于對照組(P0.05)；30代細胞中XPC蛋白顯著低于2個對照組(P0.05), ERCC1蛋白表達顯著高于對照組(P0.05)；對照組各組之間各個蛋白的表達差異均無統(tǒng)計學(xué)意義(P0.05)。 結(jié)論 HSP70參與煤焦瀝青煙提取物所致的BEAS-2B細胞惡性轉(zhuǎn)化過程；細胞惡變后,HSP70能抑制XPC的表達；在細胞染色體突變及惡性轉(zhuǎn)化過程中,DNA損傷可能是其始動因素,HSP70蛋白和XPC及ERCC1蛋白功能異�？赡苁瞧渲匾�。
[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.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R114

【參考文獻】

相關(guān)期刊論文 前10條

1 孔令文;吳慶琛;朱進國;;人支氣管上皮細胞惡性轉(zhuǎn)化過程中FHIT蛋白表達的研究[J];中國肺癌雜志;2006年02期

2 陳萬青;張思維;鄒小農(nóng);;中國肺癌發(fā)病死亡的估計和流行趨勢研究[J];中國肺癌雜志;2010年05期

3 羅元明,劉彥信,鄭德先;蛋白質(zhì)組學(xué)研究方法及其在生物醫(yī)學(xué)中的應(yīng)用[J];基礎(chǔ)醫(yī)學(xué)與臨床;2003年02期

4 郝巧玲,吳曉明,姚群峰,周宜開;核苷酸切除修復(fù)基因表達水平與肺癌易感性的關(guān)系[J];環(huán)境與職業(yè)醫(yī)學(xué);2004年03期

5 楊瑾;劉曉勇;鄒云鋒;牛丕業(yè);宮智勇;袁晶;鄔堂春;;B(a)P作用下的人支氣管上皮細胞DNA損傷與XPA、XPC蛋白表達的關(guān)系[J];環(huán)境與職業(yè)醫(yī)學(xué);2007年04期

6 王玉珍;吳逸明;周舫;李偉輝;梁守沛;許東;任文杰;;HSP70-1、HSP70-2基因多態(tài)性與肺癌易感性的相關(guān)性[J];環(huán)境與職業(yè)醫(yī)學(xué);2010年05期

7 衛(wèi)正義;樊生才;;煤焦油加工技術(shù)進展及產(chǎn)業(yè)化評述[J];煤化工;2007年01期

8 鄒小農(nóng);;中國肺癌流行病學(xué)[J];中華腫瘤防治雜志;2007年12期

9 朱進國;吳慶琛;孔令文;;人支氣管上皮細胞惡變過程中生物學(xué)特性及超微結(jié)構(gòu)的研究[J];四川腫瘤防治;2005年04期

10 李杰;劉洋;陳志文;曾益軍;周虎傳;位全芳;楊勁;;實時熒光定量PCR檢測直腸癌XPC基因表達[J];山東醫(yī)藥;2008年14期

相關(guān)博士學(xué)位論文 前2條

1 宮智勇;熱休克蛋白70在苯并（a）芘致細胞DNA損傷修復(fù)中的作用[D];華中科技大學(xué);2006年

2 段燕英;熱休克蛋白70在苯并（a）芘所致DNA損傷修復(fù)中的作用和交互作用蛋白分析[D];華中科技大學(xué);2008年

相關(guān)碩士學(xué)位論文 前2條

1 張少峰;NF-κB在腫瘤相關(guān)巨噬細胞促煤焦瀝青煙提取物誘導(dǎo)BEAS-2B惡變過程中的表達[D];鄭州大學(xué);2011年

2 李靜;熱休克蛋白70與P53相互作用在酵母雙雜交系統(tǒng)中的檢測及其對P53在細胞內(nèi)定位的影響[D];中國人民解放軍第四軍醫(yī)大學(xué);2003年

，

本文編號：1902950