本文選題：永生化人支氣管上皮細(xì)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(xì)胞適應(yīng)各種應(yīng)激反應(yīng),減輕環(huán)境有害因子對(duì)細(xì)胞的損害作用,從而增強(qiáng)機(jī)體細(xì)胞的生存能力。國(guó)內(nèi)外許多研究已經(jīng)表明,HSP70在許多腫瘤中高表達(dá),并在其發(fā)生、發(fā)展、治療及預(yù)后的整個(gè)疾病過(guò)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(guò)程中產(chǎn)生的副產(chǎn)物,成分復(fù)雜,其所致肺癌是嚴(yán)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(jī)制尚未闡釋清楚,有待進(jìn)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(xì)胞BEAS-2B的惡性轉(zhuǎn)化模型,檢測(cè)該細(xì)胞系在細(xì)胞發(fā)生惡性轉(zhuǎn)化的過(guò)程中HSP70與核苷酸切除修復(fù)因子的表達(dá)情況,探索HSP70及核苷酸切除修復(fù)因子在細(xì)胞惡性轉(zhuǎn)化過(guò)程中的作用及其可能的機(jī)制,為進(jìn)一步研究HSP70的生物學(xué)作用和煤焦瀝青的致癌機(jī)制提供一定的理論基礎(chǔ)。 方法 1.細(xì)胞惡性轉(zhuǎn)化模型的建立 實(shí)驗(yàn)分三組：即空白對(duì)照組、DMSO溶劑對(duì)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(guò)細(xì)胞活力計(jì)數(shù)法確定合適的染毒劑量,并以此劑量來(lái)誘導(dǎo)BEAS-2B細(xì)胞惡性轉(zhuǎn)化,然后觀察細(xì)胞轉(zhuǎn)化過(guò)程中細(xì)胞表型的改變情況。 2.細(xì)胞惡性轉(zhuǎn)化過(guò)程的檢測(cè) 用彗星實(shí)驗(yàn)檢測(cè)染毒后BEAS-2B細(xì)胞損傷情況,并觀察各代細(xì)胞形態(tài)的變化；用染色體核型分析和軟瓊脂集落形成實(shí)驗(yàn)檢測(cè)細(xì)胞是否發(fā)生惡性轉(zhuǎn)變。 3.HSP70蛋白表達(dá)水平與核苷酸切除修復(fù)因子表達(dá)水平的檢測(cè) Western blot法檢測(cè)HSP70及核苷酸切除修復(fù)因子著色性干皮病互補(bǔ)基因蛋白質(zhì)C(Xeroderma pigmentosum group, XPC)、切除修復(fù)交叉互補(bǔ)蛋白1(Excision repair cross complementing1, ERCC1)的表達(dá),觀察細(xì)胞受煤焦瀝青煙提取物作用后,HSP70、XPC及ERCC1在傳代過(guò)程中表達(dá)水平的動(dòng)態(tài)變化。 4.統(tǒng)計(jì)學(xué)分析 采用SPSS12.0對(duì)數(shù)據(jù)進(jìn)行統(tǒng)計(jì)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(yàn),構(gòu)成比采用卡方檢驗(yàn)：兩兩比較采用LSD法,檢驗(yàn)水準(zhǔn)a=0.05。 結(jié)果 1.細(xì)胞惡性轉(zhuǎn)化試驗(yàn) 彗星實(shí)驗(yàn)結(jié)果顯示,染毒組細(xì)胞出現(xiàn)DNA損傷,與對(duì)照組比較,染毒組細(xì)胞拖尾長(zhǎng)度較對(duì)照組均顯著增高,差異有統(tǒng)計(jì)學(xué)意義(P0.05)；表明煤焦瀝青煙提取物對(duì)BEAS-2B細(xì)胞具有遺傳毒性。染毒組20代細(xì)胞發(fā)生形態(tài)學(xué)改變,細(xì)胞胞漿豐富,體積增大,類圓形、圓形比例增加,甚至部分細(xì)胞出現(xiàn)纖維細(xì)胞樣改變；此代細(xì)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(zhǎng)實(shí)驗(yàn)顯示細(xì)胞僅有少數(shù)細(xì)胞能形成細(xì)胞集落,惡性程度不高。染毒組30代,胞體大小差異大,細(xì)胞內(nèi)顆粒物增加,面積增大,大部分細(xì)胞外形變?yōu)轭悎A形、圓形,部分細(xì)胞膜邊緣呈膜狀向往擴(kuò)展；細(xì)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(zhǎng)實(shí)驗(yàn)發(fā)現(xiàn),此代細(xì)胞可以形成大細(xì)胞集落,無(wú)接觸抑制,細(xì)胞發(fā)生了惡性轉(zhuǎn)化。 2.BEAS-2B細(xì)胞中,蛋白質(zhì)HSP70、XPC、ERCC1的表達(dá) 染毒組在0代和30代細(xì)胞HSP70的表達(dá)與對(duì)照組比較,表達(dá)水平均顯著升高(P0.05)；0代細(xì)胞XPC及ERCC1蛋白表達(dá)顯著高于對(duì)照組(P0.05)；30代細(xì)胞中XPC蛋白顯著低于2個(gè)對(duì)照組(P0.05), ERCC1蛋白表達(dá)顯著高于對(duì)照組(P0.05)；對(duì)照組各組之間各個(gè)蛋白的表達(dá)差異均無(wú)統(tǒng)計(jì)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(xì)胞惡性轉(zhuǎn)化過(guò)程；細(xì)胞惡變后,HSP70能抑制XPC的表達(dá)；在細(xì)胞染色體突變及惡性轉(zhuǎn)化過(guò)程中,DNA損傷可能是其始動(dòng)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(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R114

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本文編號(hào)：1902950