本文選題：氯乙烯 + 染色體損傷��； 參考：《復(fù)旦大學(xué)》2013年博士論文

【摘要】：氯乙烯單體(vinyl chloride monomer, VCM)是一種重要的化工原料,主要用于合成聚氯乙烯。2011年全球PVC總產(chǎn)能約5100萬噸,而我國PVC產(chǎn)能達(dá)2162.2萬噸,已成為世界第一大生產(chǎn)國和第一大消費(fèi)國。VCM是確定的人類致癌劑,可導(dǎo)致多系統(tǒng)、多器官腫瘤的發(fā)生。對其致癌機(jī)制的研究認(rèn)為主要是VCM活性代謝產(chǎn)物導(dǎo)致遺傳物質(zhì)損傷而啟動(dòng)致癌過程。 目前,發(fā)達(dá)國家VCM職業(yè)接觸限值多為1ppm (2.79mg/m3, PEL),而我國VCM時(shí)間加權(quán)平均容許濃度(PC-TWA)為10mg/m3,短時(shí)間接觸容許濃度為(PC-STEL)25mg/m3,高于西方國家衛(wèi)生標(biāo)準(zhǔn)數(shù)倍。長期和低水平接觸氯乙烯導(dǎo)致的健康危害已引起廣泛關(guān)注,尋找敏感、有效的生物標(biāo)志物對預(yù)防氯乙烯接觸導(dǎo)致的有害效應(yīng)十分必要。 在我國現(xiàn)行氯乙烯職業(yè)衛(wèi)生標(biāo)準(zhǔn)下,能否對接觸工人的遺傳物質(zhì)產(chǎn)生顯著損傷尤為引人關(guān)注。而且在同樣接觸條件下,并不是所有個(gè)體都會產(chǎn)生遺傳物質(zhì)損傷；遺傳物質(zhì)即便發(fā)生損傷,由于機(jī)體具有DNA修復(fù)能力(DNA repair capacity,DRC)而得到修復(fù)。因而DNA修復(fù)能力在維持機(jī)體基因組穩(wěn)定和正常功能方面發(fā)揮重要作用,如果DRC功能低下或缺陷,將不能及時(shí)和有效地修復(fù)受損的DNA,從而導(dǎo)致基因突變甚至細(xì)胞癌變,增加個(gè)體罹患腫瘤的危險(xiǎn)。 本課題通過橫斷面調(diào)查和追蹤研究,探討染色體損傷與氯乙烯累積接觸劑量的關(guān)系,同時(shí)應(yīng)用本課題組發(fā)展的基于微核的DNA損傷修復(fù)能力檢測方法,研究氯乙烯作業(yè)工人DNA損傷修復(fù)能力,并評價(jià)其用于預(yù)測氯乙烯致染色體損傷風(fēng)險(xiǎn)的價(jià)值,為保護(hù)易感作業(yè)工人提供科學(xué)依據(jù)。通過氯乙烯作業(yè)工人DNA細(xì)胞周期調(diào)控基因多態(tài)與VCM致遺傳損傷之間的關(guān)系研究,尋找VCM遺傳損傷的易感性生物標(biāo)志物,為闡明其毒作用機(jī)制和進(jìn)行健康風(fēng)險(xiǎn)評估提供科學(xué)依據(jù)。 本課題研究對象為某化工企業(yè)VCM接觸工人309例(男224例,女85例),該企業(yè)行政和后勤人員(內(nèi)對照)共149例(男61例,女88例),以及健康對照人群(外對照)共25例(男14例,女11例)；利用胞質(zhì)分裂阻滯微核試驗(yàn)方法(簡稱CB微核試驗(yàn))對染色體損傷情況進(jìn)行檢測, VCM接觸組外周血淋巴細(xì)胞微核細(xì)胞率(簡稱微核率)明顯高于對照組FR=3.24(95%CI2.80-3.75), p0.001,并存在劑量-反應(yīng)關(guān)系,提示在目前職業(yè)性VCM接觸水平較低的情況下,外周血淋巴細(xì)胞微核率可以作為評價(jià)低濃度VCM接觸的效應(yīng)指標(biāo)。 在2004,2007年VCM接觸工人中選取重復(fù)樣本91例,采用基準(zhǔn)劑量(BMD)法分析微核發(fā)生率與氯乙烯累積接觸劑量的關(guān)系。結(jié)果表明,按照氯乙烯累積接觸劑量計(jì)算,其基準(zhǔn)劑量95%可信區(qū)間下限(BMDL)在2004年男性和女性中分別為0.99mg/m3-year和3.38ng/m3-year;在2007年男性和女性中分別為0.63mg/m3-year和1.27mg/m3-year。提示當(dāng)總接觸劑量達(dá)到BMDL值時(shí)即引起微核損傷,為制定氯乙烯接觸限值提供了參考。 yH2AX是目前國內(nèi)外研究細(xì)胞DNA損傷應(yīng)激反應(yīng)的公認(rèn)的敏感指標(biāo),當(dāng)DNA雙鏈發(fā)生斷裂時(shí),組蛋白H2A家族成員中的H2AX第139位絲氨酸殘基迅速磷酸化,形成磷酸化的H2AX,即γ-H2AX。本研究應(yīng)用流式細(xì)胞術(shù)(FCM)對上述追蹤觀察的55例氯乙烯接觸工人,21例內(nèi)對照和25例外對照個(gè)體外周血淋巴細(xì)胞進(jìn)行DNA損傷(yH2AX)和細(xì)胞凋亡檢測。結(jié)果顯示不同VCM累積接觸水平的作業(yè)工人外周血淋巴細(xì)胞DNA損傷率和幾何平均熒光強(qiáng)度明顯高于對照組,差異有統(tǒng)計(jì)學(xué)意義(p0.05)；以對照組淋巴細(xì)胞凋亡率95%上限(4.84)為界值判斷淋巴細(xì)胞凋亡陽性,結(jié)果顯示對照組、低、高VCM累積接觸組淋巴細(xì)胞凋亡陽性率隨劑量升高而升高(p0.05)。本次研究結(jié)果表明VCM接觸可導(dǎo)致接觸工人外周血淋巴細(xì)胞DNA損傷和凋亡率升高。提示外周血淋巴細(xì)胞DNA損傷率和凋亡率可以作為評價(jià)低濃度VCM接觸的效應(yīng)指標(biāo)。 本研究還對上述追蹤觀察的部分氯乙烯接觸工人應(yīng)用基于微核的DNA損傷修復(fù)能力檢測方法開展染色體損傷修復(fù)能力研究,其中接觸組43名,同時(shí)以17名該廠行政和后勤人員作為對照。按照氯乙烯累積接觸劑量分為2組(累積接觸劑量≤10984mg為低接觸組；10984mg為高接觸組),研究顯示對照組、低、高接觸組的3AB指數(shù)分別為0.37±0.17,0.28±0.11,0.18±±0.10,存在顯著統(tǒng)計(jì)學(xué)差異p0.05),提示氯乙烯接觸會影響DNA損傷修復(fù)能力,氯乙烯累積接觸劑量越高,DNA損傷修復(fù)能力越低；另按3AB指數(shù)分成2組(3AB指數(shù)≤0.24為低損傷修復(fù)能力組；0.24為高損傷修復(fù)能力組),結(jié)果顯示,損傷修復(fù)能力高組的微核率(2.28±±2.61)低于低損傷修復(fù)能力組(4.04±2.76),存在顯著統(tǒng)計(jì)學(xué)差異p0.05),提示DNA損傷修復(fù)能力與氯乙烯作業(yè)工人染色體損傷的發(fā)生關(guān)系密切,修復(fù)能力差,染色體損傷高。通過對DNA損傷修復(fù)能力研究表明,DNA損傷修復(fù)能力與氯乙烯接觸劑量有關(guān),可作為氯乙烯接觸的效應(yīng)指標(biāo),同時(shí)又可用于易感人群的篩選,具有易感性指標(biāo)的特點(diǎn),提示DNA損傷修復(fù)能力具有重要的應(yīng)用價(jià)值和前景。 本課題還應(yīng)用PCR-RFLP和CRS-RFLP法對VCM接觸工人細(xì)胞周期調(diào)控相關(guān)基因的多態(tài)位點(diǎn)進(jìn)行檢測,VCM接觸工人309例,對照組149例。應(yīng)用Poisson回歸分析各基因多態(tài)位點(diǎn)與染色體損傷之間的關(guān)系。選擇參與細(xì)胞周期調(diào)控的常見基因p53, p21, mdm2, Gadd45a和p14ARF,對其10個(gè)常見多態(tài)位點(diǎn)(p53intron3,p53Ex4+119CG, P53IVS6+62GA,p21Ex3+7CT, mdm2Dell518, mdm2IVSl+309TG, Gadd45a g.1260TC, p14ARF g.9792GT, p14ARF g.22008GA,p14ARFg.26294GA)進(jìn)行檢測和分析。調(diào)整年齡、性別、累積接觸劑量、吸煙、飲酒等因素后,結(jié)果顯示p14ARFg.22008基因的突變位點(diǎn)是遺傳損傷的保護(hù)因素,與野生純合型比較,FR=0.77(95%CI0.63-0.93,P0.05)；而p53intron3,p21Ex3+70CT和mdm2IVS1+309TG三個(gè)基因的突變位點(diǎn)為VCM致染色體損傷的危險(xiǎn)因素,與野生純合型比較FR=1.47(95%CI1.09-1.98), FR=1.36(95%CI1.13-1.64)和FR=1.74(95%CI1.43-2.13),均具有顯著意義(p0.05)。 綜上所述,在我國現(xiàn)行職業(yè)衛(wèi)生標(biāo)準(zhǔn)下,氯乙烯接觸仍能產(chǎn)生遺傳損傷,且存在劑量-反應(yīng)關(guān)系,據(jù)此計(jì)算得到的基準(zhǔn)劑量遠(yuǎn)低于現(xiàn)行職業(yè)衛(wèi)生標(biāo)準(zhǔn)；氯乙烯接觸工人的DNA損傷修復(fù)能力降低；VCM致染色體損傷與細(xì)胞周期調(diào)控基因多態(tài)有關(guān)。
[Abstract]:Vinyl chloride monomer (VCM) is an important chemical raw material. It is mainly used in the synthesis of polyvinyl chloride (PVC) of about 51 million tons of global PVC production capacity in.2011, while our country's PVC capacity reaches 21 million 622 thousand tons. It has become the world's largest producer and the largest consumer country.VCM is a definite human carcinogen, which can lead to multisystem, multiple organ tumors. The carcinogenesis mechanism of VCM is mainly due to the initiation of carcinogenesis by the damage of genetic material induced by active metabolites.
At present, the limited value of professional contact of VCM in developed countries is 1ppm (2.79mg/m3, PEL), and the VCM time weighted mean permissible concentration (PC-TWA) is 10mg/m3, and the allowable concentration of short time contact is (PC-STEL) 25mg/m3, higher than that of the western national health standard. The health hazards caused by long and low exposure to vinyl chloride have attracted wide attention and look for the sensitivity. Therefore, effective biomarkers are necessary to prevent harmful effects caused by vinyl chloride contact.
Under the current standard of occupational hygiene of vinyl chloride in our country, it is particularly concerned about whether the genetic material of the exposed workers can be significantly damaged. And under the same contact conditions, not all individuals can produce genetic material damage. Even if the genetic material is damaged, the body has the ability of DNA repair (DNA repair capacity, DRC). To repair. Therefore, DNA repair ability plays an important role in maintaining the body's genomic stability and normal function. If DRC is ineffective or defective, the damaged DNA will not be repaired in time and effectively, resulting in gene mutation and even cell canceration, increasing the risk of cancer in the individual.
Through cross-sectional investigation and tracing study, the relationship between chromosome damage and the cumulative exposure dose of vinyl chloride was investigated. At the same time, the DNA damage repair ability of vinyl chloride workers was studied by using the microkernel based DNA damage repair ability detection method, and the risk of chloroethylene induced chromosomal damage was evaluated. In order to provide scientific basis for the workers of susceptible workers, the relationship between DNA cell cycle regulation gene polymorphism and genetic damage induced by VCM is studied to find the susceptibility biomarkers of VCM genetic damage, and to provide a scientific basis for clarifying the mechanism of its toxic action and assessing the risk of health.
The subjects were 309 cases of VCM contact workers in a chemical enterprise (224 males and 85 females), 149 cases (61 men, 88 women), and 25 cases (14 men and 11 cases) of the administrative and logistic staff (61 women), and the micronucleus test of cytoplasmic division block (CB micronucleus test) was used for chromosomal damage. The rate of micronucleus cells in peripheral blood lymphocyte in VCM contact group was significantly higher than that of the control group FR=3.24 (95%CI2.80-3.75), p0.001, and there was a dose response relationship. It was suggested that the micronucleus rate of the external Zhou Xuelin cell can be used to evaluate the effect of low concentration VCM contact in the present occupational VCM contact level. Index.
The relationship between the incidence of micronucleus and the cumulative exposure dose of vinyl chloride was analyzed by the baseline dose (BMD) method in 20042007 years of VCM workers. The results showed that the baseline dose 95% confidence interval lower limit (BMDL) was 0.99mg/m3-year and 3.38ng in men and women in 2004, respectively. /m3-year; in 2007, both men and women were 0.63mg/m3-year and 1.27mg/m3-year., respectively, suggesting that when the total contact dose reached BMDL value, it caused micronucleus damage, providing a reference for the formulation of the perchloroethylene contact limit.
YH2AX is a recognized sensitive indicator of DNA damage stress response at home and abroad. When DNA double strand breaks, H2AX 139th serine residues in histone H2A family members are rapidly phosphorylated to form phosphorylated H2AX, that is, gamma -H2AX. this study applied flow cytometry (FCM) to the trace of chloroethylene contact observed above. DNA injury (yH2AX) and cell apoptosis were detected in 21 cases of internal control and 25 exceptions in the peripheral blood lymphocytes. The results showed that the DNA damage rate and the geometric mean fluorescence intensity of peripheral blood lymphocytes of workers with different VCM accumulative contact levels were significantly higher than those of the control group (P0.05). The apoptosis rate of 95% (4.84) was the boundary value to determine the lymphocyte apoptosis positive. The results showed that the positive rate of lymphocyte apoptosis in the control group was low and high VCM accumulated contact group increased with the increase of dose (P0.05). The results of this study showed that VCM exposure could lead to the increase of DNA damage and apoptosis rate of peripheral blood gonorrhea cells in contact workers. DNA damage rate and apoptosis rate can be used as an indicator for evaluating low concentration VCM exposure.
This study also carried out a study on the ability to repair chromosomal damage by using micronucleus based DNA damage repair ability detection method based on microkernel based DNA damage repair. In this study, the contact group was divided into 2 groups (cumulative exposure dose < 10). 984mg was a low exposure group; 10984mg was a high contact group. The study showed that the 3AB index of the control group was 0.37 + 0.17,0.28 + 0.11,0.18 + 0.10, respectively, and there was a significant difference of P0.05. It suggested that vinyl chloride contact would affect the ability to repair DNA damage. The higher the dose of chloroethylene accretion contact, the lower the ability to repair DNA damage; and 3A The B index was divided into 2 groups (3AB index < 0.24 is low damage repair ability group and 0.24 is high damage repair ability group). The result showed that the micronucleus rate of high repair ability group (2.28 + 2.61) was lower than that of low injury repair ability group (4.04 + 2.76), and there was significant difference P0.05. It suggested that the repair ability of DNA injury and the chromosome of vinyl chloride workers The damage repair ability is close, the repair ability is poor and the chromosome damage is high. Through the study of DNA damage repair ability, the ability of DNA damage repair is related to the dose of vinyl chloride contact, which can be used as the effect index of vinyl chloride contact, and can also be used for screening of susceptible population, with the characteristics of susceptibility index, suggesting the ability of DNA damage repair. There is an important application value and prospect.
The PCR-RFLP and CRS-RFLP methods were used to detect the polymorphic loci of the cell cycle regulation related genes in VCM exposed workers, 309 workers in VCM contact and 149 in the control group. The relationship between polymorphic loci and chromosome damage was analyzed by Poisson regression. The common gene p53, p21, MDM2, Gadd participating in the regulation of cell cycle was selected. 45A and p14ARF were tested and analyzed for 10 common polymorphic loci (p53intron3, p53Ex4+119CG, P53IVS6+62GA, p21Ex3+7CT, mdm2Dell518, mdm2IVSl+309TG, Gadd45a g.1260TC, p14ARF g.9792GT). The results showed that the factors such as age, sex, cumulative exposure dose, smoking, drinking and other factors were shown. The mutation site of the 008 gene is a protective factor for genetic damage, compared with the wild homozygous type, FR=0.77 (95%CI0.63-0.93, P0.05), and the mutation sites of the three genes of p53intron3, p21Ex3+70CT and mdm2IVS1+309TG are the risk factors for chromosomal damage caused by VCM, compared with the wild homozygous type FR=1.47 (95%CI1.09-1.98), FR=1.36 (95%CI1.13-1.64) and FR=. 1.74 (95%CI1.43-2.13) was significant (P0.05).
To sum up, under the current standard of occupational health, chloroethylene exposure can still produce genetic damage, and there is a dose response relationship. According to this, the calculated baseline dose is far lower than the current occupational health standard; the DNA damage repair ability of vinyl chloride exposed workers is reduced; VCM induced chromosomal damage and cell cycle regulation genes are polymorphic. Close.

【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2013
【分類號】：R135.1

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