本文選題：六價(jià)鉻 + 端粒��； 參考：《鄭州大學(xué)》2012年碩士論文

【摘要】：鉻(Chromium,Cr)在自然界的主要存在形式是金屬鉻、六價(jià)鉻和三價(jià)鉻,其中六價(jià)鉻在工業(yè)生產(chǎn)中應(yīng)用最為廣泛。工人們接觸六價(jià)鉻的主要途徑是皮膚接觸和呼吸道吸入接觸,六價(jià)鉻對(duì)職業(yè)健康有著極大的危害,工人長(zhǎng)期接觸六價(jià)鉻可誘發(fā)肺癌。 端粒(Telomere)是真核生物染色體末端的DNA-蛋白結(jié)構(gòu),它的長(zhǎng)度關(guān)系著細(xì)胞的衰老與死亡,并與癌癥的發(fā)生發(fā)展有著緊密的關(guān)聯(lián)。 端粒長(zhǎng)度是由端粒結(jié)合蛋白和端粒酶所共同調(diào)控的,端粒酶通過(guò)與端粒末端結(jié)合來(lái)延伸端粒的長(zhǎng)度。端粒結(jié)合蛋白可以通過(guò)與端粒末端的特異性結(jié)合來(lái)改變端粒的結(jié)構(gòu),從而抑制端粒與端粒酶的結(jié)合,達(dá)到阻止端粒延長(zhǎng)的目的。 目的： 研究采用不同濃度的重鉻酸鉀溶液對(duì)大鼠進(jìn)行吸入染毒,分析六價(jià)鉻對(duì)大鼠端粒長(zhǎng)度及端粒結(jié)合蛋白TRF1、TRF2表達(dá)的影響,為以后進(jìn)一步研究六價(jià)鉻致癌的機(jī)制提供理論依據(jù)。 方法： 1.大鼠的急性吸入毒性試驗(yàn)選用健康成年SD大鼠40只,體重180～220g。隨機(jī)分為4組,每組雌雄各半,根據(jù)預(yù)實(shí)驗(yàn)和文獻(xiàn),將染毒劑量設(shè)計(jì)為1000mg/m3、2150mg/m3、4640mg/m3和10000mg/m3,通過(guò)吸入染毒的方式對(duì)大鼠進(jìn)行處理。染毒后觀察14d,記錄動(dòng)物的癥狀和死亡情況。對(duì)死亡動(dòng)物進(jìn)行大體解剖和病理學(xué)檢查。根據(jù)大鼠各組死亡情況,利用Horn氏法求出重鉻酸鉀對(duì)大鼠的LC5o及其95%可信區(qū)間。 2.大鼠的短期重復(fù)染毒毒性試驗(yàn)健康成年SD大鼠80只,體重180～220g,隨機(jī)分為4組,每組20只。根據(jù)所得LC50,設(shè)計(jì)高、中、低3個(gè)劑量組,分別為1/10LC50、1/31.6LC50、1/100LC50,用蒸餾水做空白對(duì)照。通過(guò)吸入染毒的方式連續(xù)染毒7d,記錄染毒時(shí)動(dòng)物的癥狀和死亡情況。 3.石墨爐原子吸收光譜法測(cè)大鼠血液中鉻的含量重復(fù)染毒毒性試驗(yàn)后,取各組大鼠血液,利用石墨爐原子吸收光譜法測(cè)定大鼠血液中鉻的含量。 4.通過(guò)實(shí)時(shí)定量PCR對(duì)大鼠端粒長(zhǎng)度進(jìn)行測(cè)定重復(fù)染毒毒性試驗(yàn)后,取各劑量組大鼠右肺,從肺組織中提取出DNA,進(jìn)行實(shí)時(shí)定量PCR分析,根據(jù)Ct值計(jì)算出大鼠的相對(duì)端粒長(zhǎng)度。 5.SP免疫組化法檢測(cè)大鼠TRF1、TRF2蛋白的表達(dá)重復(fù)染毒毒性試驗(yàn)后,取各劑量組大鼠左肺,固定包埋后,通過(guò)免疫組化法測(cè)其中TRF1、TRF2蛋白的表達(dá)。 6.統(tǒng)計(jì)分析運(yùn)用SPSS12.0統(tǒng)計(jì)軟件進(jìn)行數(shù)據(jù)分析,用t檢驗(yàn)來(lái)分析不同濃度重鉻酸鉀對(duì)大鼠血鉻含量和端粒長(zhǎng)度的影響；單因素方差分析來(lái)比較組間均值的差異,各組間兩兩比較時(shí)用Bonfferoni來(lái)校正；用Levene檢驗(yàn)來(lái)進(jìn)行方差齊性檢驗(yàn)；采用Spearman秩相關(guān)進(jìn)行相關(guān)性分析。所有統(tǒng)計(jì)檢驗(yàn)均為雙側(cè),檢驗(yàn)水準(zhǔn)為α=0.05。 結(jié)果： 1.急性吸入毒性試驗(yàn)在實(shí)驗(yàn)觀察期內(nèi),4640mg/m3劑量組,雌雄大鼠各死亡3只；10000mg/m3劑量組,10只大鼠全部死亡,其余各組無(wú)死亡。由此查Horn氏表,可得重鉻酸鉀對(duì)大鼠的LC5o為4300mg/m3,根據(jù)LC50設(shè)計(jì)短期重復(fù)染毒毒性試驗(yàn)的染毒劑量分別是430mg/m3、136mg/m3和43mg/m3。 2.短期重復(fù)染毒毒性試驗(yàn)中,中劑量組死亡2只大鼠,高劑量組死亡4只大鼠。 3.高、中、低劑量組和對(duì)照組中大鼠血鉻的含量分別為：2534.11±150.98mg/L、796.40±126.55mg/L、97.12±6.88mg/L、14.98±2.39mg/L,多組間比較差異具有統(tǒng)計(jì)學(xué)意義(F=32.63,P0.05)。 4.實(shí)時(shí)定量PCR結(jié)果：高、中、低劑量組和對(duì)照組中大鼠端粒相對(duì)長(zhǎng)度分別為0.20±0.04、0.39±0.05、0.61±0.06、1.13±0.11,多組間比較差異具有統(tǒng)計(jì)學(xué)意義(F=664.041,P0.05)。高、中、低劑量組和對(duì)照組的端粒長(zhǎng)度與血鉻含量呈負(fù)相關(guān)(r=-0.78、-0.56、-0.52、-0.45,均P0.05)。 5.免疫組化結(jié)果：高、中、低劑量組和對(duì)照組TRF1蛋白的平均光密度值依次是：0.36±0.03、0.32±0.02、0.29±0.01、0.28±0.02,組間比較差異均具有統(tǒng)計(jì)學(xué)意義(F=125.20,P0.05),TRF2蛋白的平均光密度值依次是：0.25±0.01、0.23±0.03、0.20±0.04、0.19±0.01,組間比較差異均具有統(tǒng)計(jì)學(xué)意義(F=64.71,P0.05)。其中,高、低劑量組和對(duì)照組TRF1與TRF2蛋白的表達(dá)呈正相關(guān)(r=0.77、0.59、0.51,均P0.05)；高、中、低劑量組端粒長(zhǎng)度與TRF1蛋白的表達(dá)呈負(fù)相關(guān)(r=0.77、-0.56、-0.51,均P0.05)；中、低劑量組和對(duì)照組端粒長(zhǎng)度與TRF2蛋白的表達(dá)呈正相關(guān)(r=-0.74、-0.70、-0.50,均P0.05)。 結(jié)論： 重鉻酸鉀能夠影響大鼠肺組織中端粒的功能,使其相對(duì)長(zhǎng)度變短,并使端粒結(jié)合蛋白TRF1和TRF2表達(dá)量升高。
[Abstract]:Chromium ( Cr ) is mainly in the form of metal chromium , hexavalent chromium and trivalent chromium , of which hexavalent chromium is the most widely used in industrial production . The main route for workers to contact hexavalent chromium is skin contact and inhalation exposure of respiratory tract . The hexavalent chromium has great harm to occupational health , and the workers exposed to hexavalent chromium for a long time can induce lung cancer .

Telomere ( Telomere ) is a DNA - protein structure at the end of eukaryotic chromosomes . Its length is related to aging and death of cells , and has a close relationship with the development of cancer .

Telomere length is co - regulated by telomeric binding protein and telomerase . The telomerase binding protein can extend the length of telomeric by binding to the telomeric end . Telomere - binding protein can change the structure of telomeric by binding to the specific binding of telomeric end , thus inhibiting the binding of telomeric and telomerase , and achieving the purpose of inhibiting telomeric extension .

Purpose :

The effects of hexavalent chromium on the rat Telomere length and the expression of telomeric binding proteins TRF1 and TRF2 were studied by using potassium dichromate solution at different concentrations .

Method :

1 . Forty - four healthy adult SD rats weighing 180 - 220g were randomly divided into 4 groups . The rats were randomly divided into 4 groups . According to the pre - experiment and literature , the rats were treated with the dosage of 1000 mg / m3 , 2150 mg / m3 , 4640mg / m3 and 10000mg / m3 . After exposure , the symptoms and mortality of the animals were recorded . According to the death of each group , the LC5o and 95 % confidence interval of potassium dichromate were determined by Horn ' s method .

2 . Eighty - eight healthy adult SD rats weighing 180 - 220g were randomly divided into 4 groups , 20 rats in each group .

3 . The content of chromium in blood of rats was determined by graphite furnace atomic absorption spectrometry after repeated exposure to toxicity of chromium in blood of rats by graphite furnace atomic absorption spectroscopy .

4 . After measuring the rat telomeric length by real - time quantitative PCR , DNA was extracted from lung tissue , DNA was extracted from lung tissue , and the relative telomeric length of rat was calculated according to Ct value .

5 . The expression of TRF1 and TRF2 was measured by immunohistochemistry in rats with TRF1 and TRF2 protein by SP immunohistochemical method .

6 . SPSS 12.0 was used to analyze the effects of different concentrations of potassium dichromate on the content of chromium and the length of telomeric grain in rats .
One - factor variance analysis was used to compare the differences among the groups , and Bonfferoni was used to correct the difference between the two groups .
The homogeneity test was carried out by Levene test .
Correlation analysis was carried out with the Pearson rank correlation . All statistical tests were bilateral and the test level was 偽 = 0.05 .

Results :

1 . The acute inhalation toxicity test was conducted in the dose group of 4640mg / m3 during the experimental observation period , and the female and female rats died 3 ;
In the 10000mg / m3 dosage group , 10 rats died , and the rest groups were not dead . The LC5o was 4300 mg / m 3 of potassium dichromate in rats and 430 mg / m3 , 136 mg / m3 and 43 mg / m3 respectively according to the LC 50 design .

2 . In the short - term repeat exposure toxicity test , 2 rats were killed in the middle dose group and 4 rats died in the high - dose group .

3 . The contents of chromium in blood of rats were 2534.11 鹵 150.9mg / L , 796.40 鹵 126.55mg / L , 97.12 鹵 6.88mg / L , 14.98 鹵 2.39mg / L , respectively .

4 . Real - time quantitative PCR results showed that the relative lengths of telomeric particles were 0.20 鹵 0.04 , 0.39 鹵 0.05 , 0.61 鹵 0.06 , 1.13 鹵 0.11 in the high , middle , low and low dose groups , respectively ( F = 660.041 , P0.05 ) . The telomeric lengths of the middle , low and low dose groups were negatively correlated with the content of chromium ( r = - 0.78 , - 0.56 , - 0.52 , - 0.45 , all P0.05 ) .

5 . Immunohistochemical results showed that the average optical density of TRF1 protein in high , middle , low and control groups was 0.36 鹵 0.03 , 0.32 鹵 0.02 , 0.29 鹵 0.01 , 0.28 鹵 0.02 , and the average optical density of TRF2 was 0.25 鹵 0.01 , 0.23 鹵 0.03 , 0.20 鹵 0.04 , 0.19 鹵 0.01 , respectively .
There was a negative correlation between telomeric length and TRF1 protein expression in high , middle and low dose groups ( r = 0.77 , - 0.56 , - 0.51 , all P0.05 ) .
In the middle and low dose groups and control group , the telomeric length was positively correlated with the expression of TRF2 protein ( r = - 0.74 , - 0.70 , - 0.50 , all P0.05 ) .

Conclusion :

Potassium dichromate can influence the function of telomeric function in rat lung tissue , shorten the relative length , and increase the expression of telomeric binding proteins TRF1 and TRF2 .

【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)：1736474