重铬酸钾对大鼠端粒长度及端粒结合蛋白TRF1和TRF2表达的影响

发布时间：2018-04-11 14:58

本文选题：六价铬 + 端粒　；参考：《郑州大学》2012年硕士论文

【摘要】：铬(Chromium,Cr)在自然界的主要存在形式是金属铬、六价铬和三价铬,其中六价铬在工业生产中应用最为广泛。工人们接触六价铬的主要途径是皮肤接触和呼吸道吸入接触,六价铬对职业健康有着极大的危害,工人长期接触六价铬可诱发肺癌。端粒(Telomere)是真核生物染色体末端的DNA-蛋白结构,它的长度关系着细胞的衰老与死亡,并与癌症的发生发展有着紧密的关联。端粒长度是由端粒结合蛋白和端粒酶所共同调控的,端粒酶通过与端粒末端结合来延伸端粒的长度。端粒结合蛋白可以通过与端粒末端的特异性结合来改变端粒的结构,从而抑制端粒与端粒酶的结合,达到阻止端粒延长的目的。目的：研究采用不同浓度的重铬酸钾溶液对大鼠进行吸入染毒,分析六价铬对大鼠端粒长度及端粒结合蛋白TRF1、TRF2表达的影响,为以后进一步研究六价铬致癌的机制提供理论依据。方法： 1.大鼠的急性吸入毒性试验选用健康成年SD大鼠40只,体重180～220g。随机分为4组,每组雌雄各半,根据预实验和文献,将染毒剂量设计为1000mg/m3、2150mg/m3、4640mg/m3和10000mg/m3,通过吸入染毒的方式对大鼠进行处理。染毒后观察14d,记录动物的症状和死亡情况。对死亡动物进行大体解剖和病理学检查。根据大鼠各组死亡情况,利用Horn氏法求出重铬酸钾对大鼠的LC5o及其95%可信区间。 2.大鼠的短期重复染毒毒性试验健康成年SD大鼠80只,体重180～220g,随机分为4组,每组20只。根据所得LC50,设计高、中、低3个剂量组,分别为1/10LC50、1/31.6LC50、1/100LC50,用蒸馏水做空白对照。通过吸入染毒的方式连续染毒7d,记录染毒时动物的症状和死亡情况。 3.石墨炉原子吸收光谱法测大鼠血液中铬的含量重复染毒毒性试验后,取各组大鼠血液,利用石墨炉原子吸收光谱法测定大鼠血液中铬的含量。 4.通过实时定量PCR对大鼠端粒长度进行测定重复染毒毒性试验后,取各剂量组大鼠右肺,从肺组织中提取出DNA,进行实时定量PCR分析,根据Ct值计算出大鼠的相对端粒长度。 5.SP免疫组化法检测大鼠TRF1、TRF2蛋白的表达重复染毒毒性试验后,取各剂量组大鼠左肺,固定包埋后,通过免疫组化法测其中TRF1、TRF2蛋白的表达。 6.统计分析运用SPSS12.0统计软件进行数据分析,用t检验来分析不同浓度重铬酸钾对大鼠血铬含量和端粒长度的影响；单因素方差分析来比较组间均值的差异,各组间两两比较时用Bonfferoni来校正；用Levene检验来进行方差齐性检验；采用Spearman秩相关进行相关性分析。所有统计检验均为双侧,检验水准为α=0.05。结果： 1.急性吸入毒性试验在实验观察期内,4640mg/m3剂量组,雌雄大鼠各死亡3只；10000mg/m3剂量组,10只大鼠全部死亡,其余各组无死亡。由此查Horn氏表,可得重铬酸钾对大鼠的LC5o为4300mg/m3,根据LC50设计短期重复染毒毒性试验的染毒剂量分别是430mg/m3、136mg/m3和43mg/m3。 2.短期重复染毒毒性试验中,中剂量组死亡2只大鼠,高剂量组死亡4只大鼠。 3.高、中、低剂量组和对照组中大鼠血铬的含量分别为：2534.11±150.98mg/L、796.40±126.55mg/L、97.12±6.88mg/L、14.98±2.39mg/L,多组间比较差异具有统计学意义(F=32.63,P0.05)。 4.实时定量PCR结果：高、中、低剂量组和对照组中大鼠端粒相对长度分别为0.20±0.04、0.39±0.05、0.61±0.06、1.13±0.11,多组间比较差异具有统计学意义(F=664.041,P0.05)。高、中、低剂量组和对照组的端粒长度与血铬含量呈负相关(r=-0.78、-0.56、-0.52、-0.45,均P0.05)。 5.免疫组化结果：高、中、低剂量组和对照组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,组间比较差异均具有统计学意义(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,组间比较差异均具有统计学意义(F=64.71,P0.05)。其中,高、低剂量组和对照组TRF1与TRF2蛋白的表达呈正相关(r=0.77、0.59、0.51,均P0.05)；高、中、低剂量组端粒长度与TRF1蛋白的表达呈负相关(r=0.77、-0.56、-0.51,均P0.05)；中、低剂量组和对照组端粒长度与TRF2蛋白的表达呈正相关(r=-0.74、-0.70、-0.50,均P0.05)。结论：重铬酸钾能够影响大鼠肺组织中端粒的功能,使其相对长度变短,并使端粒结合蛋白TRF1和TRF2表达量升高。
[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 伪 = 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 卤 150.9mg / L , 796.40 卤 126.55mg / L , 97.12 卤 6.88mg / L , 14.98 卤 2.39mg / L , respectively .

4 . Real - time quantitative PCR results showed that the relative lengths of telomeric particles were 0.20 卤 0.04 , 0.39 卤 0.05 , 0.61 卤 0.06 , 1.13 卤 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 卤 0.03 , 0.32 卤 0.02 , 0.29 卤 0.01 , 0.28 卤 0.02 , and the average optical density of TRF2 was 0.25 卤 0.01 , 0.23 卤 0.03 , 0.20 卤 0.04 , 0.19 卤 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 .

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R114

【参考文献】