热应激对大鼠肝脏HSP70表达及其mRNA丰度变化规律的研究
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摘要
以大鼠为试验动物,采用比色法、Western-blotting、半定量RT-PCR和Real–time PCR技术,从不同热应激强度(25℃、30℃、35℃和40℃)和40℃热应激后不同处理时间(0.5h,1.5h,2h,2.5h,4h和6h)两个角度,检测分析了大鼠血清中的丙二醛(MDA)含量、总超氧化物歧化酶(T-SOD)活力和总抗氧化能力(T-AOC)的大小,动态考察了大鼠肝脏中HSP70的表达量及其mRNA丰度变化规律,探讨HSP70mRNA水平的变化与热休克蛋白70表达的相关性,结果表明:
1.应用比色法,对热应激大鼠血清中抗氧化能力指标进行了测定。与对照组25℃相比,30℃时,大鼠血清的MDA略有下降(P>0.05)。35℃时和40℃时,血清的MDA极显著的降低(P<0.01)。大鼠血清的SOD活力和T-AOC分别呈升高和降低的趋势,各热应激组的SOD活力和T-AOC值与对照组相比,差异极显著(P<0.01)。在不同热应激时间处理下,大鼠血清的MDA先升高,在4h时达到高峰(P<0.01),之后开始下降,在6h时与0.5h相比差异不显著。大鼠血清的SOD活力和T-AOC值均处于上下波动状态,各热应激处理组的SOD活力均比0.5h极显著的升高(P<0.01)。血清的T-AOC值除在热应激处理1h后显著的升高(P<0.05)和1.5h、2h后极显著的下降外(P<0.01),其他各组无明显差异。
2.应用Western-blotting方法,对热应激大鼠肝脏中HSP70的表达量进行了测定。大鼠肝脏HSP70在25℃就有较低水平的表达,与对照组相比,在30℃时,肝脏HSP70表达量就明显的增加(P<0.05)。35℃和40℃时,表达量极显著的增加(P<0.01)。不同的热应激时间时,大鼠肝脏HSP70表达量呈先增加,到2h时表达量达到峰值(P<0.01),随着热应激时间的延长,HSP70表达量开始下降。在热应激处理4h和6h时,HSP70表达量与0.5h相比差异不显著(P>0.05)。
3.应用半定量RT-PCR技术对热应激大鼠肝脏HSP70mRNA进行了定量研究。不同强度的热应激对大鼠肝脏HSP70mRNA的表达量有显著影响,在30℃时,HSP70mRNA表达量略有下降(P>0.05)。在35℃和40℃时,表达量显著的高于对照组(P<0.05)。在不同的热应激时间里,大鼠肝脏HSP70mRNA的表达量呈先迅速增加,到热应激1h时达到最高(P<0.01)。随着热应激时间的延长,HSP70mRNA的表达量开始下降,各热应激处理组的表达量均极显著的高于0.5h组(P<0.01)。
4.应用Real-time PCR技术对热应激大鼠肝脏HSP70mRNA进行了定量研究。不同强度的热应激对大鼠肝脏HSP70mRNA的转录量有极显著的影响,各热应激处理组的HSP70mRNA的转录量极显著高于对照组(P<0.01)。在不同的热应激时间里,大鼠肝脏HSP70mRNA的转录量呈先迅速升高,到热应激1h时达到峰值(P<0.01),之后开始降低。在热应激1.5h时和2h时,极显著高于0.5h(P<0.01)。而在热应激2.5h和4h时,极显著的低于0.5h(P<0.01)。在热应激6h时,与0.5h相比差异不显著(P>0.05)。
5.通过两个等级(秩)变量间的相关分析表明,Real-time PCR和半定量RT-PCR基因检测结果极相关,两种定量PCR检测HSP70mRNA无差异,基本反映了热应激状态下HSP70mRNA转录的规律。研究表明,肝脏HSP70mRNA对热应激反映迅速,变化幅度较大,HSP70mRNA转录量在热应激反应中的总体变化趋势与HSP70表达量近似,通过mRNA的检测可在一定程度上反映HSP70的表达情况。在不同热应激强度和时间下,HSP70mRNA的转录水平呈规律性的变化,其可作为动物热应激的生物标志之一。
Rats were selected to analysis the changes of serum malondialdehyde (MDA), total superoxide superoxide dismutase (T-SOD) and total antioxidant capacity (T-AOC) and to investigate dynamically the expression of HSP70 and the discipline of abundance variation of HSP70 mRNA by using colorimetric method,western-blotting, semi-quantitative RT-PCR and Real-time PCR technology , during heat stress with different intensity( 25℃, 30℃, 35℃and 40℃)and different times(0.5,1.5,2,2.5,4 and 6 hour).The results were showed as follows:
1. This article detected Anti-oxidation indexes in the serum of the rats after heat stress using colorimetry method.Compared with the control group(25℃), The MDA contents in serum decreased slightly (P>0.05) after heat stress with 30℃.and it was significantly lower at 35℃and 40℃(P<0.01). The SOD activity in serum of rats was in rising trend and T-AOC was in declining trend.The SOD activity and T-AOC-size in every heat stress group had very significant differences compare with the control group.During heat stress with different time,The MDA conten in serum rised and reached the peak with 4 hour(P<0.01)and then decreased .There were no differences between heat stress with 6 hour and 0.5 hour.The SOD activity and T-AOC-size in serum of rats were in fluctuation. Compared with heat stress 0.5 hour,The SOD activity and T-AOC-size in every heat stress group were very significant increased(P<0.01).The T-AOC-size in serum of rats was significantly increased after heat stress with 1 hour(P<0.05)and very significantly decreased after heat stress with 1.5 hour,2 hour(P<0.01).There were not differences between other groups for T-AOC-contents in serum of rats.
2. This article detected HSP70 expression amounts in liver of rats after heat stress using western-blotting method. Result showed that HSP70 in liver of rats had a lower level of expression unde normal temperature(25℃) . Compared with the control group, HSP70 expression in liver increased obviously (P<0.05) at 30℃and increased significantly (P<0.01) at 35℃and 40℃. During heat stress with different time, the expression levels of HSP70 in liver of rats increased and reached its peak after heat stress with 2 hours(P<0.01). However, with the extension of heat stress time,HSP70 expression began to decline. The expression level with 4 hour and 6 hour were no significant differences from the HSP70 expression in heat stress with 0.5 hour(P>0.05).
3. This article researched HSP70mRNA abundances in liver of rats after heat stress using semi-quantitative RT - PCR. Results,during heat stress with different intensity had a significant effect HSP70mRNA expression levels in liver of rats.The expression level decreased slightly (P>0.05) at 30℃. At 35℃, The expression level was significantly higher than the control group(P<0.05). During heat stress with different time, the HSP70mRNA expression levels in liver of rats increased rapidly and reached the highest after heat stress with 1 hour(P<0.01).However, with the extension of heat stress time, the HSP70mRNA expression levels began to decline. The expression levels in other hour were very significant differences with the level in 0.5 hour(P<0.01).
4. The abundances of HSP70mRNA in liver of rats at different heat stress were analysed by relative quantitative RT-PCR. Results showed that different intensity of heat stress had a very significant effect on abundances of HSP70mRNA in liver of rats and the quantity of HSP70mRNA transcription of every heat stress group was significantly higher than those of the control group(P<0.01). During heat stress with different time, the quantity of HSP70mRNA transcription in liver of rats rose rapidly,and reached the peak after heat stress with 1h and then declined. The quantity of HSP70mRNA transcription in 1.5 hour and 2 hour were very significantly higher than that of in 0.5 hour. The quantity of HSP70mRNA transcription in 2.5 hour and 4 hour were very significantly lower than that of in 0.5 hour. There were no significant differences between the quantity of HSP70mRNA transcription in 6 hour and 0.5 hour (P>0.05).
5. By correlation analysis between two grades (rank) variables, genetic testing results of Real - time PCR and semi-quantitative RT-PCR were highly relevant.There were no differences between detection of HSP70mRNA by using two kinds of quantitative PCR.The laws of HSP70 mRNA transcription in heat stress were basically reflected. Studies showed that the abundances of HSP70 mRNA in liver responded quickly to heat stress,its changing range was bigger. The general trend of the HSP70mRNA transcription amounts was similar to that of HSP70 expression amounts. Through the detection of mRNA could be reflected to some extent the expression of HSP70 during heat stress. During heat stress with different intensity and time, the level of transcription could be used as the molecular biomarker of animal heat stress.
1.应用比色法,对热应激大鼠血清中抗氧化能力指标进行了测定。与对照组25℃相比,30℃时,大鼠血清的MDA略有下降(P>0.05)。35℃时和40℃时,血清的MDA极显著的降低(P<0.01)。大鼠血清的SOD活力和T-AOC分别呈升高和降低的趋势,各热应激组的SOD活力和T-AOC值与对照组相比,差异极显著(P<0.01)。在不同热应激时间处理下,大鼠血清的MDA先升高,在4h时达到高峰(P<0.01),之后开始下降,在6h时与0.5h相比差异不显著。大鼠血清的SOD活力和T-AOC值均处于上下波动状态,各热应激处理组的SOD活力均比0.5h极显著的升高(P<0.01)。血清的T-AOC值除在热应激处理1h后显著的升高(P<0.05)和1.5h、2h后极显著的下降外(P<0.01),其他各组无明显差异。
2.应用Western-blotting方法,对热应激大鼠肝脏中HSP70的表达量进行了测定。大鼠肝脏HSP70在25℃就有较低水平的表达,与对照组相比,在30℃时,肝脏HSP70表达量就明显的增加(P<0.05)。35℃和40℃时,表达量极显著的增加(P<0.01)。不同的热应激时间时,大鼠肝脏HSP70表达量呈先增加,到2h时表达量达到峰值(P<0.01),随着热应激时间的延长,HSP70表达量开始下降。在热应激处理4h和6h时,HSP70表达量与0.5h相比差异不显著(P>0.05)。
3.应用半定量RT-PCR技术对热应激大鼠肝脏HSP70mRNA进行了定量研究。不同强度的热应激对大鼠肝脏HSP70mRNA的表达量有显著影响,在30℃时,HSP70mRNA表达量略有下降(P>0.05)。在35℃和40℃时,表达量显著的高于对照组(P<0.05)。在不同的热应激时间里,大鼠肝脏HSP70mRNA的表达量呈先迅速增加,到热应激1h时达到最高(P<0.01)。随着热应激时间的延长,HSP70mRNA的表达量开始下降,各热应激处理组的表达量均极显著的高于0.5h组(P<0.01)。
4.应用Real-time PCR技术对热应激大鼠肝脏HSP70mRNA进行了定量研究。不同强度的热应激对大鼠肝脏HSP70mRNA的转录量有极显著的影响,各热应激处理组的HSP70mRNA的转录量极显著高于对照组(P<0.01)。在不同的热应激时间里,大鼠肝脏HSP70mRNA的转录量呈先迅速升高,到热应激1h时达到峰值(P<0.01),之后开始降低。在热应激1.5h时和2h时,极显著高于0.5h(P<0.01)。而在热应激2.5h和4h时,极显著的低于0.5h(P<0.01)。在热应激6h时,与0.5h相比差异不显著(P>0.05)。
5.通过两个等级(秩)变量间的相关分析表明,Real-time PCR和半定量RT-PCR基因检测结果极相关,两种定量PCR检测HSP70mRNA无差异,基本反映了热应激状态下HSP70mRNA转录的规律。研究表明,肝脏HSP70mRNA对热应激反映迅速,变化幅度较大,HSP70mRNA转录量在热应激反应中的总体变化趋势与HSP70表达量近似,通过mRNA的检测可在一定程度上反映HSP70的表达情况。在不同热应激强度和时间下,HSP70mRNA的转录水平呈规律性的变化,其可作为动物热应激的生物标志之一。
Rats were selected to analysis the changes of serum malondialdehyde (MDA), total superoxide superoxide dismutase (T-SOD) and total antioxidant capacity (T-AOC) and to investigate dynamically the expression of HSP70 and the discipline of abundance variation of HSP70 mRNA by using colorimetric method,western-blotting, semi-quantitative RT-PCR and Real-time PCR technology , during heat stress with different intensity( 25℃, 30℃, 35℃and 40℃)and different times(0.5,1.5,2,2.5,4 and 6 hour).The results were showed as follows:
1. This article detected Anti-oxidation indexes in the serum of the rats after heat stress using colorimetry method.Compared with the control group(25℃), The MDA contents in serum decreased slightly (P>0.05) after heat stress with 30℃.and it was significantly lower at 35℃and 40℃(P<0.01). The SOD activity in serum of rats was in rising trend and T-AOC was in declining trend.The SOD activity and T-AOC-size in every heat stress group had very significant differences compare with the control group.During heat stress with different time,The MDA conten in serum rised and reached the peak with 4 hour(P<0.01)and then decreased .There were no differences between heat stress with 6 hour and 0.5 hour.The SOD activity and T-AOC-size in serum of rats were in fluctuation. Compared with heat stress 0.5 hour,The SOD activity and T-AOC-size in every heat stress group were very significant increased(P<0.01).The T-AOC-size in serum of rats was significantly increased after heat stress with 1 hour(P<0.05)and very significantly decreased after heat stress with 1.5 hour,2 hour(P<0.01).There were not differences between other groups for T-AOC-contents in serum of rats.
2. This article detected HSP70 expression amounts in liver of rats after heat stress using western-blotting method. Result showed that HSP70 in liver of rats had a lower level of expression unde normal temperature(25℃) . Compared with the control group, HSP70 expression in liver increased obviously (P<0.05) at 30℃and increased significantly (P<0.01) at 35℃and 40℃. During heat stress with different time, the expression levels of HSP70 in liver of rats increased and reached its peak after heat stress with 2 hours(P<0.01). However, with the extension of heat stress time,HSP70 expression began to decline. The expression level with 4 hour and 6 hour were no significant differences from the HSP70 expression in heat stress with 0.5 hour(P>0.05).
3. This article researched HSP70mRNA abundances in liver of rats after heat stress using semi-quantitative RT - PCR. Results,during heat stress with different intensity had a significant effect HSP70mRNA expression levels in liver of rats.The expression level decreased slightly (P>0.05) at 30℃. At 35℃, The expression level was significantly higher than the control group(P<0.05). During heat stress with different time, the HSP70mRNA expression levels in liver of rats increased rapidly and reached the highest after heat stress with 1 hour(P<0.01).However, with the extension of heat stress time, the HSP70mRNA expression levels began to decline. The expression levels in other hour were very significant differences with the level in 0.5 hour(P<0.01).
4. The abundances of HSP70mRNA in liver of rats at different heat stress were analysed by relative quantitative RT-PCR. Results showed that different intensity of heat stress had a very significant effect on abundances of HSP70mRNA in liver of rats and the quantity of HSP70mRNA transcription of every heat stress group was significantly higher than those of the control group(P<0.01). During heat stress with different time, the quantity of HSP70mRNA transcription in liver of rats rose rapidly,and reached the peak after heat stress with 1h and then declined. The quantity of HSP70mRNA transcription in 1.5 hour and 2 hour were very significantly higher than that of in 0.5 hour. The quantity of HSP70mRNA transcription in 2.5 hour and 4 hour were very significantly lower than that of in 0.5 hour. There were no significant differences between the quantity of HSP70mRNA transcription in 6 hour and 0.5 hour (P>0.05).
5. By correlation analysis between two grades (rank) variables, genetic testing results of Real - time PCR and semi-quantitative RT-PCR were highly relevant.There were no differences between detection of HSP70mRNA by using two kinds of quantitative PCR.The laws of HSP70 mRNA transcription in heat stress were basically reflected. Studies showed that the abundances of HSP70 mRNA in liver responded quickly to heat stress,its changing range was bigger. The general trend of the HSP70mRNA transcription amounts was similar to that of HSP70 expression amounts. Through the detection of mRNA could be reflected to some extent the expression of HSP70 during heat stress. During heat stress with different intensity and time, the level of transcription could be used as the molecular biomarker of animal heat stress.
引文
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