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Effects of Different Intensity Cold Stimulation on Expression of CIRP in Heart, Lung, Brain and Testis of Wistar Rats

Author: ZhaoYaZuo
Tutor: LiShiZe
School: Heilongjiang Bayi Agricultural University
Course: Basic Veterinary Science
Keywords: Cold- inducible RNA binding protein Western-blot Quantitative PCR
CLC: Q75
Type: Master's thesis
Year: 2010
Downloads: 95
Quote: 0
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Abstract


Cold-inducible RNA binding protein (Cold inducible RNA-binding Protein, CIRP) is an initial cloning and identification of mouse testis cells cold shock protein, and overexpression cells from cold stress. Variety of cell stress can be induced CIRP expression, such as oxidative stress, osmotic stress and UV stress. Cold stress when CIRP as RNA molecular chaperone can promote the translation of mRNA can inhibit cell growth, may be involved in a variety of physiological processes and pathological processes in humans and animals, we can say that the cold-inducible RNA binding protein is a multifunctional protein. In recent years, studies have shown that CIRP expression of male sterility, cancer and a variety of diseases such as cancer, detect the expression of CIRP for further study its mechanism of action, function and its application in the treatment of disease providing new clues. This research from cold stimuli time and stimulation intensity of rat heart, lungs, brain and testis four tissues CIRP expression, the relationship between the analysis of of CIRP expression and cold stimuli, to verify the expression of the CIRP exists regularity. Normal rats reared at 20 ± 1 ° C, cold stimulation test will rat at -10 ± 1 ° C, -4 ± 1 ° C ± 1 ° C ± 1 ° C and 10 ± 1 ℃ five ambient temperature conditions the next, and cold stimulation at different time points after sampling. In this study, using the Western blot method and fluorescent quantitative PCR method CIRP expression in rat heart, lungs, brain and testicles four tissues from the stimulation of protein levels and mRNA levels in cold. Fluorescence quantitative PCR method using a double standard curve method the CIRP mRNA relative quantitative detection, designed two pairs of specific primers and synthetic nucleic acid sequence of the first the GenBank published on rats of CIRP and housekeeping gene GAPDH. Total RNA extracted by Trizol organizations cDNA was synthesized by reverse transcription and PCR amplification, gene purified T vector and recombinant plasmids were transformed into E. coli TG1 competent cells by the blue-white screening, PCR enzyme digestion, sequencing positive bacterium. Products according to the standards of the recombinant plasmid to establish a standard curve drawn through software analysis CIRP mRNA samples relative expression level calculated as an indicator of the level of evaluation CIRP expression ratio of the target gene CIRP and internal reference GAPDH mRNA content. Experimental results: (1) cold stimuli can induce CIRP expression with cold stimulation time the expression of different CIRP there is a certain change in the law and the four tissues of rats in the normal state where there is a certain degree of CIRP the expression. (2) the result of the Western blot method of detecting the display visible at about 18kD at a specific band, indicating that after the cold stimuli different times there are the expression of the CIRP, only the amount of each of the expression is not the same. (3) The PCR results show that good linear relationship of the target gene of CIRP and housekeeping gene GAPDH standard curve constructed; cold stimulation, the rat heart, brain and testicular CIRP expression were higher than the normal control group, and the difference between the groups not significant. 0h-6h expression levels increased after acute cold stimulation peaked at 6h, followed by the expression of a downward trend; lungs after cold stimuli the CIRP relative expression levels below the normal control group, the opposite trend with heart, lung and testicular. CIRP expression in the the chronic cold stimulation at different time points after the rat heart, lungs, brain and testicles four organizations similar difference between the groups was not significant. Cold stimuli elevated heart, brain and testicles in CIRP expression may provide a new approach for the study of the treatment of brain damage, heart disease, testicular damage, as well as male sterility disease.

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