| Cold inducible RNA-binding protein (CIRP) was originally cloned in mouse testis cells and identification of the first cold shock protein, over expressed upon cold stress. CIRP was induced expression by a wide variety of cellular stresses, such as hypoxia stress, osmotic pressure stress and UV irradiation stress et al. CIRP as an RNA chaperone, which facilitated mRNA translation, inhibited cell growth upon the perception of cold stress, and participated in many kinds of physiology and pathology process in human and animal. On the whole, CIRP was a multifunctional protein. In recent years, the study indicated that expression of CIRP related to the male sterility, tumor and cancer and so on many kinds of diseases. Therefore, detecting to the expression of CIRP will provide new clues for the mechanisms, function and application in the treatment of disease of CIRP in the future.In this study, expression of CIRP in heart, lung, brain and testis four tissue in rats at different from cold stimulation time and intensity was investigated; the relationship between expression of CIRP and cold stimulation was analyzed and whether there existed the regularity in expression of CIRP was confirmed. Wistar rats were raised at 20±1℃in normal condition and treated at -10±1℃,-4±1℃, 0±1℃, 4±1℃and 10±1℃five environment temperature, and samples were separately collected at different time intervals after cold stimulation. mRNA expression level and protein expression level of CIRP in heart, lung, brain and testis four tissues in rats after different intensity cold stimulation were detected by SYBR green I real time PCR and Western-blot method method. CIRP mRNA relative expression quantity was detected by the double standard curve in the real time PCR method. First, two pairs of specific primers were designed on the nucleic acid sequences of Housekeeping gene GAPDH and rats CIRP, which have been published in the GenBank. The total RNA was obtained from heart, lung, brain and testis four tissues in rats by Trizol method and cDNA was synthesized by reverse transcription then amplified by RCR method. PCR products were linked with vector pMD 18-T after gene purification and recombined plasmids were transformed into competence cell E. coli TG1. Recombined plasmids were extracted and positive clones were obtained by blue-white screening, PCR, double enzymes digestion, DNA sequencing. The standard curves were built by standard recombined plasmids and CIRP mRNA relative expression levels in samples were obtained by software analyzed. The results were expressed as the ratios of target gene CIRP mRNA content to reference gene GAPDH mRNA content.Experimental results indicated:⑴At normal conditional temperature, CIRP in four tissues of rats exist the basic expression level. CIRP was induced by cold stimulation, and had changed regularly with different time.⑵Western blot method results showed that a specific band was observed at about 18kDa and also indicated that CIRP were expressed at all time after cold stimulation, but the expression levels were different.⑶Real time PCR results showed that the standard curve drawn by target gene and housekeeping gene GAPDH presented a good linear tendency; compared with the control group, the expression level of CIRP in heart, brain and testis of rats was increased after cold stimulation, and there were no significant differences among groups . The expression of CIRP mRNA began to increase on 0h-6h after acute cold stimulation and peaked on 6h then started to decline in heart, brain and testis of rats. Compared with the control group, the expression of CIRP in lung of rats was degraded after cold stimulation and change tendency was opposite with the former. After chronic cold stimulation, the expression level of CIRP were approximate in heart, lung, brain and testis four tissue of rats and the difference was not significant among groups . The expression level of CIRP were increased after cold stimulation in heart, brain and testis, which will provided a new therapy way with brain damage, heart disease, testicle damage and male sterility and so on.
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