| Almost all organisms on earth, it’s physiology biochemical metabolism and behavior,such as body tempetature, blood pressure fluctuatuins, hormone levels rise and fall,sleep-wake cycle are presented about a24h regular change of cycle, called circadianrhythm. Regular circadian rhythm plays an important role in human health, Circadianrhythm disorders often lead to many diseases, such as sleep disorders, endocrine disorders,cardiovascular disease and cancer, etc. Evidences suggest that sleep-wake process which isthe most prominet performance of circadian rhythm shows gender difference. Clinicalevidence found that women prefer sleep early to bed and get up early than man and thewomen complained more sleep problems even though they spend more time on sleep thanman, in addition, the phase of women melatonin and deep body temperature which reflectthe circadian rhythm show advance than men, suggesting that there are sex difference inthe circadian rhythm of SCN. There are three levels of regulation in the circadian rhythmof mammals, the centre of circadian rhythm location in the hypothalamic Suprachiasmaticnucleus, which is only affected by light regulation, known as "pacemaker" of mammals’circadian rhythm. Therefore, to investigate whether there exists sex difference of SCNcircadian rhythm could explain the phenomenon of gender differences of sleep, bodytemperature and the secretion of melatonin.First of all, the molecular basis of circadian rhythm in SCN were a series oftranscription and translation of positive and negative feedback clock genes. Period1(Per1),Cryptochrom1(Cry1), Circadian Locomotor Output Cycles Kaout(Clock), Brain andMuscle Arnt Like protein-1(Bmal1), as the positive and negative feedback of the internalcore components of the circadian rhythm, play important role in Circadian oscillation.Thus, sex differences of clock genes expression rhythm may reflect sex difference of SCNrhythm.Secondly, previous research of our department found that with No kinas3(WNK3)which belongs to a new threonine kinase family probably involved in circadian regulationby phosphorylation clock genes. The gene of WNK3is located in X chromosome whichmay exists sex difference of its expression. Thus, gender differences of circadian rhythmmay also be derived from the gender differences of some kinase which regulates clockgene.Above all, it’s of great significance to research on sex difference of circadian rhythm and its regulating mechanism to understand the mechanism of normal circadian rhythm ofphysiological, biochemical and behavioral between woman and man. This research isdivided into two parts, first, to verify if there exist sex difference circadian rhythmexpression of the core Clock gene (Per1,Cry1, Clock, Bmal1) in rat SCN area; Second, toexplore whether there exist sex difference in the circadian rhythm expression of WNK3kinase, and screen clock gene whose expression rhythm is consistent with the WNK3rhythm.Part I: To verify if there exist sex difference circadian rhythm expression of thecore Clock genes (Per1,Cry1, Clock, Bmal1) in rat SCNObjective: Under the12h light,12h dark (12h-light:12h-dark cycle, LD)condition, to study whether the expression level of core clock genes (per1, cry1, clock,bmal1) in the hypothalamic suprachiasmatic nucleus of female and male rat exist circadianrhythm and analyze if there exist sex difference in the circadian rhythm expression of thesecore Clock genes.Methods:1), All experimental animals would be taken under the condition of LDrhythm training at least2weeks;2), male and female rat was executed in turn at0,4,8,12,16,20six time point, take hypothalamic suprachiasmatic nucleus (SCN) from the brain offemale and male rat at each time point respectively, the SCN was divided into two partsrespectively on two centrifugal pipes, store at-80degrees in refrigerator. The experimentalshould be repeated at least three times.3), Detected clock genes(per1，cry1，clock，bmal1)mRNA level in the SCN of female and male rats by real-time quantitative PCR (Real-timePCR) technology.4), Using F test to verify whether these clock genes mRNA expressionexist circadian rhythm.5), Using cosine software to obtain circadian parameters of clockgene expression, and analyze if there exist sex difference in the circadian rhythmexpression of these core Clock genesResults:1,There exist circadian rhythm of mRNA expression of core clockgenes(per1，cry1，clock，bmal1) in male and female of SD rats SCN under the condition ofLD, amplitude F test, P <0.05).2, There exist sex difference in circadian rhythm of mRNAexpression of core clock genes(cry1，bmal1) in male and female of SD rats SCN under thecondition of LD.1),Compared to male, the peak phase of female cry1mRNA expressionadvance significantly (P <0.001); but other circadian rhythm parameters such asAmplitude, Mensor, peak and valley mRNA level are consistent (P>0.05).2),Compared tomale, the peak phase of female cry1mRNA expression advance significantly (P <0.01); Peak phase of mRNA level is lower than that of male (P <0.05), the circadian rhythm ofMensor level is lower than the male,(P <0.05);but circadian rhythm parameters ofamplitude andpeak and valley mRNA level are consistent (P>0.05).3), all the circadianrhythm parameters of clock and per1mRNA expression such as peak phase, Amplitude,Mensor, peak and valley mRNA level had no statistical difference between two sexes (P>0.05).Conclusion:1,There exist circadian rhythm expression of the core Clock gene(Per1,Cry1, Clock, Bmal1) in rat SCN area, under the12h light,12h dark (12h-light:12h-dark cycle, LD) condition.2, In addition to clock and per1, there exist sex differencein circadian rhythm of mRNA expression of Cry1and Bmal1.Part II, To explore WNK3kinase circadian rhythm and sex differences and itsrelationship of circadian rhythmObjective: To explore whether there exist sex difference in the circadian rhythmexpression of WNK3kinase in the SCN of female and male rat, and screen clock genewhose expression rhythm is consistent with the WNK3rhythmMethods:1), All experimental animals would be taken under the condition of LDrhythm training at least2weeks;2), male and female rat was executed in turn at0,4,8,12,16,20six time point, take hypothalamic suprachiasmatic nucleus (SCN) from the brain offemale and male rat at each time point respectively, the SCN was divided into two partsrespectively on two centrifugal pipes, store at-80degrees in refrigerator. The experimentalshould be repeated at least three times.3), Detected WNK3mRNA level and protein levelin the SCN of female and male rats by real-time quantitative PCR (Real-time PCR)technology and Western blot technology.4), Using F test to verify whether WNK3mRNAand protein expression exist circadian rhythm.5), Using cosine software to obtaincircadian parameters of WNK3expression, and analyze if there exist sex difference in thecircadian rhythm expression of WNK3protein and mRNA.Results:1, There exist circadian rhythm of mRNA and protein expression of WNK3in male and female of SD rats SCN under the condition of LD, amplitude F test, P <0.05).2, There exist sex difference in circadian rhythm of WNK3protein expression, butnot in circadian rhythm of WNK3mRNA in male and female of SD rats SCN under thecondition of LD. There may be sex differences modification after translation process ofWNK3.1),Compared to male, the peak phase of female WNK3protein expression advancesignificantly (P <0.001); but other circadian rhythm parameters such as Amplitude, Mensor, peak and valley protein level are consistent (P>0.05).2), All the circadianrhythm parameters of WNK3mRNA expression such as peak phase, Amplitude, Mensor,peak and valley protein level had no statistical difference between two sexes (P>0.05).3,Only the circadian rhythm parameters of clock mRNA expression are consistent withWNK3mRNA expression rhythm in both male and female SD rats SCN under thecondition of LD, and only the circadian rhythm parameters of cry1mRNA expression areconsistent with WNK3protein expression rhythm in both male and female SD rats SCNunder the condition of LD.Conclusion:1, There exist circadian rhythm expression of WNK3in rat SCN, underthe12h light,12h dark (12h-light:12h-dark cycle, LD) condition.2, There exist sexdifference in circadian rhythm of WNK3protein expression, but not in circadian rhythm ofWNK3mRNA in male and female of SD rats SCN under the condition of LD.3, Circadianrhythm phase of Clock mRNA expression is well coincident with WNK3mRNAexpression, and phase of Cry1mRNA expression coincide with WNK3protein expression. |
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