| Chapter One:The location and expression of h1calponin in human uterine smooth muscle before and after deliveryObjective:Explore the change of phosphorylation level and location of h1calponin in human uterine smooth muscle before and after delivery.Methods:The uterine myometrium tissues were obtained from human uterine in non-pregnant group, term not in labor group and term in labor group. Each group included10women. With the method of western blot, the protein expression level of h1calponin and p-hl calponin was detected. The location of h1calponin in uterus smooth muscles was observed with method of immunofluorescence technique.Results:We explored the expression level of h1calponin and phospho-h1calponin in non-pregnant women, term no labor women and term labor women by the method of western blot with h1calponin and phospho-h1calponin (p-h1calponin) antibodies. The total h1calponin protein levels and phospho-hl calponin protein levels were significantly increased in myometrium from term not in labor women and term in labor women, as compared with that from the nonpregnant women(P<0.05). There was no significant difference in the ratio of p-hl calponin to h1calponin between term not in labor groups and nonpregnant groups(P>0.05). The ratio of p-hl calponin to h1calponin from term in labor women myometrium was significantly higher than that from nonpregnant women myometrium (P<0.01). The results from the immunofluorescence suggested that h1calponin was located in the cytoplasm of human uterus smooth muscles in nonpregnant women and term not in labor women, but h1calponin was located in marginal area of cytoplasm, almost close to the cell membrane.Conclusion:The up-regulation of h1calponin during pregnancy may contribute to reorganization of the myometrial cytoskeleton and maintenance the quiescence of the myometrium. Phosphorylation and migration of h1calponin play important roles to revers the quiescence and promote contraction of the uterus during labor.Chapter Two:Expression of PKC-ε and h1calponin in pregnant mice myometrium during pregnancy and labor at term and pretermObjective:To evaluate the expression and phosphorylation of PKC-ε and h1calponin in pregnant mice myometrium during pregnancy and labor at term and preterm.Methods:To induce preterm birth, pregnant mice were intubated with6g/kg ethanol on gestational day16. The mice were divided into following groups(10mice per group):nonpregnant(NP), pregnant D7(the7th day during pregnancy), pregnant D12(the12th day during pregnancy), pregnant D17(the17th day during pregnancy), pregnant D18(the18th day during pregnancy), pregnant D19(the19th day during pregnancy), in term labor (IL, after the first pup was delivered),in preterm labor. We detected the expression of h1calponin, phosphorylated h1calponin, PKC-ε and phosphorylated PKC-s in the different stages of mice during pregnancy and in labor by the method of western blot.Results:The level of the four proteins including h1calponin, phosphorylated h1calponin, PKC-s and phosphorylated PKC-s was significantly increased in pregnant mice myometrium as compared with that in nonpregnant mice(P<0.05). The ratios of phosphorylated h1calponin/hl calponin and phosphorylated PKC-s/PKC-s were reached the peak after the onset of labor in myometrium in the mice. There was no significantly difference in the ratio of phosphorylated h1calponin/total h1calponin between the in term labor mice and in preterm labor mice(P>0.05). There was no significantly difference in the ratio of phosphorylated PKC-s/total PKC-s between the in term labor mice and in preterm labor mice(P>0.05).Conclusion:The phosphorylation of PKC-ε and h1calponin both reach the peak at parturition. PKC-ε mediated h1calponin phosphorylation is suggested to contribute to the initiation of labour either at term or preterm. Chapter Three:The mechanism of PKC-ε/h1calponin pathway in regulation of pregnant mice myometrial contractilityObjective:We explore whether PKC-ε/h1calponin pathway contribute to regulation of myometrial contractility and whether PKC-ε/h1calponin pathway is a Ca2+-independent way.Methods:The myometrium strips from from mice at the19th day during pregnancy were treated with different concentration of Psi-RACK(PKC-epsilon specific activator),then we observed the contraction activity of the strips。To further investigate whether PKC-s was involved in the phosphorylation of h1calponin,10-9mol/L epsilon-V1-2(PKC-ε specific inhibitor) was administered to the tissue baths after adding10-9mol/L Psi-RACK. At the end of experiments, muscle strips were snapping frozen in liquid N2for Western Blot Analysis. To investigate whether the contraction induced by PKC-s/hl calponin pathway was Ca2+-independent,30umol/L ML-9(myosin light chain kinase specific inhibitor) was administered to the tissue baths after adding10-9mol/L Psi-RACK.Results:After the treatment of more than10-9mol/L Psi-RACK (PKC-ε activator), the contractility of myometrium strips from mice was reinforced and the level of phosphorylated h1calponin increased at the same time which could be interrupted by the specific inhibitor of PKC-ε. Meanwhile, ML-9could not suppress the contraction which stimulated by Psi-RACK。Conclusion: These data suggest that in mice myometrium, phosphorylation of h1calponin induced by the PKC-ε might regulate pregnant myometrial contractility and PKC-ε/h1calponin pathway is suggested to Ca2+-independent.
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