Study Of The Mechanism Of Action Of The SM22α Protein In Uterine Smooth Muscle Cells

Preliminary studies revealed that there were SM22a expression differences in uterus immediately prior to and after the onset of labour. It has been reported in literature that SM22a is both a calcium-binding and a cytoskeleton protein, which can promote the bundling of actin fibers into stress fibers which in turn regulates the process of smooth muscle contraction. It also has been reported that the SM22a protein can regulate the transcription of matrix metalloproteinase-9(mmp-9). All current works in literature seem to focus on vascular smooth muscle cells and it appears there is a lack of study on the role of SM22a protein in uterine smooth muscle. It must be stressed that delivery is achieved through a coordinated contraction process of all the uterine smooth muscle cells. Although delivery is a complex multi-stage process encompassing many factors such as multi-channel adjustments and molecular interactions, the contraction of the smooth muscle of the uterus plays a key role in the initiation of parturition. Therefore, the understanding of the mechanism of SM22α role in parturition has an important clinical significance and will promote the theory of delivery motivation. In this thesis, the findings from my research project on the specific role of SM22a protein in uterine smooth muscle will be explored.Chapter1:SM22a expression in the body and lower segment of the uterus, placenta and fetal membrane prior to and after the onset of labourObjective:The expression and significance of SM22a protein in the delivery process.Methods:12samples of primipara uterine body, lower segment, the placenta, and fetal membranes tissue, taken between January2008to September2009from patients in the delivery department at the Third Xiangya Hospital, were divided into two groups as "Not In Labor"(NIL) and "In Labor"(IL). Western Blot method has been used to detect the expression of the SM22a protein in the uterine body, the lower segment of the uterus, the placenta and fetal membranes prior to and after the parturition.Results:In all12samples, those from the uterine body, lower segment and the placenta all have the SM22a protein expression, whilst those from the fetal membranes do not express SM22a protein. For placenta samples, there is no statistic difference in the protein expression level of SM22a between IL and NIL group. Whilst for uterine body samples, the SM22a protein expression from the IL group is higher than that from the NIL one. For the lower uterine segment samples, on the other hand, the SM22a protein expression in the IL group is lower than that from the NIL one.Conclusion:The SM22a protein at placenta may not be involved in the onset of parturition. So, SM22α protein in the uterine body and lower segment may be involved in the onset of parturition.Chapter2:Primary culture and identification of pregnancy uterine smooth muscle cellsObjective:Preparation of vitro cultured human gestational uterine smooth muscle cells to conduct further studies of SM22a protein, Methods:Select uterine smooth muscle tissue samples from full-termed normal pregnant women undergoing cesarean delivery, primary culture uterine smooth muscle cells using tissue adherent culture and enzyme digestion methods, and then identify by Immunocytochemistry, Results: Human gestational uterine smooth muscle cells were successfully cultured.The distribution of SM22α in the uterine smooth muscle cells was similar with the actin’s as bunches by the method of Immunocytochemistry.Conclusion:Successfully prepared the gestational vitro cultured uterine smooth muscle cells. The distribution of SM22α in the uterine smooth muscle cells was similar with the actin’s as bunches. So, SM22a protein may act as cell skeleton protein in the uterine smooth muscle cells。Chapter3:pCMV-Myc-SM22a plasmid constructing and efficient siRNA screeningObjective:Obtain pCMV-Myc-SM22α plasmid including the sequence of the SM22a gene and screen high-efficient siRNA squence for vitro study, Methods:Human SM22a gene sequences can be obtained from Gene Bank, design SM22a primers and SiRNA primer by PRIMER PREMIER software, construct pCMV-Myc-SM22a plasmid by cloning technology; further express SM22a in the293FT cells by gene technology, and screen high efficient SiRNA primer; And verify high efficient SiRNA primers in vitro cultured uterine smooth muscle cells.Results:Successfully construct pCMV-Myc-SM22a plasmid and obtained high efficient SiRNA primer siRNA SM22-252Sense Chain5’-CCAUGGUCUUCAAGCAGAUTT-3’Antisense chain5’-AUCUGCUUGAAGACCAUGGAG-3’ siRNA SM22-444Sense Chain5’-CCAACUGGUUUAUGAAGAATT-3’Antisense Chain5’-UUCUUCAUAAACCAGUUGGGA-3’Conclusion:Vitro primary cultured gestational uterine smooth muscle cell gene cloning technology can only carry out on2-4generation cells. Repeatedly passaged cells is difficult to carry out cloning technology. It not only saves resources but also saves time by using easily obtainable293FT cells primer to screen, which is worthy of promotionChapter4:Impact of oxytocin, magnesium sulfate on SM22a protein expression in cultured uterine smooth muscle cells and intracellular calcium ion concentrationObjective:To study the role of SM22a protein in vitro cultured human pregnant uterus smooth muscle cells and mechanism,Methods: Stimulate vitro primary cultured uterine smooth muscle cells with oxytocin and magnesium sulfate at different times and different doses, detect cells SM22a protein expression by Western blot; then choose effective action duration, effective action drug dose acting on SM22a protein over-expression, silence SM22a protein and normal cultured vitro primary cultured uterine smooth muscle, and then detect intracellular calcium ion concentration by fluorescence quantitative.Results:Vitro experiments showed that oxytocin increases Uterine smooth muscle SM22a protein expression, in correlation with the duration and dose of oxytocin, indicating accumulation with duration and dose. Magnesium sulfate suppresses SM22a protein expression, in correlation with the duration and dose of magnesium sulfate, indicating resistance of duration and dose in vitro experiments. The concentration of intracellular calcium ion in SM22a Protein over-expression cells was significantly reduced compared with normal cultured with significant difference p<0.01. Silence SM22a protein expression increases intracellular calcium ion concentration, compared with the normal control group with very significant difference p<0.01. Magnesium sulfate reduces the concentration of intracellular calcium ion in SM22a protein over-expression cells compared with the normal culture with no significant difference p>0.05. Magnesium sulfate increases intracellular calcium ion concentration in silence SM22a protein expression compared with the normal culture with significant difference p<0.05.Oxytocin stimulation group comparisons:Oxytocin stimulation reduces intracellular calcium ion concentration in SM22a protein over-expression, compared with the normal culture with a very significant difference p <0.01. Oxytocin stimulation increases intracellular calcium ion concentration in silence SM22a protein expression, compared with the normal control group with no differences p>0.05; SM22a protein expression group comparison:Magnesium sulfate reduces the intracellular calcium ion concentration, but compared with the control group with no difference p>0.05. Oxytocin increases intracellular calcium concentration compared with the control group with difference p <0.05. Silence SM22a protein group contrast:Magnesium group significantly reduces intracellular calcium ion concentration, compared with the control group with difference p<0.01. Oxytocin increases intracellular calcium ion concentration, but compared with the control group with no significant difference p>0.05.Conclusion:. The onset of parturition is a process, in which the spontaneously secreted oxytocin stimulate the expression of SM22a protein in the uterine smooth muscle cells through quantitative change to qualitative change, and also regulated by the intra cellular calcium ion. The SM22a protein in uterine smooth muscle not only plays a skeleton role, which regulates the concentration of intra cellular calcium ion by its expression level, in order to influence the onset of labor. It is an important protein factor in the onset of labor.