| Delivery is a process of the interaction of multi-factor participation, multi-channel regulation with multi-stages. Uterine smooth muscle contraction is the key to the onset of labor. It is important to know the mechanisms of the onset of labor for understanding pathological pregnancy, reducing premature births and high risk infants. In the previous study, we have obtained the profiles of the differences of the gene expression in the corpus and lower segment of the human uterine in natural delivery and in those of non-delivery samples indicating that the gene of Calponin-1 is highly expressed in the corpus and the lower segment of the uterine with delivery status, which may be related to the mechanisms of the regulation of the uterine smooth muscle cell function. In the current study, we would like to further study the molecular mechanisms of the Calponin-1 in the regulation of uterine smooth muscle cells.Chapter I Expression and significance of Calponin-1 in human uterine smooth muscle tissues in non-labor and labor situationObjective:To investigate the significance of differential expression of Calponin-1 in human uterine smooth muscle tissues in non-labor and labor situation.Methods:Uterine smooth muscle tissues from 14 human uterine corpus and lower segment pregnancy are divided in non-labor group (NIL) and labor group (IL). Immunohistochemical technology and western-blot were used to determine the expression levels of Calponin-1 and pCalponin-1. Results:Immunohistochemistry:Calponin-1 and pCalponin-1 protein were expressed in all the uterine smooth muscle tissues of the 14 cases. (1) The average gray values of expression of Calponin-1 protein in the smooth muscle tissue of the uterine corpus in NIL and IL were 184.91±5.12 and 167.32±5.22, respectively. The average gray values of Calponin-1 protein in uterine lower segment smooth muscle tissue in NIL and IL were 174.51±4.82 and 165.42±4.52, respectively. The differences between them are significant (p< 0.05). (2) The average gray values of expression of pCalponin-1 protein in uterine corpus smooth muscle tissue in NIL and IL were 148.22±11.95 and 90.42±12.22, respectively. The average gray values of pCalponin-1 protein in uterine lower segment smooth muscle tissue in NIL and IL were 151.34±10.36 and 113.42±10.22, respectively. The differences are significant (p< 0.05).Western blot:(1) The average gray values of Calponin-1 protein in uterine corpus smooth muscle tissue in NIL and IL were 0.373±0.092 and 0.865±0.090, respectively. The expression of the difference between two situation was significant (p<0.05). The average gray values of Calponin-1 protein in uterine lower segment smooth muscle tissue in NIL and IL were 0.522±0.102 and 0.957±0.081 respectively. The differences are significant (p<0.05). (2) The average gray values of pCalponin-1 protein in uterine corpus smooth muscle tissue in NIL and IL were 0.303±0.071 and 0.532±0.063, respectively. The average gray values of pCalponin-1 protein in uterine lower segment smooth muscle tissue in NIL and IL were 0.274±0.091 and 0.567±0.085, respectively. The differences are significant (p<0.05).Conclusions: The expression of Calponin-1 and pCalponin-1 proteins in the uterine smooth muscle tissues increases after labor, which may be related to uterine smooth muscle contraction. Calponin-1 and pCalponin-1 protein possibly participated in the launch of childbirth through the adjusting uterine smooth muscle contraction.ChapterⅡEffects of the silence of Calponin-1 expression to the function of uterine smooth muscle cellObjective:To investigate the potential effect on the smooth muscle of the human uterine of the inhibition of the expression of Calponin-1 protein and its molecular mechanisms.Methods:Human uterine smooth muscle tissues were digested with enzyme, cultured and confirmed with immunocytochemistry. Calponin-1siRNA was used to silence the expression of Calponin-1 in the primary culture of uterine smooth muscle cells. MTT, flow cytometry, Transwell chamber changes, immunofluorescence were used to determine regulation of Calponin-1 on the effects of uterine smooth muscle cells. Experiments are divided into three groups:the experimental, blank control, empty vector groups.Results:(1) Primary culture and identification of uterine smooth muscle cells: immunocytochemical staining with antibodies against smooth muscle actin showed that smooth muscle cells in primary culture of the uterus staining brown.(2) siRNA-Calponin-1 adenovirus can effectively inhibit Calponin-1 mRNA and protein synthesis. The results showed that the average gray values of Calponin-1 mRNA in uterine smooth muscle cell in experimental, blank control, empty vector groups were 0.0463±0.0045,0.2513±0.0552,0.3124±0.0037, respectively. The average gray values of Calponin-1 protein were 0.1098±0.0127,0.2758±0.0384,0.3187±0.0425, respectively. The differences between experimental group and blank control group, empty vector group was statistical significance (p< 0.05). There was no significant difference between empty vector group and blank control group (p> 0.05).(3) The inhibition of the Calponin-1 expression can inhibit uterine smooth muscle cell migration without affecting its proliferation and apoptosis in vitro:Transwell chamber invasion assay showed that following 24h culture the number of cells per low magnification field of vision were 64±12,122±14,125±10 for experimental, empty vector, blank control groups, respectively. There are significant differences between experimental and vector or control groups(p<0.05). There was no significant difference between the latter two groups (p> 0.05). There is no significant difference among these three groups in cell survival rate as showed by Flow cytometry (4.22%,3.02%,4.64% for experimental, empty vector, blank control groups, respectively) (p> 0.05).(4) The inhibition of the Calponin-1 expression can cause morphologic change and rearrangement of F-actin of uterine smooth muscle cell in vitro:thinner, loosing and irregular F-actin microfibers were observed in the experimental group whereas in the empty vector and blank control groups thicker and longer F-actin microfibers were demonstrated.Fluorescence intensity of F-actin in experimental group, empty vector group, blank control group were 832.22±99.02,1123.32±102.35, 1089.19±110.53, respectively. The differences between experimental group and empty vector, blank control groups were statistically significant (p<0.01). There was no significant difference between empty vector group and blank control group (p> 0.05).Conclusion:(1) Calponin-1 siRNA plasmid can effectively silence Calponin-1 gene expression.(2) The inhibition of the Calponin-1 expression can inhibit uterine smooth muscle cell migration without affecting its proliferation and apoptosis in vitro.(3) Calponin-1 regulates uterine smooth muscle contraction by affecting morphologic change and rearrangement of F-actin of uterine smooth muscle cell in vitro.Chapter III The role of Calponin-1 expression in the regulation of uterine smooth muscle cell contraction by magnesium sulfate and oxytocinObjective:To investigate the role of the Calponin-1 expression levels in magnesium sulfate and oxytocin induced contraction in uterine smooth muscle cells.Methods:The uterine smooth muscle cells were stimulated by magnesium sulfate and oxytocin in vitro, followed by Western-blotting and RT-PCR to detect the Calponin-1 expression levels.Results:1. The effects of magnesium sulfate on Calponin-1 expression in uterine smooth muscle cells in vitro. (1) RT-PCR:following treatment of 2.0%,4.0%,8.0%,16% magnesium sulfate (MgSO4) for 30 min, the average gray values of Calponin-1 mRNA expression in control group and different concentration groups of magnesium sulfate were 0.2476±0.0431,0.2562±0.0382,0.2813±0.0243,0.3011±0.0135,0.3042±0.0119, respectively. There was no significant difference among groups (p>0,05). (2)Western Blot:following treatment of control group, 2.0%,4.0%,8.0%,16% magnesium sulfate (MgSO4) for 24h, the average gray values of Calponin-1 protein expression in corresponding concentration groups were 0.2135±0.0537,0.2572±0.0378 0.2813±0.031,0.3147±0.0161,0.3185±0.0134, respectively. There was no significant difference among groups (p>0.05).2. The effect of oxytocin on Calponin-1 expression in uterine smooth muscle cells in vitro. (1) RT-PCR:treatment with different concentrations of oxytocin (2.0%,4.0%,6.0%,8.0%) for 30 min, the average gray values of Calponin-1 mRNA expression in control group and different concentration groups of oxytocin were 0.1936±0.0283,0.4382±0.0355,0.6232±0.0491,0.3951±0.0762,0.3976±0.0647, respectively. There were significant differences between control and the test groups (p<0.05). Calponin-1mRNA level was increased most significantly in the 4.0% concentration group as demonstrated by fluorescence quantitative RT-PCR verification and semi-quantitative RT-PCR. (2) Western Blot:treatment with different concentrations of oxytocin (2.0%,4.0%,6.0%,8.0%) for 24h, the average gray values of Calponin-1 mRNA expression in control group and different concentration groups of oxytocin were 0.2095±0.0153,0.4831±0.0368,0.6197±0.0425,0.4105±0.0148,0.4112±0.0135, respectively. There was significant difference between the control group and the test groups (p<0.05), with the Calponin-1 protein expression increased most significantly in 4.0% concentration group. (3) Then uterine smooth muscle cells were treated with 4.0% oxytocin for 0h,24h,48h,96h and Calponin-1 protein levels were detected. The average gray values of Calponin-1 protein expression at different time points were 0.2014±0.0115,0.5097±0.0127,0.3865±0.0454,0.2247±0.0216, respectively. These results suggest that oxytocin increases Calponin-1 protein expression and it is most obvious at 24h time point.Conclusion:1. Oxytocin can up-regulate the Calponin-1 expression of the uterine smooth muscle cells in vitro, and is positively correlated to the concentration of oxytocin and treatment time.2.Oxytocin caused uterine contraction by increases the Calponin-1 expression in the uterine smooth muscle cells, which possibly lead to onset of labor, a new mechanism of oxytocin to promote uterine smooth muscle cell contraction.3. Magnesium sulfate does not affect expression of Calponin-1 of the uterus smooth muscle cells. We can only surmise that Calponin-1 regulate contraction of uterine smooth muscle may not be dependent on Ca2+ channel. |
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