Calponin-1介导分娩发动的分子机制研究
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摘要
分娩是由多因素作用、多途径调节、多分子参与、多阶段变化的交互作用过程。子宫平滑肌的收缩是分娩发动的关键环节。若能探明分娩发动机制对病理妊娠计划分娩、减少早产及高危儿的出生,具有重要的临床意义。在前期的研究中,我课题组已应用基因芯片技术得到了人类自然临产与未临产子宫体部与子宫下段的差异基因表达谱,发现Calponin-1等在临产后的子宫体部及下段均呈高表达,可能与调控子宫平滑肌细胞功能密切相关。Calponin-1蛋白结构已经研究清楚,但对它在平滑肌收缩功能的调节机制研究大多集中在血管平滑肌上,对其参与子宫平滑肌收缩的调节机制研究较少。本研究拟在前期工作基础上进一步探讨Calponin-1基因表达上调在分娩发动中调控子宫平滑肌细胞的分子机制。
目的：探讨Calponin-1在临产前后子宫平滑肌组织中的表达及意义。
方法：选取14例初产妇的子宫体部和子宫下段平滑肌组织,分为未临产组(NIL)和临产组(IL),采用免疫组织化学(Immunohistochemistry)和免疫印迹(Western-blot)方法检测Calponin-1蛋白(Calponin-1)和磷酸化Calponin-1蛋白(pCalponin-1)的表达。
结果：
1.免疫组织化学
14例子宫平滑肌组织中均可见Calponin-1及pCalponin-1蛋白阳性表达,阳性表达定位在平滑肌细胞胞浆,呈棕黄色。
(1)在未临产组(NIL)与临产组(IL)子宫体平滑肌组织中Calponin-1蛋白阳性表达的平均灰度值分别为：184.91±5.12和167.32±5.22,两组比较差异具有显著性(p＜0.05);在未临产组(NIL)与临产组(IL)子宫下段平滑肌组织中Calponin-1蛋白阳性表达的平均灰度值分别为：174.51±4.82和165.42±4.52,两组比较差异具有显著性(p＜0.05)。
(2)在未临产组(NIL)与临产组(IL)子宫体平滑肌组织中pCalponin-1蛋白阳性表达的平均灰度值分为：148.22±11.95和90.42±12.22,两组比较差异具有显著性(p＜0.05)；在未临产组(NIL)与临产组(IL)子宫下段平滑肌组织中pCalponin-1蛋白阳性表达的平均灰度值分别为：151.34±10.36和113.42±10.22,两组比较差异具有显著性(p＜0.05)。
2.免疫印迹
(1)在未临产组(NIL)与临产组(IL)子宫体平滑肌组织中Calponin-1蛋白的相对表达量分别为：0.373±0.092和0.865±0.090,两组比较差异具有显著性(p＜0.05)；在未临产组(NIL)与临产组(IL)子宫下段平滑肌组织中Calponin-1蛋白的相对表达量分别为：0.522±0.102和0.957±0.081,两组比较差异具有显著性(p＜0.05)。
(2) pCalponin-1蛋白表达水平检测结果显示：未临产组(NIL)和临产(IL)组子宫体平滑肌组织中pCalponin-1蛋白的相对表达量分别为：0.303±0.071和0.532±0.063,两组比较差异具有显著性(p＜0.05),未临产组(NIL)和临产(IL)组子宫下段平滑肌组织中pCalponin-1蛋白的相对表达量分别为：0.274±0.091和0.567±0.085,两组比较差异具有显著性(p＜0.05)。
结论：
临产后子宫平滑肌组织中Calponin-1及pCalponin-1蛋白的表达上调与临产后子宫平滑肌的收缩状态有关,提示临产后子宫平滑肌的收缩可能部分依赖于Calponin-1及pCalponin-1的表达。
目的：探讨Calponin-1蛋白表达抑制对妊娠子宫平滑肌细胞功能的影响,研究其在分娩发动中的分子作用机制。
方法：采用消化酶法对人子宫平滑肌组织进行子宫平滑肌细胞原代培养,并用免疫细胞化学方法进行鉴定。构建腺病毒siRNA-Calponin-1质粒转染子宫平滑肌细胞沉默Calponin-1表达,采用MTT、流式细胞术、Transwell小室的变化、免疫荧光标记和荧光显微镜观察平滑肌细胞骨架蛋白F-actin表达的变化等方法研究Calponin-1调控子宫平滑肌细胞的作用,实验分三组：实验组、空白对照组、空载体组。
结果：
1.原代子宫平滑肌细胞的培养及鉴定
培养的子宫平滑肌细胞呈典型的梭状肌细胞样生长,用平滑肌肌动蛋白抗体,经免疫细胞化学染色显示原代培养的子宫平滑肌细胞胞质呈棕色,证明获得的原代子宫平滑肌细胞符合后续实验的要求。
2. siRNA-Calponin-1腺病毒有效抑制Calponin-1 mRNA和蛋白的表达
采用RT-PCR和Western-blot方法检测pAd-mU6pro-Calponin-l-siRNA转染后子宫平滑肌细胞中Calponin-1 mRNA和蛋白的表达,结果显示：实验组、阴性对照组、空白对照组Calponin-1mRNA的相对表达量分别为0.0463±0.0045、0.2513±0.0552、0.3124±0.0037；实验组、阴性对照组、空白对照组Calponin-1蛋白的相对表达量分别为0.1098±0.0127、0.2758±0.0384、0.3187±0.0425。实验组与阴性对照组、空白对照组比较,差异有统计学意义(p＜0.05),阴性对照组与空白对照组比较,差异无统计学意义(p＞0.05)。
3.体外实验中抑制Calponin-1的表达可以抑制子宫平滑肌细胞的迁移运动而不影响其增殖和凋亡
Transwell侵袭小室测定法结果显示24小时培养后实验组、空载体组,空白对照组穿过Transwell的每低倍视野细胞数分别为64±12、122±14、125±10,实验组与空载体组、空白对照组比较,差异有统计学意义(p<0.05)；空载体组与空白对照组比较,差异无统计学意义(p>0.05)。MTT法检测转染24h、48h、72h后细胞的存活率,结果显示三组细胞存活率两两比较差异无统计学意义(p>0.05)。流式细胞仪检测结果显示,三组细胞凋亡率分别为4.22%、3.02%、4.64%,两两比较差异无统计学意义(p>0.05)。
4.体外实验中抑制Calponin-1的表达可以引起子宫平滑肌细胞骨架蛋白F-actin的改变
实验组子宫平滑肌细胞中F-actin排列紊乱、稀疏。空载体组与空白对照组子宫平滑肌细胞中F-actin微丝明显粗长,大多沿细胞纵轴呈平行排列。实验组、空载体组、空白对照组子宫平滑肌细胞中的F-actin荧光强度分别为832.22±99.02、1123.32±102.35、1089.19±110.53,实验组与空载体组、空白对照组比较差异具有显著性(p<0.01)；而空载体组与空白对照组比较差异无统计学意义(p>0.05)。
结论：
1.构建的Calponin-1siRNA干扰质粒能有效沉默Calponin-1基因表达。
2.体外实验中抑制Calponin-1的表达可以抑制子宫平滑肌细胞的迁移运动而不影响其增殖和凋亡。
3. Calponin-1通过影响子宫平滑肌细胞中骨架蛋白F-actin的改变调节子宫平滑肌的收缩。
目的：探讨硫酸镁及缩宫素调节子宫平滑肌细胞收缩与Calponin-1表达水平的相关性。
方法：用硫酸镁及缩宫素作用于体外培养的子宫平滑肌细胞,然后采用Western-blot及RT-PCR的方法对子宫平滑肌组织中Calponin-1的表达进行检测。
结果：
1.体外硫酸镁对子宫平滑肌细胞Calponin-1表达的影响
(1) RT-PCR结果硫酸镁作用子宫平滑肌细胞30分钟后,阴性对照组、2.0%、4.0%、8.0%、16%浓度硫酸镁组Calponin-lmRNA的相对表达量分别为：0.2476±0.0431、0.2562±0.0382、0.2813±0.0243、0.3011±0.0135、0.3042±0.0119,组间比较差异无统计学意义(p>0.05)。
(2) Western Blot结果硫酸镁作用子宫平滑肌细胞24h后,阴性对照组、2.0%、4.0%、8.0%、16%浓度硫酸镁组Calponin-1蛋白的相对表达量分别为：0.2135±0.0537、0.2572±0.0378、0.2813±0.0351、0.3147±0.0161、0.3185±0.0134,组间比较差异无统计学意义(p>0.05)。
2.体外缩宫素对子宫平滑肌细胞Calponin-1表达的影响
(1)RT-PCR结果缩宫素作用子宫平滑肌细胞30分钟后,阴性对照组、2.0%、4.0%、6.0%、8.0%浓度缩宫素组Calponin-1mRNA的相对表达量分别为：0.1936±0.0283、0.4382±0.0355、0.6232±0.0491、0.3951±0.0762、0.3976±0.0647,各浓度组与阴性对照组比较差异具有统计学意义(p<0.05),其中以4.0%浓度组Calponin-1mRNA表达最为显著。
(2) Western Blot结果缩宫素作用子宫平滑肌细胞24h后,阴性对照组、2.0%、4.0%、6.0%、8.0%浓度缩宫素组Calponin-1蛋白的相对表达量分别为：0.2095±0.0153、0.4831±0.0368、0.6197±0.0425、0.4105±0.0148、0.4112±0.0135,各浓度组与阴性对照组比较差异具有统计学意义(p<0.05),其中以4.0%浓度组的Calponin-1蛋白上调最为显著。我们用4.0%浓度组缩宫素作用子宫平滑肌细胞,在0h、24h、48h、96h不同时间段,用Western Blot检测Calponin-1蛋白表达水平。结果显示,0h、24h、48h、96h时间段Calponin-1蛋白的相对表达量分别为：0.2014±0.0115、0.5097±0.0127、0.3865±0.0454、0.2247±0.0216,结果提示,缩宫素上调Calponin-1蛋白表达与其作用时间具有相关性,以作用24h时Calponin-1蛋白表达上调最为明显。
结论：
1.体外缩宫素可以上调子宫平滑肌细胞Calponin-1的表达,且与其浓度及作用时间具有相关性,但与缩宫素作用浓度及作用时间并无明显正／或负相关。
2.缩宫素通过上调子宫平滑肌细胞Calponin-1的表达而引发宫·缩,导致分娩发动,这可能是缩宫素促进子宫平滑肌细胞收缩的又一新的作用途经。
3.体外硫酸镁不影响子宫平滑肌细胞Calponin-1的表达,我们推测Calponin-1调控子宫平滑肌收缩的作用可能并不依赖于Ca2+通道。
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 situation
Objective: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 cell
Objective: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 oxytocin
Objective: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.
目的：探讨Calponin-1在临产前后子宫平滑肌组织中的表达及意义。
方法：选取14例初产妇的子宫体部和子宫下段平滑肌组织,分为未临产组(NIL)和临产组(IL),采用免疫组织化学(Immunohistochemistry)和免疫印迹(Western-blot)方法检测Calponin-1蛋白(Calponin-1)和磷酸化Calponin-1蛋白(pCalponin-1)的表达。
结果：
1.免疫组织化学
14例子宫平滑肌组织中均可见Calponin-1及pCalponin-1蛋白阳性表达,阳性表达定位在平滑肌细胞胞浆,呈棕黄色。
(1)在未临产组(NIL)与临产组(IL)子宫体平滑肌组织中Calponin-1蛋白阳性表达的平均灰度值分别为：184.91±5.12和167.32±5.22,两组比较差异具有显著性(p＜0.05);在未临产组(NIL)与临产组(IL)子宫下段平滑肌组织中Calponin-1蛋白阳性表达的平均灰度值分别为：174.51±4.82和165.42±4.52,两组比较差异具有显著性(p＜0.05)。
(2)在未临产组(NIL)与临产组(IL)子宫体平滑肌组织中pCalponin-1蛋白阳性表达的平均灰度值分为：148.22±11.95和90.42±12.22,两组比较差异具有显著性(p＜0.05)；在未临产组(NIL)与临产组(IL)子宫下段平滑肌组织中pCalponin-1蛋白阳性表达的平均灰度值分别为：151.34±10.36和113.42±10.22,两组比较差异具有显著性(p＜0.05)。
2.免疫印迹
(1)在未临产组(NIL)与临产组(IL)子宫体平滑肌组织中Calponin-1蛋白的相对表达量分别为：0.373±0.092和0.865±0.090,两组比较差异具有显著性(p＜0.05)；在未临产组(NIL)与临产组(IL)子宫下段平滑肌组织中Calponin-1蛋白的相对表达量分别为：0.522±0.102和0.957±0.081,两组比较差异具有显著性(p＜0.05)。
(2) pCalponin-1蛋白表达水平检测结果显示：未临产组(NIL)和临产(IL)组子宫体平滑肌组织中pCalponin-1蛋白的相对表达量分别为：0.303±0.071和0.532±0.063,两组比较差异具有显著性(p＜0.05),未临产组(NIL)和临产(IL)组子宫下段平滑肌组织中pCalponin-1蛋白的相对表达量分别为：0.274±0.091和0.567±0.085,两组比较差异具有显著性(p＜0.05)。
结论：
临产后子宫平滑肌组织中Calponin-1及pCalponin-1蛋白的表达上调与临产后子宫平滑肌的收缩状态有关,提示临产后子宫平滑肌的收缩可能部分依赖于Calponin-1及pCalponin-1的表达。
目的：探讨Calponin-1蛋白表达抑制对妊娠子宫平滑肌细胞功能的影响,研究其在分娩发动中的分子作用机制。
方法：采用消化酶法对人子宫平滑肌组织进行子宫平滑肌细胞原代培养,并用免疫细胞化学方法进行鉴定。构建腺病毒siRNA-Calponin-1质粒转染子宫平滑肌细胞沉默Calponin-1表达,采用MTT、流式细胞术、Transwell小室的变化、免疫荧光标记和荧光显微镜观察平滑肌细胞骨架蛋白F-actin表达的变化等方法研究Calponin-1调控子宫平滑肌细胞的作用,实验分三组：实验组、空白对照组、空载体组。
结果：
1.原代子宫平滑肌细胞的培养及鉴定
培养的子宫平滑肌细胞呈典型的梭状肌细胞样生长,用平滑肌肌动蛋白抗体,经免疫细胞化学染色显示原代培养的子宫平滑肌细胞胞质呈棕色,证明获得的原代子宫平滑肌细胞符合后续实验的要求。
2. siRNA-Calponin-1腺病毒有效抑制Calponin-1 mRNA和蛋白的表达
采用RT-PCR和Western-blot方法检测pAd-mU6pro-Calponin-l-siRNA转染后子宫平滑肌细胞中Calponin-1 mRNA和蛋白的表达,结果显示：实验组、阴性对照组、空白对照组Calponin-1mRNA的相对表达量分别为0.0463±0.0045、0.2513±0.0552、0.3124±0.0037；实验组、阴性对照组、空白对照组Calponin-1蛋白的相对表达量分别为0.1098±0.0127、0.2758±0.0384、0.3187±0.0425。实验组与阴性对照组、空白对照组比较,差异有统计学意义(p＜0.05),阴性对照组与空白对照组比较,差异无统计学意义(p＞0.05)。
3.体外实验中抑制Calponin-1的表达可以抑制子宫平滑肌细胞的迁移运动而不影响其增殖和凋亡
Transwell侵袭小室测定法结果显示24小时培养后实验组、空载体组,空白对照组穿过Transwell的每低倍视野细胞数分别为64±12、122±14、125±10,实验组与空载体组、空白对照组比较,差异有统计学意义(p<0.05)；空载体组与空白对照组比较,差异无统计学意义(p>0.05)。MTT法检测转染24h、48h、72h后细胞的存活率,结果显示三组细胞存活率两两比较差异无统计学意义(p>0.05)。流式细胞仪检测结果显示,三组细胞凋亡率分别为4.22%、3.02%、4.64%,两两比较差异无统计学意义(p>0.05)。
4.体外实验中抑制Calponin-1的表达可以引起子宫平滑肌细胞骨架蛋白F-actin的改变
实验组子宫平滑肌细胞中F-actin排列紊乱、稀疏。空载体组与空白对照组子宫平滑肌细胞中F-actin微丝明显粗长,大多沿细胞纵轴呈平行排列。实验组、空载体组、空白对照组子宫平滑肌细胞中的F-actin荧光强度分别为832.22±99.02、1123.32±102.35、1089.19±110.53,实验组与空载体组、空白对照组比较差异具有显著性(p<0.01)；而空载体组与空白对照组比较差异无统计学意义(p>0.05)。
结论：
1.构建的Calponin-1siRNA干扰质粒能有效沉默Calponin-1基因表达。
2.体外实验中抑制Calponin-1的表达可以抑制子宫平滑肌细胞的迁移运动而不影响其增殖和凋亡。
3. Calponin-1通过影响子宫平滑肌细胞中骨架蛋白F-actin的改变调节子宫平滑肌的收缩。
目的：探讨硫酸镁及缩宫素调节子宫平滑肌细胞收缩与Calponin-1表达水平的相关性。
方法：用硫酸镁及缩宫素作用于体外培养的子宫平滑肌细胞,然后采用Western-blot及RT-PCR的方法对子宫平滑肌组织中Calponin-1的表达进行检测。
结果：
1.体外硫酸镁对子宫平滑肌细胞Calponin-1表达的影响
(1) RT-PCR结果硫酸镁作用子宫平滑肌细胞30分钟后,阴性对照组、2.0%、4.0%、8.0%、16%浓度硫酸镁组Calponin-lmRNA的相对表达量分别为：0.2476±0.0431、0.2562±0.0382、0.2813±0.0243、0.3011±0.0135、0.3042±0.0119,组间比较差异无统计学意义(p>0.05)。
(2) Western Blot结果硫酸镁作用子宫平滑肌细胞24h后,阴性对照组、2.0%、4.0%、8.0%、16%浓度硫酸镁组Calponin-1蛋白的相对表达量分别为：0.2135±0.0537、0.2572±0.0378、0.2813±0.0351、0.3147±0.0161、0.3185±0.0134,组间比较差异无统计学意义(p>0.05)。
2.体外缩宫素对子宫平滑肌细胞Calponin-1表达的影响
(1)RT-PCR结果缩宫素作用子宫平滑肌细胞30分钟后,阴性对照组、2.0%、4.0%、6.0%、8.0%浓度缩宫素组Calponin-1mRNA的相对表达量分别为：0.1936±0.0283、0.4382±0.0355、0.6232±0.0491、0.3951±0.0762、0.3976±0.0647,各浓度组与阴性对照组比较差异具有统计学意义(p<0.05),其中以4.0%浓度组Calponin-1mRNA表达最为显著。
(2) Western Blot结果缩宫素作用子宫平滑肌细胞24h后,阴性对照组、2.0%、4.0%、6.0%、8.0%浓度缩宫素组Calponin-1蛋白的相对表达量分别为：0.2095±0.0153、0.4831±0.0368、0.6197±0.0425、0.4105±0.0148、0.4112±0.0135,各浓度组与阴性对照组比较差异具有统计学意义(p<0.05),其中以4.0%浓度组的Calponin-1蛋白上调最为显著。我们用4.0%浓度组缩宫素作用子宫平滑肌细胞,在0h、24h、48h、96h不同时间段,用Western Blot检测Calponin-1蛋白表达水平。结果显示,0h、24h、48h、96h时间段Calponin-1蛋白的相对表达量分别为：0.2014±0.0115、0.5097±0.0127、0.3865±0.0454、0.2247±0.0216,结果提示,缩宫素上调Calponin-1蛋白表达与其作用时间具有相关性,以作用24h时Calponin-1蛋白表达上调最为明显。
结论：
1.体外缩宫素可以上调子宫平滑肌细胞Calponin-1的表达,且与其浓度及作用时间具有相关性,但与缩宫素作用浓度及作用时间并无明显正／或负相关。
2.缩宫素通过上调子宫平滑肌细胞Calponin-1的表达而引发宫·缩,导致分娩发动,这可能是缩宫素促进子宫平滑肌细胞收缩的又一新的作用途经。
3.体外硫酸镁不影响子宫平滑肌细胞Calponin-1的表达,我们推测Calponin-1调控子宫平滑肌收缩的作用可能并不依赖于Ca2+通道。
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 situation
Objective: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 cell
Objective: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 oxytocin
Objective: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.
引文
[1]Andres LB. Mechanism of labour-biochemical aspects.Br J Obstet Gynecol,2003,110 (supple20):39-45
[2]Matthias W. Role of cycokins and other inflammatory mediators. Br J Obstet Gynecol,2003,110(supple20):118-123
[3]Andrew MB, Steven T.The role of oxytocin in parturition. Br J Obstet Gynecol, 2003,110(supple20):46-51
[4]Thomas ES, Gray AJ, Robert CB, et al. Progesterone and placental hormone actions on the uterus:insights from domestic animals. Biol Reprod.2004,71:2-10
[5]Yong A, Thomson AJ, Ledingham M, et al. Immunolocalization of proinflammatory cytokins in myometrium, cervix, and fetal membranes during human parturition at term. Biol Reprod,2002,66:445-449
[6]Functional Genomics and Proteomics in term and Preterm Parturition.J Clin Endocrimal Metab,June 2002,87(6):2431-2434
[7]Chan EC, Fraser S, Yeo YS, et al. Human myometrial genes are differentially expressed in labour:a suppression subtractive hybridization study. J Clin Endocrinal metab.2002, 87(6)2435-2441
[8]Cai Z, Chiu JF, He QY. Application of proteomics in the study of tumor metastasis. Genomics Proteomics Bioinformatics.2004,2(3):152-166
[9]Shankar R,Gude N,Cullinane F,et al. An emerging role for comprehensive proteome analysis in human pregnancy research.Reproduction.2005,129(6):685-696
[10]Preterm birth:crisis and opportunity. Lancet,2006,368(9533):339
[11]Slattery MM, Morrison J J. Preterm delivery. Lancet,2002,360(9344):1489-1497
[12]Tsoi E, Fuchs IB, Rane S, et al. Sonographic measurement of cervical length in threatened preterm labor in singleton pregnancies with intact membranes. Ultrasound Obstet Gynecol.2005;25(4):353-356
[13]Shennan A, Jones G, Hawken J, et al. Fetal fibronectin test predicts delivery before 30
weeks of gestation in high risk women, but increases anxiety. BJOG. 2005 Mar; 112(3):293-298
[14]Gonzalez Bosquet E, Ferrer I, Valls C, et al.The value of interleukin-8, interleukin-6 and interleukin-1beta in vaginal wash as predictors of preterm delivery. Gynecol Obstet Invest. 2005; 59(3):175-178
[15]罗雪梅,周昌菊.孕妇血清可溶性细胞间粘附分子-1与亚临床绒毛膜羊膜炎的关系.中国实用妇科与产科杂志,2006,22(2)：112-114
[16]张卫社,周昌菊,吴新华,武招娣.临产前后子宫平滑肌组织热休克蛋白70的表达变化及其意义,中华妇产科杂志,2006,(9)：618-619
[17]马薇,周昌菊.蛋白质组学技术与妊娠相关疾病的研究进展,国外医学妇产科分册,2006,23(3)：153-156
[18]Takahashi K, Hiwada K, Kokubu T.Isolation and characterization of a 34 000-dalton calmodulin and F-actin-binding protein from chicken gizzard smooth muscle. Biochem Biophys Res Commun,1986,141(1):20-26
[19]Kim I,Leinweber BD, Morgalla M, et al. Thin and thick filament regulation of contractility in experimental cerebral vasospasm. Neurol surgery,2000,46(2):440-446
[20]Kolakowski J, Makuch R,Stepkowshi D,et al.Interaction of calponin with actin and its functional implications Biochem J,1995,306:199
[21]Gao J, Hwang JM, Jin JP.Complete nucleotide sequence, structural organization, and an alternatively spliced exon of mouse h1-calponin gene. Biochem Biophys Res Commun. 1996.5; 218(1):292-297
[22]Leinweber B, Tang J X, Stafford W F, Chalovich J M, Calponin interaction with alpha-actinin-actin:Evidence for a structural role for calponin. Biophys J,1999, 77:3208-3217
[23]岳作军,宋俊芳,黄朝峰等,绵羊钙调蛋白调节基因Calponin h 1的分子克隆.遗传.2005,27(3)：357-362
[24]张卫社,梁清华,谢庆生,等.应用cDNA微阵列筛选子宫平滑肌细胞收缩相关药物靶点.中南大学学报(医学版),2007,32(4)：579-583
[25]张卫社,吴新华,伍招娣,等.自然临产及缩宫素诱导临产子宫体部平滑肌差异基因表达谱分析.现代妇产科进展,2007,16(3)：206-209
[26]ItohT, Suzuki S, Suzuki A, Nakamura F, Naka M, Tanaka T. Effects of exogenously applied calponin on Ca2+ regulated force in skinned smooth muscle of the rabbit mesenteric artery. Pflugers Arch Eur J Physiol,1994,427:301-308
[27]Parker C A, Takahashi K, Tang J X, Tao T, Morgan K G. Cytoskeietal targeting of calponin in differentiated, contractile smooth muscle cells of the ferret. The Journal of Physiology,1998,508(1):187-198
[28]Bogatcheva N V, Gusev N B, Interaction of smooth muscle calponin with phospholipids. FEBB Letters,1955,371:123-126
[29]Wang P, Gusev N B, Interaction of smooth muscle calponin and desmin. FEBS Letters, 1996,392:255-258
[30]Fujii T, Koizumi Y, Identification of the binding regions of basic calponin on alpha and beta tubulins. J Biochem (Tokyo),1999,125:869-875
[31]Fattoum A, Roustan C, Smyczynski C, Der Terrossian E, Kassab R. Mapping the microtubule binding regions of calponin.Biochemistry,2003,42,1274-1282
[32]Francisco.Wu KC, Jin JP. Calponin in non-muscle cells.Cell Biochem Biophys.2008; 52 (3):139-148
[33]李慧,张农.调宁蛋白的研究进展.国外医学生理、病理科学与临床分册,2003,23(3)：230-233
[34]Kathleen GM, Samudra SG Mechanism and treatment progress of pulmonary artery hypertension. Chin J Med,2004,39(5):5-7
[35]卢中举,唐大椿,向继洲.平滑肌收缩调节的信号转导.生理科学进展,1977,28(4)：337-340
[36]Winder SJ, Walsh MP. Smooth muscle calponin. Inhibition of actomyosin Mg-ATPase and regulation by phosphorylation. Biol Chem,1990,265:10148-10115
[37]Winder SJ, Walsh MP. Calponin. Curr Topics Cell Reg,1993,34:155
[38]Winder SJ, Walsh MP. Calponin:thin filament-linked regulation of smooth muscle
contraction. Cell Signal,1993,5:677-697
[39]Winder SJ, Allen BG, Fraser DE, et al. Calponin phosphorylation in vitro and in intact muscle. Biochem,1993,296:827
[40]Abe M, Takahashi K, Hiwada K, et al. Light chain phosphorylation and tension in smooth muscle. Biochem,1990,108:835
[41]Yang JX, Feng XH, Zhang Y, et al. The influence of trace amount of calponin on the smooth muscle myosins in different states. Biochem Biophys Res Commun,2004, 318(4):904-910
[42]张卓然.培养细胞学与细胞培养技术.上海：上海科技出版社,2004：68-72
[43]蔡文琴.现代实用细胞与分子生物学实验技术.北京：人民军医出版社,2003：5-7
[44]Michal J. Rossi, Nasser Chegini, Byron Jmasterson. Presence of epidermal growth factor, platelet-derived growth factor, and their receptors in human myometrial tissure and smooth muscle cell:their action in smooth muscle cell in vitro[J]. Endocrinology,1992,130:1716-1727
[45]Casey ML, Paule. Mac Donald, Mitchell MD. Maintenance and characterization of human myometrial smooth muscle cells in monolayer culture. In vitro,1983,20: 396-402
[46]Manju Monga et al. Oxytocin-stimulated responses in a pregnant human immortalized myometrial cell line. Biology of Reproduction,1996,55:427-432
[47]Rossi MJ, Nasser C, Byron J. Presence of epidermal growth factor, platelet-derived growth factor, and their receptors in human myometrial tissue and smooth mascle cells. Endocrinology,1992,130:1716-1727
[48]Nowak RA, Rein MS, Heffner LJ, et al. Production of prolactin smooth muscle cells culture from human uterine fibroid tumors[J].1993,76(5):1308-1313
[49]Lee BS, Margolin SB, Nowar RA. Pirfenidone:a novel pharmacological agent that inhibits leiomyoma cell proliferation and collagen production. J Clin Endocrin Metab, 1998,1183:219-223
[50]Nesser CQTang XM, Ma CF. Regulation of transforming growth factor-expression by grannlocyte macrophage-colony-stimulating factor in leiomyoma and moymetrial and myometrial smooth mascle cells. J Clin Endocrin Metab,1999,184:4138-4143
[51]司徒镇强,吴军.细胞培养.西安：世界图书出版公司,1996：114-178
[52]李珍,赵志亮,郭述真.人子宫平滑肌细胞的原代培养.山西医药杂志,2007,36(12)：1101-1102
[53]戴凌,游兴姬,沙金燕等.人妊娠子宫平滑肌细胞的组织块培养及鉴定.医学研究杂志,2007,36(5)：51-53
[54]张荣荣,杨祖菁.人子宫平滑肌细胞的原代培养及应用.中国医师杂志,2004,11(1)7-11
[55]McNeish IA, Bell SJ, Lemoine NR. Gene therapy progress and prospects:cancer gene therapy using tumour suppressor genes [J]. Gene Ther,2004,11 (6):497-503.
[56]Arthur JF, Butterfield LH, Roth MD, et al. A comparison of gene transfer methods in human dendritic cells. Cancer Gene Ther,1997,4:17-25
[57]Van Tendeloo VF, Snoech HW, Lardon F, et al. Nonviral transfection of distinct types of human dendritic cells:highefficiency gene transfer by electroporation into hematopoietic progenitorbut not moncyte-derived dendritic cells. Gene Ther,1998,5: 700-707
[58]Ardeshna KM, Pizzey AR,Thomas NS, et al. Monocyte-derived dendritic cells do not proliferate and are not susceptible to retroviral transduction.Br J Haematol.2000,108: 817-824
[59]赵峰,周清华,陆燕蓉等.带突变基因的重组腺病毒转染树突状细胞体外诱导的抗肿瘤免疫.中华医学杂志,2004,84(17)1478-1480
[60]Sugenoya Y, Yoshimura A, Yamamura H, et al. Smooth muscle Calponin in Mesangial Cells:Regulation of expression and a role in suppressing Glomerulon ephritis. J Am Soc Nephol.2002,13:322-331
[61]Horiuchi A, Nikaido T, Taniguchi S, et al. Possible role of calponin h1 as a tumor suppressor in human uterine leiomyosarcoma. J Natl Cancer Inst,1999,91(9):790-796
[62]Gimona M, Djinovic-Carugo K, Kranewitter WJ, Winder SJ. Functional plasticity of CH domains. FEBS Lett.2002 Feb 20;513(1):98-106
[63]Danninger C, Gimona M. Live dynamics of GFP-calponin:isoform-specific modulation of the actin cytoskeleton and autoregulation by C-terminal sequences. J Cell Sci.2000 Nov;113 Pt 21:3725-3736
[64]Gimona M, Kaverinal I, Resch GP, Vignal E, Burgstaller G. Calponin repeats regulate actin filament stability and formation of podosomes in smooth muscle cells. Mol Biol Cell.2003 Jun;14(6):2482-2491
[65]Leinweber B, Tang JX, Stafford WF, Chalovich JM. Calponin interaction with alpha-actinin-actin:evidence for a structural role for calponin. Biophys J.1999 Dec;77(6):3208-3217
[66]Bartegi A, Roustan C, Kassab R, Fattoum A. Fluorescence studies of the carboxyl-terminal domain of smooth muscle calponin effects of F-actin and salts. Eur J Biochem.1999 Jun;262(2):335-341
[67]Cornwell TL,Li J,Sellak H,et al. Reorganization of myofilament proteins and decreased cGMP-dependent protein kinase in the human uterus during pregnancy[J].Clin Endocrinol Metab,2001,86(8):3981-3988
[68]Ibrahim B, Leveteau J, Guiet-Bara A, Influence of magnesium salts on the membrane potential of human endothelial placental vessel cells. Magnes Res.1995; 8(3):233-236
[69]Ibrahim B, Guiet-Bara A, Leveteau J,Membrane potential of smooth muscle cells of human placental chorionic vessels. Comparative effects of MgCl2 and MgSO4. Magnes Res.1995,8(2):127-135
[70]Fomin VP, Gibbs SG, Vanam R,Effect of magnesium sulfate on contractile force and intracellular calcium concentration in pregnant human myometrium. Am J Obstet Gynecol.2006; 194(5):1384-1390
[71]Shetty SS, Weiss GB.Effects of pH and anion substitution on magnesium accumulation in rabbit aortic smooth muscle. Blood Vessels.1989; 26(2):65-76
[72]Fetalvero KM, Zhang P, Shyu M, Young BT, et al. Prostacyclin primes pregnant human myometrium for an enhanced contractile response in parturition. Clin Invest.2008; 118(12):3966-3979
[73]Wu X, Shen H, Yu L, Peng M, Corticotropin-releasing hormone activates connexin 43 via activator protein-1 transcription factor in human myometrial smooth muscle cells. Am J Physiol Endocrinol Metab.2007; 293 (6):E1789-1794
[1]卢中举,唐大椿,向继洲.平滑肌收缩调节的信号转导[J].生理科学进展,1977,28(4)：337-340
[2]Winder SJ, Walsh MP. Smooth muscle calponin. Inhibition of actomyosin Mg-ATPase and regulation by phosphorylation. Biol Chem,1990,265:10148-10115
[3]Winder SJ,Walsh MP. Calponin:thin filament-linked regulation of smooth muscle contraction. Cell Signal,1993,5:677-697
[4]Takahashi K, Hiwada K, Kokubu T. Isolation and characterization of a 34 000-dalton calmodulin and F-actin-binding protein from chicken gizzard smooth muscle. Biochem Biophys Res Commun,1986,141(1):20-26
[5]Jin JP,Wu D,Gao JM,et al. Expression and purification of the h1 and h2 isoforms of calponin [J]. Protein Expression and purification,2003,31:231-239.chinese
[6]Matthew JD,Khromov AS,Mcduffie MJ,et al.Contractile properties and proteins of smooth muscles of a calponin knockout mouse [J]. J Physiol,2000,529:811-824
[7]Kim I,Leinweber BD, Morgalla M, et al. Thin and thick filament regulation of contractility in experimental cerebral vasospasm. Neurol surgery,2000,46(2):440-446
[8]Takahashi K, Nadal-Ginard B. Molecular cloning and sequence analysis of smooth muscle calponin. J Biol Chem,1991,266:13248
[9]Abe M, Takahashi K, Hiwada K, et al. Hascular smooth muscle calponin:A novel tropon in T-like protein. J Biochem (Tokyo),1990,107:507
[10]Menice CB, Hulvershorn J, Adam LP, et al. Calponin and mitogenactivated protein kinase signaling in differentiated vascular smooth muscle. J Biol Chem, 1997,272:25157-25161
[11]Kolakowski J, Makuch R,Stepkowshi D,et al.Interaction of calponin with actin and its functional implications Biochem J,1995,306:199
[12]Kathleen GM, Samudra SG. Mechanism and treatment progress of pulmonary artery hypertension. Chin J Med,2004,39(5):5-7
[13]Takeoka M, Ehara T, Sagara J, et al. Calponin h1 induced a flattened morphology and suppressed the growth of fibrosarcoma HT1080 cells. Eur J Cancer,2002,38 (3):436-442
[14]Dulin ON, Orlov SN, Kitchen CM, et al. G-protein-coupled-receptor activation of the smooth muscle calponin gene. Bio chem J,2001,357:587-592
[15]Winder SJ, Allen BG, Fraser DE, et al. Calponin phosphorylation in vitro and in intact muscle. Biochem,1993,296:827
[16]Abe M, Takahashi K, Hiwada K, et al. Light chain phosphorylation and tension in smooth muscle. Biochem,1990,108:835
[17]Mosunjac MB, Lewis MM, Lawson D, et al. Use of a novel marker, calponin,
for myoepithelial cells in fine-needle aspirates of papillary breast lesions. Diagn Cytopathol,2000,23 (3):151-155
[18]Winder SJ, Walsh MP. Calponin. Curr Topics Cell Reg,1993,34:155
[19]Winder SJ, Walsh MP. Calponin:thin filament-linked regulation of smooth muscle contraction. Cell Signal,1993,5:677-697
[20]Li H, Jiang T, Yang J, et al. Relationship between calponin h1 and TGFB1 in rat mesangial cells. Chin Nephrol,2005,21(4):213-218
[2l]李慧,张农.调宁蛋白的研究进展.国外医学.生理、病理科学与临床分册,2003,23(3)：230-233
[22]Dulin ON, Orlov SN, Kitchen CM, et al. G-protein-coupled-receptor activation of the smooth muscle calponin gene. Bio chem J,2001,357:587-592
[23]Gimona M et al. FEBS Lett,2002,513:98
[24]Takeoka M, Ehara T, Sagara J, et al. Calponin h1 induced a flattened morphology and suppressed the growth of fibrosarcoma HT1080 cells. Eur J Cancer,2002,38 (3):436-442
[25]Sugenoya Y, Yoshimura A, Yamamura H, et al. Smooth muscle Calponin in Mesangial Cells:Regulation of expression and a role in suppressing Glomerulon ephritis. J Am Soc Nephol.2002,13:322-331
[26]Horiuchi A, Nikaido T, Taniguchi S, et al. Possible role of calponin h1 as a tumor suppressor in human uterine leiomyosarcoma. J Natl Cancer Inst,1999,91(9):790-796
[27]Takahashi K, Hiwada K, Kokubo T et al. Isolation and characterization of a 34000 dalton calmodulin and F-actin-binding protein from chicken gizzardsmooth muscle. Biochem Biophys Res Commun,1986,141(1):20-26
[28]Negoro N, Fukui R, Tsuchikane E et al. Down expression of calponin in smooth muscle of coronary artery lesion identifies a group of lesion at high risk for restenosis after athetectomy.Circulation,1994,90:141-142
[29]Takahashi K,Fukui R,Kato O et al.Percutaneous transluminal transfer of the human Calponin gene for suppression of intimal hyperplasia following arterial balloon injury:A model for successful gene therapy for restenosis. Circulation,1993, 88:651-657
[30]苗志林,韩雅玲,王守力等.Calponin对培养的自发性高血压大鼠血管平滑肌细胞增殖的作用.解放军医学杂志,1998,23(6)：425-427
[31]陈云祥,吴幼章.Calponin表达下降与脑血管痉挛关系的实验研究[J].镇江医学院学报,2000,10(4)：662-663
[32]汪斌,丁佑铭,王卫星等.H1型调宁蛋白mRNA在胆道中的表达与妊娠豚鼠胆囊结石形成关系的实验研究.中国现代医学杂志,2006,16(20)：3061-3064,3068
[33]王新,吴开春,张家友等.类肌钙蛋白和钙调蛋白结合蛋白在胃肠运动及其病理适应性调节中的作用.中华内科杂志,2001,40(7)：459-462
[34]耿建祥,徐文华,黄书亮等p63、Calponin和SMM-HC联合标记在乳腺增生性病变中表达的比较.J Diag Pathol,2006,13(3):238
[35]Cornwell TL,Li J,Sellak H,et al. Reorganization of myofilament proteins and decreased cGMP-dependent protein kinase in the human uterus during pregnancy[J].Clin Endocrinol Metab,2001,86(8):3981-3988
[36]Gao J,Hwang JM,Jin J P.Complete nucleotide sequence,structural organization,and an alternatively spliced exon of mouse h1-calponin gene.Biochem Biophys Res Commun,1996,218(1):292-297
[37]曹圣,唐大椿.调宁蛋白的研究近况[J].微循环杂志,1997,7(1)：46-47,55
[38]卢义钦.平滑肌非钙依赖性收缩的生化机理.生命科学研究,1998,2(3)：157-162
[39]岳作军,宋俊芳,黄朝峰等.绵羊钙调蛋白调节基因Calponin h1的分子克隆[J].遗传,2005,27(3)：357-362
[40]张卫社,梁清华,谢庆生,等.应用cDNA微阵列筛选子宫平滑肌细胞收缩相关药物靶点.中南大学学报(医学版),2007,32(4)：579-583
[41]张卫社,吴新华,伍招娣等.自然临产及缩宫素诱导临产子宫体部平滑肌差异基因表达谱分析.现代妇产科进展,2007,16(3)：206-209
[42]马薇,周昌菊.蛋白质组学技术与妊娠相关疾病的研究进展.国外医学妇产科分册,2006,23(3)：153-156
[2]Matthias W. Role of cycokins and other inflammatory mediators. Br J Obstet Gynecol,2003,110(supple20):118-123
[3]Andrew MB, Steven T.The role of oxytocin in parturition. Br J Obstet Gynecol, 2003,110(supple20):46-51
[4]Thomas ES, Gray AJ, Robert CB, et al. Progesterone and placental hormone actions on the uterus:insights from domestic animals. Biol Reprod.2004,71:2-10
[5]Yong A, Thomson AJ, Ledingham M, et al. Immunolocalization of proinflammatory cytokins in myometrium, cervix, and fetal membranes during human parturition at term. Biol Reprod,2002,66:445-449
[6]Functional Genomics and Proteomics in term and Preterm Parturition.J Clin Endocrimal Metab,June 2002,87(6):2431-2434
[7]Chan EC, Fraser S, Yeo YS, et al. Human myometrial genes are differentially expressed in labour:a suppression subtractive hybridization study. J Clin Endocrinal metab.2002, 87(6)2435-2441
[8]Cai Z, Chiu JF, He QY. Application of proteomics in the study of tumor metastasis. Genomics Proteomics Bioinformatics.2004,2(3):152-166
[9]Shankar R,Gude N,Cullinane F,et al. An emerging role for comprehensive proteome analysis in human pregnancy research.Reproduction.2005,129(6):685-696
[10]Preterm birth:crisis and opportunity. Lancet,2006,368(9533):339
[11]Slattery MM, Morrison J J. Preterm delivery. Lancet,2002,360(9344):1489-1497
[12]Tsoi E, Fuchs IB, Rane S, et al. Sonographic measurement of cervical length in threatened preterm labor in singleton pregnancies with intact membranes. Ultrasound Obstet Gynecol.2005;25(4):353-356
[13]Shennan A, Jones G, Hawken J, et al. Fetal fibronectin test predicts delivery before 30
weeks of gestation in high risk women, but increases anxiety. BJOG. 2005 Mar; 112(3):293-298
[14]Gonzalez Bosquet E, Ferrer I, Valls C, et al.The value of interleukin-8, interleukin-6 and interleukin-1beta in vaginal wash as predictors of preterm delivery. Gynecol Obstet Invest. 2005; 59(3):175-178
[15]罗雪梅,周昌菊.孕妇血清可溶性细胞间粘附分子-1与亚临床绒毛膜羊膜炎的关系.中国实用妇科与产科杂志,2006,22(2)：112-114
[16]张卫社,周昌菊,吴新华,武招娣.临产前后子宫平滑肌组织热休克蛋白70的表达变化及其意义,中华妇产科杂志,2006,(9)：618-619
[17]马薇,周昌菊.蛋白质组学技术与妊娠相关疾病的研究进展,国外医学妇产科分册,2006,23(3)：153-156
[18]Takahashi K, Hiwada K, Kokubu T.Isolation and characterization of a 34 000-dalton calmodulin and F-actin-binding protein from chicken gizzard smooth muscle. Biochem Biophys Res Commun,1986,141(1):20-26
[19]Kim I,Leinweber BD, Morgalla M, et al. Thin and thick filament regulation of contractility in experimental cerebral vasospasm. Neurol surgery,2000,46(2):440-446
[20]Kolakowski J, Makuch R,Stepkowshi D,et al.Interaction of calponin with actin and its functional implications Biochem J,1995,306:199
[21]Gao J, Hwang JM, Jin JP.Complete nucleotide sequence, structural organization, and an alternatively spliced exon of mouse h1-calponin gene. Biochem Biophys Res Commun. 1996.5; 218(1):292-297
[22]Leinweber B, Tang J X, Stafford W F, Chalovich J M, Calponin interaction with alpha-actinin-actin:Evidence for a structural role for calponin. Biophys J,1999, 77:3208-3217
[23]岳作军,宋俊芳,黄朝峰等,绵羊钙调蛋白调节基因Calponin h 1的分子克隆.遗传.2005,27(3)：357-362
[24]张卫社,梁清华,谢庆生,等.应用cDNA微阵列筛选子宫平滑肌细胞收缩相关药物靶点.中南大学学报(医学版),2007,32(4)：579-583
[25]张卫社,吴新华,伍招娣,等.自然临产及缩宫素诱导临产子宫体部平滑肌差异基因表达谱分析.现代妇产科进展,2007,16(3)：206-209
[26]ItohT, Suzuki S, Suzuki A, Nakamura F, Naka M, Tanaka T. Effects of exogenously applied calponin on Ca2+ regulated force in skinned smooth muscle of the rabbit mesenteric artery. Pflugers Arch Eur J Physiol,1994,427:301-308
[27]Parker C A, Takahashi K, Tang J X, Tao T, Morgan K G. Cytoskeietal targeting of calponin in differentiated, contractile smooth muscle cells of the ferret. The Journal of Physiology,1998,508(1):187-198
[28]Bogatcheva N V, Gusev N B, Interaction of smooth muscle calponin with phospholipids. FEBB Letters,1955,371:123-126
[29]Wang P, Gusev N B, Interaction of smooth muscle calponin and desmin. FEBS Letters, 1996,392:255-258
[30]Fujii T, Koizumi Y, Identification of the binding regions of basic calponin on alpha and beta tubulins. J Biochem (Tokyo),1999,125:869-875
[31]Fattoum A, Roustan C, Smyczynski C, Der Terrossian E, Kassab R. Mapping the microtubule binding regions of calponin.Biochemistry,2003,42,1274-1282
[32]Francisco.Wu KC, Jin JP. Calponin in non-muscle cells.Cell Biochem Biophys.2008; 52 (3):139-148
[33]李慧,张农.调宁蛋白的研究进展.国外医学生理、病理科学与临床分册,2003,23(3)：230-233
[34]Kathleen GM, Samudra SG Mechanism and treatment progress of pulmonary artery hypertension. Chin J Med,2004,39(5):5-7
[35]卢中举,唐大椿,向继洲.平滑肌收缩调节的信号转导.生理科学进展,1977,28(4)：337-340
[36]Winder SJ, Walsh MP. Smooth muscle calponin. Inhibition of actomyosin Mg-ATPase and regulation by phosphorylation. Biol Chem,1990,265:10148-10115
[37]Winder SJ, Walsh MP. Calponin. Curr Topics Cell Reg,1993,34:155
[38]Winder SJ, Walsh MP. Calponin:thin filament-linked regulation of smooth muscle
contraction. Cell Signal,1993,5:677-697
[39]Winder SJ, Allen BG, Fraser DE, et al. Calponin phosphorylation in vitro and in intact muscle. Biochem,1993,296:827
[40]Abe M, Takahashi K, Hiwada K, et al. Light chain phosphorylation and tension in smooth muscle. Biochem,1990,108:835
[41]Yang JX, Feng XH, Zhang Y, et al. The influence of trace amount of calponin on the smooth muscle myosins in different states. Biochem Biophys Res Commun,2004, 318(4):904-910
[42]张卓然.培养细胞学与细胞培养技术.上海：上海科技出版社,2004：68-72
[43]蔡文琴.现代实用细胞与分子生物学实验技术.北京：人民军医出版社,2003：5-7
[44]Michal J. Rossi, Nasser Chegini, Byron Jmasterson. Presence of epidermal growth factor, platelet-derived growth factor, and their receptors in human myometrial tissure and smooth muscle cell:their action in smooth muscle cell in vitro[J]. Endocrinology,1992,130:1716-1727
[45]Casey ML, Paule. Mac Donald, Mitchell MD. Maintenance and characterization of human myometrial smooth muscle cells in monolayer culture. In vitro,1983,20: 396-402
[46]Manju Monga et al. Oxytocin-stimulated responses in a pregnant human immortalized myometrial cell line. Biology of Reproduction,1996,55:427-432
[47]Rossi MJ, Nasser C, Byron J. Presence of epidermal growth factor, platelet-derived growth factor, and their receptors in human myometrial tissue and smooth mascle cells. Endocrinology,1992,130:1716-1727
[48]Nowak RA, Rein MS, Heffner LJ, et al. Production of prolactin smooth muscle cells culture from human uterine fibroid tumors[J].1993,76(5):1308-1313
[49]Lee BS, Margolin SB, Nowar RA. Pirfenidone:a novel pharmacological agent that inhibits leiomyoma cell proliferation and collagen production. J Clin Endocrin Metab, 1998,1183:219-223
[50]Nesser CQTang XM, Ma CF. Regulation of transforming growth factor-expression by grannlocyte macrophage-colony-stimulating factor in leiomyoma and moymetrial and myometrial smooth mascle cells. J Clin Endocrin Metab,1999,184:4138-4143
[51]司徒镇强,吴军.细胞培养.西安：世界图书出版公司,1996：114-178
[52]李珍,赵志亮,郭述真.人子宫平滑肌细胞的原代培养.山西医药杂志,2007,36(12)：1101-1102
[53]戴凌,游兴姬,沙金燕等.人妊娠子宫平滑肌细胞的组织块培养及鉴定.医学研究杂志,2007,36(5)：51-53
[54]张荣荣,杨祖菁.人子宫平滑肌细胞的原代培养及应用.中国医师杂志,2004,11(1)7-11
[55]McNeish IA, Bell SJ, Lemoine NR. Gene therapy progress and prospects:cancer gene therapy using tumour suppressor genes [J]. Gene Ther,2004,11 (6):497-503.
[56]Arthur JF, Butterfield LH, Roth MD, et al. A comparison of gene transfer methods in human dendritic cells. Cancer Gene Ther,1997,4:17-25
[57]Van Tendeloo VF, Snoech HW, Lardon F, et al. Nonviral transfection of distinct types of human dendritic cells:highefficiency gene transfer by electroporation into hematopoietic progenitorbut not moncyte-derived dendritic cells. Gene Ther,1998,5: 700-707
[58]Ardeshna KM, Pizzey AR,Thomas NS, et al. Monocyte-derived dendritic cells do not proliferate and are not susceptible to retroviral transduction.Br J Haematol.2000,108: 817-824
[59]赵峰,周清华,陆燕蓉等.带突变基因的重组腺病毒转染树突状细胞体外诱导的抗肿瘤免疫.中华医学杂志,2004,84(17)1478-1480
[60]Sugenoya Y, Yoshimura A, Yamamura H, et al. Smooth muscle Calponin in Mesangial Cells:Regulation of expression and a role in suppressing Glomerulon ephritis. J Am Soc Nephol.2002,13:322-331
[61]Horiuchi A, Nikaido T, Taniguchi S, et al. Possible role of calponin h1 as a tumor suppressor in human uterine leiomyosarcoma. J Natl Cancer Inst,1999,91(9):790-796
[62]Gimona M, Djinovic-Carugo K, Kranewitter WJ, Winder SJ. Functional plasticity of CH domains. FEBS Lett.2002 Feb 20;513(1):98-106
[63]Danninger C, Gimona M. Live dynamics of GFP-calponin:isoform-specific modulation of the actin cytoskeleton and autoregulation by C-terminal sequences. J Cell Sci.2000 Nov;113 Pt 21:3725-3736
[64]Gimona M, Kaverinal I, Resch GP, Vignal E, Burgstaller G. Calponin repeats regulate actin filament stability and formation of podosomes in smooth muscle cells. Mol Biol Cell.2003 Jun;14(6):2482-2491
[65]Leinweber B, Tang JX, Stafford WF, Chalovich JM. Calponin interaction with alpha-actinin-actin:evidence for a structural role for calponin. Biophys J.1999 Dec;77(6):3208-3217
[66]Bartegi A, Roustan C, Kassab R, Fattoum A. Fluorescence studies of the carboxyl-terminal domain of smooth muscle calponin effects of F-actin and salts. Eur J Biochem.1999 Jun;262(2):335-341
[67]Cornwell TL,Li J,Sellak H,et al. Reorganization of myofilament proteins and decreased cGMP-dependent protein kinase in the human uterus during pregnancy[J].Clin Endocrinol Metab,2001,86(8):3981-3988
[68]Ibrahim B, Leveteau J, Guiet-Bara A, Influence of magnesium salts on the membrane potential of human endothelial placental vessel cells. Magnes Res.1995; 8(3):233-236
[69]Ibrahim B, Guiet-Bara A, Leveteau J,Membrane potential of smooth muscle cells of human placental chorionic vessels. Comparative effects of MgCl2 and MgSO4. Magnes Res.1995,8(2):127-135
[70]Fomin VP, Gibbs SG, Vanam R,Effect of magnesium sulfate on contractile force and intracellular calcium concentration in pregnant human myometrium. Am J Obstet Gynecol.2006; 194(5):1384-1390
[71]Shetty SS, Weiss GB.Effects of pH and anion substitution on magnesium accumulation in rabbit aortic smooth muscle. Blood Vessels.1989; 26(2):65-76
[72]Fetalvero KM, Zhang P, Shyu M, Young BT, et al. Prostacyclin primes pregnant human myometrium for an enhanced contractile response in parturition. Clin Invest.2008; 118(12):3966-3979
[73]Wu X, Shen H, Yu L, Peng M, Corticotropin-releasing hormone activates connexin 43 via activator protein-1 transcription factor in human myometrial smooth muscle cells. Am J Physiol Endocrinol Metab.2007; 293 (6):E1789-1794
[1]卢中举,唐大椿,向继洲.平滑肌收缩调节的信号转导[J].生理科学进展,1977,28(4)：337-340
[2]Winder SJ, Walsh MP. Smooth muscle calponin. Inhibition of actomyosin Mg-ATPase and regulation by phosphorylation. Biol Chem,1990,265:10148-10115
[3]Winder SJ,Walsh MP. Calponin:thin filament-linked regulation of smooth muscle contraction. Cell Signal,1993,5:677-697
[4]Takahashi K, Hiwada K, Kokubu T. Isolation and characterization of a 34 000-dalton calmodulin and F-actin-binding protein from chicken gizzard smooth muscle. Biochem Biophys Res Commun,1986,141(1):20-26
[5]Jin JP,Wu D,Gao JM,et al. Expression and purification of the h1 and h2 isoforms of calponin [J]. Protein Expression and purification,2003,31:231-239.chinese
[6]Matthew JD,Khromov AS,Mcduffie MJ,et al.Contractile properties and proteins of smooth muscles of a calponin knockout mouse [J]. J Physiol,2000,529:811-824
[7]Kim I,Leinweber BD, Morgalla M, et al. Thin and thick filament regulation of contractility in experimental cerebral vasospasm. Neurol surgery,2000,46(2):440-446
[8]Takahashi K, Nadal-Ginard B. Molecular cloning and sequence analysis of smooth muscle calponin. J Biol Chem,1991,266:13248
[9]Abe M, Takahashi K, Hiwada K, et al. Hascular smooth muscle calponin:A novel tropon in T-like protein. J Biochem (Tokyo),1990,107:507
[10]Menice CB, Hulvershorn J, Adam LP, et al. Calponin and mitogenactivated protein kinase signaling in differentiated vascular smooth muscle. J Biol Chem, 1997,272:25157-25161
[11]Kolakowski J, Makuch R,Stepkowshi D,et al.Interaction of calponin with actin and its functional implications Biochem J,1995,306:199
[12]Kathleen GM, Samudra SG. Mechanism and treatment progress of pulmonary artery hypertension. Chin J Med,2004,39(5):5-7
[13]Takeoka M, Ehara T, Sagara J, et al. Calponin h1 induced a flattened morphology and suppressed the growth of fibrosarcoma HT1080 cells. Eur J Cancer,2002,38 (3):436-442
[14]Dulin ON, Orlov SN, Kitchen CM, et al. G-protein-coupled-receptor activation of the smooth muscle calponin gene. Bio chem J,2001,357:587-592
[15]Winder SJ, Allen BG, Fraser DE, et al. Calponin phosphorylation in vitro and in intact muscle. Biochem,1993,296:827
[16]Abe M, Takahashi K, Hiwada K, et al. Light chain phosphorylation and tension in smooth muscle. Biochem,1990,108:835
[17]Mosunjac MB, Lewis MM, Lawson D, et al. Use of a novel marker, calponin,
for myoepithelial cells in fine-needle aspirates of papillary breast lesions. Diagn Cytopathol,2000,23 (3):151-155
[18]Winder SJ, Walsh MP. Calponin. Curr Topics Cell Reg,1993,34:155
[19]Winder SJ, Walsh MP. Calponin:thin filament-linked regulation of smooth muscle contraction. Cell Signal,1993,5:677-697
[20]Li H, Jiang T, Yang J, et al. Relationship between calponin h1 and TGFB1 in rat mesangial cells. Chin Nephrol,2005,21(4):213-218
[2l]李慧,张农.调宁蛋白的研究进展.国外医学.生理、病理科学与临床分册,2003,23(3)：230-233
[22]Dulin ON, Orlov SN, Kitchen CM, et al. G-protein-coupled-receptor activation of the smooth muscle calponin gene. Bio chem J,2001,357:587-592
[23]Gimona M et al. FEBS Lett,2002,513:98
[24]Takeoka M, Ehara T, Sagara J, et al. Calponin h1 induced a flattened morphology and suppressed the growth of fibrosarcoma HT1080 cells. Eur J Cancer,2002,38 (3):436-442
[25]Sugenoya Y, Yoshimura A, Yamamura H, et al. Smooth muscle Calponin in Mesangial Cells:Regulation of expression and a role in suppressing Glomerulon ephritis. J Am Soc Nephol.2002,13:322-331
[26]Horiuchi A, Nikaido T, Taniguchi S, et al. Possible role of calponin h1 as a tumor suppressor in human uterine leiomyosarcoma. J Natl Cancer Inst,1999,91(9):790-796
[27]Takahashi K, Hiwada K, Kokubo T et al. Isolation and characterization of a 34000 dalton calmodulin and F-actin-binding protein from chicken gizzardsmooth muscle. Biochem Biophys Res Commun,1986,141(1):20-26
[28]Negoro N, Fukui R, Tsuchikane E et al. Down expression of calponin in smooth muscle of coronary artery lesion identifies a group of lesion at high risk for restenosis after athetectomy.Circulation,1994,90:141-142
[29]Takahashi K,Fukui R,Kato O et al.Percutaneous transluminal transfer of the human Calponin gene for suppression of intimal hyperplasia following arterial balloon injury:A model for successful gene therapy for restenosis. Circulation,1993, 88:651-657
[30]苗志林,韩雅玲,王守力等.Calponin对培养的自发性高血压大鼠血管平滑肌细胞增殖的作用.解放军医学杂志,1998,23(6)：425-427
[31]陈云祥,吴幼章.Calponin表达下降与脑血管痉挛关系的实验研究[J].镇江医学院学报,2000,10(4)：662-663
[32]汪斌,丁佑铭,王卫星等.H1型调宁蛋白mRNA在胆道中的表达与妊娠豚鼠胆囊结石形成关系的实验研究.中国现代医学杂志,2006,16(20)：3061-3064,3068
[33]王新,吴开春,张家友等.类肌钙蛋白和钙调蛋白结合蛋白在胃肠运动及其病理适应性调节中的作用.中华内科杂志,2001,40(7)：459-462
[34]耿建祥,徐文华,黄书亮等p63、Calponin和SMM-HC联合标记在乳腺增生性病变中表达的比较.J Diag Pathol,2006,13(3):238
[35]Cornwell TL,Li J,Sellak H,et al. Reorganization of myofilament proteins and decreased cGMP-dependent protein kinase in the human uterus during pregnancy[J].Clin Endocrinol Metab,2001,86(8):3981-3988
[36]Gao J,Hwang JM,Jin J P.Complete nucleotide sequence,structural organization,and an alternatively spliced exon of mouse h1-calponin gene.Biochem Biophys Res Commun,1996,218(1):292-297
[37]曹圣,唐大椿.调宁蛋白的研究近况[J].微循环杂志,1997,7(1)：46-47,55
[38]卢义钦.平滑肌非钙依赖性收缩的生化机理.生命科学研究,1998,2(3)：157-162
[39]岳作军,宋俊芳,黄朝峰等.绵羊钙调蛋白调节基因Calponin h1的分子克隆[J].遗传,2005,27(3)：357-362
[40]张卫社,梁清华,谢庆生,等.应用cDNA微阵列筛选子宫平滑肌细胞收缩相关药物靶点.中南大学学报(医学版),2007,32(4)：579-583
[41]张卫社,吴新华,伍招娣等.自然临产及缩宫素诱导临产子宫体部平滑肌差异基因表达谱分析.现代妇产科进展,2007,16(3)：206-209
[42]马薇,周昌菊.蛋白质组学技术与妊娠相关疾病的研究进展.国外医学妇产科分册,2006,23(3)：153-156