【作者】 费奎琳；

【导师】 张卫社；

【作者基本信息】 中南大学 ， 妇产科学， 2010， 硕士

【摘要】 目的：通过特异性的siRNA分级阻断NMBR和NF-κB的表达,了解其对小鼠子宫平滑肌细胞中IL-6表达的影响。方法：1.采用产时或产后2小时之内的小鼠子宫平滑肌细胞进行原代培养及鉴定。确定NMB浓度梯度为：10-10M、10-8M、10-6M、10-4M(简称为10-10——10-4M),实验分为以下几组(1)对照组,不加任何干预因子；(2)NMB组；(3) siRNA+NMB组。2.按照细胞转染的方法分别进行细胞的NMBR siRNA和NF-κBp65 siRNA转染并检测转染效率,加入不同浓度的NMB干预。3.分别利用磷酸化ELISA、real-time PCR、ELISA法检测siRNA转染前后小鼠子宫平滑肌细胞中NF-κB p65的DNA结合活性、IL-6mRNA及蛋白表达水平。结果：1. NMBR siRNA干预对小鼠子宫平滑肌细胞NF-κB p65的DNA结合活性、IL-6表达水平的影响。NF-κB p65的DNA结合活性、IL-6的表达在10-8、10-6、10-4NMB组(三组高浓度梯度,以下均简称为10-8——10-4NMB组)均高于对照组,差异有统计学意义(P<0.05),且其值随浓度递增而递增,10-10NMB组(低浓度梯度组)与对照组比较差异无统计学意义(P>0.05), NMB各组间差异有统计学意义(P<0.05); NMB+NMBR siRNA组均低于同浓度NMB组,差异有统计学意义(P<0.05),但NMB+NMBR siRNA各组间差异无统计学意义(P>0.05)。2. NF-κB p65 siRNA干预对小鼠子宫平滑肌细胞IL-6表达水平的影响。IL-6的表达在10-8——10-4NMB组均高于对照组,差异有统计学意义(P<0.05),且其值随浓度递增而递增,10-10NMB组与对照组比较差异无统计学意义(P>0.05), NMB各组间差异有统计学意义(P<0.05); NMB+ p65 siRNA组均低于同浓度NMB组,差异有统计学意义(P<0.05),但NMB+ p65 siRNA各组间差异无统计学意义(P>0.05)。3. NMBR siRNA干预实验中NF-κB p65的DNA结合活性与IL-6mRNA的相关性分析。IL-6 mRNA的变化与p65的变化有明显的正相关(r=0.952,P<0.01)：4.分级阻断NMBR和NF-κB通路对IL-6表达影响的比较。结果示对IL-6 mRNA表达影响的差异无统计学意义(P>0.05)。结论：1.通过siRNA分级阻断NMBR和NF-κB,可下调分娩期小鼠原代子宫平滑肌细胞IL-6的表达；2.在分娩期小鼠原代子宫平滑肌细胞中,NMBR可能借助NF-κB通路,调控IL-6的表达,借以影响子宫平滑肌细胞的活动。更多还原

【Abstract】 Object:To study the influence of silencing NMBR and NF-κB by specific siRNA on expression of IL-6 in primary uterine smooth muscle cells of mice.Methods:1. Primary cells culture and identify were executed using the mice’s uterine smooth muscle cells which were intrapartum or after 2 hours of postpartum. NMB concentrations were difined as 10-10M、10-8M、10-6M、10-4M.Three groups were set up as follow:(1)control group without any interference factor;(2)NMB group;(3) siRNA+NMB group.2. NMBR siRNA and NF-κB p65 siRNA were transfected respectively into uterine smooth muscle cells.The transfection efficiency was detected.Then we added NMB of different concentrations as above.3. Detect the binding activity of NF-κB—65 DNA in uterine smooth muscle cells before and after siRNA tranfection by ELISA assay. The mRNA and protein expression of IL-6 were detected by real-time PCR and ELISA at the same time.Results:1. The influence of NMBR siRNA on the binding activity of NF-κB—p65 DNA and expression of IL-6 in uterine smooth muscle.The result was as follow:The values in NMB group(10-4、10-6、10-8) are higher than that in control group (P<0.05). Data showed the obvious correlation between the variation of the binding activity of NF-κB p65 DNA、expression of IL-6 and concentration of NMB. With the increase of NMB concentration, the binding activity of NF-κB—p65 DNA and the expression of IL-6 increased correspondingly. There is significant difference between the subgroups in NMB groups(P<0.05). The values in NMB+NMBR siRNA group are lower than those in NMB group. But there is no significant difference between the subgroups in NMB+NMBR siRNA group(P>0.05).2. The influence of NF-κB p65 siRNA on the expression of IL-6 in uterine smooth muscle. The result was as follow:The values in NMB group(10-4、10-6、10-8) are higher than that in control group (P<0.05). Data showed the obvious correlation between the mRNA expression of IL-6 and concentration of NMB.With the increase of NMB concentration, the mRNA expression of IL-6 increased correspondingly. There is significant difference between the subgroups in NMB groups(P<0.05). The values in NMB+ p65 siRNA group are lower than those in NMB group(P<0.05). But there is no significant difference between the subgroups in NMB+NMBR siRNA group(P>0.05).3. The correlation analysis between binding activity of NF-κB—p65 DNA and IL-6 by blocking of NMBR siRNA.There is a significant positive correlation between the expression of IL-6 mRNA and p65 (r=0.952, P<0.01)4. The comparison of the influence on the expression of IL-6 mRNA by blocking NMBR and NF-κB.There is no significant difference between NMBR siRNA group and NF-κB p65 siRNA group(P>0.05).Conclusion:1. The expression of IL-6 in primary uterine smooth muscle cells can be down regulated via silencing of NMBR and NF-κB by specific siRNA.2. NMBR may regulates the expression of IL-6 in uterine smooth muscle cells of mouse by NF-κB pathway and then affect the activity of uterine smooth muscle cells of mouse.更多还原