NMBR介导妊娠子宫平滑肌细胞中AP-1与NF-κB相互作用的研究

发布时间：2018-03-06 20:45

本文选题：核转录因子　切入点：激活蛋白-1　出处：《中南大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的：探索神经调节素B (Neuromedin B, NMB)—神经调节素B受体(Neuromedin B receptor, NMBR)作用的妊娠晚期人子宫下段平滑肌细胞内核转录因子(Nuclear factor kappa B, NF-κB)与激活蛋白-1(Activator protein1, AP-1)是否存在相互作用。方法： 1.通过组织块培养法获得妊娠晚期人子宫平滑肌原代培养细胞,借助免疫细胞化学方法对细胞进行鉴定并检测细胞内NMBR表达。 2.以10-6M、10-8M、10-10M浓度NMB分别诱导子宫平滑肌细胞48小时后,蛋白免疫印迹法(Western blotting, WB)检测AP-1与NF-κB蛋白在各组中的表达。分别使用anti-NF-κB p65抗体和anti-c-Jun抗体分别行免疫共沉淀(co-immunoprecipitation, co-IP),后联合WB技术,正、反验证co-IP沉淀下来的复合物中分别存在AP-1和NF-κB蛋白。 3.以NMB最佳诱导浓度分别诱导子宫平滑肌细胞12h、24h及48h。WB检测AP-1与NF-κB蛋白在各组中的表达,同理使用anti-NF-KB p65抗体和anti-c-Jun抗体分别行免疫共沉淀,正、反验证两蛋白间存在相互作用。结果： 1.获得妊娠晚期人子宫下段平滑肌原代培养细胞,至少可以传5代,经α-SMA鉴定为高纯度子宫平滑肌细胞,且证实细胞内NMBR表达阳性。 2.以10-6M、10-8M、10-10M浓度NMB诱导子宫平滑肌细胞48h后,anti-NF-κB p65抗体行免疫共沉淀,沉淀复合物以anti-c-Jun抗体作为一抗行WB鉴定,分子量36KD处出现条带；同理,经anti-c-Jun抗体富集下来的免疫共沉淀复合物,使用anti-NF-κB p65抗体作为一抗行WB鉴定,分子量65KD处出现条带。且于NMB诱导浓度为10-10M时,对应目的条带灰度值高于其它诱导浓度条带灰度值。 3.以1010MNMB诱导子宫平滑肌细胞12h、24h及48h,正反验证anti-NF-κB p65/anti-c-Jun抗体富集下来的免疫共沉淀复合物,WB检测分子量36KD/65KD均有条带出现。且于NMB诱导时间为24h时,对应目的条带灰度值高于其它诱导时间条带灰度值。结论：NMB-NMBR作用的人妊娠晚期子宫下段平滑肌细胞中存在AP-1与NF-κB相互作用,且以10-1MNMB作用24h时,二者相互作用最强。
[Abstract]:Aim: to investigate the interaction between neuromedin B, NMBM-B receptor and neuromedin B receptor (NMBR), nuclear factor kappa B (NF- 魏 B) and activator protein -1 (AP-1) in human lower uterine smooth muscle cells in late pregnancy. Methods:. 1. The primary cultured human uterine smooth muscle cells were obtained by tissue mass culture. The cells were identified by immunocytochemistry and the expression of NMBR in the cells was detected. 2. The expression of AP-1 and NF- 魏 B protein in uterine smooth muscle cells were detected by Western blotting and Western blotting after 48 hours of induction of 10 ~ (-6) MN ~ (-8) -10 ~ (-10) M NMB, respectively. Anti-NF- 魏 B p65 antibody and anti-c-Jun antibody were used to co-precipitate co-immunoprecipitation (co-IPP) and then combined with WB, respectively, to detect the expression of AP-1 and NF- 魏 B protein in each group by Western blotting and Western blotting, respectively, using anti-NF- 魏 B p65 antibody and anti-NF- 魏 B p65 antibody and anti-c-Jun antibody. AP-1 and NF- 魏 B proteins were detected in the complexes precipitated by co-IP. 3. The expression of AP-1 and NF- 魏 B protein in uterine smooth muscle cells was detected by 12h and 48h 路WB at the optimal concentration of NMB, respectively. Similarly, anti-NF-KB p65 antibody and anti-c-Jun antibody were used for immunoprecipitation, positive and negative verification of the interaction between the two proteins. Results:. 1. The primary cultured cells of human lower uterine smooth muscle cells were obtained in the third trimester of pregnancy. They were identified as high purity uterine smooth muscle cells by 伪 -SMA for at least 5 passages, and the positive expression of NMBR in the cells was confirmed. 2.The anti-NF- 魏 B p65 antibody of uterine smooth muscle cells was induced by NMB at the concentration of 10-6 MN ~ (-8) M ~ (-10 ~ (-10) M) for 48 h, and the anti-NF- 魏 B p65 antibody was used as the anti-WB antibody, and the molecular weight was 36KD. Similarly, the anti-NF- 魏 B p65 antibody was enriched by anti-c-Jun antibody. The anti-NF- 魏 B p65 antibody was used as an anti-WB assay to identify the bands with a molecular weight of 65KD. When the induced concentration of NMB was 10-10 M, the gray value of the corresponding target band was higher than that of other induced concentration bands. 3. Uterine smooth muscle cells were induced with 1010MNMB for 12h and 48h, and anti-NF- 魏 B p65 / anti-c-Jun antibody enriched by anti-NF- 魏 B p65 / anti-c-Jun antibody were positively and inversely verified. The molecular weight of 36kD / 65KD was detected by Western blot assay, and the molecular weight was detected at 24h after NMB induction. The gray value of the corresponding target band is higher than that of other induced time bands. Conclusion there is interaction between AP-1 and NF- 魏 B in the smooth muscle cells of the lower segment of the uterus treated with 10 ~ (-1) MNMBR for 24 h, and the interaction between them is the strongest when 10 ~ (-1) MNMB is used for 24 hours.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R714

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