本文選題：核轉(zhuǎn)錄因子　切入點：激活蛋白-1　出處：《中南大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：目的：探索神經(jīng)調(diào)節(jié)素B (Neuromedin B, NMB)—神經(jīng)調(diào)節(jié)素B受體(Neuromedin B receptor, NMBR)作用的妊娠晚期人子宮下段平滑肌細胞內(nèi)核轉(zhuǎn)錄因子(Nuclear factor kappa B, NF-κB)與激活蛋白-1(Activator protein1, AP-1)是否存在相互作用。 方法： 1.通過組織塊培養(yǎng)法獲得妊娠晚期人子宮平滑肌原代培養(yǎng)細胞,借助免疫細胞化學(xué)方法對細胞進行鑒定并檢測細胞內(nèi)NMBR表達。 2.以10-6M、10-8M、10-10M濃度NMB分別誘導(dǎo)子宮平滑肌細胞48小時后,蛋白免疫印跡法(Western blotting, WB)檢測AP-1與NF-κB蛋白在各組中的表達。分別使用anti-NF-κB p65抗體和anti-c-Jun抗體分別行免疫共沉淀(co-immunoprecipitation, co-IP),后聯(lián)合WB技術(shù),正、反驗證co-IP沉淀下來的復(fù)合物中分別存在AP-1和NF-κB蛋白。 3.以NMB最佳誘導(dǎo)濃度分別誘導(dǎo)子宮平滑肌細胞12h、24h及48h。WB檢測AP-1與NF-κB蛋白在各組中的表達,同理使用anti-NF-KB p65抗體和anti-c-Jun抗體分別行免疫共沉淀,正、反驗證兩蛋白間存在相互作用。 結(jié)果： 1.獲得妊娠晚期人子宮下段平滑肌原代培養(yǎng)細胞,至少可以傳5代,經(jīng)α-SMA鑒定為高純度子宮平滑肌細胞,且證實細胞內(nèi)NMBR表達陽性。 2.以10-6M、10-8M、10-10M濃度NMB誘導(dǎo)子宮平滑肌細胞48h后,anti-NF-κB p65抗體行免疫共沉淀,沉淀復(fù)合物以anti-c-Jun抗體作為一抗行WB鑒定,分子量36KD處出現(xiàn)條帶；同理,經(jīng)anti-c-Jun抗體富集下來的免疫共沉淀復(fù)合物,使用anti-NF-κB p65抗體作為一抗行WB鑒定,分子量65KD處出現(xiàn)條帶。且于NMB誘導(dǎo)濃度為10-10M時,對應(yīng)目的條帶灰度值高于其它誘導(dǎo)濃度條帶灰度值。 3.以1010MNMB誘導(dǎo)子宮平滑肌細胞12h、24h及48h,正反驗證anti-NF-κB p65/anti-c-Jun抗體富集下來的免疫共沉淀復(fù)合物,WB檢測分子量36KD/65KD均有條帶出現(xiàn)。且于NMB誘導(dǎo)時間為24h時,對應(yīng)目的條帶灰度值高于其它誘導(dǎo)時間條帶灰度值。 結(jié)論：NMB-NMBR作用的人妊娠晚期子宮下段平滑肌細胞中存在AP-1與NF-κB相互作用,且以10-1MNMB作用24h時,二者相互作用最強。
[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 偽 -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.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R714

【參考文獻】

相關(guān)期刊論文 前1條

1 張燕婉;葉玨;時那;孟憲敏;王來元;;蛋白質(zhì)免疫印跡技術(shù)的實驗研究[J];實驗技術(shù)與管理;2008年10期

，

本文編號：1576452