【摘要】：目的 1探討卵巢癌細胞SKOV3對巨噬細胞B7-H1表達的影響及其可能機制。 2進一步探討IL-12對卵巢癌相關(guān)外周血單核細胞來源巨噬細胞B7-H1表達的影響及其機制。 3探討IL-12對卵巢癌相關(guān)THP-1來源巨噬細胞B7-H1表達的影響及其可能機制。 方法 1THP-1或人外周血單核細胞經(jīng)PMA誘導分化為巨噬細胞后，與人卵巢癌細胞株SKOV3體外非接觸共培養(yǎng)24h，qPCR、western blot及流式細胞術(shù)分別檢測兩種來源巨噬細胞B7-H1的表達；進一步利用NF-κB、JAK/STAT、p38MAPK信號通路的特異性抑制劑預處理兩種來源巨噬細胞，之后再分別與SKOV3共培養(yǎng)24h，qPCR及western blot檢測B7-H1的表達。 2人外周血單核細胞來源巨噬細胞經(jīng)外源性重組人IL-12(rIL-12)或攜人全長IL-12基因的腺病毒(Ad-IL-12-GFP)處理24h，之后與SKOV3共培養(yǎng)24h。收集單核來源巨噬細胞用qPCR檢測B7-H1的相對表達量；western blot檢測B7-H1蛋白的表達及NF-κB信號通路的激活情況；同時收集上清用ELISA檢測IL-12、IFN-γ、IL-10的水平。另一方面，單獨用IFN-γ處理單核來源巨噬細胞，然后與SKOV3共培養(yǎng)，qPCR及western blot檢測B7-H1的表達。另外先用NF-κB信號通路的抑制劑Bay11-7082預處理單核來源巨噬細胞，然后再用IL-12處理，，之后共培養(yǎng)，檢測B7-H1的表達。 3同樣地，THP-1來源巨噬細胞經(jīng)rIL-12或Ad-IL-12-GFP處理24h后，與SKOV3共培養(yǎng)24h。收集THP-1來源巨噬細胞，qPCR及western blot檢測B7-H1的表達；同時收集共培養(yǎng)后的上清，ELISA分析IL-12、IL-10、IFN-γ的表達。 結(jié)果 1THP-1來源巨噬細胞及單核來源巨噬細胞與SKOV3共培養(yǎng)24h后，B7-H1在mRNA及蛋白水平都較單獨培養(yǎng)組顯著升高(p0.05)，而阻斷NF-κB、JAK/STAT、p38MAPK信號通路，B7-H1的上調(diào)被明顯抑制(p0.05)。 2與對照組相比，rIL-12或Ad-IL-12-GFP處理組的人外周單核來源巨噬細胞的B7-H1明顯升高(p0.05)，同時伴隨有IFN-γ的顯著升高(p0.05)、IL-10的顯著降低(p0.05)，以及NF-κB信號通路的激活。而在用Bay11-7082抑制NF-κB信號通路后，IL-12所致的B7-H1上調(diào)被抑制(p0.05)。經(jīng)IFN-γ處理后的外周單核來源巨噬細胞與SKOV3共培養(yǎng)后，也有B7-H1的明顯升高(p0.05)。 3不同的是，THP-1來源巨噬細胞經(jīng)過相同的處理，B7-H1的表達卻下降(p0.05)，檢測共培養(yǎng)后的上清有IL-10的顯著下降(p0.05)，但各組幾乎均檢測不到IFN-γ的表達。 結(jié)論 1卵巢癌細胞SKOV3促進了巨噬細胞B7-H1的表達，其機制可能涉及NF-κB、JAK/STAT、p38MAPK信號通路的激活。 2IL-12可上調(diào)外周單核來源巨噬細胞的B7-H1的表達，這可能主要與IL-12促進IFN-γ的分泌，并進一步激活NF-κB信號通路有關(guān)。 3IL-12下調(diào)THP-1來源巨噬細胞的B7-H1的表達，可能與IFN-γ的缺乏以及IL-12顯著抑制了IL-10的分泌有關(guān)。
[Abstract]:Objective 1 to investigate the effect of SKOV3 on the expression of B7-H1 in human ovarian cancer cells and its possible mechanism. 2 to investigate the effect of IL-12 on B7-H1 expression of macrophages derived from peripheral blood monocytes associated with ovarian cancer. 3 to investigate the effect of IL-12 on the expression of B7-H1 in macrophages derived from THP-1 and its possible mechanism. Methods after 1THP-1 or human peripheral blood monocytes were induced to differentiate into macrophages by PMA, the expression of B7-H1 in macrophages from two kinds of macrophages was detected by flow cytometry and non-contact culture with human ovarian cancer cell line SKOV3 for 24 h. Furthermore, two kinds of macrophages were pretreated with the specific inhibitor of NF- 魏 B JAK / stat p38 MAPK signaling pathway. After co-culture with SKOV3 for 24 h, the expression of B7-H1 was detected by western blot. 2 human peripheral blood monocyte derived macrophages were treated with exogenous recombinant human IL-12 (rIL-12) or adenovirus carrying human IL-12 gene (Ad-IL-12-GFP) for 24 h, and then co-cultured with SKOV3 for 24 h. Mononuclear macrophages were collected to detect the relative expression of B7-H1 by qPCR and the expression of B7-H1 protein and the activation of NF- 魏 B signal pathway by; western blot, and the levels of IL-12,IFN- 緯 and IL-10 were detected by ELISA in the supernatant. On the other hand, mononuclear macrophages were treated with IFN- 緯 alone, then co-cultured with SKOV3. The expression of B7-H1 was detected by qPCR and western blot. In addition, mononuclear macrophages were pretreated with Bay11-7082, an inhibitor of NF- 魏 B signaling pathway, then treated with IL-12, and then co-cultured to detect the expression of B7-H1. Similarly, THP-1 derived macrophages were treated with rIL-12 or Ad-IL-12-GFP for 24 hours and co-cultured with SKOV3 for 24 hours. THP-1 derived macrophages, qPCR and western blot were collected to detect the expression of B7-H1, and the supernatants of co-culture were collected. The expression of IL-12,IL-10,IFN- 緯 was analyzed by ELISA. Results after co-culture of 1THP-1 derived macrophages and mononuclear macrophages with SKOV3 for 24 hours, the level of B7-H1 in mRNA and protein was significantly higher than that in the single culture group (p0.05), but NF- 魏 B blocked the signal pathway of JAK / STATp38MAPK, and the up-regulation of B7-H1 was significantly inhibited (p0.05). 2Compared with the control group, the B7-H1 of human peripheral monocyte derived macrophages in rIL-12 or Ad-IL-12-GFP group was significantly increased (p0.05), accompanied by a significant increase in IFN- 緯 (p0.05), a significant decrease in IL-10 (p0.05), and activation of NF- 魏 B signaling pathway. The up-regulation of B7-H1 induced by IL-12 was inhibited after Bay11-7082 inhibited the NF- 魏 B signaling pathway (p0.05). The peripheral mononuclear macrophages treated with IFN- 緯 were co-cultured with SKOV3. There was also a significant increase in B7-H1 (p0.05). 3 different, the expression of B7-H1 decreased (p0.05) in macrophages derived from THP-1, and the expression of IL-10 decreased significantly in the supernatant of co-culture (p0.05), but the expression of IFN- 緯 was almost not detected in all groups. Conclusion 1Ovarian cancer cell SKOV3 can promote the expression of B7-H1 in macrophages, and its mechanism may be related to the activation of NF- 魏 B in JAK / stat p38 MAPK signaling pathway. 2IL-12 can up-regulate the expression of B7-H1 in macrophages derived from peripheral mononuclear cells. This may be related to the promotion of IFN- 緯 secretion by IL-12 and further activation of NF- 魏 B signaling pathway. 3IL-12 down-regulates the expression of B7-H1 in macrophages derived from THP-1, and may be related to the lack of IFN- 緯 and the significant inhibition of IL-10 secretion by IL-12.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R737.31

【參考文獻】

相關(guān)期刊論文 前1條

1 成鳳;匡文斌;王秦;秦曉林;范曉卿;董晉豫;梁勤東;李樸;涂植光;;攜人IL-12基因腺病毒的構(gòu)建及其對Hep3B細胞增殖及TGF-β表達的影響[J];中國免疫學雜志;2012年05期

本文編號：2264817