IL-12对卵巢癌相关巨噬细胞B7-H1表达的影响及其机制的初步探讨
发布时间：2018-10-11 18:30
【摘要】：目的 1探讨卵巢癌细胞SKOV3对巨噬细胞B7-H1表达的影响及其可能机制。 2进一步探讨IL-12对卵巢癌相关外周血单核细胞来源巨噬细胞B7-H1表达的影响及其机制。 3探讨IL-12对卵巢癌相关THP-1来源巨噬细胞B7-H1表达的影响及其可能机制。 方法 1THP-1或人外周血单核细胞经PMA诱导分化为巨噬细胞后，与人卵巢癌细胞株SKOV3体外非接触共培养24h，qPCR、western blot及流式细胞术分别检测两种来源巨噬细胞B7-H1的表达；进一步利用NF-κB、JAK/STAT、p38MAPK信号通路的特异性抑制剂预处理两种来源巨噬细胞，之后再分别与SKOV3共培养24h，qPCR及western blot检测B7-H1的表达。 2人外周血单核细胞来源巨噬细胞经外源性重组人IL-12(rIL-12)或携人全长IL-12基因的腺病毒(Ad-IL-12-GFP)处理24h，之后与SKOV3共培养24h。收集单核来源巨噬细胞用qPCR检测B7-H1的相对表达量；western blot检测B7-H1蛋白的表达及NF-κB信号通路的激活情况；同时收集上清用ELISA检测IL-12、IFN-γ、IL-10的水平。另一方面，单独用IFN-γ处理单核来源巨噬细胞，然后与SKOV3共培养，qPCR及western blot检测B7-H1的表达。另外先用NF-κB信号通路的抑制剂Bay11-7082预处理单核来源巨噬细胞，然后再用IL-12处理，，之后共培养，检测B7-H1的表达。 3同样地，THP-1来源巨噬细胞经rIL-12或Ad-IL-12-GFP处理24h后，与SKOV3共培养24h。收集THP-1来源巨噬细胞，qPCR及western blot检测B7-H1的表达；同时收集共培养后的上清，ELISA分析IL-12、IL-10、IFN-γ的表达。 结果 1THP-1来源巨噬细胞及单核来源巨噬细胞与SKOV3共培养24h后，B7-H1在mRNA及蛋白水平都较单独培养组显著升高(p0.05)，而阻断NF-κB、JAK/STAT、p38MAPK信号通路，B7-H1的上调被明显抑制(p0.05)。 2与对照组相比，rIL-12或Ad-IL-12-GFP处理组的人外周单核来源巨噬细胞的B7-H1明显升高(p0.05)，同时伴随有IFN-γ的显著升高(p0.05)、IL-10的显著降低(p0.05)，以及NF-κB信号通路的激活。而在用Bay11-7082抑制NF-κB信号通路后，IL-12所致的B7-H1上调被抑制(p0.05)。经IFN-γ处理后的外周单核来源巨噬细胞与SKOV3共培养后，也有B7-H1的明显升高(p0.05)。 3不同的是，THP-1来源巨噬细胞经过相同的处理，B7-H1的表达却下降(p0.05)，检测共培养后的上清有IL-10的显著下降(p0.05)，但各组几乎均检测不到IFN-γ的表达。 结论 1卵巢癌细胞SKOV3促进了巨噬细胞B7-H1的表达，其机制可能涉及NF-κB、JAK/STAT、p38MAPK信号通路的激活。 2IL-12可上调外周单核来源巨噬细胞的B7-H1的表达，这可能主要与IL-12促进IFN-γ的分泌，并进一步激活NF-κB信号通路有关。 3IL-12下调THP-1来源巨噬细胞的B7-H1的表达，可能与IFN-γ的缺乏以及IL-12显著抑制了IL-10的分泌有关。
[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- 纬 and IL-10 were detected by ELISA in the supernatant. On the other hand, mononuclear macrophages were treated with IFN- 纬 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- 纬 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- 纬 (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- 纬 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- 纬 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- 纬 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- 纬 and the significant inhibition of IL-10 secretion by IL-12.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.31
本文编号：2264817
[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- 纬 and IL-10 were detected by ELISA in the supernatant. On the other hand, mononuclear macrophages were treated with IFN- 纬 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- 纬 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- 纬 (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- 纬 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- 纬 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- 纬 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- 纬 and the significant inhibition of IL-10 secretion by IL-12.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.31
【参考文献】
相关期刊论文 前1条
1 成凤;匡文斌;王秦;秦晓林;范晓卿;董晋豫;梁勤东;李朴;涂植光;;携人IL-12基因腺病毒的构建及其对Hep3B细胞增殖及TGF-β表达的影响[J];中国免疫学杂志;2012年05期
本文编号：2264817
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