IL-12通过下调Stat-3诱导巨噬细胞分化为M1型并且抑制肝癌细胞生长

发布时间：2018-08-01 11:21

【摘要】：目的:研究重组腺病毒pAd5F35-IL-12成功感染人外周血单核细胞后,高表达IL-12的单核细胞是否可以诱导其成为抑制肝癌细胞生长的M1型巨噬细胞,探讨是否与Stat-3信号通路有关。方法:采用Ficoll-Paque分离液分离新鲜的人外周血单核细胞,然后让重组腺病毒pAd5F35-IL-12感染单核细胞。感染腺病毒48小时后的单核细胞和人肝癌细胞株(SMMC-7721和Hep3B)在共培养小室中进行体外共培养,每一种肝癌细胞株都分别设立3个实验组,分别是空白组(SMMC-7721和Hep3B),空载组(SMMC-7721/GFP和Hep3B/GFP),目的组(SMMC-7721/IL-12和Hep3B/IL-12)。分别检测两种肝癌细胞共培养体系中IL-12的表达情况,单核细胞分化表型及Stat-3信号通路的变化以及体内外肝癌细胞的生长情况。采用荧光显微镜观察感染了带有绿色荧光蛋白的重组腺病毒pAd5F35-IL-12的单核细胞是否发出绿色荧光,使用RT-PCR方法检测IL-12的P35、P40亚基的mRNA水平的表达情况,ELISA方法检测共培养体系培养基中IL-12 P70蛋白的表达水平。采用流式细胞术检测共培养体系中的单核细胞分化为巨噬细胞的表面分化标志cd197(m1型巨噬细胞表面分化标志)和cd206(m2型巨噬细胞表面分化标志)的平均荧光强度值mfi,分别使用定量pcr方法和elisa方法检测共培养上清中巨噬细胞表型分化相关因子il-10、il-12、tgf-β、vegf-a、mmp-9的表达情况。进一步采用westernblot方法检测共培养体系中过表达il-12的单核细胞的stat-3信号分子及其下游c-myc基因的变化情况。最后在体内外分别研究肝癌细胞的生长情况,在体外实验中,cck8法和细胞克隆实验检测肝癌细胞的增殖情况,流式细胞术检测共培养后肝癌细胞的细胞周期变化,transwell实验检测肝癌细胞的侵袭能力;体内实验中,观察nod/scid小鼠中成瘤情况和绘制肿瘤生长曲线,免疫组织化学方法检测肿瘤生长相关指标(pcna、mvd)表达情况。结果:重组腺病毒pad5f35-il-12能够感染新鲜分离的人外周血单核细胞,发出绿色荧光,分泌表达il-12p70蛋白;在单核细胞和肝癌细胞的共培养体系中,和空白组和空载组相比,目的组中过表达il-12的单核细胞cd197、il-12的表达升高,cd206、il-10、tgf-β、vegf-a、mmp-9的表达下降,stat-3和c-myc基因的mrna和蛋白水平都下降;体外实验研究结果发现共培养体系中,和空白组和空载组相比,目的组中肝癌细胞的瑞氏染色形态变化明显,细胞变圆,细胞核和细胞质之间的边界变模糊;cck-8实验和克隆形成实验中目的组的肝癌细胞株的细胞增殖能力下降,克隆形成率下降;目的组肝癌细胞周期g1期升高,s期下降;目的组肝癌细胞侵袭能力下降。动物移植瘤模型实验研究结果发现,目的组肝癌成瘤时间延长,肿瘤生长率下降,增殖细胞核抗原PCNA阳性细胞率下降,平均血管密度MVD减少。结论:重组腺病毒pAd5F35-IL-12能够有效的感染新鲜分离的人外周血单核细胞,分泌较高水平的IL-12 P70蛋白。共培养体系中过表达IL-12的单核细胞可以分化为M1型巨噬细胞,其机制可能与Stat-3和c-myc信号通路的下调有关。体内、外研究证实过表达IL-12可诱导单核细胞极化为M1型巨噬细胞,并且可以使与其共培养的肝癌细胞生长受到明显的抑制。
[Abstract]:Objective: To investigate whether the recombinant adenovirus pAd5F35-IL-12 successfully infects human peripheral blood mononuclear cells, and whether the mononuclear cells with high expression of IL-12 can induce M1 macrophages to inhibit the growth of hepatoma cells, and explore whether it is related to the Stat-3 signaling pathway. Method: to separate fresh human peripheral blood mononuclear cells by Ficoll-Paque separation solution. The recombinant adenovirus pAd5F35-IL-12 was infected with mononuclear cells. The monocyte and human hepatoma cell line (SMMC-7721 and Hep3B) infected with adenovirus 48 hours later were co cultured in the co culture compartment, and 3 experimental groups were set up in each of the hepatoma cell lines, respectively, the blank group (SMMC-7721 and Hep3B), and the empty group (SMMC-7721/GFP and Hep3B). /GFP), the target group (SMMC-7721/IL-12 and Hep3B/IL-12). The expression of IL-12 in the co culture system of two hepatoma cells, the changes of the monocyte differentiation phenotype and the Stat-3 signaling pathway and the growth of the hepatoma cells in vivo and in vitro and in vivo were detected. The recombinant adenovirus pAd5F35- with green fluorescent protein was detected by the fluorescence microscope. Whether the monocyte of IL-12 sends out green fluorescence, uses RT-PCR method to detect the expression of mRNA level of IL-12 P35, P40 subunit, and ELISA method to detect the expression level of IL-12 P70 protein in the culture medium of co culture system. Flow cytometry is used to detect the differentiation of monocytes in the co culture system to the surface differentiation marker of macrophage. The average fluorescence intensity of 97 (M1 type macrophage surface differentiation marker) and cd206 (surface differentiation marker of M2 type macrophage) was MFI. Quantitative PCR method and ELISA method were used to detect the expression of phenotypic differentiation related factors of macrophages in co culture supernatant, IL-10, IL-12, tgf- beta, VEGF-A, MMP-9. Further Westernblot method was used. The changes in the Stat-3 signal molecules of mononuclear cells expressing IL-12 and their downstream c-myc genes in the co culture system. Finally, the growth of hepatoma cells was studied in vivo and in vitro. In vitro, the proliferation of hepatoma cells was detected by CCK8 method and cell clone test. Flow cytometry was used to detect the cell size of hepatoma cells after co culture. Cell cycle changes, Transwell test was used to detect the invasion ability of liver cancer cells. In vivo, the tumor formation of nod/scid mice was observed and the tumor growth curve was plotted. The expression of tumor growth correlation index (PCNA, MVD) was detected by immunohistochemistry. Results: recombinant adenovirus pad5f35-il-12 could infect fresh isolated human peripheral blood. Nuclear cells emit green fluorescence and secrete expression of IL-12p70 protein; in the co culture system of mononuclear and hepatocellular carcinoma cells, the expression of CD197, IL-12, cd206, IL-10, tgf- beta, VEGF-A, MMP-9, and mRNA and protein levels of Stat-3 and c-myc genes are decreased in the mononuclear cells expressing IL-12 in the mononuclear and hepatocellular carcinoma cells. The results of in vitro study found that in the co culture system, compared with the blank group and the empty group, the liver cancer cells in the target group had obvious changes in the rayon staining form, the cells became round and the boundary between the nucleus and the cytoplasm became blurred, and the cell proliferation ability of the liver cancer cell lines in the CCK-8 experiment and the clone formation experiment decreased, The formation rate of lung cancer decreased, the period G1 of liver cancer cells in the target group increased, the S phase decreased, and the invasion ability of liver cancer cells in the target group decreased. The results of animal transplantation tumor model experimental study found that the tumor growth rate of liver cancer in the target group was prolonged, the tumor growth rate decreased, the rate of PCNA positive cells of proliferating cell nuclear antigen (PCNA) decreased, and the average density of blood vessel density decreased. Conclusion: recombinant human blood vessel density (MVD) decreased. Adenovirus pAd5F35-IL-12 can effectively infect fresh isolated human peripheral blood mononuclear cells and secrete a high level of IL-12 P70 protein. In co culture system, the mononuclear cells that overexpress IL-12 can differentiate into M1 type macrophages. The mechanism may be related to the lower modulation of Stat-3 and c-myc signaling pathways. In vivo, external studies have confirmed that IL-12 can be expressed. The mononuclear cells were induced to polarize M1 macrophages, and the growth of hepatoma cells co cultured with them was significantly inhibited.
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.7

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