重组腺病毒Ad-IL-12对M2型巨噬细胞表型及功能的逆转

发布时间：2017-12-31 14:32

本文关键词：重组腺病毒Ad-IL-12对M2型巨噬细胞表型及功能的逆转　出处：《重庆医科大学》2015年硕士论文　论文类型：学位论文

更多相关文章： 重组腺病毒Ad-IL-12 巨噬细胞 M2 M1 逆转

【摘要】：目的：探讨重组腺病毒Ad-IL-12转入M2型巨噬细胞后能否逆转其表型及功能和涉及的信号分子。方法：利用外源性细胞因子诱导的方法构建M2型巨噬细胞模型,在此基础上转染携带目的基因IL-12的腺病毒,作用60 h后,通过PCR技术检测M1/M2巨噬细胞极化相关指标的变化,用流式细胞术检测细胞表面胞膜分子表达的变化。通过收集条件培养基的方法,间接检测M2型巨噬细胞功能的改变,采用CCK-8方法观察条件培养基处理后肝癌细胞HepG2的增殖,流式细胞术检测条件培养基处理后肝癌细胞的周期及凋亡,最后,用western blot方法检测在此M2巨噬细胞表型逆转过程中可能的信号分子表达的变化。结果：成功构建了M2型巨噬细胞模型。从荧光显微镜图片及IL-12p35、p40的表达情况说明,携人IL-12基因的腺病毒成功转染M2型巨噬细胞且其所携带基因IL-12得到有效表达。病毒作用60h后,与空白组和空载组相比,过表达IL-12组的M2型巨噬细胞表达M1巨噬细胞极化相关指标(如促炎因子：IL-23, IL-1β等)增多,而M2巨噬细胞极化相关指标(如抗炎因子：IL-10, TGF-β)表达下降,差异有统计学意义,流式细胞术也证明实验组的胞膜分子CD86表达增高,CD206表达下降。3组不同来源的条件培养基处理肝癌细胞HepG2约72h后,CCK-8实验显示：从第3d开始,过表达IL-12组的HepG2细胞增殖开始受到明显抑制,与空白和空载组比,差异有统计学意义(P0.05),而后两者比较,差异不大(P0.05)；同样方法处理HepG272h后,流式结果显示：过表达IL-12组肝癌细胞早期凋亡明显增加(早期凋亡率达11.33±0.92%),细胞周期主要分布在G1期(占细胞总数的65.88±2.69%),与空载组的早期凋亡率(3.01±1.03%)、G1期细胞分布(48.42±2.05%),空白组的早期凋亡率(2.91±0.92%)、G1期细胞分布(44.63±2.34%)相比,差异有统计学意义,而后两者之间差异不大。Western blot结果显示：过表达IL-12组的M2型巨噬细胞信号分子p-stat3、wnt2、β-catenn、c-myc和NF-κBP50的表达量明显降低,而信号分子p-statl和NF-κBP65的表达量明显升高,与空白组和空载组比较,差异有统计学意义,而后两组之间差异不大。结论：在成功地构建M2型巨噬细胞模型基础上,通过转入携人IL-12的腺病毒,证实过表达IL-12基因可逆转M2型巨噬细胞的表型,同时伴随有其功能的改变,使M2型巨噬细胞向M1型巨噬细胞的方向转化。在此过程中,也伴随有部分信号分子的改变。
[Abstract]:Objective: to investigate whether recombinant adenovirus Ad-IL-12 can reverse the phenotype, function and signal molecules involved in M 2 macrophages. The M2 macrophage model was constructed by exogenous cytokine induction. On this basis, adenovirus carrying the target gene IL-12 was transfected. After 60 hours of exposure, the changes of polarization related indexes of M 1 / M 2 macrophages were detected by PCR technique. Flow cytometry was used to detect the changes of cellular membrane molecule expression on the cell surface, and the function of M2 macrophage was indirectly detected by the method of conditional culture medium. CCK-8 method was used to observe the HepG2 proliferation of hepatoma cells treated with conditioned medium. Flow cytometry was used to detect the cycle and apoptosis of hepatoma cells treated with conditioned medium. Western blot method was used to detect the changes of signal molecule expression during phenotypic reversal of M2 macrophages. The M _ 2 macrophage model was successfully constructed from fluorescence microscopy and IL-12p35. The expression of p40 indicated that the adenovirus carrying human IL-12 gene was successfully transfected into M2 macrophage and its gene IL-12 was effectively expressed. Compared with blank group and no-load group, M _ 2 macrophages with overexpression of IL-12 expressed more M _ 1 macrophage polarization-related indexes (such as pro-inflammatory factor: IL-23, IL-1 尾). However, the expression of anti-inflammatory factor: IL-10, TGF- 尾 in M2 macrophages decreased, and the difference was statistically significant. Flow cytometry also showed that the expression of CD86 in the membrane of experimental group increased and CD206 expression decreased. 3. The HepG2 of hepatoma cells was treated with conditioned medium from different sources for 72 hours. CCK-8 assay showed that from the 3rd day, the proliferation of HepG2 cells in overexpression IL-12 group was inhibited obviously, compared with blank group and no-load group, the difference was statistically significant (P 0.05). However, the difference between the latter two was not significant (P 0.05). After HepG272h was treated with the same method, the flow rate showed that the early apoptosis of hepatoma cells increased significantly (the rate of early apoptosis was 11.33 卤0.92) in the over-expressed IL-12 group. The cell cycle was mainly distributed in G1 phase (65.88 卤2.69% of the total cells, and 3.01 卤1.03% in the no-load group). The cell distribution in G1 phase was 48.42 卤2.05, and the early apoptotic rate was 2.91 卤0.92 in the blank group, compared with 44.63 卤2.34 in the G1 phase. The difference was statistically significant, but the difference between the latter two was not significant. The results of Western blot showed that the signal molecule p-stat3 of M2 type macrophages in IL-12 group was over-expressed. The expressions of wnt2, 尾 -catennin c-myc and NF- 魏 BP50 decreased significantly, while the expression of signal molecules p-statl and NF- 魏 BP65 increased significantly. Compared with blank group and no-load group, the difference was statistically significant, but there was no significant difference between the latter two groups. Conclusion: on the basis of successfully constructing M2 macrophage model, the adenovirus carrying human IL-12 was transferred. It is proved that overexpression of IL-12 gene can reverse the phenotype of M 2 macrophages, accompanied by changes in its function, which makes M 2 macrophages transform to M 1 type macrophages. There are also changes in some signaling molecules.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R730.5

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