RetroNectin对CIK细胞增殖、表型变化和杀伤活性影响的初步研究
发布时间：2018-08-19 17:45
【摘要】： 目的：初步研究RetroNectin对CIK细胞增值、表性变化及杀伤活性的影响，并探讨其可能机制。 方法：采集人外周血单个核细胞，标本分为两组，对照组加入IFN-γ、IL-2、IL-1α、CD3mAb等细胞因子诱导其增殖，实验组另外加入RetroNectin诱导，倒置显微镜下观察培养过程中细胞形态变化，采用活细胞计数法观察CIK细胞增殖，分别绘制细胞增值曲线；流式细胞术检测CIK细胞培养过程中的表型变化；LDH法检测CIK细胞对肿瘤细胞K562细胞和PC-3细胞的杀伤活性；采用AnnexinV／PI染色，，流式细胞术检测细胞凋亡情况；流式细胞技术检测不同培养条件下第4d、7d、11d及14d的CIK细胞周期。 结果：PBMC在体外经过多种细胞因子的诱导后，能大量扩增生成CIK细胞，培养14天，扩增倍数可达96.13±5.03倍；CIK细胞中的CD3~+CD8~+、CD3~+CD56~+细胞比例随培养时间的延长较PBMC明显增多(p＜0.05)；细胞的百分率则逐渐下降；CIK细胞对K562细胞和PC-3细胞均有较强的杀伤活性，两者相比较，差异无统计学意义(p＞0.05)，并且随着诱导时间的延长，这种杀伤活性增强，在诱导培养的第14d杀伤活性最强。经RetroNectin刺激14天后的RN-CIK细胞扩增倍数可达330.46±7.96倍，培养的第7天起明显高于普通培养方法(p＜0.05)；自培养的第7天，RN-CIK细胞中的CD25~+细胞比例较普通培养的CIK细胞明显增多(p＜0.05)。但两种培养方法对CIK细胞的CD3~+CD8~+、CD3~+CD56~+、CD3~+CD4~+表型及细胞杀伤活性的影响无明显差异(p＞0.05)。在培养的第11d，RN-CIK细胞与普通培养的CIK细胞凋亡率无明显差异(p＞0.05)。S期细胞的比例随所培养时间的延长逐渐升高，在培养的第7d达到最高，并且RN-CIK细胞中S期的细胞比例明显高于普通培养的CIK细胞组(p＜0.05)。 结论：(1)体外应用抗人CD3单抗、人重组IL-1α、人重组IFN-γ和人重组IL-2能诱导PBMC生成CIK细胞，CIK细胞对K562细胞和PC-3细胞均有较强的杀伤活性。(2)RetroNectin可以明显增加CIK细胞的扩增倍数，但是对CIK细胞的表型变化及杀伤活性影响不大。(3)RetroNectin能促进细胞增殖的可能机制：促进细胞之间的黏附，增强信号传导；诱导T细胞活化；抵抗活化细胞凋亡；促使细胞由G1期进入S期。
[Abstract]:Aim: to study the effect of RetroNectin on the proliferation, epigenetic change and cytotoxicity of CIK cells and its possible mechanism. Methods: human peripheral blood mononuclear cells (PBMC) were collected and divided into two groups. The control group was induced by IFN- 纬 -IL-2IL-2IL-1 伪 CD3mAb, and the experimental group was induced by RetroNectin. The morphological changes of the cells were observed under inverted microscope. The proliferation of CIK cells was observed by living cell count and the proliferation curve was plotted respectively. The phenotypic changes of CIK cells were detected by flow cytometry. The cytotoxicity of CIK cells to K562 cells and PC-3 cells was detected by flow cytometry. AnnexinV/PI staining and flow cytometry were used to detect the apoptosis of the cells, and the CIK cell cycle at the 4th day after 7 days and 14 days under different culture conditions was detected by flow cytometry. Results CIK cells were induced by many cytokines in vitro. After 14 days of culture, the percentage of CD3 ~ CD8 ~ + CD3 ~ CD56 ~ cells in CIK cells was 96.13 卤5.03 times higher than that in PBMC cells (p < 0. 05). The percentage of K562 cells and PC-3 cells decreased gradually, the difference was not statistically significant (p > 0. 05), and the activity increased with the prolongation of induction time. The cytotoxicity was strongest on the 14th day after induction. After 14 days of RetroNectin stimulation, the expansion of RN-CIK cells was 330.46 卤7.96-fold, which was significantly higher than that of normal culture methods on the 7th day, and the percentage of CD25~ cells in RN-CIK cells from the 7th day of culture was significantly higher than that of normal CIK cells (p < 0. 05). However, there was no significant difference between the two methods on the phenotype and cytotoxicity of CD3 ~ CD8 ~ + CD3 ~ CD56 ~ + CD3 ~ CD4 ~ in CIK cells (p > 0. 05). There was no significant difference in apoptosis rate between the cultured RN-CIK cells and the normal cultured CIK cells at the 11th day (p > 0. 05). The percentage of S phase cells increased gradually with the prolongation of the culture time, and reached the highest on the 7th day of culture. The percentage of S phase cells in RN-CIK cells was significantly higher than that in normal cultured CIK cells (p < 0. 05). Conclusion: (1) Anti-human CD3 monoclonal antibody, human recombinant IL-1 伪, human recombinant IFN- 纬 and human recombinant IL-2 can induce PBMC to produce CIK cells. (2) RetroNectin can significantly increase the expansion times of CIK cells. But it has little effect on phenotypic changes and cytotoxicity of CIK cells. (3) the possible mechanism of RetroNectin can promote cell proliferation: promote cell adhesion, enhance signal transduction, induce T cell activation, resist activated cell apoptosis; Promote cells from G1 phase to S phase.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392
本文编号：2192365
[Abstract]:Aim: to study the effect of RetroNectin on the proliferation, epigenetic change and cytotoxicity of CIK cells and its possible mechanism. Methods: human peripheral blood mononuclear cells (PBMC) were collected and divided into two groups. The control group was induced by IFN- 纬 -IL-2IL-2IL-1 伪 CD3mAb, and the experimental group was induced by RetroNectin. The morphological changes of the cells were observed under inverted microscope. The proliferation of CIK cells was observed by living cell count and the proliferation curve was plotted respectively. The phenotypic changes of CIK cells were detected by flow cytometry. The cytotoxicity of CIK cells to K562 cells and PC-3 cells was detected by flow cytometry. AnnexinV/PI staining and flow cytometry were used to detect the apoptosis of the cells, and the CIK cell cycle at the 4th day after 7 days and 14 days under different culture conditions was detected by flow cytometry. Results CIK cells were induced by many cytokines in vitro. After 14 days of culture, the percentage of CD3 ~ CD8 ~ + CD3 ~ CD56 ~ cells in CIK cells was 96.13 卤5.03 times higher than that in PBMC cells (p < 0. 05). The percentage of K562 cells and PC-3 cells decreased gradually, the difference was not statistically significant (p > 0. 05), and the activity increased with the prolongation of induction time. The cytotoxicity was strongest on the 14th day after induction. After 14 days of RetroNectin stimulation, the expansion of RN-CIK cells was 330.46 卤7.96-fold, which was significantly higher than that of normal culture methods on the 7th day, and the percentage of CD25~ cells in RN-CIK cells from the 7th day of culture was significantly higher than that of normal CIK cells (p < 0. 05). However, there was no significant difference between the two methods on the phenotype and cytotoxicity of CD3 ~ CD8 ~ + CD3 ~ CD56 ~ + CD3 ~ CD4 ~ in CIK cells (p > 0. 05). There was no significant difference in apoptosis rate between the cultured RN-CIK cells and the normal cultured CIK cells at the 11th day (p > 0. 05). The percentage of S phase cells increased gradually with the prolongation of the culture time, and reached the highest on the 7th day of culture. The percentage of S phase cells in RN-CIK cells was significantly higher than that in normal cultured CIK cells (p < 0. 05). Conclusion: (1) Anti-human CD3 monoclonal antibody, human recombinant IL-1 伪, human recombinant IFN- 纬 and human recombinant IL-2 can induce PBMC to produce CIK cells. (2) RetroNectin can significantly increase the expansion times of CIK cells. But it has little effect on phenotypic changes and cytotoxicity of CIK cells. (3) the possible mechanism of RetroNectin can promote cell proliferation: promote cell adhesion, enhance signal transduction, induce T cell activation, resist activated cell apoptosis; Promote cells from G1 phase to S phase.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392
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