培养淋巴细胞做过继性免疫治疗时细胞因子作用和基因表达特征
发布时间：2018-08-11 13:47
【摘要】： 目的:在过继免疫治疗中需要加入不同的细胞因子,本研究分析不同细胞因子刺激后淋巴细胞分化方向和相关的基因表达特征。 方法:采集正常人外周血淋巴细胞,根据细胞因子来自同一亚型的辅助淋巴细胞进行组合,之后应用不同组合的细胞因子分四组刺激外周血淋巴细胞,包括EBV多肽联合多种细胞因子刺激组(简称EBV多肽组);诱导向TH1细胞分化的IL-12,IFN-γ,IL-2,抗CD3单抗组(简称加IL-12等组),诱导TH2的IL-4,IL-5,IL-10,IL-13,IL-2,抗CD3单抗组(简称加IL-4等组),诱导分化为CTL细胞的GM-CSF,IL-15,IL-2,抗CD3单抗组(简称加GM-CSF等组),每组2管,3天换液一次,换液同时补加入相应量的细胞因子,培养的D0、1、3、7、10天,从各组的两管抽取1ml,混在一起(约5×10^6 cells),用流式细胞术(FCM)检测总T细胞(CD3+), (辅助T细胞)CD3+CD4+, (细胞毒性T细胞)CD3+CD8+, (记忆型T细胞)CD3+CD8+CD45RO+,(初始型T细胞)CD3+CD8+CD45RA+,(TH2辅助细胞)CD3+CD30+,(B细胞)CD19+,(NK细胞)CD56+,(初始调节T细胞)CD4+CD25+(,精确调节T细胞)CD4+CD25+FOXP3+在培养前后百分比的变化( EBV多肽联合细胞因子刺激组只在D0,D10天检测上述基因与抗体)。应用RT-PCR技术检测看家基因MAD1、PTEN和辅助T细胞转录调控基因T-BET(TH1)、GATA3(TH2)、细胞因子IFN-γ(TH1)、IL-4(TH2)基因表达量。 结果: 1.EBV多肽刺激组细胞临床治疗有确切疗效。培养的外周血细胞来自接受细胞过继免疫治疗的NK-T细胞淋巴瘤患者的母亲,EBV多肽组细胞培养10天回输给患者,三天后热退,EBV拷贝数从6.7×10^6拷贝/ml降为0,淋巴结肿大消失,临床治疗有效。表明成功获得杀伤EBV并抗肿瘤的特异性淋巴细胞。 2.EBV多肽组CTL细胞成为优势细胞,调节T细胞明显下降,看家基因MAD1, PTEN变化不大,辅助淋巴细胞转录调控基因T-BET,GATA3表达明显增加。EBV多肽组培养10天后CD3+CD8+细胞增加较明显(D0 21.54%→D10 58.82%),其他实验组如在加IL-4等组(D0 21.54%→D10 31.99%)与加IL-15等组(D0 21.54%→D10 36.14%)呈酌渐上升趋势,在加IL-12等组从D7天开始有明显增加(D0 21.54%→D10 42.4%),效果均不如在EBV多肽刺激组明显。说明EBV抗原肽可以有效刺激特异性CTL细胞的分化,而加IL-4等组效果较差。MAD1基因表达量均较处理前表达量减少,约为培养前的0.18-0.5倍左右;PTEN基因在加IL-15等组培养到D10天时,约为D0天的1.80倍,在EBV多肽刺激组约为D0天的1.55倍,在其他两组较培养前变化不明显(加IL-4等组D10天为D0天的1.40倍,加IL-12组D10天为D0天的1.22倍);说明多种细胞因子的刺激对其表达很可能没有明显的作用。T-BET基因在EBV多肽刺激组D10天为D0天的5.84倍,加IL-15等组(5.58倍)及加IL-12(4.6倍)等组均有明显增加,而在加IL-4等组D10天为D0天的2.79倍, GATA3基因在不同细胞因子刺激组较前均有增加,加IL-15等组D10约为D0的8.48倍,EBV多肽刺激组D10天约为D0天的8.20倍,加IL-12等组D10约为D0的8.13倍,加IL-4等组D10约为D0的7.27倍,提示细胞因子可以诱导淋巴细胞向TH1,TH2亚型方向分化,但细胞因子对该亚型之外的亚型细胞的分化也有刺激作用 3.比较加入EBV多肽和不同细胞因子培养结果,EBV抗原肽可以更有效刺激CTL生成,而IL4,IL5,lL10,IL13的TH2组作用较差。IFN-γ的表达量较细胞因子处理前变化较明显,在加IL-15等组(D10约为D0的2712倍),加IL-12等组培养到D10天时,约为D0天的2030倍,及加IL-4等组(406倍)从D7天开始较D0天有明显变化,EBV多肽刺激组(D10约为D0的61倍)均明显增加;证明用不同组合的细胞因子对IFN-γ的分泌均有明显作用。 另外CD3+CD8+CD45RO+细胞在EBV多肽刺激后增加( D0 27.92%→D10 56.24%),在加IL-12等组酌渐上升趋势(D0 27.92%→D10 51.35%),在其他两组从D1到D7天,增长不明显,在培养到D10天分别较D0天增加6.78%(加IL-4等组)、19.57%(加GM-CSF等组); CD3+CD8+CD45RA+细胞在EBV多肽刺激组培养后增加(D023.38%→D10 37.21%),在其余三组均呈酌渐上升趋势,D10天较D0天分别增加15%(加IFN-γ等组)、19.63%(加IL-4等组)、32.31%(加IL-15等组),进一步证明了细胞因子的多效性。 结论: 用EBV多肽联合细胞因子共同培养外周血单个核细胞可以获得特异性CTL细胞,IFN-γ基因表达量明显增加;辅助淋巴细胞分化相关的基因T-BET、GATA3都有明显的升高;而看家基因MAD1、PTEN基因变化不大。在不同培养环境下加入抗原肽可以更有效刺激CTL生成。
[Abstract]:Objective: Different cytokines need to be added to adoptive immunotherapy. This study analyzed the differentiation direction of lymphocytes stimulated by different cytokines and related gene expression characteristics.
METHODS: Peripheral blood lymphocytes were collected from normal subjects and combined with helper lymphocytes derived from the same subtype of cytokines. The peripheral blood lymphocytes were stimulated by cytokines of different combinations in four groups, including EBV polypeptide combined with multiple cytokine stimulation group (EBV polypeptide group), IL-12, IFN-gamma, which induced differentiation into TH1 cells. IL-2, anti-CD3 monoclonal antibody group (adding IL-12 for short), induced TH 2 IL-4, IL-5, IL-10, IL-13, IL-2, anti-CD3 monoclonal antibody group (adding IL-4 for short), induced differentiated into CTL cells GM-CSF, IL-15, IL-2, anti-CD3 monoclonal antibody group (adding GM-CSF for short), each group 2 tubes, 3 days, fluid exchange at the same time with the addition of the corresponding amount of cytokines, culture D0, 1, 3, 7, 10. One ml was taken from two tubes of each group and mixed together (about 5 *10 ^ 6 cells). Total T cells (CD3 +), helper T cells (CD3 + CD4 +), cytotoxic T cells (CD3 + CD8 +), memory T cells (memory T cells) CD3 + CD8 + CD45RO +, (initial T cells) CD3 + CD8 + CD45RA +, (TH2 helper cells) CD3 + CD30 +, (B cells) CD56 +, (NK cells) CD56 +, (initial T cells) CD3 + CD3 + CD4 +, (primary T cells) CD3 + CD3 + CD3 + CD4 +, (TH2 helper cells) CD19 +, (NK cells) CD5 +, The percentage of CD4 + CD25 + (, precisely regulates T cells) CD4 + CD25 + FOXP3 + before and after culture (EBV polypeptide combined with cytokine stimulation group only detected the above genes and antibodies at D0 and D10 days). RT-PCR was used to detect the homecare gene MAD1, PTEN and helper T cell transcription regulatory genes T-BET (TH1), GATA3 (TH2), cytokine IFN-gamma (TH1). The expression of IL-4 (TH2) gene.
Result:
1. EBV polypeptide stimulation group has a definite effect on clinical treatment. Cultured peripheral blood cells come from the mother of NK-T cell lymphoma patients receiving cell adoptive immunotherapy. The cells of EBV polypeptide group were cultured and transfused back to the patient for 10 days. After three days, the fever subsided, the copy number of EBV decreased from 6.7 *10 ^ 6 copies/ml to 0, and the lymph node enlargement disappeared. Ming successfully obtained specific lymphocytes killing EBV and tumor.
2. CTL cells became dominant cells in EBV polypeptide group, and regulatory T cells decreased significantly. The changes of housekeeping gene MAD1 and PTEN were not significant. The expression of T-BET and GATA3 in helper lymphocytes increased significantly. CD3 + CD8 + cells increased significantly in EBV polypeptide group after 10 days of culture (D0.21.54%D10.82%). Other experimental groups such as increasing IL-4 (D0.54%D10.82%). 31.99% showed a gradual upward trend with the addition of IL-15 (D0 21.54%D10 36.14%) and IL-12 (D0 21.54%D10 42.4%). The effect of MAD1 gene expression was lower than that of EBV polypeptide stimulation group. The expression of PTEN gene was about 1.80 times of D0 day, 1.55 times of D0 day in EBV polypeptide stimulation group and 1.40 times of D0 day in IL-4 group and 1.22 times of D0 day in IL-12 group. T-BET gene was 5.84 times higher in EBV polypeptide stimulation group than in D0, 5.58 times higher in IL-15 and 4.6 times higher in EBV polypeptide stimulation group, but 2.79 times higher in IL-4 and GATA3 than in D0, respectively. D10, such as IL-15, was about 8.48 times of D0, D10, D10 and D0 were about 8.20 times of EBV, 8.13 times of D0 and 7.27 times of D0 respectively, indicating that cytokines could induce lymphocytes to differentiate into TH1 and TH2 subtypes, but cytokines could also stimulate the differentiation of subtypes other than this subtype.
3. Compared with the results of EBV polypeptide and different cytokine cultures, EBV antigen peptide could stimulate CTL production more effectively, but the TH2 groups of IL4, IL5, lL10 and IL13 had less effect. The levels of IL-4, IL-4 and other cytokines (406 folds) were significantly higher in EBV polypeptide stimulation group (D10 was 61 folds of D0) than in D0 group (D7 days), which showed that different combinations of cytokines had significant effects on the secretion of IFN-gamma.
In addition, CD3 + CD8 + CD45RO + cells increased after EBV polypeptide stimulation (D0 27.92% D10 56.24%) and increased gradually with the addition of IL-12 (D0 27.92% D10 51.35%). In the other two groups, the growth was not obvious from D1 to D7 days, and increased by 6.78% (with IL-4, etc.) and 19.57% (with GM-CSF, etc.) respectively on the 10th day of culture.
CD3+CD8+CD45RA+cells increased after EBV polypeptide stimulation (D023.38%D10 37.21%) and showed a gradual upward trend in the other three groups, D10 days increased by 15% (adding IFN-gamma etc.), 19.63% (adding IL-4 etc.), 32.31% (adding IL-15 etc.) respectively, which further proved the multipotency of cytokines.
Conclusion:
Specific CTL cells can be obtained from peripheral blood mononuclear cells co-cultured with EBV polypeptides and cytokines, and the expression of IFN-gamma gene is significantly increased; T-BET and GATA3 genes related to helper lymphocyte differentiation are significantly increased; while MAD1 and PTEN genes have little change. Effective stimulation of CTL production.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392
本文编号：2177166
[Abstract]:Objective: Different cytokines need to be added to adoptive immunotherapy. This study analyzed the differentiation direction of lymphocytes stimulated by different cytokines and related gene expression characteristics.
METHODS: Peripheral blood lymphocytes were collected from normal subjects and combined with helper lymphocytes derived from the same subtype of cytokines. The peripheral blood lymphocytes were stimulated by cytokines of different combinations in four groups, including EBV polypeptide combined with multiple cytokine stimulation group (EBV polypeptide group), IL-12, IFN-gamma, which induced differentiation into TH1 cells. IL-2, anti-CD3 monoclonal antibody group (adding IL-12 for short), induced TH 2 IL-4, IL-5, IL-10, IL-13, IL-2, anti-CD3 monoclonal antibody group (adding IL-4 for short), induced differentiated into CTL cells GM-CSF, IL-15, IL-2, anti-CD3 monoclonal antibody group (adding GM-CSF for short), each group 2 tubes, 3 days, fluid exchange at the same time with the addition of the corresponding amount of cytokines, culture D0, 1, 3, 7, 10. One ml was taken from two tubes of each group and mixed together (about 5 *10 ^ 6 cells). Total T cells (CD3 +), helper T cells (CD3 + CD4 +), cytotoxic T cells (CD3 + CD8 +), memory T cells (memory T cells) CD3 + CD8 + CD45RO +, (initial T cells) CD3 + CD8 + CD45RA +, (TH2 helper cells) CD3 + CD30 +, (B cells) CD56 +, (NK cells) CD56 +, (initial T cells) CD3 + CD3 + CD4 +, (primary T cells) CD3 + CD3 + CD3 + CD4 +, (TH2 helper cells) CD19 +, (NK cells) CD5 +, The percentage of CD4 + CD25 + (, precisely regulates T cells) CD4 + CD25 + FOXP3 + before and after culture (EBV polypeptide combined with cytokine stimulation group only detected the above genes and antibodies at D0 and D10 days). RT-PCR was used to detect the homecare gene MAD1, PTEN and helper T cell transcription regulatory genes T-BET (TH1), GATA3 (TH2), cytokine IFN-gamma (TH1). The expression of IL-4 (TH2) gene.
Result:
1. EBV polypeptide stimulation group has a definite effect on clinical treatment. Cultured peripheral blood cells come from the mother of NK-T cell lymphoma patients receiving cell adoptive immunotherapy. The cells of EBV polypeptide group were cultured and transfused back to the patient for 10 days. After three days, the fever subsided, the copy number of EBV decreased from 6.7 *10 ^ 6 copies/ml to 0, and the lymph node enlargement disappeared. Ming successfully obtained specific lymphocytes killing EBV and tumor.
2. CTL cells became dominant cells in EBV polypeptide group, and regulatory T cells decreased significantly. The changes of housekeeping gene MAD1 and PTEN were not significant. The expression of T-BET and GATA3 in helper lymphocytes increased significantly. CD3 + CD8 + cells increased significantly in EBV polypeptide group after 10 days of culture (D0.21.54%D10.82%). Other experimental groups such as increasing IL-4 (D0.54%D10.82%). 31.99% showed a gradual upward trend with the addition of IL-15 (D0 21.54%D10 36.14%) and IL-12 (D0 21.54%D10 42.4%). The effect of MAD1 gene expression was lower than that of EBV polypeptide stimulation group. The expression of PTEN gene was about 1.80 times of D0 day, 1.55 times of D0 day in EBV polypeptide stimulation group and 1.40 times of D0 day in IL-4 group and 1.22 times of D0 day in IL-12 group. T-BET gene was 5.84 times higher in EBV polypeptide stimulation group than in D0, 5.58 times higher in IL-15 and 4.6 times higher in EBV polypeptide stimulation group, but 2.79 times higher in IL-4 and GATA3 than in D0, respectively. D10, such as IL-15, was about 8.48 times of D0, D10, D10 and D0 were about 8.20 times of EBV, 8.13 times of D0 and 7.27 times of D0 respectively, indicating that cytokines could induce lymphocytes to differentiate into TH1 and TH2 subtypes, but cytokines could also stimulate the differentiation of subtypes other than this subtype.
3. Compared with the results of EBV polypeptide and different cytokine cultures, EBV antigen peptide could stimulate CTL production more effectively, but the TH2 groups of IL4, IL5, lL10 and IL13 had less effect. The levels of IL-4, IL-4 and other cytokines (406 folds) were significantly higher in EBV polypeptide stimulation group (D10 was 61 folds of D0) than in D0 group (D7 days), which showed that different combinations of cytokines had significant effects on the secretion of IFN-gamma.
In addition, CD3 + CD8 + CD45RO + cells increased after EBV polypeptide stimulation (D0 27.92% D10 56.24%) and increased gradually with the addition of IL-12 (D0 27.92% D10 51.35%). In the other two groups, the growth was not obvious from D1 to D7 days, and increased by 6.78% (with IL-4, etc.) and 19.57% (with GM-CSF, etc.) respectively on the 10th day of culture.
CD3+CD8+CD45RA+cells increased after EBV polypeptide stimulation (D023.38%D10 37.21%) and showed a gradual upward trend in the other three groups, D10 days increased by 15% (adding IFN-gamma etc.), 19.63% (adding IL-4 etc.), 32.31% (adding IL-15 etc.) respectively, which further proved the multipotency of cytokines.
Conclusion:
Specific CTL cells can be obtained from peripheral blood mononuclear cells co-cultured with EBV polypeptides and cytokines, and the expression of IFN-gamma gene is significantly increased; T-BET and GATA3 genes related to helper lymphocyte differentiation are significantly increased; while MAD1 and PTEN genes have little change. Effective stimulation of CTL production.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392
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