培養(yǎng)淋巴細(xì)胞做過繼性免疫治療時細(xì)胞因子作用和基因表達(dá)特征
發(fā)布時間：2018-08-11 13:47
【摘要】： 目的:在過繼免疫治療中需要加入不同的細(xì)胞因子,本研究分析不同細(xì)胞因子刺激后淋巴細(xì)胞分化方向和相關(guān)的基因表達(dá)特征。 方法:采集正常人外周血淋巴細(xì)胞,根據(jù)細(xì)胞因子來自同一亞型的輔助淋巴細(xì)胞進(jìn)行組合,之后應(yīng)用不同組合的細(xì)胞因子分四組刺激外周血淋巴細(xì)胞,包括EBV多肽聯(lián)合多種細(xì)胞因子刺激組(簡稱EBV多肽組);誘導(dǎo)向TH1細(xì)胞分化的IL-12,IFN-γ,IL-2,抗CD3單抗組(簡稱加IL-12等組),誘導(dǎo)TH2的IL-4,IL-5,IL-10,IL-13,IL-2,抗CD3單抗組(簡稱加IL-4等組),誘導(dǎo)分化為CTL細(xì)胞的GM-CSF,IL-15,IL-2,抗CD3單抗組(簡稱加GM-CSF等組),每組2管,3天換液一次,換液同時補(bǔ)加入相應(yīng)量的細(xì)胞因子,培養(yǎng)的D0、1、3、7、10天,從各組的兩管抽取1ml,混在一起(約5×10^6 cells),用流式細(xì)胞術(shù)(FCM)檢測總T細(xì)胞(CD3+), (輔助T細(xì)胞)CD3+CD4+, (細(xì)胞毒性T細(xì)胞)CD3+CD8+, (記憶型T細(xì)胞)CD3+CD8+CD45RO+,(初始型T細(xì)胞)CD3+CD8+CD45RA+,(TH2輔助細(xì)胞)CD3+CD30+,(B細(xì)胞)CD19+,(NK細(xì)胞)CD56+,(初始調(diào)節(jié)T細(xì)胞)CD4+CD25+(,精確調(diào)節(jié)T細(xì)胞)CD4+CD25+FOXP3+在培養(yǎng)前后百分比的變化( EBV多肽聯(lián)合細(xì)胞因子刺激組只在D0,D10天檢測上述基因與抗體)。應(yīng)用RT-PCR技術(shù)檢測看家基因MAD1、PTEN和輔助T細(xì)胞轉(zhuǎn)錄調(diào)控基因T-BET(TH1)、GATA3(TH2)、細(xì)胞因子IFN-γ(TH1)、IL-4(TH2)基因表達(dá)量。 結(jié)果: 1.EBV多肽刺激組細(xì)胞臨床治療有確切療效。培養(yǎng)的外周血細(xì)胞來自接受細(xì)胞過繼免疫治療的NK-T細(xì)胞淋巴瘤患者的母親,EBV多肽組細(xì)胞培養(yǎng)10天回輸給患者,三天后熱退,EBV拷貝數(shù)從6.7×10^6拷貝/ml降為0,淋巴結(jié)腫大消失,臨床治療有效。表明成功獲得殺傷EBV并抗腫瘤的特異性淋巴細(xì)胞。 2.EBV多肽組CTL細(xì)胞成為優(yōu)勢細(xì)胞,調(diào)節(jié)T細(xì)胞明顯下降,看家基因MAD1, PTEN變化不大,輔助淋巴細(xì)胞轉(zhuǎn)錄調(diào)控基因T-BET,GATA3表達(dá)明顯增加。EBV多肽組培養(yǎng)10天后CD3+CD8+細(xì)胞增加較明顯(D0 21.54%→D10 58.82%),其他實(shí)驗(yàn)組如在加IL-4等組(D0 21.54%→D10 31.99%)與加IL-15等組(D0 21.54%→D10 36.14%)呈酌漸上升趨勢,在加IL-12等組從D7天開始有明顯增加(D0 21.54%→D10 42.4%),效果均不如在EBV多肽刺激組明顯。說明EBV抗原肽可以有效刺激特異性CTL細(xì)胞的分化,而加IL-4等組效果較差。MAD1基因表達(dá)量均較處理前表達(dá)量減少,約為培養(yǎng)前的0.18-0.5倍左右;PTEN基因在加IL-15等組培養(yǎng)到D10天時,約為D0天的1.80倍,在EBV多肽刺激組約為D0天的1.55倍,在其他兩組較培養(yǎng)前變化不明顯(加IL-4等組D10天為D0天的1.40倍,加IL-12組D10天為D0天的1.22倍);說明多種細(xì)胞因子的刺激對其表達(dá)很可能沒有明顯的作用。T-BET基因在EBV多肽刺激組D10天為D0天的5.84倍,加IL-15等組(5.58倍)及加IL-12(4.6倍)等組均有明顯增加,而在加IL-4等組D10天為D0天的2.79倍, GATA3基因在不同細(xì)胞因子刺激組較前均有增加,加IL-15等組D10約為D0的8.48倍,EBV多肽刺激組D10天約為D0天的8.20倍,加IL-12等組D10約為D0的8.13倍,加IL-4等組D10約為D0的7.27倍,提示細(xì)胞因子可以誘導(dǎo)淋巴細(xì)胞向TH1,TH2亞型方向分化,但細(xì)胞因子對該亞型之外的亞型細(xì)胞的分化也有刺激作用 3.比較加入EBV多肽和不同細(xì)胞因子培養(yǎng)結(jié)果,EBV抗原肽可以更有效刺激CTL生成,而IL4,IL5,lL10,IL13的TH2組作用較差。IFN-γ的表達(dá)量較細(xì)胞因子處理前變化較明顯,在加IL-15等組(D10約為D0的2712倍),加IL-12等組培養(yǎng)到D10天時,約為D0天的2030倍,及加IL-4等組(406倍)從D7天開始較D0天有明顯變化,EBV多肽刺激組(D10約為D0的61倍)均明顯增加;證明用不同組合的細(xì)胞因子對IFN-γ的分泌均有明顯作用。 另外CD3+CD8+CD45RO+細(xì)胞在EBV多肽刺激后增加( D0 27.92%→D10 56.24%),在加IL-12等組酌漸上升趨勢(D0 27.92%→D10 51.35%),在其他兩組從D1到D7天,增長不明顯,在培養(yǎng)到D10天分別較D0天增加6.78%(加IL-4等組)、19.57%(加GM-CSF等組); CD3+CD8+CD45RA+細(xì)胞在EBV多肽刺激組培養(yǎng)后增加(D023.38%→D10 37.21%),在其余三組均呈酌漸上升趨勢,D10天較D0天分別增加15%(加IFN-γ等組)、19.63%(加IL-4等組)、32.31%(加IL-15等組),進(jìn)一步證明了細(xì)胞因子的多效性。 結(jié)論: 用EBV多肽聯(lián)合細(xì)胞因子共同培養(yǎng)外周血單個核細(xì)胞可以獲得特異性CTL細(xì)胞,IFN-γ基因表達(dá)量明顯增加;輔助淋巴細(xì)胞分化相關(guān)的基因T-BET、GATA3都有明顯的升高;而看家基因MAD1、PTEN基因變化不大。在不同培養(yǎng)環(huán)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.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R392
本文編號：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.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R392
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