IL-23单独或联合IL-2对人外周血单个核细胞增殖及抗白血病活性的研究
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摘要
目的:白血病是血液系统常见的恶性肿瘤,目前发病率呈逐年增高的趋势。尽管目前临床上可以通过化疗、骨髓移植等手段使部分患者达到完全缓解,但多数患者终因耐药、复发等情况导致治疗失败。因此,需要寻找更好的治疗方法,以提高疗效,改善患者的预后和生活质量。越来越多的研究证据表明,免疫因素在控制造血系统恶性肿瘤中起重要作用,免疫治疗也逐渐成为恶性血液病治疗中的热点。
IL-23(interleukin-23, IL-23)是2000年发现的一种细胞因子,属于IL-12家族,主要由活化的树突状细胞(dendritic cell, DC)和巨噬细胞(Macrophage, Mφ)产生,能促进记忆T细胞的增殖、诱导T细胞和自然杀伤细胞(natural killer cell, NK cell)产生干扰素-γ(interferon-γ, IFN-γ),在机体的免疫防御、肿瘤免疫及某些自身免疫性疾病中起着重要作用。IL-2是由T细胞产生的一种自分泌生长因子,能刺激细胞毒性T淋巴细胞(cytotoxic Tlymphocyte, CTL)的增殖与分化,增强NK细胞的细胞毒活性。现已证实,IL-2在体内具有显著的抗肿瘤作用,但该作用依赖于外源性大剂量IL-2的持续输注且常伴有严重的毒副作用。为减轻毒副作用,近年来相继有不少关于小剂量IL-2与其他细胞因子联合对T细胞体外增殖及功能影响的报道,且多呈协同增强效应。
IL-23能促进T细胞产生IFN-γ,而且因其诱导产生的IFN-γ较少而不会在临床应用时产生严重的毒副反应,因此有可能成为肿瘤治疗中一种更为安全的选择。目前国内外尚无有关IL-23与IL-2联合对白血病细胞影响的报道,本课题拟通过上述两种细胞因子在不同浓度单独或联合应用对人外周血单个核细胞增殖及抗白血病活性影响的研究,希望为白血病的免疫治疗提供新的线索,以进一步提高临床恶性血液病的治疗效果。
方法:用Ficoll密度梯度离心法分离人外周血单个核细胞(peripheral blood mononuclear cells, PBMC),以含不同浓度IL-23单独或联合IL-2的培养液培养后,用MTT比色法测定PBMC的增殖情况及PBMC对白血病K562细胞株的杀伤活性,采用流式细胞术分析诱导前后PBMC的细胞表型变化。所得结果运用SPSS16.0统计软件包处理,以α=0.05为显著性检验水准。
结果:1. IL-23单独或联合IL-2对PBMC增殖的影响:IL-23各浓度组2、10、50 ng/mL,及IL-23(ng/mL)+IL-2(IU/mL)组2 +10、10+10、50+10、2+100、10+100、50+100,作用于PBMC 1d后测吸光度A值分别为0.536±0.047、0.628±0.018、0.660±0.036、0.643±0.030、0.710±0.031、0.768±0.022、0.732±0.020、0.798±0.047、0.864±0.053;3d后测A值分别为0.580±0.014、0.654±0.037、0.706±0.014、0.690±0.014、0.741±0.014、0.808±0.021、0.791±0.008、0.831±0.023、0.886±0.030;5d后A值分别为0.642±0.009、0.727±0.025、0.796±0.008、0.788±0.013、0.841±0.009、0.893±0.023、0.897±0.023、0.958±0.012、1.077±0.151。统计学分析显示,细胞因子各浓度组分别培养PBMC1d、3d、5d后均能增强PBMC的增殖作用,各个时间点相比较,差异有显著性(P<0.05);各浓度组间比较,单独应用IL-23各浓度组2、10、50 ng/mL之间增殖作用有显著性差异(P<0.05);相同浓度的IL-23(分别为2、10、50 ng/mL)单独应用与分别联合不同浓度IL-2(分别为10 IU/mL、100 IU/mL)组之间相比增殖作用有显著性差异(P<0.05),即IL-23浓度相同时,在一定范围内,随IL-2浓度的增加增殖作用也显著性增加。同样,当IL-2浓度相同时,联合不同浓度IL-23,随IL-23浓度的增加,各组间增殖作用也有显著性差异(P<0.05),即IL-23对PBMC的增殖有促进作用,与IL-2联合可以增强这种促进作用,在一定浓度范围内,随着作用浓度增加和作用时间延长,IL-23联合IL-2对PBMC有明显的促进增殖作用。
2. IL-23单独或联合IL-2作用后的PBMC对白血病K562细胞株杀伤活性的影响:取各浓度组IL-23(2、10、50 ng/mL)及IL-23 (ng/mL)+IL-2 (IU/mL)组(2 +10、10+10、50+10、2+100、10+100、50+100)处理1d后的PBMC,作用于K562细胞,检测其杀伤率分别为:(8.6±0.42) %、(11.8±0.59) %、(15.2±0.57)%、(10.9±1.50) %、(13.0±0.91) %、(17.9±0.65) %、(16.4±1.07) %、(20.2±1.19)%、(23.5±0.77) %;细胞因子处理3d后的PBMC对K562细胞的杀伤率分别为:(18.7±1.40) %、(23.1±0.25) %、(25.4±0.56) %、(21.4±1.06) %、(24.3±0.47) %、(30.7±0.51) %、(26.4±0.59) %、(32.0±0.98) %、(39.9±1.59)%;细胞因子处理5d后的PBMC对K562细胞的杀伤率分别为:(26.2±0.37) %、(30.1±0.68) %、(32.9±0.54)%、(29.9±0.73)%、(34.5±0.64)%、(38.4±1.83) %、(35.9±1.13) %、(43.4±3.92) %、(52.2±3.38) %;统计学分析显示,细胞因子各浓度组作用后的PBMC分别作用K562细胞1d、3d、5d后杀伤率明显增加,各个时间点间比较,差异有显著性(P<0.05);各浓度组间比较,单独应用IL-23各浓度组2、10、50 ng/mL作用后的PBMC杀伤活性有显著性差异(P<0.05);相同浓度的IL-23单独作用与联合不同浓度IL-2作用后的PBMC相比较,随IL-2浓度的增加,杀伤活性有显著性差异(P<0.05);相同浓度的IL-2联合不同浓度的IL-23作用于PBMC,随IL-23浓度的增加,PBMC的杀伤活性也有显著性差异(P<0.05)。IL-23能促进PBMC对K562细胞的杀伤活性,IL-23与IL-2联合具有协同作用,可以增强杀伤活性。在一定浓度和时间范围内,增加IL-2作用浓度和作用时间,可以进一步增强PBMC的杀伤活性。
3. IL-23单独或联合IL-2诱导前后PBMC的细胞表型的变化:IL-23 50 ng/mL与IL-23+IL-2(50ng/mL+100 IU/mL)组作用于PBMC 5d后,流式细胞术检测其细胞表型分别为,CD3+细胞为(80.7±4.37)%和(83.2±4.04)%,CD16+CD56+细胞为(8.4±0.28)%和(12.7±0.9)%,CD4+细胞为(45.2±1.4)%和(47.0±1.72)%,CD8+细胞为(34.5±2.53)%和(35.4±2.11)%;CD4+/CD8+比值分别为1.31和1.33,对照组为1.27。经单因素方差分析,以上两组与对照组相比对PBMC细胞表型的增加有显著性差异(P<0.05);两组间在CD16+CD56+细胞的表达上有显著性差异(P<0.05);两组间对CD3+细胞、CD4+细胞、CD8+细胞的表达以及CD4+/CD8+比值的变化均无显著性差异(P>0.05)。IL-23+IL-2(50+100)组可以更强的促进CD16+CD56+细胞的表达。
结论:1. IL-23能促进PBMC的增殖,联合IL-2后可以进一步促进PBMC的增殖,二者对PBMC的增殖有协同作用,并呈时间和浓度依赖性。
2. IL-23能促进PBMC对K562细胞的杀伤活性,IL-23与IL-2联合可以增强PBMC的杀伤活性,并呈时间和浓度依赖性。
3. IL-23单独或联合IL-2诱导后PBMC中CD3、CD16/56、CD4、CD8抗原的表达均增加,CD4+/CD8+比值不变,二者联合后可以进一步增加CD16/56抗原的表达。推测其抗白血病活性与诱导T细胞和NK细胞的增殖分化有关。
Objective: Leukemia is one of the common cancers in hematological system with an increasing incidence. At present ,though some leukemia patients could avhieve complete remission through chemotherapeutics and bone marrow transplantation, finally drug resistance and relapse would lead to treatment failure in majority of patients. Therefore, it is urgently needed to explore more effective treatments to improve the patient’s prognosis and quality of life. There is growing body of research evidence that the immune factors in the control of hematopoietic malignancies play an important role. Immune therapy is becoming hot spots in the treatment of hematologic malignancies.
IL-23(interleukin-23) found in the year 2000 is a cytokine excreted by activated dendritic cell (DC) and macrophage cells, it belongs to IL-12 family. It can promote proliferation of memory T cell and induce T cells and natural killer cell (NK cells) to produce interferon-γ(IFN-γ). Therefore IL-23 takes a very important part in immune defence, tumor immunity and some autoimmunity diseases. IL-2 is a self-secreted growth factor produced by T cells, it can stimulate the proliferation and differentiation of cytotoxic Tlymphocyte (CTL) and enhance the cytotoxicity of NK cell. It has now been confirmed that IL-2 has significant anti-tumor effect in vivo, but the effect depends on continuous infusion of exogenous high-dose IL-2 and associated with serious side effects. To reduce the toxic side effects, in recent years, there is a lot of reports about the low-dose IL-2 and other cytokines on proliferation and function of T cells in vitro, and most show synergistic effect.
IL-23 can promote T cells to produce less IFN-γ, and will not have serious side effects in the clinical application, so it is possible to become a safer choice of cancer therapy. Now there are no reports on the antileukemia effect of IL-23 combined with IL-2 on leukemia cells at home and abroad. In this study, We investigated these two kinds of cytokines in different concentrations either alone or in combination, to study their impacts on proliferation and anti-leukemia activity of human peripheral blood mononuclear cell. We want to provide a new treatment for leukemia, and improve the clinical outcome of hematological malignancies.
Materials and method: We separated human peripheral blood mononuclear cells (PBMC) by ficoll density gradient centrifugation, and cultured them with the culture medium Containing different concentrations of IL-23 alone or in combination with IL-2 . Cell proliferation and PBMC’s killing activity for leukemia cell lines K562 was investigated by MTT assay. Changes in cell phenotype of PBMC before or after induction were detected by Flow cytometry (FCM). The results were analyzed by SPSS16.0, and we established the standard of statistic significance asα=0.05.
Results: 1. Proliferation of human PBMC activated by IL-23 alone or combined with IL-2: Absorbance (A) of PBMC after treated with various concentrations of IL-23 ng/mL ( 2、10、50) and IL-23 ng/mL +IL-2 IU/mL (2 +10、10+10、50+10、2+100、10+100、50+100) for 1 day were 0.536±0.047、0.628±0.018、0.660±0.036 and 0.643±0.030、0.710±0.031、0.768±0.022、0.732±0.020、0.798±0.047、0.864±0.053, respectively; Absorbance (A) of PBMC after treated with various concentrations of cytokines for 3 days were 0.580±0.014、0.654±0.037、0.706±0.014 and 0.690±0.014、0.741±0.014、0.808±0.021、0.791±0.008、0.831±0.023、0.886±0.030, respectively; Absorbance (A) of PBMC after treated with various concentrations of cytokines for 5 days were 0.642±0.009、0.727±0.025、0.796±0.008 and 0.788±0.013、0.841±0.009、0.893±0.023、0.897±0.023、0.958±0.012、1.077±0.151, respectively. Cell proliferation in experiment groups after treated for 1d, 3d, 5d were significantly different (P<0.05) , at the same time, there was a significant difference between concentration groups and time groups (P<0.05). IL-23 can promote the proliferation of PBMC, IL-23 combined with IL-2 can further enhance the proliferation. Within in a certain concentration range, with increasing concentration and time, IL-23 combined with IL-2 can significantly promote the proliferation of PBMC in a time-and concentration-dependant manner.
2. Anti-leukemia cell line K562 activity of human PBMC activated by IL-23 alone or combine with IL-2: The rate of killing K562 cells of PBMC after treated with various concentrations of IL-23 ng/mL ( 2、10、50) and IL-23 ng/mL +IL-2 IU/mL (2 +10、10+10、50+10、2+100、10+100、50+100) for 1 day were(8.6±0.42) %、(11.8±0.59) %、(15.2±0.57)% and (10.9±1.50) %、(13.0±0.91) %、(17.9±0.65) %、(16.4±1.07) %、(20.2±1.19)%、(23.5±0.77) %, respectively; The rate of killing K562 cells of PBMC after treated with various concentrations of IL-23 and IL-23 +IL-2 for 3 days were:(18.7±1.40) %、(23.1±0.25) %、(25.4±0.56) % and (21.4±1.06) %、(24.3±0.47) %、(30.7±0.51) %、(26.4±0.59) %、(32.0±0.98) %、(39.9±1.59)%, respectively; The rate of killing K562 cells of PBMC after treated with various concentrations of IL-23 and IL-23 +IL-2 for 5 days were:(26.2±0.37) %、(30.1±0.68) %、(32.9±0.54)% and (29.9±0.73)%、(34.5±0.64)%、(38.4±1.83) %、(35.9±1.13) %、(43.4±3.92) %、(52.2±3.38) %, respectively. The rates of killing K562 cells of PBMC in experiment groups after treated for 1d, 3d, 5d were significantly different (P<0.05) , at the same time, there was a significant difference between concentration groups and time groups (P<0.05). IL-23 can promote the rate of killing K562 cells of PBMC, IL-23 combined with IL-2 can further enhance the rate. Within a certain concentration range, with increasing concentration and time, IL-23 combined with IL-2 can significantly promote the rate of killing K562 cells by PBMC in a time-and concentration-dependant manner.
3. Flow cytometric analysis of PBMC surface markers activated by IL-23 alone or combined with IL-2 : Surface markers of PBMC after treated with IL-23 50ng/mL and IL-23 50ng/mL +IL-2 100IU/mL for 5 days were: CD3+ cells were (80.7±4.37)% and (83.2±4.04)%,CD16+CD56+ cells were (8.4±0.28)% and (12.7±0.9)%,CD4+ cells were (45.2±1.4)% and (47.0±1.72)%,CD8+ cells were (34.5±2.53)% and (35.4±2.11)%. CD4/CD8 ratio were 1.31and 1.33,while the control was 1.27. In both sets of pairs of PBMC as compared with the control group, the increase in cell phenotype were significantly different (P<0.05). The expression of CD16+CD56+ cells between the two groups have significant difference (P<0.05), but there was no significant difference between the two groups of the expression of CD3+ cells、CD4+ cells and CD8+ cells (P>0.05).
Conclusions: 1. IL-23 can promote the proliferation of PBMC, and IL-23 combined with IL-2 can further enhance the proliferation with a concentration and time dependent fashion.
2. IL-23 can promote the rate of killing K562 cells by PBMC, and IL-23 combined with IL-2 can further enhance the rate. Within a certain concentration range, with increasing concentration and time, IL-23 combined with IL-2 can significantly promote the rate of killing K562 cells of PBMC.
3. Surface markers of PBMC after treated with IL-23 alone or combined with IL-2 were increased, and IL-23 combined with IL-2 can further enhance the antigen expression of CD16/56, while CD4/CD8 ratio remains unchanged. We speculate that there was a relationship between Anti-leukemia activity and the proliferation and differentiation of T cells and NK cells.
IL-23(interleukin-23, IL-23)是2000年发现的一种细胞因子,属于IL-12家族,主要由活化的树突状细胞(dendritic cell, DC)和巨噬细胞(Macrophage, Mφ)产生,能促进记忆T细胞的增殖、诱导T细胞和自然杀伤细胞(natural killer cell, NK cell)产生干扰素-γ(interferon-γ, IFN-γ),在机体的免疫防御、肿瘤免疫及某些自身免疫性疾病中起着重要作用。IL-2是由T细胞产生的一种自分泌生长因子,能刺激细胞毒性T淋巴细胞(cytotoxic Tlymphocyte, CTL)的增殖与分化,增强NK细胞的细胞毒活性。现已证实,IL-2在体内具有显著的抗肿瘤作用,但该作用依赖于外源性大剂量IL-2的持续输注且常伴有严重的毒副作用。为减轻毒副作用,近年来相继有不少关于小剂量IL-2与其他细胞因子联合对T细胞体外增殖及功能影响的报道,且多呈协同增强效应。
IL-23能促进T细胞产生IFN-γ,而且因其诱导产生的IFN-γ较少而不会在临床应用时产生严重的毒副反应,因此有可能成为肿瘤治疗中一种更为安全的选择。目前国内外尚无有关IL-23与IL-2联合对白血病细胞影响的报道,本课题拟通过上述两种细胞因子在不同浓度单独或联合应用对人外周血单个核细胞增殖及抗白血病活性影响的研究,希望为白血病的免疫治疗提供新的线索,以进一步提高临床恶性血液病的治疗效果。
方法:用Ficoll密度梯度离心法分离人外周血单个核细胞(peripheral blood mononuclear cells, PBMC),以含不同浓度IL-23单独或联合IL-2的培养液培养后,用MTT比色法测定PBMC的增殖情况及PBMC对白血病K562细胞株的杀伤活性,采用流式细胞术分析诱导前后PBMC的细胞表型变化。所得结果运用SPSS16.0统计软件包处理,以α=0.05为显著性检验水准。
结果:1. IL-23单独或联合IL-2对PBMC增殖的影响:IL-23各浓度组2、10、50 ng/mL,及IL-23(ng/mL)+IL-2(IU/mL)组2 +10、10+10、50+10、2+100、10+100、50+100,作用于PBMC 1d后测吸光度A值分别为0.536±0.047、0.628±0.018、0.660±0.036、0.643±0.030、0.710±0.031、0.768±0.022、0.732±0.020、0.798±0.047、0.864±0.053;3d后测A值分别为0.580±0.014、0.654±0.037、0.706±0.014、0.690±0.014、0.741±0.014、0.808±0.021、0.791±0.008、0.831±0.023、0.886±0.030;5d后A值分别为0.642±0.009、0.727±0.025、0.796±0.008、0.788±0.013、0.841±0.009、0.893±0.023、0.897±0.023、0.958±0.012、1.077±0.151。统计学分析显示,细胞因子各浓度组分别培养PBMC1d、3d、5d后均能增强PBMC的增殖作用,各个时间点相比较,差异有显著性(P<0.05);各浓度组间比较,单独应用IL-23各浓度组2、10、50 ng/mL之间增殖作用有显著性差异(P<0.05);相同浓度的IL-23(分别为2、10、50 ng/mL)单独应用与分别联合不同浓度IL-2(分别为10 IU/mL、100 IU/mL)组之间相比增殖作用有显著性差异(P<0.05),即IL-23浓度相同时,在一定范围内,随IL-2浓度的增加增殖作用也显著性增加。同样,当IL-2浓度相同时,联合不同浓度IL-23,随IL-23浓度的增加,各组间增殖作用也有显著性差异(P<0.05),即IL-23对PBMC的增殖有促进作用,与IL-2联合可以增强这种促进作用,在一定浓度范围内,随着作用浓度增加和作用时间延长,IL-23联合IL-2对PBMC有明显的促进增殖作用。
2. IL-23单独或联合IL-2作用后的PBMC对白血病K562细胞株杀伤活性的影响:取各浓度组IL-23(2、10、50 ng/mL)及IL-23 (ng/mL)+IL-2 (IU/mL)组(2 +10、10+10、50+10、2+100、10+100、50+100)处理1d后的PBMC,作用于K562细胞,检测其杀伤率分别为:(8.6±0.42) %、(11.8±0.59) %、(15.2±0.57)%、(10.9±1.50) %、(13.0±0.91) %、(17.9±0.65) %、(16.4±1.07) %、(20.2±1.19)%、(23.5±0.77) %;细胞因子处理3d后的PBMC对K562细胞的杀伤率分别为:(18.7±1.40) %、(23.1±0.25) %、(25.4±0.56) %、(21.4±1.06) %、(24.3±0.47) %、(30.7±0.51) %、(26.4±0.59) %、(32.0±0.98) %、(39.9±1.59)%;细胞因子处理5d后的PBMC对K562细胞的杀伤率分别为:(26.2±0.37) %、(30.1±0.68) %、(32.9±0.54)%、(29.9±0.73)%、(34.5±0.64)%、(38.4±1.83) %、(35.9±1.13) %、(43.4±3.92) %、(52.2±3.38) %;统计学分析显示,细胞因子各浓度组作用后的PBMC分别作用K562细胞1d、3d、5d后杀伤率明显增加,各个时间点间比较,差异有显著性(P<0.05);各浓度组间比较,单独应用IL-23各浓度组2、10、50 ng/mL作用后的PBMC杀伤活性有显著性差异(P<0.05);相同浓度的IL-23单独作用与联合不同浓度IL-2作用后的PBMC相比较,随IL-2浓度的增加,杀伤活性有显著性差异(P<0.05);相同浓度的IL-2联合不同浓度的IL-23作用于PBMC,随IL-23浓度的增加,PBMC的杀伤活性也有显著性差异(P<0.05)。IL-23能促进PBMC对K562细胞的杀伤活性,IL-23与IL-2联合具有协同作用,可以增强杀伤活性。在一定浓度和时间范围内,增加IL-2作用浓度和作用时间,可以进一步增强PBMC的杀伤活性。
3. IL-23单独或联合IL-2诱导前后PBMC的细胞表型的变化:IL-23 50 ng/mL与IL-23+IL-2(50ng/mL+100 IU/mL)组作用于PBMC 5d后,流式细胞术检测其细胞表型分别为,CD3+细胞为(80.7±4.37)%和(83.2±4.04)%,CD16+CD56+细胞为(8.4±0.28)%和(12.7±0.9)%,CD4+细胞为(45.2±1.4)%和(47.0±1.72)%,CD8+细胞为(34.5±2.53)%和(35.4±2.11)%;CD4+/CD8+比值分别为1.31和1.33,对照组为1.27。经单因素方差分析,以上两组与对照组相比对PBMC细胞表型的增加有显著性差异(P<0.05);两组间在CD16+CD56+细胞的表达上有显著性差异(P<0.05);两组间对CD3+细胞、CD4+细胞、CD8+细胞的表达以及CD4+/CD8+比值的变化均无显著性差异(P>0.05)。IL-23+IL-2(50+100)组可以更强的促进CD16+CD56+细胞的表达。
结论:1. IL-23能促进PBMC的增殖,联合IL-2后可以进一步促进PBMC的增殖,二者对PBMC的增殖有协同作用,并呈时间和浓度依赖性。
2. IL-23能促进PBMC对K562细胞的杀伤活性,IL-23与IL-2联合可以增强PBMC的杀伤活性,并呈时间和浓度依赖性。
3. IL-23单独或联合IL-2诱导后PBMC中CD3、CD16/56、CD4、CD8抗原的表达均增加,CD4+/CD8+比值不变,二者联合后可以进一步增加CD16/56抗原的表达。推测其抗白血病活性与诱导T细胞和NK细胞的增殖分化有关。
Objective: Leukemia is one of the common cancers in hematological system with an increasing incidence. At present ,though some leukemia patients could avhieve complete remission through chemotherapeutics and bone marrow transplantation, finally drug resistance and relapse would lead to treatment failure in majority of patients. Therefore, it is urgently needed to explore more effective treatments to improve the patient’s prognosis and quality of life. There is growing body of research evidence that the immune factors in the control of hematopoietic malignancies play an important role. Immune therapy is becoming hot spots in the treatment of hematologic malignancies.
IL-23(interleukin-23) found in the year 2000 is a cytokine excreted by activated dendritic cell (DC) and macrophage cells, it belongs to IL-12 family. It can promote proliferation of memory T cell and induce T cells and natural killer cell (NK cells) to produce interferon-γ(IFN-γ). Therefore IL-23 takes a very important part in immune defence, tumor immunity and some autoimmunity diseases. IL-2 is a self-secreted growth factor produced by T cells, it can stimulate the proliferation and differentiation of cytotoxic Tlymphocyte (CTL) and enhance the cytotoxicity of NK cell. It has now been confirmed that IL-2 has significant anti-tumor effect in vivo, but the effect depends on continuous infusion of exogenous high-dose IL-2 and associated with serious side effects. To reduce the toxic side effects, in recent years, there is a lot of reports about the low-dose IL-2 and other cytokines on proliferation and function of T cells in vitro, and most show synergistic effect.
IL-23 can promote T cells to produce less IFN-γ, and will not have serious side effects in the clinical application, so it is possible to become a safer choice of cancer therapy. Now there are no reports on the antileukemia effect of IL-23 combined with IL-2 on leukemia cells at home and abroad. In this study, We investigated these two kinds of cytokines in different concentrations either alone or in combination, to study their impacts on proliferation and anti-leukemia activity of human peripheral blood mononuclear cell. We want to provide a new treatment for leukemia, and improve the clinical outcome of hematological malignancies.
Materials and method: We separated human peripheral blood mononuclear cells (PBMC) by ficoll density gradient centrifugation, and cultured them with the culture medium Containing different concentrations of IL-23 alone or in combination with IL-2 . Cell proliferation and PBMC’s killing activity for leukemia cell lines K562 was investigated by MTT assay. Changes in cell phenotype of PBMC before or after induction were detected by Flow cytometry (FCM). The results were analyzed by SPSS16.0, and we established the standard of statistic significance asα=0.05.
Results: 1. Proliferation of human PBMC activated by IL-23 alone or combined with IL-2: Absorbance (A) of PBMC after treated with various concentrations of IL-23 ng/mL ( 2、10、50) and IL-23 ng/mL +IL-2 IU/mL (2 +10、10+10、50+10、2+100、10+100、50+100) for 1 day were 0.536±0.047、0.628±0.018、0.660±0.036 and 0.643±0.030、0.710±0.031、0.768±0.022、0.732±0.020、0.798±0.047、0.864±0.053, respectively; Absorbance (A) of PBMC after treated with various concentrations of cytokines for 3 days were 0.580±0.014、0.654±0.037、0.706±0.014 and 0.690±0.014、0.741±0.014、0.808±0.021、0.791±0.008、0.831±0.023、0.886±0.030, respectively; Absorbance (A) of PBMC after treated with various concentrations of cytokines for 5 days were 0.642±0.009、0.727±0.025、0.796±0.008 and 0.788±0.013、0.841±0.009、0.893±0.023、0.897±0.023、0.958±0.012、1.077±0.151, respectively. Cell proliferation in experiment groups after treated for 1d, 3d, 5d were significantly different (P<0.05) , at the same time, there was a significant difference between concentration groups and time groups (P<0.05). IL-23 can promote the proliferation of PBMC, IL-23 combined with IL-2 can further enhance the proliferation. Within in a certain concentration range, with increasing concentration and time, IL-23 combined with IL-2 can significantly promote the proliferation of PBMC in a time-and concentration-dependant manner.
2. Anti-leukemia cell line K562 activity of human PBMC activated by IL-23 alone or combine with IL-2: The rate of killing K562 cells of PBMC after treated with various concentrations of IL-23 ng/mL ( 2、10、50) and IL-23 ng/mL +IL-2 IU/mL (2 +10、10+10、50+10、2+100、10+100、50+100) for 1 day were(8.6±0.42) %、(11.8±0.59) %、(15.2±0.57)% and (10.9±1.50) %、(13.0±0.91) %、(17.9±0.65) %、(16.4±1.07) %、(20.2±1.19)%、(23.5±0.77) %, respectively; The rate of killing K562 cells of PBMC after treated with various concentrations of IL-23 and IL-23 +IL-2 for 3 days were:(18.7±1.40) %、(23.1±0.25) %、(25.4±0.56) % and (21.4±1.06) %、(24.3±0.47) %、(30.7±0.51) %、(26.4±0.59) %、(32.0±0.98) %、(39.9±1.59)%, respectively; The rate of killing K562 cells of PBMC after treated with various concentrations of IL-23 and IL-23 +IL-2 for 5 days were:(26.2±0.37) %、(30.1±0.68) %、(32.9±0.54)% and (29.9±0.73)%、(34.5±0.64)%、(38.4±1.83) %、(35.9±1.13) %、(43.4±3.92) %、(52.2±3.38) %, respectively. The rates of killing K562 cells of PBMC in experiment groups after treated for 1d, 3d, 5d were significantly different (P<0.05) , at the same time, there was a significant difference between concentration groups and time groups (P<0.05). IL-23 can promote the rate of killing K562 cells of PBMC, IL-23 combined with IL-2 can further enhance the rate. Within a certain concentration range, with increasing concentration and time, IL-23 combined with IL-2 can significantly promote the rate of killing K562 cells by PBMC in a time-and concentration-dependant manner.
3. Flow cytometric analysis of PBMC surface markers activated by IL-23 alone or combined with IL-2 : Surface markers of PBMC after treated with IL-23 50ng/mL and IL-23 50ng/mL +IL-2 100IU/mL for 5 days were: CD3+ cells were (80.7±4.37)% and (83.2±4.04)%,CD16+CD56+ cells were (8.4±0.28)% and (12.7±0.9)%,CD4+ cells were (45.2±1.4)% and (47.0±1.72)%,CD8+ cells were (34.5±2.53)% and (35.4±2.11)%. CD4/CD8 ratio were 1.31and 1.33,while the control was 1.27. In both sets of pairs of PBMC as compared with the control group, the increase in cell phenotype were significantly different (P<0.05). The expression of CD16+CD56+ cells between the two groups have significant difference (P<0.05), but there was no significant difference between the two groups of the expression of CD3+ cells、CD4+ cells and CD8+ cells (P>0.05).
Conclusions: 1. IL-23 can promote the proliferation of PBMC, and IL-23 combined with IL-2 can further enhance the proliferation with a concentration and time dependent fashion.
2. IL-23 can promote the rate of killing K562 cells by PBMC, and IL-23 combined with IL-2 can further enhance the rate. Within a certain concentration range, with increasing concentration and time, IL-23 combined with IL-2 can significantly promote the rate of killing K562 cells of PBMC.
3. Surface markers of PBMC after treated with IL-23 alone or combined with IL-2 were increased, and IL-23 combined with IL-2 can further enhance the antigen expression of CD16/56, while CD4/CD8 ratio remains unchanged. We speculate that there was a relationship between Anti-leukemia activity and the proliferation and differentiation of T cells and NK cells.
引文
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