马钱子碱对再生障碍性贫血患者T细胞分泌功能及T-bet表达的影响
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摘要
马钱子碱对再生障碍性贫血患者T淋巴细胞分泌功能及T-bet表达的影响
目的通过观察不同浓度马钱子碱对再生障碍性贫血患者(简称再障)外周血T淋巴细胞分泌TNF-α、IFN-γ、IL-4的水平及T-bet mRNA表达的影响,探讨中药马钱子治疗再生障碍性贫血的作用机制。
方法再障患者及健康志愿者各10例。采集各研究对象外周血10ml,采用密度梯度离心法及免疫磁珠法分离、纯化外周血T淋巴细胞。获得的T淋巴细胞需进行活细胞计数及流式细胞仪纯度测定,活细胞比例及纯度均在95%以上者进入下一步实验。设健康志愿者T淋巴细胞为正常对照组,再障T淋巴细胞为实验组。正常对照组不作干预,将实验组T淋巴细胞随机分为四组,分别加入0ug/ml、100ug/ml、200ug／ml、400ug／ml浓度的马钱子碱进行干预(各组均加入终浓度为100ug／ml的PHA)。培养72h后,收集各组T淋巴细胞培养上清液,酶联免疫吸附试验(ELISA)检测其中TNF-α、IFN-γ及IL-4的水平；收集各组T淋巴细胞,提取RNA,采用逆转录—聚合酶链式反应法(RT-PCR)检测各组T-bet mRNA的表达水平。
结果
1.与正常对照组相比,再障空白组T淋巴细胞培养上清液中TNF-α、IFN-γ的水平均明显增高,而IL-4水平则明显降低(p均<0.01)；再障空白组T-bet mRNA表达水平显著高于正常对照组(p<0.01)。
2.再障100ug/ml、200ug/ml、400ug／ml马钱子碱干预组TNF-α、IFN-γ水平较空白组均明显降低,差异均具统计学意义；且随着马钱子碱作用浓度的增高,TNF-α及IFN-γ水平均逐渐下降,各干预组间比较差别均具统计学意义,且TNF-α及IFN-γ均于400ug/ml组降至正常水平。
3.再障不同浓度马钱子碱干预组之间比较,IL-4水平无明显差异(p>0.05)。
4.再障100ug/ml、200ug／ml、400ug/ml马钱子碱干预组T-bet mRNA表达水平较空白组明显降低(p均<0.01)；与100ug/ml组相比,200ug/ml组、400ug／ml组T-bet mRNA表达水平均降低(p均<0.01)；且200ug／ml组、400ug/ml组T-bet mRNA表达均降至正常水平。
5.再障T-bet mRNA表达水平与IFN-γ水平呈正相关(r=0.795,p<0.01)。
结论
1.再障患者外周血T淋巴细胞分泌TNF-α、IFN-γ水平较正常人明显升高,而IL-4水平则明显降低。
2.再障患者外周血T淋巴细胞T-bet mRNA表达水平明显高于正常人。
3.马钱子碱可降低再障患者造血负调控因子TNF-α、IFN-γ的水平及转录因子T-betmRNA的表达水平,且作用呈剂量依赖性,其中,IFN-γ的产生可能主要由T-bet调控。
4.马钱子碱可能通过上述机制在再障的治疗过程中发挥作用,为马钱子的临床应用提供了科学依据。
Effects of brucine on secretion of T lymphocytes and expression of T-bet in patients with aplastic anemia
Objective
By observing the effects of brucine on levels of TNF-α、IFN-γ、IL-4,were secreted by peripheral blood T lymphocytes and expression of T-bet in patients with aplastic anemia, to investigate the mechanism of nux vomica in the treatment for aplastic anemia.
Method
Peripheral blood T lymphocytes were isolated, purified from 10 patients with aplastic anemia and 10 healthy volunteers. Collected 10 ml Peripheral blood and separated T lymphocytes by density gradient centrifugation method and immunomagnetic beads. Variable count and purity measurement by flow cytometry were necessary to further research (>95%). T lymphocytes from healthy volunteers were normal control groups and no interference. T lymphocytes from patients were experimental groups, were divided into 4 groups randomly, black group, 100ug/ml,200ug/ml and 400ug/ml brucine treated group (100ug/ml PHA was added to all groups) respectively. After cultured for 72 hours, the levels of TNF-a, IFN-y, IL-4 in the culture supernatant were detected by mean of Enzyme-linked immunosorbant assay (ELISA); and collected T lymphocytes and abstracted RNA in all groups. The expression levels of T-bet were detected by mean of Reverse transcription-polymerase chain reaction (RT-PCR).
Results
1. Compared with normal control, the levels of TNF-α(p<0.01) and IFN-γ(p<0.01) in the culture supernatant of blank group were significantly increased, and IL-4 were significantly decreased (p<0.01). The expression level of T-bet in blank group was higher than that of normal control (p<0.01).
2. The levels of TNF-αand IFN-γin brucine treated groups were lower than blank group, and depended on concentration of brucine; The difference among brucine treated groups were significant. Both of them in 400ug/ml group were same as that of normal control.
3. The levels of IL-4 were invariable in brucine treated groups (p>0.05).
4. Compared with normal control, the expression levels of T-bet in 100ug/ml (p<0.01), 200ug/ml (p<0.01) and 400ug/ml group (p<0.01) were significantly decreased; 200ug/ml (p<0.01) and 400ug/ml group (p<0.01) were lower than 100ug/ml group; The difference among normal control,200ug/ml and 400ug/ml group was no significant.
5. In experimental groups, the expression levels of T-bet were positive correlation with the levels of IFN-y(r=0.795, p<0.01).
Conclusion
1. The levels of TNF-a, IFN-y in patients with aplastic anemia were higher than healthy person, but the levels of IL-4 were lower.
2. The level of T-bet mRNA in patients with aplastic anemia was higher than healthy person.
2. Brucine could reduce the levels of negative regulation factors TNF-a and IFN-y and the expression of T-bet in patients with aplastic anemia, and the effects depended on concentration of brucine, and the level of IFN-y maybe mainly regulated by T-bet.
3. Above that was not only the evidence for brucine in the treatment for aplastic anemia, but also the scientific evidence for use of nux vomica in clinic.
目的通过观察不同浓度马钱子碱对再生障碍性贫血患者(简称再障)外周血T淋巴细胞分泌TNF-α、IFN-γ、IL-4的水平及T-bet mRNA表达的影响,探讨中药马钱子治疗再生障碍性贫血的作用机制。
方法再障患者及健康志愿者各10例。采集各研究对象外周血10ml,采用密度梯度离心法及免疫磁珠法分离、纯化外周血T淋巴细胞。获得的T淋巴细胞需进行活细胞计数及流式细胞仪纯度测定,活细胞比例及纯度均在95%以上者进入下一步实验。设健康志愿者T淋巴细胞为正常对照组,再障T淋巴细胞为实验组。正常对照组不作干预,将实验组T淋巴细胞随机分为四组,分别加入0ug/ml、100ug/ml、200ug／ml、400ug／ml浓度的马钱子碱进行干预(各组均加入终浓度为100ug／ml的PHA)。培养72h后,收集各组T淋巴细胞培养上清液,酶联免疫吸附试验(ELISA)检测其中TNF-α、IFN-γ及IL-4的水平；收集各组T淋巴细胞,提取RNA,采用逆转录—聚合酶链式反应法(RT-PCR)检测各组T-bet mRNA的表达水平。
结果
1.与正常对照组相比,再障空白组T淋巴细胞培养上清液中TNF-α、IFN-γ的水平均明显增高,而IL-4水平则明显降低(p均<0.01)；再障空白组T-bet mRNA表达水平显著高于正常对照组(p<0.01)。
2.再障100ug/ml、200ug/ml、400ug／ml马钱子碱干预组TNF-α、IFN-γ水平较空白组均明显降低,差异均具统计学意义；且随着马钱子碱作用浓度的增高,TNF-α及IFN-γ水平均逐渐下降,各干预组间比较差别均具统计学意义,且TNF-α及IFN-γ均于400ug/ml组降至正常水平。
3.再障不同浓度马钱子碱干预组之间比较,IL-4水平无明显差异(p>0.05)。
4.再障100ug/ml、200ug／ml、400ug/ml马钱子碱干预组T-bet mRNA表达水平较空白组明显降低(p均<0.01)；与100ug/ml组相比,200ug/ml组、400ug／ml组T-bet mRNA表达水平均降低(p均<0.01)；且200ug／ml组、400ug/ml组T-bet mRNA表达均降至正常水平。
5.再障T-bet mRNA表达水平与IFN-γ水平呈正相关(r=0.795,p<0.01)。
结论
1.再障患者外周血T淋巴细胞分泌TNF-α、IFN-γ水平较正常人明显升高,而IL-4水平则明显降低。
2.再障患者外周血T淋巴细胞T-bet mRNA表达水平明显高于正常人。
3.马钱子碱可降低再障患者造血负调控因子TNF-α、IFN-γ的水平及转录因子T-betmRNA的表达水平,且作用呈剂量依赖性,其中,IFN-γ的产生可能主要由T-bet调控。
4.马钱子碱可能通过上述机制在再障的治疗过程中发挥作用,为马钱子的临床应用提供了科学依据。
Effects of brucine on secretion of T lymphocytes and expression of T-bet in patients with aplastic anemia
Objective
By observing the effects of brucine on levels of TNF-α、IFN-γ、IL-4,were secreted by peripheral blood T lymphocytes and expression of T-bet in patients with aplastic anemia, to investigate the mechanism of nux vomica in the treatment for aplastic anemia.
Method
Peripheral blood T lymphocytes were isolated, purified from 10 patients with aplastic anemia and 10 healthy volunteers. Collected 10 ml Peripheral blood and separated T lymphocytes by density gradient centrifugation method and immunomagnetic beads. Variable count and purity measurement by flow cytometry were necessary to further research (>95%). T lymphocytes from healthy volunteers were normal control groups and no interference. T lymphocytes from patients were experimental groups, were divided into 4 groups randomly, black group, 100ug/ml,200ug/ml and 400ug/ml brucine treated group (100ug/ml PHA was added to all groups) respectively. After cultured for 72 hours, the levels of TNF-a, IFN-y, IL-4 in the culture supernatant were detected by mean of Enzyme-linked immunosorbant assay (ELISA); and collected T lymphocytes and abstracted RNA in all groups. The expression levels of T-bet were detected by mean of Reverse transcription-polymerase chain reaction (RT-PCR).
Results
1. Compared with normal control, the levels of TNF-α(p<0.01) and IFN-γ(p<0.01) in the culture supernatant of blank group were significantly increased, and IL-4 were significantly decreased (p<0.01). The expression level of T-bet in blank group was higher than that of normal control (p<0.01).
2. The levels of TNF-αand IFN-γin brucine treated groups were lower than blank group, and depended on concentration of brucine; The difference among brucine treated groups were significant. Both of them in 400ug/ml group were same as that of normal control.
3. The levels of IL-4 were invariable in brucine treated groups (p>0.05).
4. Compared with normal control, the expression levels of T-bet in 100ug/ml (p<0.01), 200ug/ml (p<0.01) and 400ug/ml group (p<0.01) were significantly decreased; 200ug/ml (p<0.01) and 400ug/ml group (p<0.01) were lower than 100ug/ml group; The difference among normal control,200ug/ml and 400ug/ml group was no significant.
5. In experimental groups, the expression levels of T-bet were positive correlation with the levels of IFN-y(r=0.795, p<0.01).
Conclusion
1. The levels of TNF-a, IFN-y in patients with aplastic anemia were higher than healthy person, but the levels of IL-4 were lower.
2. The level of T-bet mRNA in patients with aplastic anemia was higher than healthy person.
2. Brucine could reduce the levels of negative regulation factors TNF-a and IFN-y and the expression of T-bet in patients with aplastic anemia, and the effects depended on concentration of brucine, and the level of IFN-y maybe mainly regulated by T-bet.
3. Above that was not only the evidence for brucine in the treatment for aplastic anemia, but also the scientific evidence for use of nux vomica in clinic.
引文
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[11]Deng XK,Yin W,Li WD,et al.The anti-tumor effects os alkaloids from the seeds of Strychnos nux-vomicaon HepG2 cells and its possible mechanism. J Ethnopharmacol,2006,106(2):179-186.
[12]余志艳,李平.马钱子碱对乳腺癌细胞MDA-MB-231作用的实验研究.安徽医药,2008,12(9)：779-782.
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[21]张斌严,魏武,申徐良.复方马钱子散治疗慢性再生障碍性贫血疗效观察.山西医药杂志,1991,20：348.
[22]李文佳.中西医结合治疗再生障碍性贫血35例.陕西中医,2003,24(3)：203-204.
[23]毕玲玲,马琴,王树庆,等.补肾活血通络法对儿童慢性再生障碍性贫血骨髓CD34+细胞凋亡影响的临床研究.新中医,2005,37(1)：28-29.
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