P物质对NK92MI细胞NKG2A和NKG2D分子表达的影响

发布时间：2018-05-10 13:21

  本文选题：物质 + NK92MI　； 参考：《中国医科大学》2008年硕士论文

【摘要】： 前言 P物质(Substance P,SP)是神经内分泌系统反应时分泌的一种生物活性多肽,广泛分布于细的初级传入神经纤维内。躯体和精神刺激作用于神经系统时,既可通过神经的直接作用影响免疫系统的功能活动,也可通过释放内分泌激素或其它细胞因子对免疫功能进行调节,而经典神经递质、神经肽则是直接作用于免疫活性细胞和其他参与免疫反应细胞的重要调节物质。SP的生物活性是通过与速激肽受体(NK-R)的亚型NK-1(neuorkinin-1),NK-2,NK-3的结合表现出来的。 SP与临床的多种疾病有关,SP在不同条件下所引起的作用具有复杂性和多样性。进一步研究SP在神经内分泌-免疫网络中的具体作用机制会对炎性疾病及免疫性疾病的病理生理本质有更新的认识,以期对临床防治有所帮助。 NK细胞是天然免疫系统中一类十分重要的淋巴细胞,在机体抗感染、抗肿瘤、免疫调节及造血系统等方面发挥重要作用。NK细胞通过细胞介导的细胞毒杀伤非己细胞,并通过释放多种细胞因子(如IFN,GM-CSF和TNF)和趋化因子调节天然免疫和获得性免疫。NK细胞的杀伤活性由其抑制性受体和活化受体调控。 SP在体内体外均可调节多种免疫功能,但SP对NK细胞功能影响的报道较少。为此,我们检测了SP是否可以促进NK细胞增殖和杀伤活性,并测定SP对NKG2A/D表达的影响,以研究SP对NK细胞功能的影响及可能的作用机制。 材料和方法 1、细胞培养和处理,收集对数期细胞加不同浓度P物质培养24h; 2、MTT法检测不同浓度SP(10~(-6)mol/L,10~(-8)mol/L,10~(-9)mol/L,10~(-10)mol/L,10~(-12)mol/L,10~(-14)mol/L)对NK92-MI细胞增殖和细胞周期的影响; 3、MTT释放法测定不同浓度P物质对NK92-MI细胞杀伤活性的影响; 4、流式细胞术检测比较NK92-MI细胞抑制性受体NKG2A和活化性受体NKG2D的膜表达情况; 5、RT-PCR检测比较NKG2A和NKG2D的mRNA表达水平。 结果 1、10~(-6)M-10~(-10)M浓度的SP对NK92-MI细胞的增殖和活力有促进作用,与对照组比较有显著性差异。 2、SP在10~(-6)mol/L,10~(-8)mol/L,10~(-14)mol/L浓度时,作用24h后,实验组的DNA合成前期细胞所占百分比(G1%)下降,而DNA合成期细胞所占百分比(S%)增高,反应增殖活力的增殖指数PrI值有明显增加:10~(-4)mol/L的SP使S期细胞比例下调,G1期百分比增加。 3、效靶比为4:1时,各浓度SP;NNK细胞杀伤活性显示有增强作用,此作用强度随SP浓度的增加而降低。效靶比1:1时,SP对NK92-MI细胞杀伤活性的影响不明显。 4、SP与NK92-MI细胞共孵育24h,FCAS检测结果表明,NKG2A在10~(-8)-10~(-12)M浓度SP作用下NKG2A表达均有上调,当SP在10~(-14)M浓度时,NKG2A的表达下调,但无统计学意义。NKG2D各组SP浓度较对照组均有不同程度上调。 5、各浓度SP与NK92-MI细胞共孵育24h,采用RT-PCR方法对部分功能受体mRNA表达变化情况进行检测,结果显示NKG2A各浓度SP组较对照组mRNA均有不同程度增强;各浓度组NKG2D的mRNA表达均有上调。 结论 一定范围浓度的SP对NK92MI细胞的增殖有促进影响(存在剂量依赖性)。一定浓度的SP能够增强NK92-MI细胞的杀伤活性,各浓度SP均上调NKG2D的膜表达水平和mRNA水平(两者变化趋势完全一致),上调作用随浓度增大而增强。10~(-8)M、10~(-10)M和10~(-12)M组NKG2A膜表达和mRNA水平均有上调,10~(-6)M组mRNA水平上调而膜表达无明显变化。低浓度的SP(10~(-14)M)不能上调NKG2A的膜表达,而NKG2D表达上调,可能为此浓度SP促杀伤活性显著增强的原因之一。
[Abstract]:Foreword


Substance P ( SP ) is a kind of bioactive polypeptide secreted by neuroendocrine system . It is widely distributed in the primary afferent nerve fiber . The body and the spirit stimulate the function of the immune system through the direct action of nerve , but the classical neurotransmitters and neuropeptides act directly on immune active cells and other important regulating substances involved in the immune response cells . The biological activity of SP is demonstrated by the combination of the subtypes of NK - 1 , NK - 2 , NK - 3 with tachykinin receptor ( NK - R ) .


SP is associated with a variety of clinical diseases , and SP plays a role in complexity and diversity under different conditions . Further research on the specific mechanism of SP in neuroendocrine - immune network can be helpful for clinical prevention and treatment .


NK cells are a very important lymphocyte in innate immune system . NK cells play an important role in anti - infection , anti - tumor , immunoregulation and hematopoietic system . NK cells can kill non - hexyl cells through cell - mediated cytotoxicity and regulate innate immunity and acquired immunity by releasing various cytokines ( such as IFN , GM - CSF and TNF ) and chemokine . The killing activity of NK cells is regulated by inhibitory receptors and activated receptors .


The effects of SP on NK cell proliferation and killing activity were detected , and the effects of SP on NKG2A / D expression were measured to investigate the effects of SP on NK cell function and possible mechanism .


Materials and Methods


1 , cell culture and treatment , collection of logarithmic phase cells plus different concentration of substance P for 24h ;


2 . The effects of different concentrations of SP ( 10 ~ ( -6 ) mol / L , 10 ~ ( -8 ) mol / L , 10 ~ ( -9 ) mol / L , 10 ~ ( -10 ) mol / L , 10 ~ ( -12 ) mol / L , 10 ~ ( -14 ) mol / L ) on the proliferation and cell cycle of NK92 - MI cells were determined by MTT assay .


3 . The effects of different concentrations of substance P on the cytotoxicity of NK92 - MI cells were determined by MTT assay .


4 . Flow cytometry was used to detect the expression of NKG2A and NKG2D in NKG2A and NKG2D .


5 . The mRNA expression level of NKG2A and NKG2D was detected by RT - PCR .


Results


The effects of SP on the proliferation and viability of NK92 - MI cells in the concentration of 1 , 10 ~ ( -6 ) M - 10 ~ ( -10 ) M were significantly different from those in the control group .


After 24h , the percentage of DNA synthesis ( G1 % ) in the experimental group decreased , while the percentage of the cells in the experimental group ( S % ) increased , and the proliferation index PrI of the cells increased significantly : 10 - ( -4 ) mol / L SP decreased the proportion of S - phase cells and the percentage of G1 phase increased .


3 . When the target ratio was 4 鈭，

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