TLR4信号调节小鼠B淋巴细胞发育的实验研究

发布时间：2018-06-02 13:18

本文选题：immature + B细胞　；参考：《第四军医大学》2008年博士论文

【摘要】： 目的:B淋巴细胞起源于多能造血干细胞,在整个发育过程当中经历了多个发育阶段,造血干细胞必须成功经过各个发育阶段后,才能成为一个正常发挥免疫应答功能的成熟B淋巴细胞。但是B细胞发育的精确调控机制可能会因许多病理条件而被扰乱,特别值得关注的是炎症的影响。Toll样受体是一种能够识别多种病原相关分子的蛋白,一旦识别病原相关分子之后,它们可以启动一系列反应激活免疫系统清除入侵病原微生物,从而构成机体免疫的第一道防线;TLR诱导产生炎性细胞因子,而许多这类因子对B细胞发育均有抑制作用。这两个原本独立的现象提示TLR信号与B细胞发育间存在某种关联。考虑到TLR信号对成熟B细胞功能具有直接而深刻的影响,一个显而易见的问题是TLR信号是否对发育中的B细胞也有类似的直接作用。在本试验中,我们检测了TLR4在各个阶段B细胞中的表达水平,检测了正常C3H/He小鼠和C3H/HeJ小鼠之间B细胞分布的区别,同时通过体内和体外试验检测了TLR4信号对发育中的B细胞的影响。方法:通过实时荧光定量PCR检测TLR4在各个B细胞亚群中的表达水平;对C57BL/6小鼠腹膜内注射LPS,在不同时间点取骨髓通过细胞计数及流式检测B细胞及各个亚群的变化;流式细胞仪分析正常C3H/He小鼠和C3H/HeJ小鼠骨髓内全B细胞以及B细胞亚群的比例; FACs分选出的未成熟B细胞体外培养,LPS处理后通过CFSE染色检测增殖代数,通过流式检测培养细胞表面IgD、CD21、CD23、CD86的表达;FACs分选出的未成熟B细胞体外培养,分别用LPS、anti-μ或者LPS+anti-μ处理之后,流式检测细胞分化和凋亡的相关指标。结果:(1)实时荧光定量PCR结果显示小鼠骨髓中四个B细胞亚群均表达TLR4,并且mature B细胞的表达水平明显高于前三个亚群,而脾来源的mature B细胞的表达水平又明显高于骨髓来源的mature B细胞;(2)对C57BL/6小鼠应用LPS处理后,发现处理后的小鼠骨髓细胞总数、全B细胞、pre B细胞和immature B细胞计数在第1周会明显下降,随后会出现反弹性的异常增高,在第3周的时候逐渐恢复正常水平;(3)在C3H/HeJ小鼠骨髓中,全B细胞、pro B、pre B细胞占总骨髓细胞的比例均明显高于正常的C3H/He小鼠,而immature B和mature B细胞的比例没有统计学意义的差别;(4)Immature B细胞体外培养体系中应用LPS刺激后,IgD、CD21、CD23、CD86的阳性率均明显高于阴性对照组;(5)Immature B细胞体外培养体系中应用LPS、anti-μ或者LPS+anti-μ刺激后,发现LPS+anti-μ组的凋亡细胞比例明显低于anti-μ组,而其IgD阳性率则明显低于LPS组。结论:(1)TLR4的表达水平在新生B淋巴细胞迁出骨髓后会有一个大幅度的提高,这种提高可能是受到外周环境当中的微生物组分和各种细胞因子的影响;(2)当小鼠体内TLR4信号过强时,骨髓内的B淋巴细胞也会出现异常发育,至于这种异常是由于早期造血干细胞向淋系的发育受阻还是由于B淋巴细胞的发育加速,经过我们的分析,这两种原因可能在这一过程中都起着一定的作用;(3)在TLR4功能缺失的小鼠体内B淋巴细胞的发育是异常的,主要是阻滞在pro B、pre B和immature B细胞三个早期的阶段;(4)TLR4信号可以使immature B细胞向成熟方向分化,并可以帮助自身免疫性B淋巴细胞逃避阴性选择过程免于凋亡;(5)LPS促进immature B细胞分化成熟这一作用可以被anti-μ抑制掉。以上试验结果提示TLR4信号在体内外均可促进immature B细胞的发育,而机体产生B淋巴细胞是一个极其复杂的过程,受着多种不同因素的影响。
[Abstract]:Objective: B lymphocytes originate from multipotent hematopoietic stem cells and undergo multiple developmental stages during the whole development process. The hematopoietic stem cells must successfully pass through various developmental stages to become a mature B lymphocyte that normally plays the immune response function, but the precise regulation mechanism of B cell development may be caused by many pathological strips. The.Toll like receptor is a protein that recognizes a variety of pathogenic molecules. Once the pathogenic molecules are identified, they can activate a series of responses to activate the immune system to clear the pathogenic microbes, thus forming the first line of defense for the immune system; TLR induced production. Inflammatory cytokines, and many of these factors inhibit the development of B cells. These two original independent phenomena suggest that there is a certain association between the TLR signal and the development of B cells. Considering that the TLR signal has a direct and profound effect on the function of mature B cells, the obvious question is whether the TLR signal is also the B cells in the developing B cells. In this experiment, we detected the expression level of TLR4 in B cells at various stages, detected the difference in the distribution of B cells between normal C3H/He mice and C3H/HeJ mice, and detected the effect of TLR4 signals on the developing B cells by both in vivo and in vitro tests.
Methods: the expression level of TLR4 in each B cell subgroup was detected by real time fluorescence quantitative PCR; LPS was injected into the peritoneum of C57BL/6 mice. The changes of the bone marrow through cell count and flow cytometry were taken at different time points. The flow cytometry was used to analyze the total B and B cells in the bone marrow of normal C3H/He mice and C3H/HeJ mice. The proportion of subgroups; the immature B cells selected by FACs were cultured in vitro, and the proliferation algebra was detected by CFSE staining after LPS treatment. The expression of IgD, CD21, CD23 and CD86 on the surface of the cells was cultured by flow cytometry, and the immature B cells isolated by FACs were cultured in vitro, and the cell differentiation and withering were detected with LPS, anti-, or micron, respectively. Related indicators of death.
Results: (1) real time fluorescence quantitative PCR results showed that four B cell subsets in mouse bone marrow expressed TLR4, and the expression level of mature B cells was significantly higher than that of the first three subgroups, while the expression level of mature B cells in the spleen was significantly higher than that of mature B cells derived from bone marrow; (2) after the application of LPS treatment to C57BL/6 mice, the treatment was found after treatment. The total number of bone marrow cells, total B cells, pre B cells and immature B cells decreased significantly at first weeks, followed by an abnormal increase in anti elasticity, and gradually restored to normal levels at third weeks. (3) the proportion of all B cells in the bone marrow of C3H/HeJ mice, pro B, pre B cells were significantly higher than that of normal C3H/He. There was no significant difference in the proportion of immature B and mature B cells; (4) the positive rates of IgD, CD21, CD23, CD86 were significantly higher than those in the negative control group after the application of LPS stimulation in the culture system of Immature B cells. The percentage of apoptotic cells was significantly lower than that of group anti-, while the positive rate of IgD was significantly lower than that of LPS group.
Conclusion: (1) the expression level of TLR4 can be greatly improved after the migration of B lymphocytes from the newborn cells, which may be influenced by the microbiological components and various cytokines in the peripheral environment. (2) the abnormal development of the B lymphocytes in the bone marrow is also developed when the TLR4 signal is too strong in the mice, as for this abnormality. It is due to the obstruction of the development of the early hematopoietic stem cells to the drenching system or the acceleration of the development of B lymphocytes. Through our analysis, these two reasons may play a role in this process. (3) the development of B lymphocytes in the mice with TLR4 dysfunction is abnormal, mainly blocking the Pro B, pre B and immature B fine. Three early stages of the cell; (4) the TLR4 signal can differentiate the immature B cells from the mature direction and help the autoimmune B lymphocytes escape the negative selection process from apoptosis; (5) the effect of LPS to promote the differentiation and maturation of immature B cells can be suppressed by anti- us. The results suggested that the TLR4 signal can be promoted in vivo and in vitro. The development of immature B cells and the generation of B lymphocytes in the body are very complex processes, which are affected by many different factors.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R392

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