模拟微重力对单核细胞TF表达的影响及其机制研究

发布时间：2018-06-06 04:02

本文选题：模拟微重力 + RCCS　；参考：《南方医科大学》2011年硕士论文

【摘要】：研究背景与目的随着空间站的建立以及人类登月计划的实施,空间环境对机体的影响已逐渐成为科学领域中一个新的研究热点,空间环境是一种与地球完全不同的环境,微重力是空间环境的主要特点之一,指特殊空间环境中物体处于10-2-10-5G的重力状态。微重力对生物的作用效应可以通过空间飞行搭载实验研究。但是这种实验条件机会少,条件苛刻,且耗费巨大,不能满足对空间效应深入研究的要求。地基模拟装置的研发弥补了这个缺陷,由美国宇航局(NASA)开发研制的旋转细胞培养系统(Rotary Cell Culture System,RCCS)是目前公认的模拟微重力环境的培养装置,它是一种在地面条件下模拟微重力环境的细胞培养装置,实验结果和真实微重力有很好的相关性,可以用来做关于微重力的研究。地球上的生物都是在1G重力环境中生长和发育的,因此重力环境的改变势必对其生理系统产生巨大的影响。微重力状态下,由于心血管系统中流体静压改变和血液头向分布,将导致机体出现一系列病理性的变化,如航天“贫血症”的发生,近年来国内外关于微重力状态下心血管系统的改变也主要集中在血液病方面,至于微重力状态下凝血功能的改变,则研究甚少。在机体止凝血过程中,凝血因子之一的组织因子(Tissue factor, TF)发挥着举足轻重的作用,当细胞表面表达的TF暴露于血浆蛋白时,TF就会和与其有高亲和力的因子Ⅶ(factorⅦ,FⅦ)相粘连。游离的因子Ⅶa(FⅦa)和/或已形成的TF-Ⅶa复合物可激活TF-Ⅶ复合物转变成TF-Ⅶa复合物,而且TF-Ⅶa复合物可进一步激活游离FⅦ。这些机制被称为TF介导FⅦ自身激活。TF-Ⅶa复合物可迅速催化因子X的激活。另外,TF-Ⅶa能以较低的速率激活因子Ⅸ,激活的因子Ⅸa在辅因子Ⅷ的存在下可激活因子X转变成因子Xa,这些过程最终导致凝血酶产生。凝血酶进而催化纤维蛋白原转变成纤维蛋白,形成血栓。即TF可同时激活凝血因子Ⅸ和Ⅹ,启动内、外源性凝血途径,在血栓形成过程中起着重要作用。血液系统中的单核细胞是重要的免疫细胞,但在近年的研究中发现它是血源性TF的主要来源,在动脉粥样硬化、冠心病等疾病中与血栓形成有着密切的关系。在生理条件下,机体的单核细胞不表达TF,但在受到内毒素、肿瘤坏死因子、IL-1等炎症介质的刺激时可大量的表达TF并激活凝血途径。脂多糖(Lipopolysaccharide,LPS)活化核因子-κB(NF-κB)这条信号通路中现已有明确的结论,LPS首先与LPS结合蛋白(LPB)结合,再传递给CD14分子,形成LPS-LBP-CD 14复合物,该复合物与Toll样受体4(Toll-like receptor 4,TLR4)-MD2相互作用,通过激活细胞内的信号通路而最终导致NF-κb、活化蛋白-1(AP-1)等核转录因子的活化。由此可见,在LPS刺激单核细胞合成TF的信号通路中,CD14和TLR4起着关键的作用。模拟微重力对于已经适应地球1G重力的单核细胞而言,本身就是一个刺激因素。在这个刺激因素下,单核细胞TF的表达是否增强?若是培养基中加入LPS,是否会联合模拟微重力增强单核细胞TF的表达?其具体机制又是如何?国内外现在未见类似的报道,因此这是开展本课题研究的主要目的。本课题研究将分为以下三个部分： (1)利用RCCS模拟微重力环境,分析单独模拟微重力与模拟微重力联合LPS作用对单核细胞细胞TF mRNA水平和蛋白水平表达的影响。 (2)检测模拟微重力对单核细胞表面标志CD14表达的影响,为阐述模拟微重力对THP-1细胞TF表达的影响机制提供一定的理论依据。 (3)分析模拟微重力状态下单核细胞TLR4 mRNA表达和TLR4阳性细胞率的改变,进一步探讨模拟微重力影响单核细胞TF表达的可能机制。方法 (1)模拟微重力对单核细胞TF表达的影响利用RCCS模拟微重力环境,实验刺激分为单独模拟微重力与模拟微重力联合LPS,分别在在培养0h、2h、4h、8h、12h各取出10ml细胞悬液进行Real timePCR基因分析,培养0h、4h、8h、12h、24h各取出10ml细胞悬液进行Western blotting蛋白水平分析,观察模拟微重力对单核细胞TF表达的影响。 (2)模拟微重力对单核细胞CD14表面标志的影响模拟微重力条件下,在培养0h、2h、4h、8h、12h时各取出10ml细胞悬液,利用流式细胞术检测单核细胞表面标志物CD14的表达量。 (3)模拟微重力对单核细胞TLR4 mRNA表达和TLR4阳性细胞率的影响模拟微重力环境中,实验刺激分为单独模拟微重力与模拟微重力联合LPS,在培养0h、2h、4h、8h、12h各取出10ml细胞悬液,利用Real time PCR技术和流式细胞术对各实验组中TLR4 mRNA相对表达量和TLR4细胞阳性率分别进行检测分析。 (4)统计学分析数据以均数±标准差(x±s)表示,两组间相同时间点表面标志物阳性率、细胞阳性率的比较采用独立样本t检验,多组间不同时间点mRNA相对表达量、蛋白相对表达量比较采用重复测量数据的方差分析,方差齐时多重比较用Bonferroni法,方差不齐时则用Welch近似法,多重比较用Dunnett T3法,选取检验水准a=0.05,采用SPSS13.0统计软件计算。结果 (1)模拟微重力对单核细胞TF表达的影响分别于培养的0h、2h、4h、8h、12h收集总RNA, TF mRNA相对表达量分析结果显示,五个时间点静止组和模拟微重力组之间THP-1细胞TF mRNA相对表达量不存在显著性差异(P0.05),但LPS组和LPS联合模拟微重力组在培养2h和培养4h时,两组问TF mRNA相对表达量存在显著性差异(t值分别为13.037和12.118,P值均为0.000)；LPS组和静止组相比,TF mRNA相对表达量均有显著性升高(t值分别为-20.151、-19.465和-23.925,P值为0.002、0.000和0.000)。在培养的Oh、4h、8h、12h、24h收集各组细胞的总蛋白进行Western blotting检测,光密度扫描蛋白相对比值分析发现,在培养的五个时间点,静止组和模拟微重力组之间TF蛋白表达量并无显著性差异；各时间点与0h相比蛋白表达量也无差异。加入LPS后,LPS组12h和24h两个时间点TF蛋白表达量显著高于LPS联合模拟微重力组(t值分别为9.369和38.622,P值为0.001和0.000)。 (2)模拟微重力对单核细胞CD14表面标志的影响以未加任何刺激的THP-1细胞CD14表面标志阳性率为基础水平,流式分析结果显示,静止组和模拟微重力组之间,培养0h、2h、4h、8h、12h五个时间点时,CD14表面标志阳性率均不存在显著性差异。 (3)模拟微重力对单核细胞TLR4表达的影响 Real time PCR检测结果显示,未加LPS时,培养至8h和12h模拟微重力TLR4mRNA的相对表达量低于静止组(t值分别为5.843和8.198,P值分别为0.004和0.001)。加入LPS后,LPS组和LPS联合模拟微重力组之间在培养2h、4h和8h时,单核细胞TLR4 mRNA相对表达量存在显著性差异(t值分别为3.560,14.962和4.260,P值为0.024,0.000和0.013)。TLR4阳性细胞率检测结果显示,未加入LPS刺激时,静止组和模拟微重力组TLR4阳性细胞率均不存在统计学差异(P0.05)。加入LPS后,在培养至4h和8h时,LPS组和模拟微重力联合LPS组相比较,前者TLR4阳性细胞率显著高于后者,两者间有统计学差异(t=7.257和8.915,P=0.002和0.001)。结论 (1)在生理条件下,单核细胞低表达TF时,模拟微重力对TF表达的影响并不显著,但在LPS刺激单核细胞高效表达TF后,无论是在基因水平还是蛋白水平,模拟微重力都显著性地抑制单核细胞TF表达的增加。 (2)CD14是LPS刺激单核细胞TF表达信号通路中的主要物质之一,流式分析结果显示,未加任何刺激的静止培养状态下,单核细胞CD14表面标志阳性率高达98%,模拟微重力对其表达量并没有显著性的影响。 (3)TLR4是LPS刺激单核细胞TF表达的主要受体之一。实验证实模拟微重力能显著性抑制LPS刺激后单核细胞TLR4 mRNA的表达和TLR4阳性细胞率的增加,与模拟微重力对LPS刺激单核细胞TF表达影响相一致,提示模拟微重力可能通过抑制TLR4的表达而抑制LPS刺激后单核细胞TF表达的增加。创新点 (1)本实验首次检测分析模拟微重力对单核细胞TF表达的影响,明确模拟微重力可显著抑制LPS刺激后单核细胞TF表达的增加。 (2)本研究初步证实模拟微重力可通过抑制单核细胞TLR4表达而抑制LPS刺激后单核细胞TF表达的增加。
[Abstract]:Research background and purpose
With the establishment of the space station and the implementation of the human landing program, the influence of space environment on the body has gradually become a new research hotspot in the field of science. The space environment is a completely different environment from the earth. Microgravity is one of the main characteristics of the space environment. It refers to the gravity of the object in the special space environment in the 10-2-10-5G. The effect of microgravity on biological effects can be studied by space flight loading experiment. However, this experimental condition is less opportunity, harsh and costly, and can not meet the requirement of deep study on space effect. The research and development of foundation simulation device make up for this defect, and the culture of rotating cells developed by NASA (NASA) Rotary Cell Culture System (RCCS) is a widely recognized culture device for simulated microgravity environment at present. It is a cell culture device for simulating microgravity environment under ground conditions. The experimental results have a good correlation with real microgravity. It can be used for the study of micro gravity.
The life of the earth is growing and developing in the 1G gravity environment, so the change of the gravity environment is bound to have a great influence on its physiological system. In the state of microgravity, the hydrostatic pressure changes in the cardiovascular system and the distribution of the blood head will lead to a series of pathological changes in the body, such as the hair of the space "anemia". In recent years, the changes of cardiovascular system in the state of microgravity are mainly concentrated in the blood disease, and the change of coagulation function in microgravity is very little.
Tissue factor (TF), one of the coagulation factors, plays an important role in the body anticoagulant process. When the TF is exposed to the plasma protein on the surface of the cell, TF will adhere to the factor VII (factor VII, F VII) with its high affinity. The dissociated factor VII a (F VII a) and / or the formed TF- a complex can be stimulated. The active TF- VII complex is transformed into a TF- VII a complex, and the TF- VII a complex can further activate the free F VII. These mechanisms are called TF mediated F VII self activated.TF- VII a complex, which can catalyze factor X activation rapidly. Furthermore, TF- VII can activate factor IX at a lower rate and activates the activation factor in the presence of cofactor VIII. The subX changes into factor Xa, which eventually leads to the production of thrombin. Thrombin then catalyzes the transformation of fibrinogen into fibrin to form thrombus. That is, TF activates coagulation factor IX and the thrombus at the same time. The exogenous coagulation pathway plays an important role in the formation of thrombus.
Mononuclear cells in the blood system are important immune cells, but in recent years it is found that it is the main source of blood derived TF. It has a close relationship with thrombosis in atherosclerosis, coronary heart disease and other diseases. Under physiological conditions, the mononuclear cells of the body do not express TF, but they are subjected to endotoxin, tumor necrosis factor, IL-1 and so on. The stimulation of the inflammatory medium can express TF and activate the coagulation pathway. The Lipopolysaccharide (LPS) activation nuclear factor kappa B (NF- kappa B) is a clear conclusion that LPS first combines with LPS binding protein (LPB) and then transferred to CD14 molecules to form a LPS-LBP-CD 14 complex, the complex and Toll like receptor 4. Ike receptor 4, TLR4) -MD2 interact with the activation of NF- kappa B, activation protein -1 (AP-1) and other nuclear transcription factors by activating intracellular signaling pathways. This shows that CD14 and TLR4 play a key role in the signaling pathway of LPS stimulated mononuclear cells to synthesize TF.
Simulated microgravity is a stimulus itself to the mononuclear cells that have adapted to earth's 1G gravity. Is the expression of monocyte TF enhanced under this stimulus? If LPS is added to the medium, will it combine to simulate microgravity enhanced mononuclear cell TF? How is the specific mechanism? Similar reports, so this is the main purpose of this research project.
This research will be divided into three parts:
(1) using RCCS to simulate microgravity environment, the effects of microgravity and simulated microgravity on the expression of TF mRNA and protein level in monocyte cells were analyzed by simulated microgravity and simulated microgravity (LPS).
(2) the effect of simulated microgravity on the expression of CD14 on the surface of mononuclear cells was detected to provide a theoretical basis for explaining the mechanism of the influence of simulated microgravity on the expression of TF in THP-1 cells.
(3) to analyze the changes in the expression of TLR4 mRNA and the rate of TLR4 positive cells in mononuclear cells under simulated microgravity, and further explore the possible mechanism of the influence of simulated microgravity on the expression of TF in mononuclear cells.
Method
(1) the effect of simulated microgravity on the expression of TF in mononuclear cells
Using RCCS to simulate microgravity environment, the experimental stimulation was divided into single simulated microgravity and simulated microgravity LPS, and the Real timePCR gene was analyzed in 0h, 2h, 4h, 8h, 12h respectively. The effect on the expression of TF in monocyte.
(2) the effect of simulated microgravity on the surface markers of mononuclear cell CD14 in simulated microgravity conditions, 10ml cell suspensions were removed at 0h, 2h, 4h, 8h, 12h, and the expression of the surface marker of mononuclear cells (CD14) was detected by flow cytometry.
(3) the effects of simulated microgravity on the expression of TLR4 mRNA and the rate of TLR4 positive cells in mononuclear cells were simulated in microgravity environment. The experimental stimulation was divided into single simulated microgravity and simulated microgravity LPS combined with 0h, 2h, 4h, 8h, and 12h to remove the 10ml cell suspension. The expression level and the positive rate of TLR4 cells were detected and analyzed.
(4) statistical analysis
The positive rate of the surface markers at the same time point between the two groups was expressed with the mean number of standard deviation (x + s). The comparison of the positive rate of the cell positive rate was compared with the independent sample t test, the relative expression of mRNA in different groups of different time points, the relative expression of the protein was compared with the variance analysis of the repeated measurement data, the multiple comparison of the variance was Bonferroni, and the variance was not. At the same time, the Welch approximation method was used, and the Dunnett T3 method was used for multiple comparisons, and the test level a=0.05 was selected and calculated by SPSS13.0 statistical software.
Result
(1) the effects of simulated microgravity on the expression of TF in mononuclear cells were collected in 0h, 2h, 4h, 8h, 12h, respectively. The relative expression of TF mRNA showed that there was no significant difference in the relative expression of TF mRNA between the five time points and the simulated microgravity groups, but the group and the combined simulated microgravity group were in the culture. When 2H and 4H were cultured, there were significant differences in the relative expression of TF mRNA in the two groups (T values were 13.037 and 12.118, P values were 0), and the LPS group was significantly higher in TF mRNA relative expression (t value was -20.151, -19.465, and 0). The total protein of the cell was detected by Western blotting, and the relative ratio analysis of the light density scanning protein found that there was no significant difference in the expression of TF protein between the stationary group and the simulated microgravity group at five time points, and there was no difference in the protein expression at each time point compared with the 0h. After adding LPS, the LPS group 12h and 24h had two time points TF eggs. The expression level of white was significantly higher than that of LPS combined simulated microgravity group (t value was 9.369 and 38.622, P value was 0.001 and 0 respectively).
(2) effect of simulated microgravity on CD14 surface markers of monocytes
The positive rate of CD14 surface markers in THP-1 cells without any stimulation was based on the base level. Flow analysis showed that there was no significant difference in the positive rate of CD14 surface markers between the five time points between 0h, 2h, 4h, 8h and 12h between the stationary group and the simulated microgravity group.
(3) the effect of simulated microgravity on the expression of TLR4 in monocytes
The results of Real time PCR detection showed that the relative expression of TLR4mRNA in 8h and 12h simulated microgravity TLR4mRNA was lower than that in the rest group (t value was 5.843 and 8.198, P was 0.004 and 0.001 respectively). The detection results of the positive cell rate (t value of 3.560,14.962 and 4.260, P value 0.024,0.000 and 0.013).TLR4 positive cell rate showed that there was no statistical difference between the static group and the simulated microgravity group TLR4 positive cell rate without LPS stimulation (P0.05). When LPS was added to 4h and 8h, the LPS group was compared with the simulated microgravity group. Compared with the latter, the rate of TLR4 positive cells was significantly higher than that of the latter, and there was statistical difference between them (t=7.257 and 8.915, P=0.002 and 0.001).
conclusion
(1) under the physiological conditions, the effect of simulated microgravity on the expression of TF was not significant when the monocytes were low expression of TF, but after LPS stimulated the mononuclear cells to express TF efficiently, the simulated microgravity significantly inhibited the increase of TF expression in mononuclear cells, both at the gene level and protein level.
(2) CD14 is one of the main substances in the LPS stimulation monocyte TF expression signaling pathway. Flow analysis results show that the positive rate of CD14 surface markers in monocyte is up to 98% without any stimulation in static culture, and the simulated microgravity has no significant effect on its expression.
(3) TLR4 is one of the main receptors of TF expression in LPS stimulated monocyte. It is proved that simulated microgravity can significantly inhibit the expression of TLR4 mRNA and the increase of TLR4 positive cell rate after LPS stimulation, which is consistent with the effect of simulated microgravity on the TF expression of LPS stimulated mononuclear cells, suggesting that simulated microgravity may inhibit the expression of TLR4 by the simulated microgravity. It inhibited the increase of TF expression in monocytes after LPS stimulation.
innovation point
(1) the effects of simulated microgravity on the expression of TF in mononuclear cells were analyzed for the first time. It was clear that simulated microgravity could significantly inhibit the increase of TF expression in mononuclear cells after LPS stimulation.
(2) this study preliminarily confirmed that simulated microgravity could inhibit the increase of TF expression in monocytes after LPS stimulation by inhibiting the expression of TLR4 in monocytes.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R85

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