本文選題：模擬微重力 + RCCS　； 參考：《南方醫(yī)科大學(xué)》2011年碩士論文

【摘要】：研究背景與目的 隨著空間站的建立以及人類登月計(jì)劃的實(shí)施,空間環(huán)境對(duì)機(jī)體的影響已逐漸成為科學(xué)領(lǐng)域中一個(gè)新的研究熱點(diǎn),空間環(huán)境是一種與地球完全不同的環(huán)境,微重力是空間環(huán)境的主要特點(diǎn)之一,指特殊空間環(huán)境中物體處于10-2-10-5G的重力狀態(tài)。微重力對(duì)生物的作用效應(yīng)可以通過(guò)空間飛行搭載實(shí)驗(yàn)研究。但是這種實(shí)驗(yàn)條件機(jī)會(huì)少,條件苛刻,且耗費(fèi)巨大,不能滿足對(duì)空間效應(yīng)深入研究的要求。地基模擬裝置的研發(fā)彌補(bǔ)了這個(gè)缺陷,由美國(guó)宇航局(NASA)開(kāi)發(fā)研制的旋轉(zhuǎn)細(xì)胞培養(yǎng)系統(tǒng)(Rotary Cell Culture System,RCCS)是目前公認(rèn)的模擬微重力環(huán)境的培養(yǎng)裝置,它是一種在地面條件下模擬微重力環(huán)境的細(xì)胞培養(yǎng)裝置,實(shí)驗(yàn)結(jié)果和真實(shí)微重力有很好的相關(guān)性,可以用來(lái)做關(guān)于微重力的研究。 地球上的生物都是在1G重力環(huán)境中生長(zhǎng)和發(fā)育的,因此重力環(huán)境的改變勢(shì)必對(duì)其生理系統(tǒng)產(chǎn)生巨大的影響。微重力狀態(tài)下,由于心血管系統(tǒng)中流體靜壓改變和血液頭向分布,將導(dǎo)致機(jī)體出現(xiàn)一系列病理性的變化,如航天“貧血癥”的發(fā)生,近年來(lái)國(guó)內(nèi)外關(guān)于微重力狀態(tài)下心血管系統(tǒng)的改變也主要集中在血液病方面,至于微重力狀態(tài)下凝血功能的改變,則研究甚少。 在機(jī)體止凝血過(guò)程中,凝血因子之一的組織因子(Tissue factor, TF)發(fā)揮著舉足輕重的作用,當(dāng)細(xì)胞表面表達(dá)的TF暴露于血漿蛋白時(shí),TF就會(huì)和與其有高親和力的因子Ⅶ(factorⅦ,FⅦ)相粘連。游離的因子Ⅶa(FⅦa)和/或已形成的TF-Ⅶa復(fù)合物可激活TF-Ⅶ復(fù)合物轉(zhuǎn)變成TF-Ⅶa復(fù)合物,而且TF-Ⅶa復(fù)合物可進(jìn)一步激活游離FⅦ。這些機(jī)制被稱為T(mén)F介導(dǎo)FⅦ自身激活。TF-Ⅶa復(fù)合物可迅速催化因子X(jué)的激活。另外,TF-Ⅶa能以較低的速率激活因子Ⅸ,激活的因子Ⅸa在輔因子Ⅷ的存在下可激活因子X(jué)轉(zhuǎn)變成因子X(jué)a,這些過(guò)程最終導(dǎo)致凝血酶產(chǎn)生。凝血酶進(jìn)而催化纖維蛋白原轉(zhuǎn)變成纖維蛋白,形成血栓。即TF可同時(shí)激活凝血因子Ⅸ和Ⅹ,啟動(dòng)內(nèi)、外源性凝血途徑,在血栓形成過(guò)程中起著重要作用。 血液系統(tǒng)中的單核細(xì)胞是重要的免疫細(xì)胞,但在近年的研究中發(fā)現(xiàn)它是血源性TF的主要來(lái)源,在動(dòng)脈粥樣硬化、冠心病等疾病中與血栓形成有著密切的關(guān)系。在生理?xiàng)l件下,機(jī)體的單核細(xì)胞不表達(dá)TF,但在受到內(nèi)毒素、腫瘤壞死因子、IL-1等炎癥介質(zhì)的刺激時(shí)可大量的表達(dá)TF并激活凝血途徑。脂多糖(Lipopolysaccharide,LPS)活化核因子-κB(NF-κB)這條信號(hào)通路中現(xiàn)已有明確的結(jié)論,LPS首先與LPS結(jié)合蛋白(LPB)結(jié)合,再傳遞給CD14分子,形成LPS-LBP-CD 14復(fù)合物,該復(fù)合物與Toll樣受體4(Toll-like receptor 4,TLR4)-MD2相互作用,通過(guò)激活細(xì)胞內(nèi)的信號(hào)通路而最終導(dǎo)致NF-κb、活化蛋白-1(AP-1)等核轉(zhuǎn)錄因子的活化。由此可見(jiàn),在LPS刺激單核細(xì)胞合成TF的信號(hào)通路中,CD14和TLR4起著關(guān)鍵的作用。 模擬微重力對(duì)于已經(jīng)適應(yīng)地球1G重力的單核細(xì)胞而言,本身就是一個(gè)刺激因素。在這個(gè)刺激因素下,單核細(xì)胞TF的表達(dá)是否增強(qiáng)?若是培養(yǎng)基中加入LPS,是否會(huì)聯(lián)合模擬微重力增強(qiáng)單核細(xì)胞TF的表達(dá)?其具體機(jī)制又是如何?國(guó)內(nèi)外現(xiàn)在未見(jiàn)類似的報(bào)道,因此這是開(kāi)展本課題研究的主要目的。 本課題研究將分為以下三個(gè)部分： (1)利用RCCS模擬微重力環(huán)境,分析單獨(dú)模擬微重力與模擬微重力聯(lián)合LPS作用對(duì)單核細(xì)胞細(xì)胞TF mRNA水平和蛋白水平表達(dá)的影響。 (2)檢測(cè)模擬微重力對(duì)單核細(xì)胞表面標(biāo)志CD14表達(dá)的影響,為闡述模擬微重力對(duì)THP-1細(xì)胞TF表達(dá)的影響機(jī)制提供一定的理論依據(jù)。 (3)分析模擬微重力狀態(tài)下單核細(xì)胞TLR4 mRNA表達(dá)和TLR4陽(yáng)性細(xì)胞率的改變,進(jìn)一步探討模擬微重力影響單核細(xì)胞TF表達(dá)的可能機(jī)制。 方法 (1)模擬微重力對(duì)單核細(xì)胞TF表達(dá)的影響 利用RCCS模擬微重力環(huán)境,實(shí)驗(yàn)刺激分為單獨(dú)模擬微重力與模擬微重力聯(lián)合LPS,分別在在培養(yǎng)0h、2h、4h、8h、12h各取出10ml細(xì)胞懸液進(jìn)行Real timePCR基因分析,培養(yǎng)0h、4h、8h、12h、24h各取出10ml細(xì)胞懸液進(jìn)行Western blotting蛋白水平分析,觀察模擬微重力對(duì)單核細(xì)胞TF表達(dá)的影響。 (2)模擬微重力對(duì)單核細(xì)胞CD14表面標(biāo)志的影響模擬微重力條件下,在培養(yǎng)0h、2h、4h、8h、12h時(shí)各取出10ml細(xì)胞懸液,利用流式細(xì)胞術(shù)檢測(cè)單核細(xì)胞表面標(biāo)志物CD14的表達(dá)量。 (3)模擬微重力對(duì)單核細(xì)胞TLR4 mRNA表達(dá)和TLR4陽(yáng)性細(xì)胞率的影響模擬微重力環(huán)境中,實(shí)驗(yàn)刺激分為單獨(dú)模擬微重力與模擬微重力聯(lián)合LPS,在培養(yǎng)0h、2h、4h、8h、12h各取出10ml細(xì)胞懸液,利用Real time PCR技術(shù)和流式細(xì)胞術(shù)對(duì)各實(shí)驗(yàn)組中TLR4 mRNA相對(duì)表達(dá)量和TLR4細(xì)胞陽(yáng)性率分別進(jìn)行檢測(cè)分析。 (4)統(tǒng)計(jì)學(xué)分析 數(shù)據(jù)以均數(shù)±標(biāo)準(zhǔn)差(x±s)表示,兩組間相同時(shí)間點(diǎn)表面標(biāo)志物陽(yáng)性率、細(xì)胞陽(yáng)性率的比較采用獨(dú)立樣本t檢驗(yàn),多組間不同時(shí)間點(diǎn)mRNA相對(duì)表達(dá)量、蛋白相對(duì)表達(dá)量比較采用重復(fù)測(cè)量數(shù)據(jù)的方差分析,方差齊時(shí)多重比較用Bonferroni法,方差不齊時(shí)則用Welch近似法,多重比較用Dunnett T3法,選取檢驗(yàn)水準(zhǔn)a=0.05,采用SPSS13.0統(tǒng)計(jì)軟件計(jì)算。 結(jié)果 (1)模擬微重力對(duì)單核細(xì)胞TF表達(dá)的影響分別于培養(yǎng)的0h、2h、4h、8h、12h收集總RNA, TF mRNA相對(duì)表達(dá)量分析結(jié)果顯示,五個(gè)時(shí)間點(diǎn)靜止組和模擬微重力組之間THP-1細(xì)胞TF mRNA相對(duì)表達(dá)量不存在顯著性差異(P0.05),但LPS組和LPS聯(lián)合模擬微重力組在培養(yǎng)2h和培養(yǎng)4h時(shí),兩組問(wèn)TF mRNA相對(duì)表達(dá)量存在顯著性差異(t值分別為13.037和12.118,P值均為0.000)；LPS組和靜止組相比,TF mRNA相對(duì)表達(dá)量均有顯著性升高(t值分別為-20.151、-19.465和-23.925,P值為0.002、0.000和0.000)。在培養(yǎng)的Oh、4h、8h、12h、24h收集各組細(xì)胞的總蛋白進(jìn)行Western blotting檢測(cè),光密度掃描蛋白相對(duì)比值分析發(fā)現(xiàn),在培養(yǎng)的五個(gè)時(shí)間點(diǎn),靜止組和模擬微重力組之間TF蛋白表達(dá)量并無(wú)顯著性差異；各時(shí)間點(diǎn)與0h相比蛋白表達(dá)量也無(wú)差異。加入LPS后,LPS組12h和24h兩個(gè)時(shí)間點(diǎn)TF蛋白表達(dá)量顯著高于LPS聯(lián)合模擬微重力組(t值分別為9.369和38.622,P值為0.001和0.000)。 (2)模擬微重力對(duì)單核細(xì)胞CD14表面標(biāo)志的影響 以未加任何刺激的THP-1細(xì)胞CD14表面標(biāo)志陽(yáng)性率為基礎(chǔ)水平,流式分析結(jié)果顯示,靜止組和模擬微重力組之間,培養(yǎng)0h、2h、4h、8h、12h五個(gè)時(shí)間點(diǎn)時(shí),CD14表面標(biāo)志陽(yáng)性率均不存在顯著性差異。 (3)模擬微重力對(duì)單核細(xì)胞TLR4表達(dá)的影響 Real time PCR檢測(cè)結(jié)果顯示,未加LPS時(shí),培養(yǎng)至8h和12h模擬微重力TLR4mRNA的相對(duì)表達(dá)量低于靜止組(t值分別為5.843和8.198,P值分別為0.004和0.001)。加入LPS后,LPS組和LPS聯(lián)合模擬微重力組之間在培養(yǎng)2h、4h和8h時(shí),單核細(xì)胞TLR4 mRNA相對(duì)表達(dá)量存在顯著性差異(t值分別為3.560,14.962和4.260,P值為0.024,0.000和0.013)。TLR4陽(yáng)性細(xì)胞率檢測(cè)結(jié)果顯示,未加入LPS刺激時(shí),靜止組和模擬微重力組TLR4陽(yáng)性細(xì)胞率均不存在統(tǒng)計(jì)學(xué)差異(P0.05)。加入LPS后,在培養(yǎng)至4h和8h時(shí),LPS組和模擬微重力聯(lián)合LPS組相比較,前者TLR4陽(yáng)性細(xì)胞率顯著高于后者,兩者間有統(tǒng)計(jì)學(xué)差異(t=7.257和8.915,P=0.002和0.001)。 結(jié)論 (1)在生理?xiàng)l件下,單核細(xì)胞低表達(dá)TF時(shí),模擬微重力對(duì)TF表達(dá)的影響并不顯著,但在LPS刺激單核細(xì)胞高效表達(dá)TF后,無(wú)論是在基因水平還是蛋白水平,模擬微重力都顯著性地抑制單核細(xì)胞TF表達(dá)的增加。 (2)CD14是LPS刺激單核細(xì)胞TF表達(dá)信號(hào)通路中的主要物質(zhì)之一,流式分析結(jié)果顯示,未加任何刺激的靜止培養(yǎng)狀態(tài)下,單核細(xì)胞CD14表面標(biāo)志陽(yáng)性率高達(dá)98%,模擬微重力對(duì)其表達(dá)量并沒(méi)有顯著性的影響。 (3)TLR4是LPS刺激單核細(xì)胞TF表達(dá)的主要受體之一。實(shí)驗(yàn)證實(shí)模擬微重力能顯著性抑制LPS刺激后單核細(xì)胞TLR4 mRNA的表達(dá)和TLR4陽(yáng)性細(xì)胞率的增加,與模擬微重力對(duì)LPS刺激單核細(xì)胞TF表達(dá)影響相一致,提示模擬微重力可能通過(guò)抑制TLR4的表達(dá)而抑制LPS刺激后單核細(xì)胞TF表達(dá)的增加。 創(chuàng)新點(diǎn) (1)本實(shí)驗(yàn)首次檢測(cè)分析模擬微重力對(duì)單核細(xì)胞TF表達(dá)的影響,明確模擬微重力可顯著抑制LPS刺激后單核細(xì)胞TF表達(dá)的增加。 (2)本研究初步證實(shí)模擬微重力可通過(guò)抑制單核細(xì)胞TLR4表達(dá)而抑制LPS刺激后單核細(xì)胞TF表達(dá)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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R85

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本文編號(hào)：1984979