發(fā)布時間：2018-08-09 11:02

【摘要】： 研究背景 細胞極性是多種不同類型細胞的基本特征，對多數(shù)細胞的功能發(fā)揮是必需的。細胞極性是指細胞為了行使特殊的生理功能或產生定向的分化，在胞內的信號分子不對稱分布致使細胞在表型上的不對稱。具體表現(xiàn)為細胞的形狀、細胞內的蛋白質、脂質以及產生極性所必需的細胞骨架等的不對稱分布，細胞結構出現(xiàn)重構的變化。細胞極性形成的過程通常是由肌動蛋白細胞骨架和細胞皮質所介導，在這一進程中，細胞膜首先形成縐褶，同時在縐褶處肌動蛋白聚集增加，從而形成一個前極具有片足后極出現(xiàn)尾足的極化形態(tài)。細胞極性在細胞遷移、軸突生長、胚胎發(fā)育、血管發(fā)生、傷口修復等細胞基本生命活動過程中發(fā)揮著重要的調控作用。 中性粒細胞極性對于其生理功能的發(fā)揮有著重要的意義。臨床上多種疾病如敗血癥、哮喘、缺血／再灌注損傷和器官移植的排斥反應、類風濕性關節(jié)炎及一些腫瘤的發(fā)生和轉移都與中性粒細胞極性功能的增強和減弱有關。當機體遭受外來細菌和其他病原體入侵時，中性粒細胞充當了機體的第一道防線，這種作用是建立在細胞的極性和趨化性基礎上的。在各種趨化物的作用下，中性粒細胞憑借胞內分子對極化信號的優(yōu)化和放大，能夠準確向炎癥部位形成極性化，，并最終向此處遷移，而中性粒細胞極性是這種定向遷移功能的前提條件。 目前有關中性粒細胞極性形成機制的研究主要包括參與胞內極性形成的信號傳導通路的研究，參與胞內鈣離子(Ca~(2+))信號調節(jié)的鈣庫操縱性鈣離子流入(SOCE)機制的探討以及胞膜上的特殊脂質結構脂筏在其中的作用。但中性粒細胞極性各種機制之間的關系，尤其是參與SOCE機制的瞬時受體電位離子通道(TRP)在極化過程中的明確作用，以及其與脂筏的相互聯(lián)系還有待于進一步探討。 目的 1．采用4℃溫度對急性分離的人外周血嗜中性粒細胞進行預處理，探討溫度處理對細胞的極性和膜通道有何影響。 2．探討均一濃度的趨化劑fMLP對中性粒細胞極性形成的影響。 3．探討fMLP誘導的中性粒細胞胞內游離鈣離子濃度([Ca~(2+)]_i)變化與細胞極性化過程啟動的關系。 4．探討TRPC1和脂筏在fMLP誘導的中性粒細胞極性中的作用及二者之間的關聯(lián)性。 方法 1．采用重復梯度密度離心的方法分離中性粒細胞，進行臺盼藍和瑞氏染色，分析細胞的活性和純度。分離后的細胞進行4℃溫度預處理。通過倒置顯微鏡觀察fMLP誘導的中性粒細胞的變化情況，采用單通道和全細胞模式膜片鉗記錄中性粒細胞的鉀通道的電流活動。 2．采用激光共聚焦顯微鏡測量中性粒細胞[Ca~(2+)]_i。 3．應用免疫熒光方法檢測對照組、fMLP刺激組和脂筏破壞后fMLP刺激組的TRPC1的表達情況。 4．使用超高速冷凍離心機分離脂筏，并應用Western-blotting方法檢測對照組、fMLP刺激組和脂筏破壞后fMLP刺激組的TRPC1蛋白在脂筏中的含量變化。 結果 1．中性粒細胞分離后，對細胞進行臺盼藍染色，細胞的活性大于94％，瑞氏—姬姆薩染色，細胞純度大于95％。對分離后的細胞進行4℃溫度預處理，結果表明4℃溫度預處理的細胞出現(xiàn)片足的時間(45.93±7.87msec，n=35)和常溫組(33.75±6.02msec，n=28)相比有顯著性差異(P＜0.01)。4℃溫度預處理的細胞極性周期所用時間(538.26±27.74sec，n=23)和常溫組(260.25±25.87sec，n=23)相比有顯著性差異(P＜0.01)。全細胞模式記錄溫度預處理和常溫對照組中性粒細胞的電壓依賴性鉀電流，兩組的Ⅰ-Ⅴ曲線，電流幅度均隨鉗制電位升高而增高，電流幅度沒有顯著性差異(n=5，P＞0.05)。采用單通道記錄BKCa電流，兩組細胞的電導值大約為210pS(n=3，P＞0.05)。在鉗制電位為40mV、60mV、80mV下，溫度處理組和常溫組的NPo分別進行t檢驗，兩組之間均沒有顯著性差異(n=30，P＞0.05)。 2．在三種濃度(10nM，50nM，100nM)下的fMLP激活中性粒細胞，極化中性粒細胞的50％極化率所用的時間(51.16±6.49sec，45.83±4.95sec，37.33±5.16sec；n=6，P＜0.01)，偽足的平均極化長度(1.62±0.050μm，1.73±0.036μm，1.81±0.027μm；n=30，P＜0.01)以及極性振蕩周期(209.37±17.70sec，n=52；234.00±21.42sec，n=50；286.15±19.44sec，n=52；P＜0.01)均具有顯著性差異。三種濃度下的偽足延伸率(0.067±0.0073μm／sec，0.107±0.0086μm／sec，0.120±0.0132μm／sec；n=30，P＜0.01)，縮短率(0.051±0.0050μm／sec，0.091±0.0051μtm／sec，0.110±0.0142μm／sec；n=30，P＜0.01)和變化率(0.057±0.0071μm／sec，0.099±0.0059μm／sec，0.114±0.0124μm／sec；n=30，P＜0.01))也均具有顯著性差異。 3．加入fMLP(100nM)后對應于[Ca~(2+)]_i變化的不同時相，即靜息期(Osec)、快速上升期(10sec)、快速下降期(150sec)、慢速下降期(250sec)和終末期五個階段，而中性粒細胞表現(xiàn)為極性化和去極性化狀態(tài)的交替出現(xiàn)。 4．SKF96365(10μM)和mβCD(10mM)能抑制fMLP誘導中性粒細胞[Ca~(2+)]_i增加及中性粒細胞的極化形態(tài)的形成。 5．正常中性粒細胞的TRPC1是在膜均勻的表達，而fMLP(100nM)組TRPC1明顯聚集到細胞的片足位置，但fMLP+mβCD組TRPC1沒有出現(xiàn)聚集現(xiàn)象。同時使用Western-blotting技術檢測三組的TRPC1蛋白在脂筏和非脂筏的含量變化，在非脂筏層，對照組(289.00±8.00)和fMLP+mβCD組(283.33±6.02)的TRPC1的表達比fMLP組(76.00±7.00)顯著增高，差異具有統(tǒng)計學意義(n=3，P＜0.01)；而在脂筏層，fMLP組(215.00±6.55)的TRPC1的表達比對照組(0.00±0.00)和fMLP+mβCD組(21.33±5.68)顯著增高，差異具有統(tǒng)計學意義(n=3，P＜0.01)。 結論 1．采用4℃溫度預處理分離后的中性粒細胞，發(fā)現(xiàn)細胞極性明顯抑制，而離子通道的功能活動不受影響，因此4℃溫度預處理可以減少中性粒細胞自發(fā)極化的發(fā)生，為后續(xù)實驗提供良好的細胞來源； 2．建立了一組評價細胞極性變化的指標，根據(jù)指標所得數(shù)據(jù)發(fā)現(xiàn)中性粒細胞極性形成對fMLP有時間和濃度的依賴性； 3．本實驗顯示不同濃度趨化劑fMLP誘導的[Ca~(2+)]_i變化沒有顯著性差異，而中性粒細胞極性化周期與fMLP濃度有關，進一步揭示了[Ca~(2+)]_i的升高與中性粒細胞極性化啟動機制有關； 4．證明了SOCE機制和脂筏參與了fMLP誘導中性粒細胞的極性形態(tài)的產生及[Ca~(2+)]_i升高過程，進一步闡明了中性粒細胞極性化過程中的關鍵信號分子(Ca~(2+)]_i、TRPC1)在信號轉導過程中的作用； 5．在可掌握的文獻范圍內本研究首次報道TRPC1在參與中性粒細胞極化時是結合在脂筏上而發(fā)揮作用的。
[Abstract]:Research background
Cell polarity is the basic feature of a variety of different types of cells and is necessary for the function of most cells. Cell polarity refers to the asymmetric distribution of signal molecules in the cell in order to exercise special physiological functions or to produce directional differentiation. The asymmetric distribution of proteins, lipids, and cytoskeletons necessary for the generation of polarity, and the changes in the structure of cells. The process of cell polarity is usually mediated by the actin cytoskeleton and cell cortex. In this process, the membrane is first crepe fold, and the actin aggregation at the crepe fold increases. The polarization form of the tail of a front pole is formed. Cell polarity plays an important regulatory role in cell migration, axon growth, embryo development, angiogenesis, wound repair and other cell basic activities.
Neutrophil polarity plays an important role in its physiological function. Many diseases such as septicaemia, asthma, ischemia / reperfusion injury and rejection of organ transplantation, rheumatoid arthritis and the occurrence and metastasis of some tumors are related to the enhancement and weakening of the neutrophil polarity function. When bacteria and other pathogens invade, neutrophils act as the first line of defense, based on the polarity and chemotaxis of the cells. Under the action of various chemotaxis, the neutrophils can be accurately polarized to the inflammatory sites by the optimization and amplification of the intracellular molecules. Migration is eventually here, and the polarity of neutrophils is a prerequisite for this directional migration function.
Current studies on the mechanism of neutrophil polarity formation mainly include the study of signal transduction pathways involved in the formation of intracellular polarity, the mechanism of calcium ion inflow (SOCE) regulated by intracellular calcium (Ca~ (2+)) signal and the role of lipid rafts with special lipid structure on the membrane. The relationship between various mechanisms, especially the clear role of the transient receptor potential ion channel (TRP) involved in the SOCE mechanism in the polarization process, and the interaction with the lipid rafts, remains to be further explored.
objective
1. The human peripheral blood neutrophils were pretreated at 4 C to investigate the effect of temperature on cell polarity and membrane channel.
2. to explore the effect of homogeneous concentration of chemoattractant fMLP on the polarity formation of polymorphonuclear neutrophils.
3. To investigate the relationship between the changes of intracellular free calcium concentration ([Ca~ (2+)]_i) in neutrophils induced by fMLP and the initiation of cell polarization.
4. to explore the role of TRPC1 and lipid rafts in the polarity of fMLP induced neutrophils and the correlation between the two.
Method
1. the neutrophils were separated by repeated gradient density centrifugation. Trypan blue and Rayleigh staining were used to analyze the activity and purity of the cells. The cells after the separation were pretreated at 4 degrees centigrade. The changes of neutrophils induced by fMLP were observed by inverted microscope, and the neutrophils were recorded by single channel and whole cell mode patch clamp. The current activity of the potassium channel of the cell.
2. laser confocal microscopy was used to measure neutrophil [Ca~ (2+)]_i..
3. immunofluorescence assay was used to detect the expression of TRPC1 in the control group, fMLP stimulation group and fMLP stimulation group after lipid rafting.
4. the lipid rafts were separated by ultra high speed freezer centrifuge, and the control group was detected by Western-blotting method. The content of TRPC1 protein in the lipid raft was changed by the fMLP stimulation group and the fMLP stimulation group after the lipid raft was destroyed.
Result
1. neutrophils were stained with trypan blue, the cell activity was more than 94%, and the cell purity was more than 95%., and the cell purity was more than 95%.. The cells were pretreated at 4 degrees centigrade after the separation. The results showed that the time (45.93 + 7.87msec, n=35) and normal temperature group (33.75 + 6.02msec, n=28) of the pretreated cells at 4 C Compared with the significant difference (P < 0.01), the time used for the cell polar cycle (538.26 + 27.74sec, n=23) and the normal temperature group (260.25 + 25.87sec, n=23) had significant difference (P < 0.01). The temperature preconditioning and the voltage dependent potassium current of the neutrophils in the normal temperature control group and the two group of I - V curved (P < 0.01). Line, the amplitude of current increased with the increase of the clamp potential (n=5, P > 0.05). Using a single channel to record BKCa current, the conductance value of the two groups of cells was about 210pS (n=3, P > 0.05). Under the clamp potential of 40mV, 60mV, and 80mV, the t test was carried out between the temperature treatment group and the normal temperature group, and there was no significant difference between the two groups. Sexual differences (n=30, P > 0.05).
2. the time (51.16 + 6.49sec, 45.83 + 4.95sec, 37.33 + 5.16sec, n=6, P < 0.01) for the activation of neutrophils at the three concentrations (10nM, 50nM, 100nM), the average polarization length of the pseudo foot (1.62 + 0.050 mu m, 1.73 + 0.036 mu m, 1.81 + 0.027 Mu m), and polarity oscillation cycle 9.37 + 17.70sec, n=52; 234 + 21.42sec, n=50; 286.15 + 19.44sec, n=52; P < 0.01) have significant differences. The elongation rate of pseudo foot under three concentrations (0.067 + 0.0073, M / sec, 0.107 + 0.0086 mu m / sec, 0.120 + 0.0132 mu / M / 0.01) There were also significant differences in the rates of change (0.057.0071/ sec, 0.099.0059/ sec, 0.114.0124/ sec, n = 30, P < 0.01).
3. after adding fMLP (100nM) to the different phases of [Ca~ (2+)]_i, that is, resting period (Osec), rapid rise period (10sec), rapid descent (150sec), slow descent (250sec) and end stage five stages, and neutrophils appear to be polar and depolarization state alternately.
4. SKF96365 (10 mu M) and M beta CD (10 mM) could inhibit the increase of neutrophil [Ca~ (2+)]_iinduced by fMLP and the formation of polarized morphology of neutrophils.
5. the TRPC1 of normal neutrophils was expressed evenly in the membrane, while the TRPC1 in the group of fMLP (100nM) was obviously aggregated to the cell foot position of the cells, but the TRPC1 in the fMLP+m beta CD group did not appear to be aggregated. At the same time, the content of TRPC1 protein in the lipid rafts and non fat rafts in the three groups was detected by Western-blotting technology, in the non fat raft and in the control group (289 + 8). The expression of TRPC1 in group fMLP+m beta CD (283.33 + 6.02) was significantly higher than that in group fMLP (76 + 7), and the difference was statistically significant (n=3, P < 0.01), while in lipid rafts, the expression of TRPC1 in group fMLP (215 + 6.55) was significantly higher than that of the control group (0 + 0) and fMLP+m beta CD group (21.33 + 5.68), and the difference was statistically significant (n=3, P < 0.01).
conclusion
1. when the neutrophils were pretreated at 4 C, it was found that the cell polarity was obviously inhibited and the functional activity of the ion channel was not affected. Therefore, the temperature preconditioning at 4 C could reduce the spontaneous polarization of neutrophils, and provide a good cell source for the follow-up experiment.
2. A set of indices were established to evaluate the changes of cell polarity. According to the data obtained from these indices, it was found that the formation of neutrophil polarity depended on the concentration and time of fMLP.
3. the experiment showed that there was no significant difference in the changes of [Ca~ (2+)]_i induced by different concentrations of chemokine fMLP, but the polarity of neutrophils was related to the concentration of fMLP, which further revealed that the increase of [Ca~ (2+)]_i was related to the mechanism of neutrophil polarity initiation.
4. it was demonstrated that the SOCE mechanism and lipid rafts participated in the formation of the polar morphology of fMLP induced neutrophils and the process of [Ca~ (2+)]_i increase, and further elucidated the role of the key signal molecules (Ca~ (2+)]_i, TRPC1) in the process of signal transduction in the process of neutrophil polarity.
5. In the available literature, we report for the first time that TRPC1 binds to lipid rafts when it participates in neutrophil polarization.
【學位授予單位】：南方醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2007
【分類號】：R392

【引證文獻】

相關期刊論文 前1條

1 鄒珍友;黃艷;王勇;張慧;戴威;許露露;程彥偉;汪素美;;趨化劑對細胞偽足極性生長的影響研究[J];現(xiàn)代農業(yè)科技;2009年01期

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