肺炎鏈球菌觸發(fā)肺型上皮細(xì)胞微絲肌動(dòng)蛋白重排的鈣信

1、肺炎鏈球菌觸發(fā)肺型上皮細(xì)胞微絲肌動(dòng)蛋白重排的鈣信號轉(zhuǎn)導(dǎo)機(jī)制 作者：徐邦牢1；尹一兵2(1廣州市第一人民醫(yī)院檢驗(yàn)科，廣東 廣州 510180；2重慶醫(yī)科大學(xué)檢驗(yàn)系， 重慶 400016)摘要：目的通過體外實(shí)驗(yàn)研究肺炎鏈球菌(S.pn)是否可通過肺型上皮細(xì)胞(A549)鈣信號轉(zhuǎn)導(dǎo)途徑觸發(fā)微絲肌動(dòng)蛋白(F-actin)細(xì)胞骨架重排，進(jìn)而導(dǎo)致S.pn對A549細(xì)胞的侵襲。 方法 采用F-actin特異性FITC-phalloidin熒光染料，觀察S.pn作用A549細(xì)胞前后F-actin細(xì)胞骨架重排情況，以重排百分率表示；用F-actin細(xì)胞骨架重排抑制劑細(xì)胞松弛素D預(yù)處理A549細(xì)胞，觀察S.pn對

2、A549細(xì)胞的侵襲；使用Ca2+信號轉(zhuǎn)導(dǎo)抑制劑dantrolene預(yù)處理A549細(xì)胞，觀察其與F-actin細(xì)胞骨架重排百分率間是否存在劑量依賴關(guān)系；用Fura-2/AM熒光探針負(fù)載A549細(xì)胞后測定S.pn粘附A549細(xì)胞30、60、90 min的胞內(nèi)Ca2+i。 結(jié)果 S.pn作用A549細(xì)胞后，經(jīng)FITC-phalloidin熒光染色，F(xiàn)-actin細(xì)胞骨架呈黃綠色塊狀聚集；F-actin細(xì)胞骨架重排抑制劑細(xì)胞松弛素D可明顯降低S.pn對A549細(xì)胞的侵襲，在其濃度為0.25 g/ml時(shí)，未得到可測的細(xì)菌數(shù)；Ca2+信號轉(zhuǎn)導(dǎo)抑制劑可部分抑制A549細(xì)胞F-actin細(xì)胞骨架重排，且與F-

3、actin細(xì)胞骨架重排百分率間存在量效關(guān)系；S.pn粘附A549細(xì)胞30、60、90 min后的胞內(nèi)Ca2+i高于對照(187.417.3 nmol/L)，并達(dá)到飽和，分別為(487.538.1)、(548.235.6)和(557.247.5) nmol/L。 結(jié)論 S.pn可通過Ca2+細(xì)胞信號轉(zhuǎn)導(dǎo)途徑觸發(fā)A549細(xì)胞F-actin細(xì)胞骨架重排，進(jìn)而導(dǎo)致S.pn侵襲A549細(xì)胞。關(guān)鍵詞：鏈球菌，肺炎；肺/細(xì)胞學(xué)；上皮細(xì)胞；微絲肌動(dòng)蛋白；鈣信號；細(xì)胞支架中圖分類號：R378.14文獻(xiàn)標(biāo)識碼：A文章編號：1000-2588(2004)02-0168-04Calcium signaling eve

4、nts in Streptococcus pneumoniae invasion of human type pneumocytesXU Bang-lao1； YIN Yi-bing21Department of Clinical Laboratory, First Municipal Hospital of Guangzhou, Guangzhou 510180, China; 2Department of Laboratory Medicine, Chongqing University of Medical Sciences, Chongqing 400016, ChinaAbstrac

5、t: Objective To study whether Streptococcus pneumoniae (S.pn) can provoke filamentous actin (F-actin) rearran- gements in vitro through calcium signaling pathways in type pneumocytes(A549 cells), resulting in S.pn invasion of the cells. Method After FITC-phalloidin labeling of F-actin, F-actin rearr

6、angements were observed by S.pn adhesion to typepneumocyte A549 cells. S.pn invasion of A549 cells was determined by pretreating A549 cells with cytochalasin D. To investigate whether F-actin rearrangements could be blocked by Ca2+ inhibitors, A549 cells were pretreated with Ca2+ inhibitors dantrole

7、ne, and loaded in Fura-2/AM probe to determine the concentration of cytosolic free calcium by S.pn adhesion to A549 cells after 30, 60, and 90 min respectively. Results Intact S.pn can promote F-actin rearrangements. Cytochalasin D was able to prevent S.pn invasion of A549 cells. No invasion of A549

8、 cell can be determined at 0.25 g/ml of cytochalasin D. One subset of the inhibitors of Ca2+ signal transduction molecules blocked F-actin rearrangements dose-dependently, and S.pn adhesion of A549 cells for 30, 60, and 90 min increased cytosolic free calcium, reaching(487.538.1),(548.235.6) and(557

9、.247.5) nmol/L, respectively. They were higher than of the control group. Conclusion S.pn can provoke F-actin rearrangements through Ca2+ signaling pathways, which further leads to S.pn invasion of A549 cells.Key words: Streptococcus pneumoniae; lung/cytology; epithelial cells; filamentous actin; ca

10、lcium signaling; cytoskeleton收稿日期：2003-10-12基金項(xiàng)目：國家自然科學(xué)基金(30170050)Supported by National Natural Science Foundation of China(30170050)作者簡介：徐邦牢(1969-)，男，2002年畢業(yè)于重慶醫(yī)科大學(xué)，碩士，主管技師，電話E-mail: xubanglao 肺炎鏈球菌(Streptococcus pneumoniae，S.pn)是一種常見的革蘭氏陽性致病菌，所致臨床發(fā)病率和死亡率均較高1。目前，由于多糖莢膜疫苗免疫原性較弱及耐藥現(xiàn)象的出

11、現(xiàn)，使預(yù)防及治療具有一定的局限性。有文獻(xiàn)資料報(bào)道，腸致病性大腸埃希氏菌及沙門氏菌等革蘭氏陰性致病菌粘附宿主細(xì)胞可使胞內(nèi)Ca2+i增加，由此觸發(fā)微絲肌動(dòng)蛋白(F-actin)細(xì)胞骨架重排，進(jìn)而侵襲宿主細(xì)胞23。Ca2+除具有信號轉(zhuǎn)導(dǎo)功能外，還具有直接參與由細(xì)胞內(nèi)肌動(dòng)蛋白引起的興奮-收縮耦聯(lián)效應(yīng)這一重要的生理功能?；诖?，本研究旨在探討S.pn是否通過鈣信號轉(zhuǎn)導(dǎo)觸發(fā)F-actin細(xì)胞骨架重排，進(jìn)而侵襲肺型上皮細(xì)胞(A549)。這將為最終闡明S.pn的致病機(jī)制提供科學(xué)的理論依據(jù)，為開發(fā)新一代治療S.pn感染性疾病的藥物提供新思路。1材料和方法432PO4 0.6 g，KCl 0.4 g，NaHCO3

12、 0.42 g，MgSO47H2O 0.197 g，Na2HPO412H22HPO4620=0.4，約108菌落形成單位/ml)。用于整個(gè)實(shí)驗(yàn)時(shí)，先離心(2 500 r/min、10 min)，再用預(yù)冷的PBS洗3次，不含雙抗RPMI-1640懸浮至108252孵箱中溫育2 h后，用無菌PBS輕輕洗3次；加入含1%FCS、10 U/ml青霉素、200 U/ml慶大霉素的RPMI-1640培養(yǎng)液；37 、5%CO2孵箱孵育1 h，以充分殺死胞外S.pn；用無菌PBS洗3次，加入100 l 0.25%胰酶使貼壁細(xì)胞懸浮，再加入400 l 0.025% TritonX-100裂解細(xì)胞3 min，10

13、倍稀釋后吸取100 l涂布瓊脂血平板6；37 、5% CO22+i測定 用0.25%胰酶消化貼壁單層A549，D-Hanks液洗3次；用2 ml負(fù)載液懸浮細(xì)胞，加入10 l Fura-2/AM溶液(終濃度為5 mol/L)，混勻，37 水浴避光振蕩40 min。將負(fù)載后細(xì)胞用含0.2% BSA的D-Hanks液洗3次，用細(xì)胞內(nèi)鈣測定液懸浮細(xì)胞。錐蟲藍(lán)染色檢查細(xì)胞活力90%7，并調(diào)整細(xì)胞數(shù)至2106/ml。測定前將儀器比色器恒定在37 ，分別掃描標(biāo)準(zhǔn)品及負(fù)載后細(xì)胞懸液的激發(fā)波長和發(fā)射波長以判斷負(fù)載是否成功，選擇負(fù)載后細(xì)胞懸液的激發(fā)波長和發(fā)射波長為測定波長。胞內(nèi)Ca2+i由下式計(jì)算：Ca2+i(n

14、mol/L)=Kd(F-Fmin)/(Fmax-F)，式中Kd為Fura-2/Ca2+的解離常數(shù)，在37 時(shí)為224 nmol/L，F(xiàn)為在不同條件下測定的熒光強(qiáng)度值；Fmax為加入終濃度為0.1% TrtonX-100后的最大熒光強(qiáng)度值，F(xiàn)min為在Fmax基礎(chǔ)上再加入終濃度為5 mmol/L EGTA時(shí)的最小熒光強(qiáng)度值82+i測定 將A549接種于24孔培養(yǎng)板孔。單層貼壁(約5105/孔)后吸出孔內(nèi)液體，各孔加入無雙抗RPMI-1640培養(yǎng)液懸浮的 S.pn菌液(約108菌落形成單位/ml)，37 、5% CO2分別溫育30、60、90 min，收集12孔細(xì)胞于一塑料試管中。收集細(xì)胞方法：在

15、溫育后用無菌PBS(pH 7.4)洗滌細(xì)胞6次；用含0.25%膠原酶的RPMI-1640溫育消化細(xì)胞10 min；再用含5%FCS的PBS洗2次，負(fù)載并測定計(jì)算A549細(xì)胞內(nèi)Ca2+2結(jié)果2.1完整的S.pn觸發(fā)A549細(xì)胞F-actin細(xì)胞骨架重排S.pn作用A549細(xì)胞后，F(xiàn)-actin經(jīng)FITC-phalloidin染色呈圓形深黃色聚集(圖1)；對照細(xì)胞無上述變化，呈現(xiàn)均勻黃色熒光外觀(圖2)。 圖1完整的S.pn作用A549細(xì)胞2 h后F-actin在熒光顯微鏡下呈黃色并圓形聚集(原放大倍數(shù)：400)Fig.1Round aggregation of yellow F-actin in

16、 A549 cells observed under fluoroscopy after intact S.pn adhesion ofA549 for 2 h(Original magnification:400)圖2對照A549細(xì)胞內(nèi)F-actin在熒光顯微鏡下呈黃色并均勻分布(原放大倍數(shù)：400)Fig.2Even distribution of yellow F-actin in control A549 cells observed under fluoroscopy(Original magnification:400)2+2+抑制劑dantrolene預(yù)處理A549細(xì)胞后的F-a

17、ctin的重排百分率與對照組(dantrolene濃度為0 mol/L)比較有顯著差異(P0.001)。F-actin細(xì)胞骨架重排與信號轉(zhuǎn)導(dǎo)抑制劑dantrolene間存在劑量依賴關(guān)系。圖3顯示用Ca2+抑制劑dantrolene 預(yù)處理A549細(xì)胞后，在熒光顯微鏡下F-actin細(xì)胞骨架重排被部分抑制；圖4顯示DMSO對重排無影響。 圖3Dantrolene預(yù)處理后F-actin聚集被部分抑制(原放大倍數(shù)：200)Fig.3F-actin aggregation partly inhibited by 25 mol/L dantrolene(Original magnification:20

18、0)圖4DMSO作用A549細(xì)胞后F-actin不發(fā)生聚集(原放大倍數(shù)：200)Fig.4F-actin without aggregation after DMSO treatment of A549 cells(Original magnification:200)2+i細(xì)胞外Ca2+i為1.3 mmol/L時(shí)，對照A549細(xì)胞的胞內(nèi)Ca2+i為(194.117.4)nmol/L(n=4)；測定液中不含Ca2+并加入0.1 mmol/L EGTA時(shí)，A549細(xì)胞的胞內(nèi)Ca2+2+i測定 S.pn粘附A549細(xì)胞30、60、90 min后的胞內(nèi)Ca2+i高于對照(187.417.3 nmol

19、/L)，P0.001并達(dá)到飽和，分別為(487.538.1)、(548.235.6)和(557.247.5) nmol/L。3討論病原菌侵襲宿主細(xì)胞是傳染過程建立的重要環(huán)節(jié)之一，病原菌通常借助宿主細(xì)胞F-actin細(xì)胞骨架的高度可塑性及其運(yùn)動(dòng)功能而侵襲細(xì)胞9。研究S.pn是否通過F-actin細(xì)胞骨架侵襲宿主細(xì)胞及其信號轉(zhuǎn)導(dǎo)機(jī)制，對于闡明其致病機(jī)制有重要意義。3.1S.pn觸發(fā)A549細(xì)胞F-actin細(xì)胞骨架重排及F-actin細(xì)胞骨架重排在S.pn侵襲A549細(xì)胞中的作用本研究證實(shí)完整的S.pn可觸發(fā)A549細(xì)胞F-actin細(xì)胞骨架重排，這與文獻(xiàn)所報(bào)道的關(guān)于革蘭氏陰性病原菌作用于宿主細(xì)胞

20、后的結(jié)果有類似之處，也是到目前為止，首次描述S.pn觸發(fā)A549細(xì)胞F-actin細(xì)胞骨架重排的形態(tài)學(xué)特征。為進(jìn)一步研究F-actin細(xì)胞骨架重排在S.pn侵襲A549細(xì)胞中的作用，我們用不同濃度的細(xì)胞松弛素D預(yù)處理A549細(xì)胞，觀察侵襲數(shù)的變化，發(fā)現(xiàn)隨抑制劑濃度增大，侵襲數(shù)明顯降低，并在濃度為0.25 g/ml時(shí)，未得到可測的侵襲數(shù)，提示F-actin細(xì)胞骨架重排在S.pn侵襲A549細(xì)胞中起著非常重要的作用，它可直接影響S.pn侵襲A549細(xì)胞。3.2S.pn觸發(fā)A549細(xì)胞F-actin細(xì)胞骨架重排的鈣信號轉(zhuǎn)導(dǎo)機(jī)制目前為止，文獻(xiàn)中尚無關(guān)于S.pn可觸發(fā)A549細(xì)胞F-actin細(xì)胞骨架重

21、排機(jī)制的報(bào)道。我們通過研究發(fā)現(xiàn)S.pn可通過鈣信號轉(zhuǎn)導(dǎo)途徑觸發(fā)A549細(xì)胞F-actin細(xì)胞骨架重排，F(xiàn)-actin細(xì)胞骨架重排百分率與該抑制劑間存在劑量依賴關(guān)系，二者變化呈高度負(fù)相關(guān)，相關(guān)系數(shù)為r=-0.86。S.pn作用于A549細(xì)胞可引起胞內(nèi)Ca2+i的變化，S.pn粘附A549細(xì)胞30、60、90 min后胞內(nèi)Ca2+i均比對照組約高3倍，由此提示S.pn粘附A549細(xì)胞可使胞內(nèi)Ca2+i增加，F(xiàn)-actin細(xì)胞骨架發(fā)生重排是由S.pn粘附A549細(xì)胞后觸發(fā)鈣信號轉(zhuǎn)導(dǎo)途徑引起的。其最可能的機(jī)制為：S.pn粘附A549細(xì)胞，使胞內(nèi)Ca2+i增加，而Ca2+可影響多種F-actin結(jié)合蛋白

22、活性1011。S.pn很可能通過鈣信號轉(zhuǎn)導(dǎo)使多種F-actin結(jié)合蛋白活性發(fā)生變化，活性變化大小與F-actin細(xì)胞骨架重排百分率成比例，但有關(guān)S.pn通過Ca2+信號轉(zhuǎn)導(dǎo)途徑引起與F-actin細(xì)胞骨架重排相關(guān)的A549細(xì)胞是何種蛋白的變化，以及其與A549細(xì)胞內(nèi)Ca2+振蕩的調(diào)節(jié)或相關(guān)關(guān)系還有待進(jìn)一步研究。參考文獻(xiàn)：1Gillespie SH. Aspects of pneumococcal infection including bacterial virulence, host response and vaccinationJ. J Med Microbiol, 1989, 28(9

23、): 237-48.2Dytoc M, Fedorko L, Sherman PM. Signal transduction in human epithelial cells infected with attaching and effacing Escherichia coli in vitroJ. Gastroenterology, 1999, 106(21): 1150-61.3Philpott DJ, Ismaili A, Dytoc MT. Increased adherence of Escheri- chia coli RDEC-1 to human tissue cultu

24、re cells results in the activation of host signaling pathwaysJ. J Infect Dis, 1995, 172(13): 136-43.4Lacks SA. Study of genetic material determining an enzyme activity in pneumococcusJ. Biochem Biophys Acta, 1960, 39(7): 508-17.5Peiffer I, Servin AL, Bernet-Camard MF. Piracy of decay-accelera- ting

25、factor (CD55) by the diffusely adhering strain Escherichia coli C1845 promotes cytoskeletal F-actin rearrangements in cultured human intestinal INT407 cellsJ. Infect Immun, 1998, 66(9): 4036-42.6Paton JC. Molecular analysis of the pathogenicity of streptococcus pneumoniae: the role of pneumococcal p

26、roteinsJ. Annu Rev Micro- biol, 1993, 47(22): 89-115.7劉振偉. 用Fura-2測定缺氧時(shí)海馬細(xì)胞內(nèi)游離鈣離子濃度的變化J. 細(xì)胞生物學(xué)雜志 (J Cytobiol), 1994, 16(3): 141-3.8Dana JP, Ismaili A, Dytoc MT, et al. Increased adherence of Escheri- chia coli RDEC-1 to human tissue culture cells results in the activa- tion of host signaling pathway

27、sJ. J Infect Dis, 1998, 172(1): 136-43.9Mitchell TJ. Celluar microbiology: an integrated approach to under- standing pathogenesis of infectionJ. J Cell Sci, 2000, 113(pt19): 3355-6.10楊建一. 醫(yī)學(xué)細(xì)胞生物學(xué)M. 北京: 人民衛(wèi)生出版社, 2000. 127-48.11Finlay BB, Cossart P. Exploitation of mammalian host cell function by bact

28、erial pathogensJ. Science, 1997, 276(5313): 718-25.參考文獻(xiàn)：1Gillespie SH. Aspects of pneumococcal infection including bacterial virulence, host response and vaccinationJ. J Med Microbiol, 1989, 28(9): 237-48.2Dytoc M, Fedorko L, Sherman PM. Signal transduction in human epithelial cells infected with at

29、taching and effacing Escherichia coli in vitroJ. Gastroenterology, 1999, 106(21): 1150-61.3Philpott DJ, Ismaili A, Dytoc MT. Increased adherence of Escheri- chia coli RDEC-1 to human tissue culture cells results in the activation of host signaling pathwaysJ. J Infect Dis, 1995, 172(13): 136-43.4Lacks SA. Study of genetic material determining an enzyme activity in pneumococcusJ. Biochem Biophys Acta, 1960, 39(7): 508-17.5Peiffer I, Servin AL, Bernet-Camard MF