中国病理生理杂志
中國病理生理雜誌
중국병리생리잡지
Chinese Journal of Pathophysiology
2015年
9期
1680-1687
,共8页
钱江%吴健%安弘%房祥峰%李东风%杨士芳%孟锦绣%高兴林
錢江%吳健%安弘%房祥峰%李東風%楊士芳%孟錦繡%高興林
전강%오건%안홍%방상봉%리동풍%양사방%맹금수%고흥림
抗原85B%胸腺基质淋巴细胞生成素%髓样树突状细胞%胸腺基质淋巴细胞生成素受体%OX40L
抗原85B%胸腺基質淋巴細胞生成素%髓樣樹突狀細胞%胸腺基質淋巴細胞生成素受體%OX40L
항원85B%흉선기질림파세포생성소%수양수돌상세포%흉선기질림파세포생성소수체%OX40L
Antigen 85B%Thymic stromal lymphopoietin%Myeloid dendritic cells%Thymic stromal lymphopoi-etin receptor%OX40L
目的：研究抗原85B（ Ag85B）体外诱导小鼠未成熟的髓样树突状细胞（ mDCs）的成熟以及对胸腺基质淋巴细胞生成素（TSLP）介导下mDCs表达TSLP受体（TSLPR）和OX40L的影响，探究Ag85B抑制哮喘气道炎症的可能机制。方法：应用重组小鼠GM－CSF和IL－4体外诱生C57BL／6小鼠未成熟的mDCs，并运用免疫磁珠分离的方法纯化，采用光镜和扫描电镜、流式细胞术进行形态学观察和细胞表型鉴定；分别用0、50、100、200μg／L不同浓度的Ag85B或TSLP作用于纯化并鉴定后的mDCs，培养24 h，流式细胞术检测细胞表面分子CD80、CD86、TSL－PR和OX40L的表达，选取最佳的Ag85B或TSLP处理浓度。随后将 mDCs随机分为空白对照组、Ag85B处理组、TSLP处理组和Ag85B＋TSLP处理组，培养24 h后检测mDCs的促炎表面分子TSLPR和OX40L的表达。结果：体外诱导培养7 d，倒置相差显微镜下可见细胞表面呈现不规则树突样突起，扫描电镜下见细胞类圆形，表面有少量皱褶和较少分叉的树突状突起，符合未成熟mDCs的形态学特点；纯化后的mDCs表达表面分子CD11c的细胞较表达共刺激分子CD80和CD86的细胞多，符合未成熟mDCs 的表型特征。与空白对照组比较，50～200μg／L的Ag85B处理组mDCs表达CD80和CD86的细胞比率显著增高（P＜0．05），表达TSLPR和OX40L的细胞比率无显著差异。与空白对照组相比较，50、100和200μg／L浓度的TSLP处理组的mDCs表达CD80和CD86的细胞比率均显著增加（ P＜0．05）；与空白对照组和50μg／L TSLP处理组相比较，100μg／L和200μg／L TSLP处理组的mDCs表达TSLPR和OX40L的细胞比率均显著升高（ P＜0．05）。选取200μg／L作为Ag85B和TSLP的优化作用浓度，结果发现Ag85B处理组和Ag85B＋TSLP处理组的mDCs表达TSLPR和OX40L的细胞比率较TSLP处理组均显著降低（ P＜0．05），与空白对照组比较差异不显著。结论：Ag85B可通过上调mDCs 表达共刺激分子CD80和CD86促进其成熟，同时下调TSLP介导的mDCs表达促炎表面分子TSLPR和OX40L，推测Ag85B可能通过TSLP介导的mDCs途径抑制气道炎症。
目的：研究抗原85B（ Ag85B）體外誘導小鼠未成熟的髓樣樹突狀細胞（ mDCs）的成熟以及對胸腺基質淋巴細胞生成素（TSLP）介導下mDCs錶達TSLP受體（TSLPR）和OX40L的影響，探究Ag85B抑製哮喘氣道炎癥的可能機製。方法：應用重組小鼠GM－CSF和IL－4體外誘生C57BL／6小鼠未成熟的mDCs，併運用免疫磁珠分離的方法純化，採用光鏡和掃描電鏡、流式細胞術進行形態學觀察和細胞錶型鑒定；分彆用0、50、100、200μg／L不同濃度的Ag85B或TSLP作用于純化併鑒定後的mDCs，培養24 h，流式細胞術檢測細胞錶麵分子CD80、CD86、TSL－PR和OX40L的錶達，選取最佳的Ag85B或TSLP處理濃度。隨後將 mDCs隨機分為空白對照組、Ag85B處理組、TSLP處理組和Ag85B＋TSLP處理組，培養24 h後檢測mDCs的促炎錶麵分子TSLPR和OX40L的錶達。結果：體外誘導培養7 d，倒置相差顯微鏡下可見細胞錶麵呈現不規則樹突樣突起，掃描電鏡下見細胞類圓形，錶麵有少量皺褶和較少分扠的樹突狀突起，符閤未成熟mDCs的形態學特點；純化後的mDCs錶達錶麵分子CD11c的細胞較錶達共刺激分子CD80和CD86的細胞多，符閤未成熟mDCs 的錶型特徵。與空白對照組比較，50～200μg／L的Ag85B處理組mDCs錶達CD80和CD86的細胞比率顯著增高（P＜0．05），錶達TSLPR和OX40L的細胞比率無顯著差異。與空白對照組相比較，50、100和200μg／L濃度的TSLP處理組的mDCs錶達CD80和CD86的細胞比率均顯著增加（ P＜0．05）；與空白對照組和50μg／L TSLP處理組相比較，100μg／L和200μg／L TSLP處理組的mDCs錶達TSLPR和OX40L的細胞比率均顯著升高（ P＜0．05）。選取200μg／L作為Ag85B和TSLP的優化作用濃度，結果髮現Ag85B處理組和Ag85B＋TSLP處理組的mDCs錶達TSLPR和OX40L的細胞比率較TSLP處理組均顯著降低（ P＜0．05），與空白對照組比較差異不顯著。結論：Ag85B可通過上調mDCs 錶達共刺激分子CD80和CD86促進其成熟，同時下調TSLP介導的mDCs錶達促炎錶麵分子TSLPR和OX40L，推測Ag85B可能通過TSLP介導的mDCs途徑抑製氣道炎癥。
목적：연구항원85B（ Ag85B）체외유도소서미성숙적수양수돌상세포（ mDCs）적성숙이급대흉선기질림파세포생성소（TSLP）개도하mDCs표체TSLP수체（TSLPR）화OX40L적영향，탐구Ag85B억제효천기도염증적가능궤제。방법：응용중조소서GM－CSF화IL－4체외유생C57BL／6소서미성숙적mDCs，병운용면역자주분리적방법순화，채용광경화소묘전경、류식세포술진행형태학관찰화세포표형감정；분별용0、50、100、200μg／L불동농도적Ag85B혹TSLP작용우순화병감정후적mDCs，배양24 h，류식세포술검측세포표면분자CD80、CD86、TSL－PR화OX40L적표체，선취최가적Ag85B혹TSLP처리농도。수후장 mDCs수궤분위공백대조조、Ag85B처리조、TSLP처리조화Ag85B＋TSLP처리조，배양24 h후검측mDCs적촉염표면분자TSLPR화OX40L적표체。결과：체외유도배양7 d，도치상차현미경하가견세포표면정현불규칙수돌양돌기，소묘전경하견세포류원형，표면유소량추습화교소분차적수돌상돌기，부합미성숙mDCs적형태학특점；순화후적mDCs표체표면분자CD11c적세포교표체공자격분자CD80화CD86적세포다，부합미성숙mDCs 적표형특정。여공백대조조비교，50～200μg／L적Ag85B처리조mDCs표체CD80화CD86적세포비솔현저증고（P＜0．05），표체TSLPR화OX40L적세포비솔무현저차이。여공백대조조상비교，50、100화200μg／L농도적TSLP처리조적mDCs표체CD80화CD86적세포비솔균현저증가（ P＜0．05）；여공백대조조화50μg／L TSLP처리조상비교，100μg／L화200μg／L TSLP처리조적mDCs표체TSLPR화OX40L적세포비솔균현저승고（ P＜0．05）。선취200μg／L작위Ag85B화TSLP적우화작용농도，결과발현Ag85B처리조화Ag85B＋TSLP처리조적mDCs표체TSLPR화OX40L적세포비솔교TSLP처리조균현저강저（ P＜0．05），여공백대조조비교차이불현저。결론：Ag85B가통과상조mDCs 표체공자격분자CD80화CD86촉진기성숙，동시하조TSLP개도적mDCs표체촉염표면분자TSLPR화OX40L，추측Ag85B가능통과TSLP개도적mDCs도경억제기도염증。
AIM:To investigate the maturation of mice immature myeloid dendritic cells (mDCs) induced by antigen(Ag)85B of mycobacterium tuberculosis, and the expression of TSLPR and OX40L mediated by TSLP in vitro. METHODS:Recombinant mouse GM-CSF ( rmGM-CSF) and rmIL-4 were used to induce bone marrow precursor cells of C57BL/6 mice to differentiate into immature mDCs in vitro.mDCs were identified followed by purification using CD 11c binding magnetic beads .The morphological characteristic of mDCs was observed under inverted phase-contrast microscope and scanning electron microscope .The surface phenotypes of mDCs were determined by flow cytometry .To obtain the opti-mal concentrations of Ag85B and TSLP, immature mDCs were cultured with different concentrations of Ag 85B or TSLP at 0 (control group), 50, 100 and 200 μg/L for 24 h, and the expression of cell surface molecules CD 80, CD86, TSLPR and OX40L was detected by flow cytometry.In addition, the expression of TSLPR and OX40L in Ag85B and TSLP-co-stimula-ted mDCs was determined by flow cytometry .RESULTS:After 7 d of culture in vitro, the cells showed irregular dendritic protrusions under the inverted-phase contrast microscope , and had wrinkles and dendritic splits under scanning electron mi-croscope , conformed to the morphological characteristics of immature mDCs .The mDCs cells expressed higher level of spe-cific marker CD11c, lower level of co-stimulatory molecules CD80 and CD86, which conformed to the phenotype of imma-ture mDCs.The CD80 +and CD86 +cell ratios of mDCs displayed significant increases in 50, 100 and 200μg/L Ag85B or TSLP groups compared with control group (P<0.05).The ratios of TSLPR +and OX40L+cells did not differ among dif-ferent concentrations of Ag 85B groups.The ratios of TSLPR +and OX40L+cells were significantly increased in 100 μg/L and 200μg/L TSLP groups compared with control group and 50μg/L TSLP group (P<0.05).Under the circumstance of optimal Ag85B or TSLP treatment concentration at 200 μg/L, there was significantly decreased in TSLPR and OX 40L cell ratio of mDCs in Ag85B group or Ag85B combined with TSLP group when compared with TSLP group (P<0.05), and no significant difference among Ag85B group, Ag85B combined with TSLP group and control group was observed .CONCLU-SION: Ag85B enhances mDCs maturation by up-regulating the expression of co-stimulatory molecules CD80 and CD86, and inhibit the expression of pro-inflammatory specific molecules TSLPR and OX40L on TSLP-activated mDCs, indicating that Ag85B modifies the development of asthmatic airway inflammation through the pathway of TSLP -activated mDCs.
