胃肠病学
胃腸病學
위장병학
CHINESE JOURNAL OF GASTROENTEROLOGY
2014年
11期
644-649
,共6页
淋巴细胞功能相关抗原-1%炎症性肠病%Na?veT细胞%Th17细胞%细胞因子类
淋巴細胞功能相關抗原-1%炎癥性腸病%Na?veT細胞%Th17細胞%細胞因子類
림파세포공능상관항원-1%염증성장병%Na?veT세포%Th17세포%세포인자류
Lymphocyte Function-Associated Antigen-1%InfIammatory BoweI Disease%Na?veTCeIIs%Th17 CeIIs%Cytokines
背景：淋巴细胞功能相关抗原-1（ LFA-1）参与T细胞的活化和功能调节，与炎症性肠病的发病密切相关。目的：观察LFA-1基因缺失（ LFA-1-/-）对小鼠Na?ve T细胞体外向Th17细胞分化的影响。方法：繁殖LFA-1-/-子代小鼠，提取鼠尾DNA，PCR法鉴定基因型。LFA-1-/-子代小鼠为实验组，野生型（ WT）C57BL/6J小鼠为对照组，磁珠分选脾脏单个核细胞中的CD4+CD62L+ Na?ve T细胞并检测其纯度。体外建立不同Th17细胞诱导分化体系[转化生长因子-β（ TGF-β）、TGF-β+白细胞介素-6（ IL-6）和 TGF-β+IL-6+IL-23]，以流式细胞术检测两组分选得到的Na?ve T细胞在不同体系中诱导出的Th17细胞比率，荧光定量PCR法和ELISA法检测Th17细胞特异性转录因子ROR-γt和特异性标记物 IL-17A表达。结果：15只子代小鼠均为 LFA-1-/-小鼠，磁珠分选得到的 CD4+CD62L+Na?ve T细胞纯度大于95%。低剂量TGF-β+IL-6即能诱导出Th17细胞，在此基础上加入IL-23能促进更多Th17细胞产生。与WT对照组相比，LFA-1-/-组Na?ve T细胞在TGF-β+IL-6+IL-23体系中诱导产生Th17细胞的效应更为明显（17.2%±1.4%对5.7%±0.2%，P＜0.001），ROR-γt、IL-17A mRNA表达上调（P＜0.001），细胞培养上清液中IL-17A浓度升高（ P＜0.01）。结论：LFA-1基因缺失能促进小鼠Na?ve T细胞体外向Th17细胞分化。
揹景：淋巴細胞功能相關抗原-1（ LFA-1）參與T細胞的活化和功能調節，與炎癥性腸病的髮病密切相關。目的：觀察LFA-1基因缺失（ LFA-1-/-）對小鼠Na?ve T細胞體外嚮Th17細胞分化的影響。方法：繁殖LFA-1-/-子代小鼠，提取鼠尾DNA，PCR法鑒定基因型。LFA-1-/-子代小鼠為實驗組，野生型（ WT）C57BL/6J小鼠為對照組，磁珠分選脾髒單箇覈細胞中的CD4+CD62L+ Na?ve T細胞併檢測其純度。體外建立不同Th17細胞誘導分化體繫[轉化生長因子-β（ TGF-β）、TGF-β+白細胞介素-6（ IL-6）和 TGF-β+IL-6+IL-23]，以流式細胞術檢測兩組分選得到的Na?ve T細胞在不同體繫中誘導齣的Th17細胞比率，熒光定量PCR法和ELISA法檢測Th17細胞特異性轉錄因子ROR-γt和特異性標記物 IL-17A錶達。結果：15隻子代小鼠均為 LFA-1-/-小鼠，磁珠分選得到的 CD4+CD62L+Na?ve T細胞純度大于95%。低劑量TGF-β+IL-6即能誘導齣Th17細胞，在此基礎上加入IL-23能促進更多Th17細胞產生。與WT對照組相比，LFA-1-/-組Na?ve T細胞在TGF-β+IL-6+IL-23體繫中誘導產生Th17細胞的效應更為明顯（17.2%±1.4%對5.7%±0.2%，P＜0.001），ROR-γt、IL-17A mRNA錶達上調（P＜0.001），細胞培養上清液中IL-17A濃度升高（ P＜0.01）。結論：LFA-1基因缺失能促進小鼠Na?ve T細胞體外嚮Th17細胞分化。
배경：림파세포공능상관항원-1（ LFA-1）삼여T세포적활화화공능조절，여염증성장병적발병밀절상관。목적：관찰LFA-1기인결실（ LFA-1-/-）대소서Na?ve T세포체외향Th17세포분화적영향。방법：번식LFA-1-/-자대소서，제취서미DNA，PCR법감정기인형。LFA-1-/-자대소서위실험조，야생형（ WT）C57BL/6J소서위대조조，자주분선비장단개핵세포중적CD4+CD62L+ Na?ve T세포병검측기순도。체외건립불동Th17세포유도분화체계[전화생장인자-β（ TGF-β）、TGF-β+백세포개소-6（ IL-6）화 TGF-β+IL-6+IL-23]，이류식세포술검측량조분선득도적Na?ve T세포재불동체계중유도출적Th17세포비솔，형광정량PCR법화ELISA법검측Th17세포특이성전록인자ROR-γt화특이성표기물 IL-17A표체。결과：15지자대소서균위 LFA-1-/-소서，자주분선득도적 CD4+CD62L+Na?ve T세포순도대우95%。저제량TGF-β+IL-6즉능유도출Th17세포，재차기출상가입IL-23능촉진경다Th17세포산생。여WT대조조상비，LFA-1-/-조Na?ve T세포재TGF-β+IL-6+IL-23체계중유도산생Th17세포적효응경위명현（17.2%±1.4%대5.7%±0.2%，P＜0.001），ROR-γt、IL-17A mRNA표체상조（P＜0.001），세포배양상청액중IL-17A농도승고（ P＜0.01）。결론：LFA-1기인결실능촉진소서Na?ve T세포체외향Th17세포분화。
BacKground:Lymphocyte function-associated antigen-1( LFA-1 ) pIays a cruciaI roIe in the pathogenesis of infIammatory boweI disease by reguIating the activation and function of T ceIIs. Aims:To investigate the effect of LFA-1 deficiency( LFA-1-/-)on differentiation of mice na?ve T ceIIs into Th17 ceIIs in vitro. Methods:LFA-1-/- mice were breeded and the progeny genome DNA was extracted from the taiIs for genotyping by PCR. CD4+CD62L+ na?ve T ceIIs were separated from spIenic mononucIear ceIIs of LFA-1-/- progeny mice and wiId type ( WT ) C57BL/6J mice, respectiveIy,by magnetic-activated ceII sorting( MACS),and then the purity of separated ceIIs was determined. Na?ve T ceIIs obtained were cuItured in different inducing systems[ transforming growth factor-β( TGF-β),TGF-β + interIeukin-6 (IL-6),and TGF-β + IL-6 + IL-23]in vitro for Th17 ceII differentiation;ceIIs in each inducing system were coIIected for anaIyzing the ratio of Th17 ceIIs by fIow cytometry,and the expressions of Th17-specific transcription factor ROR-γt and Th17-specific marker IL-17A were measured by qRT-PCR and ELISA methods. Results:AII fifteen progeny mice were identified as LFA-1-/- genotype. Purity of CD4+CD62L+ na?ve T ceIIs separated by MACS was above 95%. Th17 ceIIs couId be induced by Iow-dose TGF-βcombined with IL-6,and the differentiation ratio was increased obviousIy when IL-23 was added. In inducing system containing TGF-β,IL-6 and IL-23,na?ve T ceIIs from LFA-1-/- mice produced more Th17 ceIIs than those from WT mice(17. 2% ± 1. 4% vs. 5. 7% ± 0. 2%,P<0. 001),expressions of ROR-γt mRNA and IL-17A mRNA were up-reguIated(P<0. 001),and IL-17A concentration in ceII cuIture supernatant was increased(P<0. 01). Conclusions:Deficiency of LFA-1 promotes the differentiation of mice na?ve T ceIIs into Th17 ceIIs in vitro.
