中国危重病急救医学
中國危重病急救醫學
중국위중병급구의학
CHINESE CRITICAL CARE MEDICINE
2011年
4期
208-212,后插2
,共6页
T淋巴细胞%超抗原%肠毒素%趋化因子受体%肺动脉内皮细胞
T淋巴細胞%超抗原%腸毒素%趨化因子受體%肺動脈內皮細胞
T림파세포%초항원%장독소%추화인자수체%폐동맥내피세포
T cell%Superantigen%Enterotoxin%Chemokine receptor%Pulmonary artery endothelial cell
目的 观察革兰阳性菌肠毒素活化的T淋巴细胞(T细胞)对人肺动脉内皮细胞(HPAEC)的损伤作用,初步探讨其损伤机制.方法 将葡萄球菌肠毒素B(SEB)活化后的T细胞上清加入HPAEC,检测内皮细胞趋化因子的分泌情况;用Transwell小室观察内皮细胞分泌的趋化因子对T细胞趋化的影响.用10 ng/ml SEB刺激的T细胞与HPAEC共培养,检测T细胞与内皮细胞的黏附率;用原位末端缺刻标记法(TUNEL)检测内皮细胞凋亡情况.结果 不同浓度T细胞培养上清刺激的HPAEC都表现出趋化因子随刺激时间延长而释放增加的趋势,72 h后1×10-2、1×10-1、1×100T细胞中单核细胞趋化蛋白-1(MCP-1,ng/ml)分别为1.240±0.103、4.200±0.305、6.500±0.500,巨噬细胞炎性蛋白-1α(MIP-1α,ng/ml)分别为0.210±0.015、0.287±0.012、0.531±0.037,正常T细胞表达和分泌因子(Rantes,ng/ml)分别为1.420±0.074、7.634±0.630、15.700±1.300,其中Rantes表现出早期(6 h)快速释放和后期(12、24、48、72 h)延迟释放的特点.与未经SEB作用的对照组相比,超抗原组的T细胞从Transwell小室上室趋化移动至聚碳酸酯膜的数量(个)显著增多(86.38±14.50比16.50±2.50,P＜0.01＝;与T细胞组比较,超抗原组24 h T细胞黏附率显著增加[(15.50±1.08)%比(1.60±0.22)%,P＜0.01＝,加入1 μg/ml甲基化Rantes组T细胞黏附率[(4.39±0.66)%]较超抗原组显著下降(P＜0.01＝;黏附到下层HPAEC的T细胞趋化因子受体5(CCR5)/CD4较100 ng/ml SEB诱导3 d的单个核细胞(PBMC)增加了约2.5倍,CCR5/CD8增加了约2.8倍;与正常培养的HPAEC组比较,超抗原组HPAEC细胞凋亡指数增加[(32.50±4.50)%比(3.50±0.50)%,P＜0.01＝.结论 SEB活化的T细胞增加了对HPAEC的趋化和黏附,进一步导致HPAEC的损伤;这种趋化作用的增强与SEB活化的T细胞表面CCR5表达上调有关.
目的 觀察革蘭暘性菌腸毒素活化的T淋巴細胞(T細胞)對人肺動脈內皮細胞(HPAEC)的損傷作用,初步探討其損傷機製.方法 將葡萄毬菌腸毒素B(SEB)活化後的T細胞上清加入HPAEC,檢測內皮細胞趨化因子的分泌情況;用Transwell小室觀察內皮細胞分泌的趨化因子對T細胞趨化的影響.用10 ng/ml SEB刺激的T細胞與HPAEC共培養,檢測T細胞與內皮細胞的黏附率;用原位末耑缺刻標記法(TUNEL)檢測內皮細胞凋亡情況.結果 不同濃度T細胞培養上清刺激的HPAEC都錶現齣趨化因子隨刺激時間延長而釋放增加的趨勢,72 h後1×10-2、1×10-1、1×100T細胞中單覈細胞趨化蛋白-1(MCP-1,ng/ml)分彆為1.240±0.103、4.200±0.305、6.500±0.500,巨噬細胞炎性蛋白-1α(MIP-1α,ng/ml)分彆為0.210±0.015、0.287±0.012、0.531±0.037,正常T細胞錶達和分泌因子(Rantes,ng/ml)分彆為1.420±0.074、7.634±0.630、15.700±1.300,其中Rantes錶現齣早期(6 h)快速釋放和後期(12、24、48、72 h)延遲釋放的特點.與未經SEB作用的對照組相比,超抗原組的T細胞從Transwell小室上室趨化移動至聚碳痠酯膜的數量(箇)顯著增多(86.38±14.50比16.50±2.50,P＜0.01＝;與T細胞組比較,超抗原組24 h T細胞黏附率顯著增加[(15.50±1.08)%比(1.60±0.22)%,P＜0.01＝,加入1 μg/ml甲基化Rantes組T細胞黏附率[(4.39±0.66)%]較超抗原組顯著下降(P＜0.01＝;黏附到下層HPAEC的T細胞趨化因子受體5(CCR5)/CD4較100 ng/ml SEB誘導3 d的單箇覈細胞(PBMC)增加瞭約2.5倍,CCR5/CD8增加瞭約2.8倍;與正常培養的HPAEC組比較,超抗原組HPAEC細胞凋亡指數增加[(32.50±4.50)%比(3.50±0.50)%,P＜0.01＝.結論 SEB活化的T細胞增加瞭對HPAEC的趨化和黏附,進一步導緻HPAEC的損傷;這種趨化作用的增彊與SEB活化的T細胞錶麵CCR5錶達上調有關.
목적 관찰혁란양성균장독소활화적T림파세포(T세포)대인폐동맥내피세포(HPAEC)적손상작용,초보탐토기손상궤제.방법 장포도구균장독소B(SEB)활화후적T세포상청가입HPAEC,검측내피세포추화인자적분비정황;용Transwell소실관찰내피세포분비적추화인자대T세포추화적영향.용10 ng/ml SEB자격적T세포여HPAEC공배양,검측T세포여내피세포적점부솔;용원위말단결각표기법(TUNEL)검측내피세포조망정황.결과 불동농도T세포배양상청자격적HPAEC도표현출추화인자수자격시간연장이석방증가적추세,72 h후1×10-2、1×10-1、1×100T세포중단핵세포추화단백-1(MCP-1,ng/ml)분별위1.240±0.103、4.200±0.305、6.500±0.500,거서세포염성단백-1α(MIP-1α,ng/ml)분별위0.210±0.015、0.287±0.012、0.531±0.037,정상T세포표체화분비인자(Rantes,ng/ml)분별위1.420±0.074、7.634±0.630、15.700±1.300,기중Rantes표현출조기(6 h)쾌속석방화후기(12、24、48、72 h)연지석방적특점.여미경SEB작용적대조조상비,초항원조적T세포종Transwell소실상실추화이동지취탄산지막적수량(개)현저증다(86.38±14.50비16.50±2.50,P＜0.01＝;여T세포조비교,초항원조24 h T세포점부솔현저증가[(15.50±1.08)%비(1.60±0.22)%,P＜0.01＝,가입1 μg/ml갑기화Rantes조T세포점부솔[(4.39±0.66)%]교초항원조현저하강(P＜0.01＝;점부도하층HPAEC적T세포추화인자수체5(CCR5)/CD4교100 ng/ml SEB유도3 d적단개핵세포(PBMC)증가료약2.5배,CCR5/CD8증가료약2.8배;여정상배양적HPAEC조비교,초항원조HPAEC세포조망지수증가[(32.50±4.50)%비(3.50±0.50)%,P＜0.01＝.결론 SEB활화적T세포증가료대HPAEC적추화화점부,진일보도치HPAEC적손상;저충추화작용적증강여SEB활화적T세포표면CCR5표체상조유관.
Objective To observe the injurious effect of T cell activated by Staphylococcus enterotoxin B (SEB) on human pulmonary artery endothelial cell (HPAEC) and explore its possible mechanism. Methods HPAEC was cocultured with SEB-activated T cells supernatant, and the secretion of chemotactic factors from HPAEC was examined. The Transwell inserts was used in chemoattraction assays. After HPAECs were cocultured with T cells and 10 ng/ml SEB for 3 days, HPAEC damage was monitored by microscopy and the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. Results Three kinds of tested chemokines showed a time-dependent increase in all supernatant of HPAEC incubated with different concentrations of T cells. After 72 hours, the monocyte chemoattractant protein-1 (MCP-1,ng/ml) in 1 × 10-2, 1 × 10-1 , 1× 100 T cell supernatant groups was 1. 240± 0. 103, 4. 200± 0. 305, 6. 500±0. 500, respectively, macrophage inflammatory protein-1a (MIP-1α, ng/ml) was 0. 210 ± 0. 015, 0. 287 ±0. 012, 0. 531 ± 0. 037, respectively, and Rantes (ng/ml) was 1. 420 ± 0. 074, 7. 634 ± 0. 630, 15. 700 ±1. 300, respectively. Rantes presented a two-phase secretion mode : in early 6 hours it increased swiftly, but relatively slow at 12, 24, 48, 72 hours. T cell adherent to polycarbonate membrane increased after SEB stimulation in superantigen group compared with control group without SEB stimulation (86. 38± 14. 50 vs.16. 50± 2. 50, P＜ 0. 01 ). When 10 ng/ml SEB activated T cell was cocultured with HPAEC, more of originally suspended cultured T cells adhered to HPAEC monolayer [(15. 50±1.08)% vs. (1.60±0. 22)%,P＜0.01], whereas the cell adhesion ratio decreased markedly in 1 μg/ml Met-Rantes group [(4. 39±0. 66)%, P ＜ 0. 01 ). FACs test of HPAEC-adherent T cell showed lymphocyte chemokine receptor 5 (CCR5)/CD4 and CCR5/CD8 increased over 2. 5 folds and 2.8 folds compared with 100 ng/ml SEB activated T cell. Cell death rate of HPAEC was increased when cocultured with SEB-activated T cell in superantigen group compared with HPAEC normal incubation group [(32. 50±4. 50)% vs. (3. 50±0. 50)%, P＜0. 01].Conclusion Increased chemoattraction and adherence of SEB-activated T cells to HPAEC could damage HPAEC; this effect was possibly due to up-regulation of CCR5 on T cell.
