中华烧伤杂志
中華燒傷雜誌
중화소상잡지
Chinese Journal of Burns
2015年
5期
354-360
,共7页
刘大东%庄明峰%张景丽%陈静家%孙炳伟
劉大東%莊明峰%張景麗%陳靜傢%孫炳偉
류대동%장명봉%장경려%진정가%손병위
脓毒症%血小板%外源性一氧化碳释放分子2%糖原合酶激酶3β
膿毒癥%血小闆%外源性一氧化碳釋放分子2%糖原閤酶激酶3β
농독증%혈소판%외원성일양화탄석방분자2%당원합매격매3β
Sepsis%Blood platelets%Exogenous carbon monoxide-releasing molecules 2%Glycogen synthase kinase 3β
目的 探讨外源性一氧化碳释放分子2(CORM-2)对LPS所致健康人外周血中血小板过度活化的作用及分子机制.方法 采集1名健康成年人的静脉血,离心分离出富血小板血浆(PRP),分装于硅化后的试管,按随机数字表法分为正常对照组、LPS组、无活性CORM-2(iCORM-2)组、10 μmol/L CORM-2组、50 μmol/L CORM-2组,每组3管.正常对照组不进行任何处理;LPS组接受20 mL 10 μg/mL的LPS刺激;iCORM-2组、10 μmol/L CORM-2组、50 μmol/L CORM-2组在接受上述LPS刺激的同时分别接受50 μmol/L iCORM-2、10μmol/L CORM-2、50 μmol/L CORM-2的干预,用量均为20 mL.培养30 min后玻瓶法检测血小板黏附率;免疫荧光法检测血小板侵袭至纤维蛋白原的数量;比浊法检测血小板聚集率;取PRP制备贫血小板血浆(PPP)及血小板,化学荧光素法检测PPP及血小板内ATP含量;流式细胞仪检测血小板膜糖蛋白(GP)Ⅰ bα和GPⅥ表达;蛋白质印迹法、免疫沉淀法分别检测血小板糖原合酶激酶3β (GSK-3β)及磷酸化GSK-3β表达.以上指标均重复测定3次.对数据行单因素方差分析、SNK检验.结果 与正常对照组比较,LPS组、iCORM-2组PRP中血小板黏附率、侵袭至纤维蛋白原上的血小板数量、血小板聚集率、血小板GP Ⅰ bα和GPⅥ表达量及PPP中ATP含量均明显增加,血小板内ATP含量均明显减少,P值均小于0.05.与LPS组比较,iCORM-2组上述各指标无明显变化(P值均大于0.05);10 μmol/L CORM-2组、50 μmol/LCORM-2组血小板内ATP含量明显增加,其余上述各指标均明显减少,P值均小于0.05.正常对照组、LPS组、iCORM-2组、10 μmol/L CORM-2组、50μmol/L CORM-2组PRP中血小板GSK-3β表达量分别为0.550±0.060、1.437 ±0.214、1.210±0.108、0.720 ±0.010、0.670 ±0.010,磷酸化GSK-3β表达量分别为0.950±0.070、1.607±0.121、1.420±0.040、1.167±0.015、0.513 ±0.122.LPS组、iCORM-2组PRP中血小板GSK-3β表达量及磷酸化GSK-3β表达量较正常对照组明显增加(P值均小于0.05),iCORM-2组PRP中血小板GSK-3β表达量及磷酸化GSK-3β表达量与LPS组相近(P值均大于0.05),10 μmol/L CORM-2组、50μmol/L CORM-2组PRP中血小板GSK-3β表达量及磷酸化GSK-3β表达量较LPS组明显减少(P值均小于0.05).结论 LPS刺激可致健康人外周血中血小板异常活化,CORM-2的干预能够有效地抑制血小板的过度活化,其机制可能涉及GP介导的GSK-3β的磷酸化.
目的 探討外源性一氧化碳釋放分子2(CORM-2)對LPS所緻健康人外週血中血小闆過度活化的作用及分子機製.方法 採集1名健康成年人的靜脈血,離心分離齣富血小闆血漿(PRP),分裝于硅化後的試管,按隨機數字錶法分為正常對照組、LPS組、無活性CORM-2(iCORM-2)組、10 μmol/L CORM-2組、50 μmol/L CORM-2組,每組3管.正常對照組不進行任何處理;LPS組接受20 mL 10 μg/mL的LPS刺激;iCORM-2組、10 μmol/L CORM-2組、50 μmol/L CORM-2組在接受上述LPS刺激的同時分彆接受50 μmol/L iCORM-2、10μmol/L CORM-2、50 μmol/L CORM-2的榦預,用量均為20 mL.培養30 min後玻瓶法檢測血小闆黏附率;免疫熒光法檢測血小闆侵襲至纖維蛋白原的數量;比濁法檢測血小闆聚集率;取PRP製備貧血小闆血漿(PPP)及血小闆,化學熒光素法檢測PPP及血小闆內ATP含量;流式細胞儀檢測血小闆膜糖蛋白(GP)Ⅰ bα和GPⅥ錶達;蛋白質印跡法、免疫沉澱法分彆檢測血小闆糖原閤酶激酶3β (GSK-3β)及燐痠化GSK-3β錶達.以上指標均重複測定3次.對數據行單因素方差分析、SNK檢驗.結果 與正常對照組比較,LPS組、iCORM-2組PRP中血小闆黏附率、侵襲至纖維蛋白原上的血小闆數量、血小闆聚集率、血小闆GP Ⅰ bα和GPⅥ錶達量及PPP中ATP含量均明顯增加,血小闆內ATP含量均明顯減少,P值均小于0.05.與LPS組比較,iCORM-2組上述各指標無明顯變化(P值均大于0.05);10 μmol/L CORM-2組、50 μmol/LCORM-2組血小闆內ATP含量明顯增加,其餘上述各指標均明顯減少,P值均小于0.05.正常對照組、LPS組、iCORM-2組、10 μmol/L CORM-2組、50μmol/L CORM-2組PRP中血小闆GSK-3β錶達量分彆為0.550±0.060、1.437 ±0.214、1.210±0.108、0.720 ±0.010、0.670 ±0.010,燐痠化GSK-3β錶達量分彆為0.950±0.070、1.607±0.121、1.420±0.040、1.167±0.015、0.513 ±0.122.LPS組、iCORM-2組PRP中血小闆GSK-3β錶達量及燐痠化GSK-3β錶達量較正常對照組明顯增加(P值均小于0.05),iCORM-2組PRP中血小闆GSK-3β錶達量及燐痠化GSK-3β錶達量與LPS組相近(P值均大于0.05),10 μmol/L CORM-2組、50μmol/L CORM-2組PRP中血小闆GSK-3β錶達量及燐痠化GSK-3β錶達量較LPS組明顯減少(P值均小于0.05).結論 LPS刺激可緻健康人外週血中血小闆異常活化,CORM-2的榦預能夠有效地抑製血小闆的過度活化,其機製可能涉及GP介導的GSK-3β的燐痠化.
목적 탐토외원성일양화탄석방분자2(CORM-2)대LPS소치건강인외주혈중혈소판과도활화적작용급분자궤제.방법 채집1명건강성년인적정맥혈,리심분리출부혈소판혈장(PRP),분장우규화후적시관,안수궤수자표법분위정상대조조、LPS조、무활성CORM-2(iCORM-2)조、10 μmol/L CORM-2조、50 μmol/L CORM-2조,매조3관.정상대조조불진행임하처리;LPS조접수20 mL 10 μg/mL적LPS자격;iCORM-2조、10 μmol/L CORM-2조、50 μmol/L CORM-2조재접수상술LPS자격적동시분별접수50 μmol/L iCORM-2、10μmol/L CORM-2、50 μmol/L CORM-2적간예,용량균위20 mL.배양30 min후파병법검측혈소판점부솔;면역형광법검측혈소판침습지섬유단백원적수량;비탁법검측혈소판취집솔;취PRP제비빈혈소판혈장(PPP)급혈소판,화학형광소법검측PPP급혈소판내ATP함량;류식세포의검측혈소판막당단백(GP)Ⅰ bα화GPⅥ표체;단백질인적법、면역침정법분별검측혈소판당원합매격매3β (GSK-3β)급린산화GSK-3β표체.이상지표균중복측정3차.대수거행단인소방차분석、SNK검험.결과 여정상대조조비교,LPS조、iCORM-2조PRP중혈소판점부솔、침습지섬유단백원상적혈소판수량、혈소판취집솔、혈소판GP Ⅰ bα화GPⅥ표체량급PPP중ATP함량균명현증가,혈소판내ATP함량균명현감소,P치균소우0.05.여LPS조비교,iCORM-2조상술각지표무명현변화(P치균대우0.05);10 μmol/L CORM-2조、50 μmol/LCORM-2조혈소판내ATP함량명현증가,기여상술각지표균명현감소,P치균소우0.05.정상대조조、LPS조、iCORM-2조、10 μmol/L CORM-2조、50μmol/L CORM-2조PRP중혈소판GSK-3β표체량분별위0.550±0.060、1.437 ±0.214、1.210±0.108、0.720 ±0.010、0.670 ±0.010,린산화GSK-3β표체량분별위0.950±0.070、1.607±0.121、1.420±0.040、1.167±0.015、0.513 ±0.122.LPS조、iCORM-2조PRP중혈소판GSK-3β표체량급린산화GSK-3β표체량교정상대조조명현증가(P치균소우0.05),iCORM-2조PRP중혈소판GSK-3β표체량급린산화GSK-3β표체량여LPS조상근(P치균대우0.05),10 μmol/L CORM-2조、50μmol/L CORM-2조PRP중혈소판GSK-3β표체량급린산화GSK-3β표체량교LPS조명현감소(P치균소우0.05).결론 LPS자격가치건강인외주혈중혈소판이상활화,CORM-2적간예능구유효지억제혈소판적과도활화,기궤제가능섭급GP개도적GSK-3β적린산화.
Objective To explore the effects of exogenous carbon monoxide-releasing molecules 2 (CORM-2) on LPS-induced abnormal activation of platelets in peripheral blood of healthy human donors and its possible molecular mechanism.Methods Venous blood samples were collected from a healthy volunteer, and platelet-rich plasma (PRP) from the blood were isolated by differential centrifugation.The PRP was subpackaged into siliconized test tubes and then divided into control group, LPS group, inactive CORM-2 (iCORM-2) group, 10 μmol/L CORM-2 group, and 50 μmol/L CORM-2 group according to the random number table, with 3 tubes in each group.The PRP in control group did not receive any treatment.The PRP in LPS group received LPS (20 mL, 10 g/mL) stimulation, and the PRP in iCORM-2 group, 10 μmol/L CORM-2 group, and 50 μmol/L CORM-2 group underwent the same LPS stimulation and treatment of 50 μmol/L iCORM-2, 10 μmol/L CORM-2, and 50 μmol/L CORM-2, respectively, with the dosage of 20 mL.After being cultured for 30 min, the platelet adhesion rate was determined by glass bottle method, the number of platelet spreading on fibrinogen was determined with immunofluorescent method, and the platelet aggregation rate was measured by turbidimetric method.The platelet poor plasma (PPP) was prepared from PRP, the levels of ATP in PPP and platelets were determined by chemical fluorescein method.The expressions of platelet glycoprotein Ⅰ bα (GP Ⅰ bα) and GPⅥ were analyzed by flow cytometer.The expressions of glycogen synthase kinase 3β (GSK-3β) and phosphorylated GSK-3β were determined by Western blotting and immunoprecipitation, respectively.Measurement of the above indices was repeated for 3 times.Data were processed with one-way analysis of variance and SNK test.Results Compared with those in control group, the platelet adhesion rates, numbers of platelets spreading on fibrinogen, platelet aggregation rates, expressions of GP Ⅰ bα and GP Ⅵ in PRP, levels of ATP in PPP in LPS and iCORM-2 groups were significantly increased, while levels of ATP in platelets were significantly decreased (with P values below 0.05).Compared with those in LPS group, the former 7 indices in iCORM-2 group showed no significant differences (with P values above 0.05) , while the levels of ATP in platelets in the 10 μmol/L CORM-2 and 50 μmol/L CORM-2 groups were significantly increased, and the other 6 indices in 10 μmol/L CORM-2 and 50 μmol/L CORM-2 groups were significantly decreased (with P values below 0.05).The expression levels of GSK-3β of the platelets in PRP in control, LPS, iCORM-2, 10 μmol/L CORM-2, and 50 μ mol/L CORM-2 groups were 0.550 ± 0.060, 1.437 ± 0.214, 1.210 ± 0.108, 0.720 ± 0.010, and 0.670 ± 0.010, respectively, and the expression levels of the phosphorylated GSK-3β of the platelets in PRP in the above 5 groups were 0.950 ±0.070, 1.607 ±0.121, 1.420 ±0.040, 1.167 ±0.015, and 0.513 ±0.122, respectively.Compared with those in control group, both the expression levels of GSK-3β and phosphorylated GSK-3β of the platelets in PRP in LPS and iCORM-2 groups were significantly increased (with P values below 0.05).The expression levels of GSK-3β and phosphorylated GSK-3β of the platelets in PRP between LPS group and iCORM-2 group were similar (with P values above 0.05).The expression levels of GSK-3β and phosphorylated GSK-3β of the platelets in PRP in 10 μmol/L CORM-2 and 50 μmol/LCORM-2 groups were significantly decreased compared with those in LPS group (with P values below 0.05).Conclusions LPS stimulation can abnormally activate the platelets in peripheral blood of healthy human,but the abnormal activation can be inhibited by CORM-2 intervention, and the mechanism of the latter may involve the phosphorylation of GSK-3β mediated by GP.
