目的 初步探讨热休克蛋白70 (heat shock protein 70,HSP7O)抑制脓毒症血清所致心肌细胞凋亡及其机制.方法 将原代培养心肌细胞分组:正常对照组、正常大鼠血清组、脓毒症血清组、转空载体对照组和转HSP70基因组,转HSP70基因组以重组质粒pcDNA3.1-HSP70转染后36 h后验证基因和蛋白表达；各组以相应血清混合培养2h;分别行Hoechst 33258染色后计数以及DNA“梯状”条带检测心肌细胞凋亡,再应用Western-blot分析HSP70过表达对Caspase-3,8,9活化和Bid裂解的影响情况.结果 处理后12、24、36 h凋亡率,转HSP70基因组心肌细胞为(12.48±2.39)％、(23.96±3.12)％、(25.40±3.96)％,明显低于脓毒症血清组[(28.66±2.24)％、( 55.76±5.69)％、(46.89±8.74)％,t =5.851、5.932、6.027,P＜0.01],亦明显低于转空载体组[(34.25±3.42)％、(50.71±6.38)％、(47.62±5.74)％,t=5.876、5.903、6.122,P＜0.01],但明显高于正常对照组和正常血清组(3.13％ ～6.75％,t=6.324、6.578、6.137、5.987、6.032、6.871,P＜0.01)；在Caspase-3、8、9活化表达中,P11、P20和P10条带比值,转HSP70基因组分别为(12.5276±2.1247、9.3481±4.5423、16.1349±6.0641),低于脓毒症血清组(27.1324±2.1564、25.5643±4.3018、36.5647±6.7135,t=5.856、5.902、5.891,P＜0.01),低于转空载体组(28.0314±2.0367、25.6413±4.1356、34.5648±5.9473,t=3.861、3.933、4.281,P＜0.05),高于正常对照组(8.0324±1.5234、5.1246±1.3274、2.0314±0.6423,t=3.286、3.867、4.031,P＜0.05)和正常血清组(8.5649±1.2136、6.0324±1.0214、3.2146±0.1325,t=5.898、5.969、6.879,P＜0.01)；tBid条带比值,转HSP70基因组(12.0316±2.3641)低于脓毒症血清组(27.0536±5.3214,t=3.274,P＜0.05)和转空载体组(27.1034±3.6741,t=3.301,P＜0.05),但高于正常对照组(6.0347±2.1304,t=5.924,P ＜0.01)和正常血清组(7.3121±1.3021,t=5.871,P＜0.01).结论 HSP70通过干预死亡受体通路和线粒体通路而抑制脓毒症血清所致的细胞凋亡.
目的 初步探討熱休剋蛋白70 (heat shock protein 70,HSP7O)抑製膿毒癥血清所緻心肌細胞凋亡及其機製.方法 將原代培養心肌細胞分組:正常對照組、正常大鼠血清組、膿毒癥血清組、轉空載體對照組和轉HSP70基因組,轉HSP70基因組以重組質粒pcDNA3.1-HSP70轉染後36 h後驗證基因和蛋白錶達；各組以相應血清混閤培養2h;分彆行Hoechst 33258染色後計數以及DNA“梯狀”條帶檢測心肌細胞凋亡,再應用Western-blot分析HSP70過錶達對Caspase-3,8,9活化和Bid裂解的影響情況.結果 處理後12、24、36 h凋亡率,轉HSP70基因組心肌細胞為(12.48±2.39)％、(23.96±3.12)％、(25.40±3.96)％,明顯低于膿毒癥血清組[(28.66±2.24)％、( 55.76±5.69)％、(46.89±8.74)％,t =5.851、5.932、6.027,P＜0.01],亦明顯低于轉空載體組[(34.25±3.42)％、(50.71±6.38)％、(47.62±5.74)％,t=5.876、5.903、6.122,P＜0.01],但明顯高于正常對照組和正常血清組(3.13％ ～6.75％,t=6.324、6.578、6.137、5.987、6.032、6.871,P＜0.01)；在Caspase-3、8、9活化錶達中,P11、P20和P10條帶比值,轉HSP70基因組分彆為(12.5276±2.1247、9.3481±4.5423、16.1349±6.0641),低于膿毒癥血清組(27.1324±2.1564、25.5643±4.3018、36.5647±6.7135,t=5.856、5.902、5.891,P＜0.01),低于轉空載體組(28.0314±2.0367、25.6413±4.1356、34.5648±5.9473,t=3.861、3.933、4.281,P＜0.05),高于正常對照組(8.0324±1.5234、5.1246±1.3274、2.0314±0.6423,t=3.286、3.867、4.031,P＜0.05)和正常血清組(8.5649±1.2136、6.0324±1.0214、3.2146±0.1325,t=5.898、5.969、6.879,P＜0.01)；tBid條帶比值,轉HSP70基因組(12.0316±2.3641)低于膿毒癥血清組(27.0536±5.3214,t=3.274,P＜0.05)和轉空載體組(27.1034±3.6741,t=3.301,P＜0.05),但高于正常對照組(6.0347±2.1304,t=5.924,P ＜0.01)和正常血清組(7.3121±1.3021,t=5.871,P＜0.01).結論 HSP70通過榦預死亡受體通路和線粒體通路而抑製膿毒癥血清所緻的細胞凋亡.
목적 초보탐토열휴극단백70 (heat shock protein 70,HSP7O)억제농독증혈청소치심기세포조망급기궤제.방법 장원대배양심기세포분조:정상대조조、정상대서혈청조、농독증혈청조、전공재체대조조화전HSP70기인조,전HSP70기인조이중조질립pcDNA3.1-HSP70전염후36 h후험증기인화단백표체；각조이상응혈청혼합배양2h;분별행Hoechst 33258염색후계수이급DNA“제상”조대검측심기세포조망,재응용Western-blot분석HSP70과표체대Caspase-3,8,9활화화Bid렬해적영향정황.결과 처리후12、24、36 h조망솔,전HSP70기인조심기세포위(12.48±2.39)％、(23.96±3.12)％、(25.40±3.96)％,명현저우농독증혈청조[(28.66±2.24)％、( 55.76±5.69)％、(46.89±8.74)％,t =5.851、5.932、6.027,P＜0.01],역명현저우전공재체조[(34.25±3.42)％、(50.71±6.38)％、(47.62±5.74)％,t=5.876、5.903、6.122,P＜0.01],단명현고우정상대조조화정상혈청조(3.13％ ～6.75％,t=6.324、6.578、6.137、5.987、6.032、6.871,P＜0.01)；재Caspase-3、8、9활화표체중,P11、P20화P10조대비치,전HSP70기인조분별위(12.5276±2.1247、9.3481±4.5423、16.1349±6.0641),저우농독증혈청조(27.1324±2.1564、25.5643±4.3018、36.5647±6.7135,t=5.856、5.902、5.891,P＜0.01),저우전공재체조(28.0314±2.0367、25.6413±4.1356、34.5648±5.9473,t=3.861、3.933、4.281,P＜0.05),고우정상대조조(8.0324±1.5234、5.1246±1.3274、2.0314±0.6423,t=3.286、3.867、4.031,P＜0.05)화정상혈청조(8.5649±1.2136、6.0324±1.0214、3.2146±0.1325,t=5.898、5.969、6.879,P＜0.01)；tBid조대비치,전HSP70기인조(12.0316±2.3641)저우농독증혈청조(27.0536±5.3214,t=3.274,P＜0.05)화전공재체조(27.1034±3.6741,t=3.301,P＜0.05),단고우정상대조조(6.0347±2.1304,t=5.924,P ＜0.01)화정상혈청조(7.3121±1.3021,t=5.871,P＜0.01).결론 HSP70통과간예사망수체통로화선립체통로이억제농독증혈청소치적세포조망.
Objective To investigate the mechanism of HSP70 that inhibits myocardial cell apoptosis in sepsis.Methods Myocardial cells in primary culture were randomly divided into control group,normal serum group,sepsis serum group,transported empty vector group and transported HSP70 group.The myocardial cells in transported HSP70 group have been transported by pcDNA3.1-HSP70 for 36 hours.The myocardial cells in every group have been cultured by respective serum for 2 hours and dyed by Hoechst 33258,and then calculate the rate of myocardial cells apoptosis.Using Western-blot to investigate the effect of overexpression of HSP70 on Caspase-3,8,9's activation and Bid's cracking.Results The rate of myocardial cells apoptosis after dealing in transported HSP70 group [ ( 12.48 ± 2.39 ) ％,( 23.96 ± 3.12 ) ％,( 25.40 ± 3.96) ％ ] is lower than in sepsis serum group [ ( 28.66 ± 2.24 ) ％,( 55.76 ± 5.69 ) ％,( 46.89±8.74)％,t =5.856,5.932,6.027,P ＜0.01,n =3] and lower than in transported empty vector group [(34.25±3.42)％,(50.71±6.38)％,(47.62+5.74)％,t =5.876,5.903,6.122,P ＜0.01,n =3],is higher than in control group,and in normal serum group(3.13％ ～ 6.75％ ,t =6.324,6.578,6.137,5.987,6.032,6.871,P ＜ 0.01,n =3 ).When Caspase-3,8,9 activating,the gray-scale of P11,P20 and P10 in transported HSP70 group( 12.5276 ± 2.1247,9.3481 ± 4.5423,16.1349 ± 6.0641 ) is lighter than that in sepsis serum group ( 27.1324 ± 2.1564,25.5643 ± 4.3018,36.5647 ± 6.7135,t =5.856,5.902,5.891,P ＜ 0.01,n =3 ) and lighter than in transported empty vector group (28.0314 ±2.0367,25.6413 ±4.1356,34.5648 ±5.9473,t =3.861,3.933,4.281,P ＜0.05,n =3),is deeper than in control group(8.0324 ± 1.5234,5.1246 ± 1.3274,2.0314 ±0.6423,t =3.286,3.867,4.031,P＜0.05,n =3) and in normal serum group(8.5649 ± 1.2136,6.0324 ± 1.0214,3.2146 ±0.1325,t =5.898,5.969,6.879,P ＜0.01,n =3).The gray-scale of tBid in transported HSP70 group( 12.0316 ±2.3641 ) is lighter than in sepsis serum group(27.0536 ± 5.3214),t =3.274 ( P ＜ 0.05,n =3 ) and lighter than in transported empty vector group(27.1034 ± 3.6741,t =3.301,P ＜ 0.05,n =3 ),is deeper than in control group ( 6.0347 ± 2.1304,t =5.924,P ＜ 0.01,n =3 ) and in normal serum group ( 7.3121± 1.3021,t =5.871,P ＜ 0.01,n =3 ).Conclusions HSP70 inhibit myocardial cells apoptosis in sepsis by intervened the death receptor pathway and mitochondrial pathway.
