热休克蛋白70抑制大鼠脓毒症血清诱导的心肌细胞凋亡及其信号转导通路的研究

Studies on the mechanism of heat shock protein 70 that inhibits sepsis - induced myocardial cell apoptosis and - signaling transduction pathway

目的初步探讨热休克蛋白70（heat shock protein 70，HSP70）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36h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Hoechst33258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、36h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（27．13244-2．1564、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，￡=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HSPT0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HSPT0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。

Objective To investigate the mechanism of HSP70 that inhibits myocardial cell apopto- sis in sepsis. Methods Myocardial ceils in primary culture were randomly divided into control group, nor- mal 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 ceils in every group have been cultured by respective serum for 2 hours and dyed by Hoechst 33258, and then calculate the rate of myocardial ceils 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 myocar-dial 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 Pll, 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 ±1. 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 ）. ConelusiolaS HSP70 inhibit myocardial cells apoptosis in sepsis by intervened the death receptor pathway and mitochondrial pathway.