脓毒症淋巴细胞凋亡和内质网应激状态以及与预后关系的研究

A study of lymphocyte apoptosis and endoplasmic reticulum stress in the development of sepsis and their association with outcome in septic patients

目的 探讨淋巴细胞凋亡和内质网应激途径在脓毒症发生中的作用及其与预后的关系。方法 采用前瞻性队列研究方法，选择2012年1月至2014年4月同济大学附属东方医院急诊重症加强治疗病房（ICU）住院治疗的脓毒症患者共70例，于入ICU后1、3和7d取外周血，采用流式细胞仪检测外周血淋巴细胞凋亡率，采用反转录一聚合酶链反应（RT—PCR）检测内质网特异性葡萄糖调节蛋白78（GRP78）和核转录因子CHOP的mRNA表达。采用Spearman相关分析CHOPmRNA表达与外周血淋巴细胞凋亡率的相关性。根据患者28d预后分为死亡组（23例）和生存组（47例）；选取同期20例健康志愿者作为健康对照组。结果 ①淋巴细胞凋亡率：与健康对照组[（2．86±O．66）％]比较，脓毒症生存组和死亡组患者人ICU1、3、7d外周血淋巴细胞凋亡率均明显升高[生存组分别为（12．44±4．43）％、（8．57±3．38）％、（6．78±3．35）％，死亡组分别为（14．42±2．01）％、（11．32±2．53）％、（8．87±3．62）％，均P〈O．01]，以1d时升高最为显著，随后逐渐下降；生存组与死亡组1d时差异无统计学意义，但3d、7d时生存组较死亡组明显降低（均P〈0．05）。②CHOPmRNA表达（2^△△C1）：与健康对照组[（2．56±1．09）×10^-3]比较，脓毒症生存组和死亡组患者入ICU1、3、7dCHOPmRNA表达水平均明显升高[生存组分别为（5．83±1．96）×10^-3、（4．24±1.60）×10^-3、（4．15±1．64）×10^-3，死亡组分别为（37．20±20．70）×10^-3、（18．80±13．90）×10^-3、（9．28±7．78）×10^-3，均P〈O．01]；虽然死亡组人ICU1、3、7d较生存组分别升高了6．38、4．43、2．24倍（P值分别为0．000、0．000、0．001），但下降也较迅速。③GRP78mRNA表达（2^△△C1）：与健康对照组[（3．31±2．04）×10^-3]比较，脓毒症生存组和死亡组患者人ICU1d时GRP78mRNA表达即明显升高[（5．83±2．00）×10^-3、（11．30±6．48）×10^-3，均P〈O．01]，随后逐渐下降；入ICU3d、7d时，生存组已降至健康对照组水平[3d：（3．99±1．60）×10^-3，7d：（3．30±1．35）×10^-3，均P〉O．05]，而死亡组GRP78mRNA表达虽逐步下降，但仍显著高于健康对照组[3d：（7．27±3．64）×10^-3，7d：（5．23±1．94）×10^-3，均P〈0．01]。④Spearman相关分析：脓毒症患者外周血CHOPmRNA表达水平与外周血淋巴细胞凋亡率呈显著正相关（r=O．414，P=0．000）。结论 淋巴细胞凋亡增加和内质网应激途径被活化在脓毒症发生发展中发挥了作用，并与预后相关。

Objective To investigate the role of lymphocyte apoptosis and endoplasmic reticulum stress （ERS） on the development of sepsis and their association with the prognosis of sepsis patients. Methods A prospective cohort study was conducted. Seventy septic patients admitted to intensive care unit （ICU） of Shanghai East Hospital of Tongji University were enrolled. Blood samples were collected on days 1, 3 and 7 to measure percentage of circulating apoptotic lymphocyte with flow cytometry analysis. The relative expressions of endoplasmic reticulum specific glucose regulated protein 78 （GRP78） mRNA and transcription factor CHOP mRNA were measured by real-time reverse transcription-polymerase chain reaction （RT-PCR）. The correlation between CHOP mRNA expression and percentage of circulating apoptotic lymphocyte was analyzed by Spearman relative analysis. The patients were divided into death （n = 23） and survival subgroups （n = 47）. Twenty healthy volunteers during the same period were selected as the healthy control group. Results ① Rate of lymphocyte apoptosis： compared with healthy control group [ （2.86 ± 0.66）% ], septic patients, either survival or death subgroup, exhibited higher rate of lymphocyte apoptosis on days 1, 3 and 7 [ survival subgroup： （ 12.44 ± 4.43 ）%, （ 8.57 ± 3.38 ）%, （ 6.78 ± 3.35 ）%; death subgroup： （ 14.42 ±2.01 ）%, （ 11.32± 2.53 ）%, （8.87±3.62）%, all P 〈 0.01 ], and it was obvious on day 1, and the phenomenon became less marked gradually. The rate of circulating apoptotie lymphocytes did not differ between the death and survival subgroups on day 1, but there was a significant difference in the rate on day 3 and day 7 （both P 〈 0.05 ）. ② The expression of CHOP mRNA （2^△△Ct）： compared with that in healthy controls [ （2.56 ± 1.09）^ 10-3 ], CHOP mRNA expression was increased on days 1, 3 and 7 in septic patients [ survival subgroup： （5.83 ± 1.96） ×10^-3, （4.24 ± 1.60） ×10^-3, （4.15±1.64） ×10^-3, death subgroup： （ 37.20± 20.70 ） ×10^-3, （ 18.80 ± 13.90 ）×10^-3, （ 9.28 ± 7.78 ） ×10^-3, all P 〈 0.01 ], and it was more obvious in the death subgroup, as it was increased by 6.38, 4.43, and 2.24 folds （P values was 0.000, 0.000, and 0.001 ）, but it decreased rapidly in death subgroup. ③ The expression of GRP78 mRNA （2-^△△Ct）： compared with healthy eontrols [ （3.31± 2.04 ）×10^-3 ], the expression of GRP78 mRNA in both survival and death subgroups increased in septic patients on day 1 [ （ 5.83± 2.00） ×10^-3, （ 11.30± 6.48 ） ×10^-3, both P 〈 0.01 ], and they decreased subsequently. The expression of GRP78 mRNA in the survival subgroup declined to the levels of the healthy control group on day 3 and day 7 [ 3 days： （3.99 ± 1.60） ×10^-3, 7 days： （3.30 ± 1.35 ）×10^-3, both P 〉 0.05 ], and GRP78 mRNA expression in the death subgroup was gradually lowered, but it was still higher than that in the healthy control group [ 3 days： （7.27 ± 3.64） ×10^-3, 7 days： （5.23 ±1.94） ×10^-3, both P 〈 0.01 ]. ④ Spearman relative analysis showed that the express!on of CHOP mRNA was positively correlated with the rate of lymphocyte apoptosis （ r = 0.414, P = 0.000 ）. Conclusion The increase in the rate of lymphocyte apoptosis and activation of ERS play an important role in the development of sepsis, and it is associated with worse outcome in the septic patients.