目的 初步评价乌司他丁对严重烧伤患者免疫功能的影响. 方法 选取笔者单位2013年3月-2015年10月收治的符合入选标准的严重烧伤患者40例,采用随机数字表法并结合患者个人意愿分为常规治疗组20例和乌司他丁治疗组20例.常规治疗组患者入院后给予抗休克、抗感染、清创、植皮、营养支持等治疗,乌司他丁治疗组患者在上述治疗基础上,入院后第1天开始静脉滴注乌司他丁,每日2次,每次8×105 U,连续7d.治疗1、3、5、7d,抽取2组患者外周静脉血;另选20名健康志愿者为健康对照组,于试验开始后第1天统一抽取其外周静脉血.流式细胞仪检测CD4+ CD25+调节性T淋巴细胞(Treg)比例,酶标仪检测T淋巴细胞增殖活性(以吸光度值表示),ELISA法检测T淋巴细胞培养上清液中IL-2含量及血清中IL-4、y干扰素含量,流式细胞仪检测CD14+单核细胞人类白细胞DR抗原(HLA-DR)表达.对数据行重复测量方差分析、X2检验、LSD-t检验. 结果 (1)与健康对照组健康受试者比较,常规治疗组患者CD4+ CD25+ Treg比例于治疗1~7d显著升高(t值为13.303 ~ 26.043,P值均小于0.01).与常规治疗组比较,乌司他丁治疗组患者CD4+ CD25+ Treg比例于治疗5、7d显著降低(t值分别为8.317、15.071,P值均小于0.01).(2)治疗1、3、5、7d,常规治疗组患者T淋巴细胞增殖活性分别为0.71 ±0.11、0.61 ±0.15、0.54±0.12、0.67±0.17,明显低于健康对照组健康受试者的1.21±0.22(t值为8.686~11.957,P值均小于0.01).治疗3、5、7d,乌司他丁治疗组患者T淋巴细胞增殖活性分别为0.81±0.11、0.85±0.14、1.08±0.13,明显高于常规治疗组(t值为4.808~ 8.568,P值均小于0.01).(3)与健康对照组健康受试者比较,常规治疗组患者T淋巴细胞培养上清液中IL-2含量于治疗1~7d明显降低(t值为8.073 ~9.288,P值均小于0.01),血清IL-4含量于治疗1~7d明显升高(t值为18.926 ~41.451,P值均小于0.01),血清γ干扰素含量于治疗1~7d明显降低(t值为4.543~27.659,P值均小于0.01).与常规治疗组比较,乌司他丁治疗组患者T淋巴细胞培养上清液中IL-2含量于治疗3~7d明显升高(t值为6.507~8.869,P值均小于0.01),血清IL-4含量于治疗3~7d明显降低(t值为6.922 ~8.843,P值均小于0.01),血清γ干扰素含量于治疗5、7d明显升高(t值分别为5.369、13.521,P值均小于0.01).(4)常规治疗组患者治疗1、3、5、7 d HLA-DR表达阳性的CD14+单核细胞所占比例分别为(28±6)%、(25±7)%、(25±7)%、(39±10)%,明显低于健康对照组健康受试者的(87±8)%(t值为16.323~25.645,P值均小于0.01).治疗3、5、7d,乌司他丁治疗组患者HLA-DR表达阳性的CD14+单核细胞所占比例分别为(40±6)%、(42±9)%、(49±10)%,明显高于常规治疗组(t值为3.071 ~7.324,P值均小于0.01). 结论 乌司他丁治疗能下调严重烧伤患者CD4+ CD25+ Treg比例,明显改善T淋巴细胞及Th功能,CD14+单核细胞HLA-DR表达增加,从而改善其免疫功能.
Objective To primarily evaluate the effects of ulinastatin on immune function of patients with severe burn injury.Methods Forty patients with severe burn admitted to our ward from March 2013 to October 2015,conforming to the study criteria,were divided into conventional treatment group (CT,n =20) and ulinastatin treatment group (UT,n =20) according to the random number table and patient's consent.After admission,patients in group CT received antishock treatment,antibiotic treatment,debridement,skin grafting,and nutrition support,etc.On the basis of the above-mentioned treatment,patients in group UT received intravenous drip of ulinastatin from first day after admission twice a day,with a dosage of 8 × 105 U every time,for 7 days in addition.Peripheral venous blood samples were collected from patients in groups CT and UT on post treatment day (PTD) 1,3,5 and 7,respectively.Twenty healthy volunteer were selected as health control group (HC),and peripheral venous blood samples were collected on the first day of the study.Percentage of CD4 + CD25 + regulatory T lymphocytes (Tregs) was determined by flow cytometer.The proliferative activity of T lymphocytes was detected by microplate reader (denoted as absorbance value).Content of interleukin 2 (IL-2) in culture supernatant of T lymphocytes,and content of IL-4 and γ interferon (IFN-γ) in serum were detected by enzyme-linked immunosorbent assay.Expression of human leukocyte antigen-DR (HLA-DR) on CD14 + monocytes was determined by flow cytometer.Data were processed with analysis of variance for repeated measurement,chi-square test,and LSD-t test.Results (1) Compared with that of volunteer in group HC,the percentage of CD4 + CD25 + Tregs of patients in group CT was significantly increased from PTD 1 to 7 (with t values from 13.303 to 26.043,P values below 0.01).Compared with that in group CT,the percentage of CD4 + CD25 + Tregs of patients in group UT was significantly decreased on PTD 5 and 7 (with t values respectively 8.317 and 15.071,P values below 0.01).(2) The proliferative activity of T lymphocytes of patients in group CT on PTD 1,3,5,and 7 was respectively 0.71 ± 0.11,0.61 ± 0.15,0.54 ± 0.12,and 0.67 ± 0.17,which was significantly lower than that in group HC (1.21 ± 0.22,with t values from 8.686 to 11.957,P values below 0.01).The proliferative activity of T lymphocytes of patients in group UT on PTD 3,5,and 7 were respectively 0.81 ± 0.11,0.85 ± 0.14,and 1.08 ±0.13,which was significantly higher than that in group CT (with t values from 4.808 to 8.568,P values below 0.01).(3) Compared with those of volunteer in group HC,content of IL-2 in culture supernatant of T lymphocytes of patients in group CT was significantly decreased from PTD 1 to 7 (with t values from 8.073 to 9.288,P values below 0.01),content of IL-4 in serum of patients in group CT was significantly increased from PTD 1 to 7 (with t values from 18.926 to 41.451,P values below 0.01),and content of IFN-γ in serum of patients in group CT was significantly decreased from PTD 1 to 7 (with t values from 4.543 to 27.659,P values below 0.01).Compared with those in group CT,content of IL-2 in culture supernatant of T lymphocytes of patients in group UT was significantly increased from PTD 3 to 7 (with t values from 6.507 to 8.869,P values below 0.01),content of IL-4 in serum of patients in group UT was significantly decreased from PTD 3 to 7 (with t values from 6.922 to 8.843,P values below 0.01),and content of IFN-γ in serum of patients in group UT was significantly increased on PTD 5 and 7 (with t values respectively 5.369 and 13.521,P values below 0.01).(4) The percentages of CD14 + monocytes with positive expression of HLA-DR of patients in group CT on PTD 1,3,5,and 7 were respectively (28 ±6)%,(25 ±7)%,(25 ± 7)%,and (39 ± 10)%,which were significantly lower than the percentage of volunteer in group HC [(87 ± 8) %,with t values from 16.323 to 25.645,P values below 0.01].The percentages of CD14 + monocytes with positive expression of HLA-DR of patients in group UT on PTD 3,5,and 7 were respecfively (40 ± 6) %,(42 ± 9) %,and (49 ± 10) %,which were significantly higher than those in group CT (with t values from 3.071 to 7.324,P values below 0.01).Conclusions On the basis of CT,additional ulinastatin intervention can decrease CD4 + CD25 + Tregs percentage,improve the immune function of T lymphocytes and T helper cells,and increase expression of HLA-DR on CD14 + monocytes of patients with severe burn injury,thus improve the immune function of patients.