【作者】 赵丽芸；

【导师】 陈镜合；

【作者基本信息】 广州中医药大学 ， 中医内科学， 2006， 博士

【摘要】 研究目的: 研究严重脓毒症(severe sepsis,简称 SS)患者血清促炎/抗炎细胞因子浓度、单核细胞HLA—DR表达、TLR2和TLR4基因表达动态变化规律及其临床意义:通过对患者中医证候的分析,初步揭示严重脓毒症的病机特点,并从免疫调理、Toll样受体基因表达调控的角度,探讨中药“复苏饮”防治严重脓毒症的临床疗效及其作用机制,为中西医治疗脓毒症、降低病死率提供新的思路和方法。 研究方法: 采用前瞻性随机对照研究。2004年4月至2005年11月广州中医药大学第一附属医院ICU收治的符合ACCP/SCCM会议诊断标准的SS患者被随机分为两组:F组(20例,西医常规治疗加复苏饮)、A组(20例,西医常规治疗加安慰剂),A组按预后又分为生存组(9例)和死亡组(11例),另选取15名健康者作为正常对照组。所有患者入ICU后即进行中医辨证,确定中医证型,并在纳入研究时(治疗前)、治疗后第3天和第7天分别进行APACHEⅡ评分,同期抽取外周静脉血10ml,采用ELISA法测定血清TNF-a、IL—6、IL—10浓度,流式细胞术检测外周血单核细胞HLA—DR表达率及应用RT—PCR法测定外周血单核细胞TLR2、TLR4mRNA表达。 结果: 1、中医证候分析:SS患者各中医证型中单纯实证者占10%,虚实夹杂者为85%,单纯虚证者占5%,邪实中以热毒、腑实、瘀血为主,而正虚方面以气阴两虚最为常见。 2、28天病死率的比较:F组病死率30%(6/20);A组病死率55%(11/20),F组病死率明显低于A组(P<0.05)。 3、细胞因子的变化:治疗前治疗前A组和F组所有SS患者血清细胞因子TNF-a、IL—6、IL—10浓度均明显高于正常水平,组间无显著差别(P>0.05);治疗第3天F组TNF-a、IL—6明显下降,IL—10变化不明显,组间比较TNF-a、IL—6差异有显著性(P<0.05);第7天两组TNF-a、IL—6、IL—10均下降明显,但A组下降幅度不及F组,组间比较仍有显著性差异。从患者的预后来看,第3天、第7天存活组3种细胞因子浓度均明显下降,并显著低于同期死亡组(P<0.05)。 4、单核细胞HLA—DR表达率的变化:治疗前A组和F组SS病人外周血单核细胞HLA—DR表达率明显低于正常对照组(P<0.01),组间无显著差别;第7天两组HLA—DR表达均明显回升,但A组仍明显低于F组(P<0.01)。在预后方面,存活组单核细胞HLA—DR表达率明显高于同期死亡组(P<0.01),且治疗后存活组HLA—DR表达率回升明显,而死亡组则无明显变化。 5、单核细胞Toll样受体2和4基因表达的变化:治疗前两组患者单核细胞TLR2、更多还原

【Abstract】 Objective:To study the dynamic changes and the corresponding clinical significance of serum concentrations of pro-and anti-inflammatory cytokines, monocyte human leukocyte antigen-DR (HLA-DR) , TLR2 and TLR4 mRNA expression in patients with severe sepsis;To investigate pathogenic features of severe sepsis by analyzing TCM syndrome;and explore the efficacy of "fusu yin" in treating severe sepsis (SS) as well as its mechanisms from the view of immunomodification and regulating TLR2 and TLR4 mRNA expression, which may provide a new way and thought for the treatment of SS with the combination of Chinese medicine and western medicine, reducing the fatality of SS as well.Methods:Our study was performed at the First Affiliated Hospital of Guangzhou University of TCM. From April 2004 to November 2005 patients admitted to the intensive care unit (ICU) , who fulfilled with ACCP/SCCM Consensus Conference criteria of severe sepsis were enrolled in this prospective study. Subjects in this study were randomly divided into group F and group A. 20 cases in group F were treated with Fusu yin capsule and routine western therapy, however 20 cases in group A were treated with placebo and routine western therapy . Another group of 15 healthy individuals were also included in the study as controls. Group A was divided into two subgroups according to the progrosis: survival group (n=9) and the death group (n=11). The traditional Chinese medical (TCM) features of the different symptom types in SS patients were investigated on admission . And on admission (before treatment), on Days 3 and day7 after starts of treatment, Acute Physiology and Chronic Health Evaluation (APACHE Ⅱ) scoreswere also recorded, then 10 ml peripheral blood samples were obtained from patients .The serum concentrations of TNF-a^ IL—6? IL—10 were measured by means of ELISA, HLA-DR expression was assessed by means of flow cytometry , and the expression of TLR2 and TLR4 mRNA in monocyte were observed by method of RT- PCR.Results:K The type of TCM syndrome: In SS patients, the percentage of the excess syndrome is 10%, which of the excess & deficiency syndrome is 85% and deficiency syndrome is 5%. Heat toxin constipation blood stasis are the main pathogenic factors, and the deficiency of qi and yin was mostly seen in SS.2^ 28-day all-cause mortality: The mortality rate was 30% (6/20) in group F, while that of in group A was 55% (11/20) . There was a significant difference between two groups.3n Changes of cytokines: On admission, patients with severe sepsis had significantly higher concentrations of all measured cytokines compared to normal controls (P <0. 01) , and there is no significant difference between the two treatment group;On day 3, TNF-a^ IL—6 levels in group F decreased significantly (P<0. 05) , while IL—10 level had no remarkable change. The difference of TNF-a^ IL —6 levels between F and A group were significant (P<0. 05);On day 7, the levels of TNF-a^ IL-6 and IL—10 in two groups declined , but the dereasing degree of concentrations in group A was slighter than that in group F, so there was also significantly different between two group. In accordance with progrosis, surviors had lower concentrations of all measured cytokines than nonsurviors on day 3 and day 7 (P<0. 05) .4, Monocyte HLA-DR expression: On admission HLA-DR expression were obviously lower in SS than those in normals (P<0.01) , while there is no significant difference between the two treatment group. On day 7, HLA-DR expression in two treatment were obviously higher (P<0. 01) . After treatment monocyte HLA-DR expression in surviors increased remarkably, but there was no significant change in nonsurviors. Surviors had higher monocyte HLA-DR expression than nonsurviors at the same time points (P<0. 01) .5^ monocyte TLR2 and TLR4 mRNA expression: On admission TLR2 and TLR4 mRNA expression were less in SS patients than those in normals (P<0.01) , and there is also no significant difference between the two treatment group. Aftertreatment TLR2 and TLR4 mRNA expression in group F increased remarkably on day3, while these changes occurred in group A until on day7. TLR2 and TLR4 mRNA expression in group A were lower than those in group F on day3 and day7. TLR2 and TLR4 mRNA expression in surviors reached normal levels on day7(P>0. 05) , but there was no significant increase in nonsurviors. It was . found that mRNA expression was higher in surviors than in nonsurviors at every observation points (P<0. 01) .6> APACHEIIscore: Before treatment there is no significant difference between group F with group A. Scores had a decrease trend declined in two groups during the treatment. On day3 and day 7, compared with group A, scores declined significantly in group F(P<0. 01). APACHEII scores gradually decreased with the patients’ condition improved, vice versa. Nonsurviors had higher scores from day 3 after treatment than surviors (P<0.05) .7, Correlation analysis: At the same time points, there was a significant positive correlation between APACHEII score and serum cytokine concentrations (P<0. 05) , in addition, TLR2 mRNA expression was also positively correlated with monocyte HLA-DR expression (P<0. 01), so as TLR4 mRNA expression (P<0. 01).Conclusions:K Based on the analysis of TCM syndrome and tutor’ s clinical experiences, we believe that the pathogenic feature is deficiency in origin and excess in superficiality. It is to say the fundamental is the deficiency of qi and yin , while the superficiality is heat, toxin, blood stasis, which manifests body resistance weakened and pathogenic factors prevailing, so strengthening healthy qi to eliminate pathogenic factors is identified as the basic therapeutic principle. We bring forward therapeutic principles, such as " tonifying qi and yin, clearing away heat-toxin, promoting blood circulation to dissipate blood stasis, removing stasis by purgation" .2, Combined the syndrome differentiation of TCM with APACHE II score may help to describe the condition of patients with SS more objectively.3, Significantly lower HLA-DR expression was found in the patients with SS, while the serum concentrations of pro-and anti-inflammatory cytokines were higher than those in healthy controls, which was associated with increased mortality . Our study demonstrated patients with SS were in a status of immunological disturbance which is characteristics by specific depression ofimmune function and nonspecific increase activation of inflammatory reaction . Higher APACHEII score indicated worse patients’ condition, poorer outcome and higher mortality rate. Dynamic monitoring serum concentrations of cytokines and HLA-DR expression, which especially combined with APACHEII score, would be helpful to evaluating the condition and prognosis of patients with SS.4> Decreaesd TLR2 and TLR4 mRNA expression were closely associated with patients’ poor outcome. And there were significant positive correlations between TLR2/4 mRNA expression and monocyte HLA-DR expression (P<0. 01), which suggests TLR2/4 mRNA expression may reflect host’ immune status to a certain extent.5> It could effectively regulate immune function, alleviate symptoms, decrease the mortality and improve the prognosis of SS patients by using Fusu yin capsule and the conventional western therapy. The mechanism may be related to decreasing levels of pro-and anti-inflammatory cytokines, up-regulating the monocyte HLA-DR expression, TLR2 and TLR4 mRNA expression as well as strengthening the host’ s response to stress.更多还原