CD4+白细胞介素-17+辅助性T细胞对香烟暴露小鼠肺部炎症及肺气肿的作用

目的 观察香烟烟雾暴露小鼠肺实质中CD4+白细胞介素(IL)-17+辅助性T细胞(Th17)数量及活性的表达,探讨其在香烟暴露小鼠肺部CD4+ γ-干扰素+(Th1)炎症及肺气肿中的作用及相关机制.方法 将40只雄性Balb/c小鼠按随机数字表法分为4组:对照12周(C12)组、对照24周(C24)组、烟雾暴露12周(S12)组、烟雾暴露24周(S24)组,每组10只.香烟烟雾暴露法建立小鼠肺气肿模型.HE染色观察小鼠肺气肿的改变,计算平均内衬间隔和肺泡破坏指数(DI);流式细胞术检测小鼠肺实质中CD4+IL-17+T(Th17)细胞、CD4+γ-干扰素+T(Th1)细胞、CD4+IL-17+γ-干扰素+T(Th17/Th1)细胞、CD8+γ-干扰素+T(Tc1)细胞、CD8+IL-21R+细胞及CD4+IL-17+IL-21+细胞比例;荧光定量PCR法检测小鼠肺实质中维甲酸相关孤独受体(RORγt)和IL-17的mRNA表达,并分析这些指标的相互关系.结果 S12组和S24组的平均内衬间隔[(39±4)μm和(47±7)μm]和DI(39.1±1.6和45.2±3.1)明显高于C12组[(32±4)μm和28.2±1.6]和C24组[(33±3)μm和28.9±2.1],且以S24组的增高更为明显,差异均有统计学意义(t值为4.378～15.188,均P＜0.05);S12组和S24组Th17细胞比例[(3.3±1.1)%和(7.2±2.2)%]均明显高于C12组和C24组[(1.8±0.8)%和(2.0±0.6)%];S12组和S24组RORγtmRNA表达量[(25±4)和(35±3)]及IL-17的mRNA表达量[(26±3)和(36±3)]亦明显高于C12组[(10±5)和(13±5)]和C24组[(11±7)和(8±6)],以S24组增高更为明显,差异均有统计学意义(P＜0.05);S12组和S24组Th1细胞比例[(10.0±3.7)%和(26.2 ±6.0)%]、Th17/Th1细胞比例[(0.61±0.30)%和(1.82±0.52)%]及Tc1细胞比例[(17.0±4.5)%和(26.8±8.5)%]均明显高于C12组[(3.8±1.7)%、(0.27±0.17)%和(4.8±1.9)%]和C24组[(4.2±1.3)%、(0.28±0.11)%和(5.2±1.0)%],以S24组增高更为明显,差异均有统计学意义(P＜0.05);S12组和S24组小鼠Th17细胞与Th1、Tc1细胞比例、平均内衬间隔、DI值均呈显著正相关(r值为0.519～0.797,均P＜0.01);Th17/Th1细胞比例与平均内衬间隔、DI值呈显著正相关(r值分别为0.742和0.802,均P＜0.01);S12组和S24组CD4+IL-17+IL-21+细胞比例[(0.19±0.04)和(0.55±0.24)]明显高于C12组和C24组[(0.07±0.03)和(0.08±0.03)],S24组增高更为明显,差异均有统计学意义(P＜0.05).S12组和S24组的CD8+IL-21R+细胞比例[(2.94±1.26)和(4.12±2.26)]高于C12组和C24组[(1.22±0.31)和(1.34±0.18)](P＞0.05);S12组及S24组小鼠CD4+IL-17+IL-21+细胞比例与Th1、Tc1细胞比例、平均内衬间隔和DI值均呈显著正相关(r值为0.694～0.754,均P＜0.05);S12及S24组小鼠CD8+IL-21R+细胞比例与平均内衬间隔和DI呈显著正相关(r值分别为0.516和0.725均P＜0.05).结论 香烟暴露导致肺气肿小鼠肺内Th17细胞数量及活性上调,并随烟雾暴露时间延长而增强;Th17细胞通过IL-21及IL-21R在肺部Th1/Tc1炎症中起重要促进作用;这对探讨COPD肺部炎症和肺气肿发生机制以及新的治疗靶点具有重要意义.

Abstract：

Objective To evaluate the expression and the role of Th 17 in cigarette smoke-induced lung inflammation and emphysema in mice.Methods Forty male BALB/c mice were randomly divided into 4 groups, including a control group C12, a control group C24, a smoke-exposure 12 week group (S12) and a smoke-exposure 24 week group S24 (n = 10 each).Morphological changes were evaluated by mean linear intercepts and destructive index (DI).The proportion of CD4+ IL-17 + Th17, CD4+ IFN-γ+ Th1, CD4+ IL-17 +IFN-γ+ T( Th17/ Th1 ), CD8+ IFN-γ+ Tc1, CD8+ IL-21R + and CD4+ IL-17 + IL-21 + T cells in lungs of mice was determined by flow cytometry.The mRNA expressions of RORγt and IL-17 were evaluated by real-time PCR.Results Mean linear intercepts and DI were significantly higher in S12 and S24 groups [(39 ± 4)μm, (47 ±7) μm], (39.1 ± 1.6, 45.2 ±3.1 ) as compared to C12[(32 ±4) μm,28.2 ± 1.6] and C24groups [(33 ± 3 ) μm ,28.9 ± 2.1], all P ＜ O.05.The percentage of Th17 of S12 and S24 groups [(3.3 ±1.1 )%, (7.2 ±2.2)%] was significantly increased as compared with that of C12 and C24 groups [( 1.8± 0.8) %, (2.0 ± 0.6) %], all P ＜ 0.05.The mRNA levels of RORγt [( 25 ± 4), ( 35 ± 3 )] and IL-17 [(26 ± 3), (36 ± 3 )] in S12 and S24 groups were higher than in C12 [(10 ± 5 ), (13 ± 5 )] and C24 groups [( 11 ± 7 ), (8 ± 6)], all P ＜ 0.05.The percentage of Th 1, Th17/Th1 and Tc1 cells of S12 and S24 groups [(10.0 ±3.7)%, (26.2 ±6.0)%], [(0.61 ±0.30)%, (1.82 ±0.52)%], [(17.0±4.5 ) %, ( 26.8 ± 8.5 ) %] was significantly increased as compared with that of C12 [( 3.8 ± 1.7 ) %,(0.27±0.17)%, (4.8 ±1.9)%] and C24 groups [(4.2±1.3)%, (0.28±0.11)%, (5.2±1.0)%], all P＜0.05.Moreover, the frequency of Th17 cells had a positive correlation with Th1, Tc1 cells and emphysematous lesions ( r =0.519 - 0.797, all P ＜ 0.01 ).In addition, a positive correlation between Th17/Th1 cells and emphysematous lesions was also found (r =0.742, 0.802, all P ＜0.01 ).The percentage of CD4+ IL-17+ IL-21 +T cells was significantly increased in S12 and S24 groups [(0.19 ±0.04) %, (0.55 ± 0.24) %] compared to controls [(0.07 ± 0.03 ) %, (0.08 ± 0.03 ) %], all P ＜ 0.05.Meanwhile, as compared with that of the controls [( 1.22 ± 0.31 ), ( 1.34 ± 0.18 )], the percentage of CD8+ IL-21 R + T cells was also increased in SI 2 and S24 groups [( 2.94 ± 1.26 ), (4.12 ± 2.26 )], but there were no differences among smoke-exposure groups ( P ＞0.05 ).The frequency of CD4+ IL-17 + IL-21 + T cells had a positive correlation with Th 1, Tc1 cells and emphysematous lesions (r = 0.694 -0.754, all P ＜0.05).And the frequency of CD8+ IL-21R+ T cells also had a positive correlation with emphysematous lesions ( r = 0.516, 0.725, all P ＜ 0.05).Conclusions Cigarette smoke increased the expression and the activity of Th17 in mice.Th17 may play a potential (active) role in the development of lung inflammation through IL-21/IL-21R pathway.     

慢性阻塞性肺疾病合并心血管疾病的危险因素及预后

目的 探讨慢性阻塞性肺疾病(COPD)合并心血管疾病(CVD)的危险凶素及预后.方法选取2004年至2009年我院因COPD急性加重住院的患者为研究对象,按是否合并CVD进行分组,所有患者均详细记录病史、体格榆查结果,记录血常规、血脂、血糖、心肌酶等化验及肺功能、心电图、超声心动网等检查.对结果采用SPSS 16.0软件进行分析.结果两组患者在年龄、性别、吸烟史方面差异无统计学意义,COPD+CVD组患者体质量指数(BMI)均值(24.29±4.07)kg/m2,高于COPD组(P=0.011);COPD+CVD组合并高血压、高脂血症、糖尿病的比例与COPD组相比[(64.70%vs 58.24%)、(17.65%vs 9.89%)、(26.47%vs 8.79%)],只有合并糖尿病的比例差异具有统计学意义(P=0.023);COPD+CVD组FEV1/FVC、FEV1%pred(吸入支气管扩张剂后)均值低于COPD组[(46.64±8.10)%vs(50.79±10.94)%、(44.62±9.80)%vs(50.21±13.76)%],差异有统计学意义(P值均<0.05),且COPD+CVD组肺功能Ⅲ～Ⅳ级的比例高于COPD组(76.47%vs 49.50%)(P=0.016);COPD+CVD组患者PaO2低于C()PD组[(67.67±10.31)mm Hg vs(73.74±13.67)mm Hg](P=0.038);COPD+CVD组在与炎症相关的指标如白细胞、中性粒细胞比例、C反应蛋白(CRP)、超敏CRP及纤维蛋白原水平上均高于COPD组(P值均<0.05),而在血脂,血糖水平上的差异无统计学意义(P值均>0.05);COPD+CVD组患者平均住院大数、平均再住院次数、死亡患者比例高于COPD组[(21.12±9.83)d vs(17.75±7.37)d、(1.59±1.67)次vs(O.90±1.17)次、20.59%vs 7.69%],且差异有统计学意义(P值均<0.05).Logistic回归分析表明,BMI、是否合并糖尿病、肺功能、血氧水平及与炎症相关的指标都和COPD合并CVD存在一定相关性,但经多因素Logistic回归分析,只有CRP水平与COPD合并CVD 独立相关(P=O.026).结论传统的CVD危险冈素及肺功能的下降都在COPD合并CVD中起一定的作用,这些作用的机制可能与全身炎症和缺氧等因素有关.合并CVD的COPD患者预后差.

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Objective To discussion the risk factors and prognosis of chronic obstructive pulmonary disease (COPD) with cardiovascular disease (CVD). Methods We choose the hospitalized patients diagnosis of AECOPD ( n = 125) during 2004-2009 and group them according to the combination of CVD or not. The history, physical examination, laboratory tests, lung function, ECG and echocardiography were recorded. All data were analysis by SPSS 16.0. Results Of the two groups, there are no significant difference in age, sex and smoking history. The BMI of the group COPD+ CVD is (24. 29± 4.07) kg/m2,higher than the group of COPD( P =0. 011). About combined disease, the percentage of hypertension of the COPD+CVD group, hypcrlipidemia, and diabetes, which are higher than the COPD group[ (64.70 %vs 58.24%),(17.65% vs 9.89%),(26.47% vs 8.79%)], but only the differences in the percentage of diabetes is significant statistically( P =0. 023). There are significant differences in lung function and the percentage of GOLD Ⅲ-Ⅳ between the two groups. The PaO2 level of the group COPD+CVD is lower than the COPD group [(67.67±10.31) mm Hg vs (73.74±13.76) mg Hg](P =0.038). About laboratory tests, there are significant differences in the inflammation-related indicators such as the count of white blood cell, the proportion of neutrophils, CRP, high sensitivity CRP and Fib( P<0.05) but no significant differences in blood glucose and lipids. The average length of stay, re-hospitalization and the proportion of patients died of COPD+ CVD group are higher than group COPD[( 21.12±9.83) days vs (17.75±7.3) days, (1.59 ± 1.67) times vs (0.90±0.17) times, 20.59~ vs 7.69%]. Logisticregression analysis showed that BMI, combined with diabetes or not, lung function, blood oxygen levels and inflammation-related indicators are all related with the increase of CVD in COPD. Multivariate Logistic regression analysis show that only CRP level is associated with the increase of CVD in COPD independently( P = 0. 026). Conclusions Traditional cardiovascular risk factors and the decline of lung function are both play a role in the increase risk of CVD in COPD. The mechanisms of these effects may be related to systemic inflammation and hypoxia etc. The prognosis is poor in COPD patients when they combined with CVD.     

肌抑制素与慢性阻塞性肺疾病患者的营养不良

目的 调查慢性阻塞性肺疾病(COPD)稳定期患者的营养状况,通过检测外周血清中肌抑制素的水平,探讨肌抑制素与COPD患者营养不良的关系.方法 选71例COPD稳定期患者和60例老年健康对照者,参照营养不良多参数评分(MNI)对所有受试者的营养状况进行总体评价;采用酶联免疫吸附法检测受试者血清中肌抑制素、TNFα、C反应蛋白(CRP)水平.结果 COPD患者MNI显著升高,其中MNI≥5分者55例(77%),MNI＜5分者16例.MNI≥5分者血清中肌抑制素水平为(12.18±4.76)μg/L,较MNI＜5分者[(9.73±2.85)μg/L]和健康对照者[(7.93±2.35)μg/L]显著升高(P＜0.001).COPD患者TNFα显著升高,与健康对照者比差异有统计学意义(P＜0.05).COPD患者血清肌抑制素水平与MNI、TNFα水平呈正相关(r=0.438,P=0.000;r=0.234,P=0.041).结论 COPD患者普遍存在较严重的营养不良,血清肌抑制素水平明显升高,营养不良与血清肌抑制素升高密切相关.

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Objective To investigate the prevalence and severity of malnutrition in patients with stable chronic obstructive pulmonary disease (COPD) , analyze serum levels of myostatin, tumor necrosis factor alpha (TNFα) and C reactive protein (CRP) , and investigate the relationship between serum myostatin and malnutrition in COPD. Methods Seventy-one patients with stable COPD and 60 age-matched healthy volunteers were recruited in this study. Pulmonary function was tested in all of the subjects and the severity of malnutrition was evaluated by a multiple-parameter malnutritional index (MNI). Based on the MNI scores, patients with COPD were divided into group Ⅰ (MNI≥5 score) and group Ⅱ (MNI ＜ 5 score) , the former represents the patients with severe or very severe malnutrition while the latter represents the patients with mild or without malnutrition. Serum concentration of myostatin, TNFα and CRP were measured by enzyme-linked immunosorbent assay. Results The MNI score was significantly elevated in patients with COPD [(7. 75 ±3. 86)score] compared with the controls [(1. 13 ±0. 96)score; P＜0.001],and 55 patients (77%) in COPD group Ⅰ showed MNI ≥ 5 (9. 30 ± 3. 01) score. Serum myostatin concentration was significantly elevated in COPD group Ⅰ [(12. 18 ±4. 76)μg/L] than in COPD group Ⅱ [(9. 73 ±2.85) μgL] and controls [(7.93 ±2.35) μg/L], with each P ＜ 0.001. Serum TNFα concentration was also significantly elevated in patients with COPD compared with the controls (P ＜ 0. 001).Pearson correlation analysis showed that serum myostatin levels were significantly correlated with MNI scores (r = 0. 438, P - 0. 000) and TNFa levels (r = 0. 234, P = 0. 041) in COPD group (combined group I and Ⅱ) while MNI scores were correlated inversely with BMI in COPD group (r = - 0. 530, P = 0. 000) . After stratified with subgroups, the correlation between myostatin levels and MNI scores was more significant and the correlation coefficient was higher (r =0.464, P =0.000) in COPD group I patients. Moreover,myostatin levels were inversely correlated with BMI (r = - 0. 287, P = 0. 034) and forced expiratory volume in one second of the predicted value (r = - 0. 264, P = 0. 049) in COPD group I patients. Conclusions Malnutrition commonly and substantially exists in patients with COPD; serum myostatin concentration is significantly elevated and is correlated with the severity of malnutrition in the patients. The elevation of serum myostatin may contribute to malnutrition in COPD patients.     

免疫性血小板减少性紫癜35例患者外周血免疫细胞亚群的检测及其临床意义

目的 探讨免疫细胞亚群的变化在免疫性血小板减少性紫癜(ITP)发病机制中的作用及其临床意义.方法 应用流式细胞术检测35例ITP患者治疗前、后及20例正常对照者免疫细胞亚群各指标的变化,包括CD3+、CD4+、CD8+、CD56+、CD19+淋巴细胞及CD4+/CD8+比值.结果 ITP患者CD3+ T淋巴细胞百分比(61.58±6.45)%、CD4+ T淋巴细胞百分比(28.38±4.89)%、CD4+/CD8+比值(0.99±0.22)较对照组[(67.85±4.68)%、(38.00±3.37)%、1.54±0.13]均减低(P值均＜0.05),治疗后3项指标[(69.41±5.03)%、(38.17±3.18)%、1.60±0.15]均升高至正常水平;CD8+ T淋巴细胞百分比(29.20±4.50)%及CD19+ B淋巴细胞百分比(17.74±4.14)%较对照组[(24.82±2.93)%、(12.09±3.51)%]升高(P值均＜0.05),治疗后2项指标[(24.06±3.02)%、(10.90±3.55)%]均降至正常水平;ITP患者CD56+细胞百分比治疗前(15.80±2.85)%、治疗后(15.16±2.77)%与对照组(16.36±2.75)%差异无统计学意义(P＞0.05).结论 免疫细胞亚群紊乱参与了ITP的发病,对其检测可作为ITP的辅助诊断,在指导治疗方面可能有一定的意义.

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Objective To explore the clinical significance of immunocyte subsets before and after immunosuppressive therapy in the peripheral blood of patients with immune thrombocytopenic purpura (ITP).MethodsThe percentages of immunocyte subsets in the peripheral blood of 35 patients with ITP and 20 healthy controls were detected by flow cytometry,including CD3+,CD4+,CD8+,CD56+,CD19+ lymphocytes and CD4+/CD8+.Results The percentages of CD3+ T lymphocyte (61.58 ± 6.45 ) %,CD4+ T lymphocyte (28.38 ±4.89)% and the ratio of CD4+/CD8+ 0.99 0.22 in patients with ITP were lower than those in healthy controls[( 67.85 ± 4.68 ) %,( 38.00 ± 3.37 ) %,1.54 ± 0.13,all P ＜ 0.05].After immunosuppressive therapy,the percentages of CD3+ T lymphocyte ( 69.41 ± 5.03 ) %,CD4+ T lymphocyte (38.17 ±3.18)% and the ratio of CD4+/CD8+ 1.60 ±0.15 recovered to control levels.The percentages of CD8+ T lymphocyte (29.20 ±4.50)% and CD19+B lymphocyte ( 17.74 ±4.14)% were higher than those in healthy controls[( 24.82 ± 2.93 ) % and ( 12.09 ± 3.51 ) %,all P ＜ 0.05].After the immunosuppressive therapy,the percentages of CD8+ T lymphocyte ( 24.06 ± 3.02 ) % and CD19+ B lymphocyte ( 10.90 ± 3.55 ) %recovered to control levels.There were no significant difference of the percentage of CD56+ lymphocyte among ITP patients ( 15.80 ± 2.85 )%,ITP patients after immunosuppressive therapy ( 15.16 ± 2.77 )% and healthy controls ( 16.36 ± 2.75 ) %.ConclusionThe aberrant immunocyte subsets are involved in the pathogenesis of ITP,and detection of immunocyte subsets might be helpful for the diagnosis and determination of therapeutic outcome of ITP.     

耐多药和广泛耐药肺结核患者外周血中免疫细胞的表达及其意义

目的 探讨耐多药(MDR)和广泛耐药(XDR)肺结核患者外周血CD3+CD16+CD56+自然杀伤(NK)T细胞、CD3-CD16+CD56+NK细胞和T细胞亚群的表达状态及其意义.方法 对2008年1月至2009年6月在上海市肺科医院住院的316例肺结核患者进行回顾性分析,其中男204例,女112例;年龄17～88岁,平均(44±16)岁;初治119例,复治197例.根据耐药情况分为MDR组146例,其中男102例,女44例,年龄19～84岁,平均(42±16)岁;XDR组77例,其中男42例,女35例,年龄18～88岁,平均(50±16)岁;敏感组93例,其中男60例,女33例,年龄17～83岁,平均(43±19)岁.另按照空洞所占肺野范围分为1～2个肺野组(132例)、3～4个肺野组(49例)和5～6个肺野组(9例).采用流式细胞仪抗体双标法检测患者外周血中NKT细胞、NK细胞和T细胞亚群表达率.两组间比较采用Wilcoxon秩和检验,多组间比较采用Kruskal-Wallis秩和检验.结果 XDR组NKT细胞和NK细胞表达率的中位数(四分位间距)[11%(6%～16%)和7%(4%～12%)]明显高于MDR组[8%(5%～14%)和6%(4%～11%)]和敏感组[7%(4%～11%)和5%(3%～9%)],3组间两两比较,差异均有统计学意义(H值分别为6.478和8.369,均P＜0.05);XDR组男性NKT细胞和NK细胞表达率[(14±9)%和(11±7)%]明显高于女性[(9±5)%和(6±4)%],CD4T细胞表达率和CD4/CD8[(38±10)%和1.9±1.3]明显低于女性[(44±10)%和2.2±0.7],两两比较,差异均有统计学意义(z值为-2.91～2.41,P＜0.05和P＜0.01);1～2个肺野组CD4 T细胞表达率最高[(42±9)%],CD8 T细胞表达率最低[(22±8)%];5～6个肺野组CD4 T细胞表达率最低[(36±11)%],CD8 T细胞表达率最高[(28±12)%],CD4/CD8最低(1.5±0.8),差异均有统计学意义(H值为8.404～16.175,均P＜0.01).结论 NKT细胞和NK细胞表达率随结核病耐药程度加重而升高,但T细胞亚群表达率不随耐药程度加重而变化.肺结核患者的空洞范围越大,其外周血中CD4T细胞和CD4/CD8值越低,CD8T细胞值越高.XDR男性肺结核患者的细胞免疫功能损害更为明显.

Abstract：

Objective To explore the expressions and the significance of CD3+ CD16+CD56+ NKT cells,CD3-CD16+CD56+ NK cells and T lymphocyte subsets in peripheral blood of patients with multi-drug resistant ( MDR-TB ) and extensively drug-resistant ( XDR-TB ) pulmonary tuberculosis. Methods The data of 316patients with pulmonary tuberculosis hospitalized in Shanghai Pulmonary Hospital from January 2008 to June 2009 were retrospectively analyzed, of whom 119 were newly diagnosed, and 197 were retreated patients.There were 204 males and 112 females, aged from 17 -88 years, mean ( 44 ± 16 ) years. According to the results of drug-resistance, these patients were divided into a MDR group, an XDR group and a sensitive group. There were 146 patients in the MDR group, with 102 males and 44 females, aged from 19 - 84years, mean ( 42 ± 16 ) years. There were 77 patients in the XDR group, with 42 males and 35 females,aged from 18 -88 years, mean (50 ± 16) years. There were 93 patients in the susceptible group, with 60males and 33 females, aged from 17-83 years, mean (43 ± 19) years. According to the distribution of cavitation in lung fields, these patients were also divided into 1 -2 lung field affected group (n = 132), 3 -4 lung field affected group ( n = 49 ) and 5 - 6 lung field affected group ( n = 9 ). The frequencies of NKT cells, NK cells and T cells from whole blood were tested by flow cytometry. Rank test (SAS software) was used for statistic analyses. Results The expression rate of NKT cells and NK cells was the highest in the XDR group [11%(6% - 16%) and 7% (4% - 12%)], as compared to the MDR group [8% (5% -14% ) and 6% (4% - 11% ) ], and the susceptible group [7% (4% - 11% ) and 5% (3% -9% ) ], the difference being statistically significant ( H = 6. 478 - 8. 369, P ＜ 0. 05 ). The expression rate of the NKT (14 ± 9)% and NK cells (11 ± 7)% in males of the XDR group was significantly higher than that in females [ NKT (9 ±5)% and NK cell (6 ±4)% ], while CD4(38 ± 10)% and CD4/CD8( 1.9 ± 1.3) were significantly lower than those of the females [ CD4 (44 ± 10) % and CD4/CD8 ( 2. 2 ± 0. 7 ) ], the difference being statistically significant (z = - 2. 91 - - 2. 79, P ＜ 0. 05, P ＜ 0. 01 ). The expression rate of CD4(42 ± 9)% was the highest, but CD8 (22 ± 8)% was the lowest in the 1 -2 lung field group. While in the 5-6 lung field group, the expression rate of CD4 (36 ± 11 )% was the lowest, CD8 (28 ± 12)% was the highest, and CD4/CD8 (1. 5 ±0. 8) was the lowest, the difference being statistically significant (H = 8. 404 -16. 175, P ＜0. 01 ). Conclusions With the increasing level of drug resistance, the expression rate of NKT cells and NK cells increased, while the expression of T cell subsets did not change. The value of CD4 and CD4/CD8 in peripheral blood decreased, but CD8 increased as the extent of cavitation increased in these patients. The impairment of cellular immune function in XDR-TB was more prominent in male patients.     

Effects of smoking cessation on expressions of nuclear factor-κB, matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 in airway epithelial cells of smoking rats

Objective To investigate the effects of smoking and smoking cessation on airway inflammation and remodeling in chronic obstructive pulmonary diseases through detecting mRNA and protein expressions of nuclear factor-κB (NF-κB), cell matrix metalloproteinase-9 (MMP-9) and cellular tissue inhibitor of metalloproteinase-1 (TIMP-1) in airway epithelial cells of smoking and smoking cessation rats. Methods Twenty-four male Wistar rats were randomly divided into control group, smoking group and smoking cessation group,eight in each group. Hybridization in situ and immunohistochemistry were used to detect mRNA and protein expressions of NF-κB, MMP-9 and TIMP-1 in airway epithelial cells of rats. Results ① Compared with control group (0.29 ± 0.06,0.29±0.06), mRNA and protein expressions of NF-κB in smoking group (0.45±0.04,0.41±0.03) and smoking cessation group (0.40±0.05,0.37±0.03) were higher (all P＜0.05). The mRNA and protein expressions of NF-κB in smoking cessation group were lower than those in smoking group (all P ＜0.05). ②Compared with control group (0.30±0.06,0.30±0.06) ,mRNA and protein expressions of MMP-9 in smoking group (0.52±0.03,0.51±0.07) and smoking cessation group (0.38±0.03,0.33±0.02) were higher (all P＜0.05). The mRNA and protein expressions of MMP-9 in smoking cessation group were lower than those in smoking group (all P＜0.05). ③Compared with control group (0.26±0.04, 0.26±0.04), mRNA and protein expressions of TIMP-1 in smoking group (0.49±0.05,0.37±0.03) and smoking cessation group (0.42±0.04,0.35±0.03) were higher (all P ＜0.05). The mRNA and protein expressions of TIMP-1 in smoking cessation group were lower than those in smoking group (all P ＜ 0.05). ④ Compared with control group (1.00±0.02,1.00±0.02), MMP-9/TIMP-1 mRNA and protein expressions were larger than one in smoking group (1.07±0.14, 1.37±0.19), and less than one in smoking cessation group (0.92±0.13,0.94±0.10) (all P ＜0.05). ⑤The mRNA and protein expressions of NF-κB and MMP-9in each group were positively correlation (r=0.87,0.66,all P ＜0.05). Conclusions In airway epithelial cells of smoking rats, mRNA and protein expressions of NF-κB, MMP-9 and TIMP-1 increase, and MMP-9/TIMP-1 is larger than one. After stoping smoking, mRNA and protein expressions of NF-κB,MMP-9 and TIMP-1 decrease, and MMP-9/TIMP-1 is less than one. This experiment explains that smoking can cause airway inflammation and remodeling, smoking cessation can reduce airway inflammation and remodeling.     

Effects of smoking cessation on expressions of nuclear factor-κB, matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 in airway epithelial cells of smoking rats

Objective To investigate the effects of smoking and smoking cessation on airway inflammation and remodeling in chronic obstructive pulmonary diseases through detecting mRNA and protein expressions of nuclear factor-κB (NF-κB), cell matrix metalloproteinase-9 (MMP-9) and cellular tissue inhibitor of metalloproteinase-1 (TIMP-1) in airway epithelial cells of smoking and smoking cessation rats. Methods Twenty-four male Wistar rats were randomly divided into control group, smoking group and smoking cessation group,eight in each group. Hybridization in situ and immunohistochemistry were used to detect mRNA and protein expressions of NF-κB, MMP-9 and TIMP-1 in airway epithelial cells of rats. Results ① Compared with control group (0.29 ± 0.06,0.29±0.06), mRNA and protein expressions of NF-κB in smoking group (0.45±0.04,0.41±0.03) and smoking cessation group (0.40±0.05,0.37±0.03) were higher (all P＜0.05). The mRNA and protein expressions of NF-κB in smoking cessation group were lower than those in smoking group (all P ＜0.05). ②Compared with control group (0.30±0.06,0.30±0.06) ,mRNA and protein expressions of MMP-9 in smoking group (0.52±0.03,0.51±0.07) and smoking cessation group (0.38±0.03,0.33±0.02) were higher (all P＜0.05). The mRNA and protein expressions of MMP-9 in smoking cessation group were lower than those in smoking group (all P＜0.05). ③Compared with control group (0.26±0.04, 0.26±0.04), mRNA and protein expressions of TIMP-1 in smoking group (0.49±0.05,0.37±0.03) and smoking cessation group (0.42±0.04,0.35±0.03) were higher (all P ＜0.05). The mRNA and protein expressions of TIMP-1 in smoking cessation group were lower than those in smoking group (all P ＜ 0.05). ④ Compared with control group (1.00±0.02,1.00±0.02), MMP-9/TIMP-1 mRNA and protein expressions were larger than one in smoking group (1.07±0.14, 1.37±0.19), and less than one in smoking cessation group (0.92±0.13,0.94±0.10) (all P ＜0.05). ⑤The mRNA and protein expressions of NF-κB and MMP-9in each group were positively correlation (r=0.87,0.66,all P ＜0.05). Conclusions In airway epithelial cells of smoking rats, mRNA and protein expressions of NF-κB, MMP-9 and TIMP-1 increase, and MMP-9/TIMP-1 is larger than one. After stoping smoking, mRNA and protein expressions of NF-κB,MMP-9 and TIMP-1 decrease, and MMP-9/TIMP-1 is less than one. This experiment explains that smoking can cause airway inflammation and remodeling, smoking cessation can reduce airway inflammation and remodeling.     

CD4+白细胞介素-17+辅助性T细胞对香烟暴露小鼠肺部炎症及肺气肿的作用

Objective To evaluate the expression and the role of Th 17 in cigarette smoke-induced lung inflammation and emphysema in mice.Methods Forty male BALB/c mice were randomly divided into 4 groups, including a control group C12, a control group C24, a smoke-exposure 12 week group (S12) and a smoke-exposure 24 week group S24 (n = 10 each).Morphological changes were evaluated by mean linear intercepts and destructive index (DI).The proportion of CD4+ IL-17 + Th17, CD4+ IFN-γ+ Th1, CD4+ IL-17 +IFN-γ+ T( Th17/ Th1 ), CD8+ IFN-γ+ Tc1, CD8+ IL-21R + and CD4+ IL-17 + IL-21 + T cells in lungs of mice was determined by flow cytometry.The mRNA expressions of RORγt and IL-17 were evaluated by real-time PCR.Results Mean linear intercepts and DI were significantly higher in S12 and S24 groups [(39 ± 4)μm, (47 ±7) μm], (39.1 ± 1.6, 45.2 ±3.1 ) as compared to C12[(32 ±4) μm,28.2 ± 1.6] and C24groups [(33 ± 3 ) μm ,28.9 ± 2.1], all P ＜ O.05.The percentage of Th17 of S12 and S24 groups [(3.3 ±1.1 )%, (7.2 ±2.2)%] was significantly increased as compared with that of C12 and C24 groups [( 1.8± 0.8) %, (2.0 ± 0.6) %], all P ＜ 0.05.The mRNA levels of RORγt [( 25 ± 4), ( 35 ± 3 )] and IL-17 [(26 ± 3), (36 ± 3 )] in S12 and S24 groups were higher than in C12 [(10 ± 5 ), (13 ± 5 )] and C24 groups [( 11 ± 7 ), (8 ± 6)], all P ＜ 0.05.The percentage of Th 1, Th17/Th1 and Tc1 cells of S12 and S24 groups [(10.0 ±3.7)%, (26.2 ±6.0)%], [(0.61 ±0.30)%, (1.82 ±0.52)%], [(17.0±4.5 ) %, ( 26.8 ± 8.5 ) %] was significantly increased as compared with that of C12 [( 3.8 ± 1.7 ) %,(0.27±0.17)%, (4.8 ±1.9)%] and C24 groups [(4.2±1.3)%, (0.28±0.11)%, (5.2±1.0)%], all P＜0.05.Moreover, the frequency of Th17 cells had a positive correlation with Th1, Tc1 cells and emphysematous lesions ( r =0.519 - 0.797, all P ＜ 0.01 ).In addition, a positive correlation between Th17/Th1 cells and emphysematous lesions was also found (r =0.742, 0.802, all P ＜0.01 ).The percentage of CD4+ IL-17+ IL-21 +T cells was significantly increased in S12 and S24 groups [(0.19 ±0.04) %, (0.55 ± 0.24) %] compared to controls [(0.07 ± 0.03 ) %, (0.08 ± 0.03 ) %], all P ＜ 0.05.Meanwhile, as compared with that of the controls [( 1.22 ± 0.31 ), ( 1.34 ± 0.18 )], the percentage of CD8+ IL-21 R + T cells was also increased in SI 2 and S24 groups [( 2.94 ± 1.26 ), (4.12 ± 2.26 )], but there were no differences among smoke-exposure groups ( P ＞0.05 ).The frequency of CD4+ IL-17 + IL-21 + T cells had a positive correlation with Th 1, Tc1 cells and emphysematous lesions (r = 0.694 -0.754, all P ＜0.05).And the frequency of CD8+ IL-21R+ T cells also had a positive correlation with emphysematous lesions ( r = 0.516, 0.725, all P ＜ 0.05).Conclusions Cigarette smoke increased the expression and the activity of Th17 in mice.Th17 may play a potential (active) role in the development of lung inflammation through IL-21/IL-21R pathway.     

CD+4 Foxp3+调节性T细胞对断烟大鼠肺部炎症及肺气肿的作用

目的 观察香烟暴露及断烟后大鼠Treg细胞的变化,探讨Treg细胞与断烟后大鼠肺部炎症反应和肺气肿持续存在的关系.方法 将50只雄性Wistar大鼠按随机数字表法分为5组:健康对照组、香烟暴露组和断烟组,其中健康对照组和香烟暴露组又分为12周组(对照1组和实验1组)和24周组(对照2组和实验2组).香烟烟雾暴露法建立大鼠肺气肿模型.HE染色观察大鼠肺气肿的改变,酶联免疫吸附法检测大鼠BALF中白细胞介素(IL-8)和肿瘤坏死因子-α(TNF-α)水平,流式细胞术检测大鼠外周血和肺实质中CD4+Foxp3调节性T细胞(Treg细胞)比例,实时定量PCR法检测大鼠外周血和肺实质中Foxp3的mRNA表达.多组间比较采用单因素方差分析,组内两两比较采用SNK和Games-Howell检验.结果 实验1组和实验2组平均内衬间隔(MLI)为(64.9±5.3)μm和(77.9±11.5)μm,均明显高于对照1组和对照2组的(39.0±3.8)μm和(40.3±2.7)μm,差异均有统计学意义(P＜0.01);断烟组MLI[(71.5 ±5.8)μm]介于实验1组和实验2组之间,但肺气肿程度较实验1组明显加重,差异有统计学意义(P＜0.01).实验1组和实验2组IL-8水平[(68±17)ng/L和(85±16)ng/L]及TNF-α水平[(14.1±1.8)ng/L和(20.1±8.7)ng/L]均明显高于对照1组和对照2组[(44±8)ng/L、(43±9)ng/L及(6.3±2.3)ng/L、(5.8±1.6)ng/L],差异均有统计学意义(P＜0.05).实验1组和实验2组肺实质中CD+4Foxp3+Treg细胞[(6.6±0.8)%和(5.3±0.9)%]明显少于对照1组和对照2组[(9.0±1.0)%和(9.6±0.9)%],差异均有统计学意义(P＜0.01);断烟组肺实质中CD+4Foxp3+Treg细胞[(7.2±0.6)%]与实验1组比较虽无明显减少,但未能恢复至正常水平.实验1组和实验2组肺实质中Foxp3的mRNA表达量(17±7和9±7)明显低于对照1组和对照2组(39±6和42±7),差异均有统计学意义(P＜0.01);断烟组肺实质中Foxp3的mRNA表达量(21±9)与实验1组比较未见明显减少,但未能恢复至正常水平.结论 香烟暴露肺气肿大鼠肺内调节性T细胞下调可能导致大鼠肺内炎症反应放大,并在断烟后持续存在,提示调节性T细胞下调可能是大鼠肺气肿持续进展的原因之一.     

慢性阻塞性肺疾病急性加重期患者血浆选择素相关蛋白水平的观察

目的 探讨整合素相关蛋白CD_(47)和P-选择素在COPD发病中的作用.方法 病例来自2008年10月至2009年3月在遵义医学院附属医院呼吸一科住院的COPD患者35例,其中男26例,女9例;年龄55～84岁,平均(65±7)岁.未经任何治疗时为急性加重期(AECOPD)组,经过抗感染、解痉平喘、祛痰、吸氧和支持等治疗,达到临床缓解时为稳定期组.同期体检的健康志愿者加名为对照组,其中男14名,女6名;年龄45～80岁,平均(59±7)岁.采用流式细胞术检测所有研究对象外周血浆中CD_(47)和P-选择素水平,并检测血小板计数.采用SNK-q检验和Pearson回归分析进行相关性分析.结果 AECOPD组CD_(47)阳性率为(93±4)%,明显高于稳定期组的(72±11)%和对照组的(67±10)%,差异均有统计学意义(q值分别为11.26和13.32,均P＜0.01),稳定期组与对照组的差异无统计学意义(q=1.73,P＞0.05);AECOPD组P-选择素阳性率为[(35±11)%],明显高于稳定期组[(12±8)%]和对照组[(10±4)%],差异均有统计学意义(q值分别为9.93和12.19,均P＜.0.05),稳定期组也明显高于对照组,差异有统计学意义(q=1.90,P＜0.05);AECOPD组血小板计数为(188±56)×10~9/L,与稳定期组的(213±57)×10~9/L和对照组的(204±51)×10~9/L比较,差异无统计学意义(F=1.74,P＞0.05);AECOPD组CD_(47)与P-选择素呈正相关(r=0.77,P＜0.01),稳定期及对照组CD_(47)与P-选择素均无相关性(r值分别为-0.04和-0.15,均P＞0.05).结论 CD_(47)和P-选择素作为血小板活化标志物,在AECOPD时明显增高,表明AECOPD存在血小板活化,提示血小板作为一种炎性细胞参与AECOPD发病,可能以其活化形式发挥重要作用.CD_(47)和P-选择素可作为判断AECOPD严重程度及血小板活化程度的指标.     

白介素17抗体减轻吸烟所致慢性阻塞性肺疾病模型小鼠的气道炎症

目的 研究白介素17(interhukin-17,IL-17)抗体在吸烟所致慢性阻塞性肺疾病(chronic obstructive pulmonary disease,COPD)模型小鼠气道炎症中的作用.方法 C67/BL6雄性小鼠随机分为COPD组(8只)、COPD+IL-17抗体干预组(简称COPD+干预组,8只)和正常对照组(10只).对支气管肺泡灌洗液(BALF)进行细胞计数、染色和分类;用酶联免疫吸附试验检测小鼠肺组织匀浆IL-17水平,观察各组小鼠气道病理改变.结果 COPD组与COPD+干预组相比肺功能差异无统计学意义.COPD组、COPD+干预组小鼠与正常对照组相比BALF细胞总数显著增高[分别为(19.64±1.89)×104/ml,(15.47±2.99)×104/ml和(5.13±1.00)×104/ml,P＜0.01];COPD+干预组较COPD组细胞总数下降(P＜0.01);中性粒细胞比例[分别为(8.58+6.77)%,(22.98±8.46)%]及绝对值[(1.28±0.96)×104/ml,(4.53±1.73)×104/ml]显著下降(P值均＜0.01).肺组织HE染色病理评分COPD+干预组(73.25±18.58)较cOPD组(106.13±36.27)炎症有所减轻(P＜0.05).COPD+干预组小鼠肺组织匀浆中IL-17含量(0.084±0.041)pg/mg pro与COPD组(0.221±0.081)pg/mg pro相比显著降低(P＜0.01).结论 吸烟所致COPD小鼠模型中IL-17参与了中性粒细胞引起的气道炎症,抑制IL-17的表达,可以减少气道内中性粒细胞的数量,减轻气道炎症.     

卡介苗免疫小鼠T淋巴细胞亚群表达穿孔素与抗结核作用的相关性

目的 探讨卡介苗免疫小鼠抗结核作用和T细胞亚群表达穿孔素的相关性.方法 120只清洁级KM雄性小鼠分为2个对照组(各20只)、2个卡介苗组(各20只)、攻毒组(20只)和结核病组(20只).2个卡介苗组与攻毒组小鼠腹部皮内接种卡介苗,接种3个月后攻毒组和结核病组小鼠接受MTB攻击,同时对照1组和卡介苗1组小鼠取血后处死.攻毒组和结核病组小鼠于接受MTB攻毒1个月后与对照2组和卡介苗2组小鼠取血后处死.观察所有小鼠的肺、肝和脾组织病理学改变,并经组织匀浆涂片查找抗酸杆菌和MTB培养.流式细胞仪检测分析血液标本中表达穿孔素的T细胞亚群计数及其占总淋巴细胞百分率.多组间免疫学指标的比较采用单因素方差分析,两组间比较采用t检验.结果 结核病组小鼠的肺、肝和脾组织均有结核病表现,1个月内死亡11只,其他3组小鼠均未发现结核病表现,也无死亡鼠.卡介苗组小鼠表达穿孔素的CD8+T细胞数[(5.9±0.9)×103]明显高于对照组[(4.8±0.8)×103],差异有统计学意义(F=42.24,P＜0.01);结核病组小鼠表达穿孔素的CD+百分率[(5.6±0.9)%]明显低于卡介苗组[(7.3±1.1)%],差异有统计学意义(F=35.51,P＜0.05);攻毒组小鼠获得卡介苗免疫后,在致病量MTB攻击下表达穿孔素的CD3+、CD8+和CD4+ CD8+ T细胞数[(20.1±5.5)×103、(8.7±0.4)×103和72±19]及其占总淋巴细胞百分率[(23.3±3.3)%、(10.7±1.6)%和(0.084±0.015)%]均明显高于对照组[(11.1 ±3.0)×103、(4.8±0.8)×103和30±7及(14.9±1.7)%、(6.7±0.9)%和(0.040±0.006)%]、卡介苗组[(13.0±3.2)×103、(5.9±0.9)× 103和36±5及(15.5±1.7)%、(7.3±1.1)%和(0.044±0.007)%]及结核病组[(12.6±1.6)×103、(5.0±0.1)×103和31±3及(14.0±1.7)%、(5.6±0.9)%和(0.035±0.005)%],CD4+/CD8+比值(0.54±0.17)明显低于对照组(0.76±0.22),差异均有统计学意义(F值为4.54～74.98,均P＜0.05或P＜0.01).结论 卡介苗免疫主要诱导表达穿孔素的CD8+ T细胞水平升高,在致病量MTB攻击下卡介苗免疫小鼠不发生结核病,可能与表达穿孔素的CD3+、CD8+和CD4+ CD8+ T细胞水平升高相关;穿孔素表达水平高低可能是判断宿主抗结核免疫力强弱的重要标志之一.     

吸烟对慢性阻塞性肺疾病患者稳定期支气管肺泡灌洗液中T细胞功能影响的临床研究

目的 评价吸烟对慢性阻塞性肺疾病(chronic obstructive pulmonary disease,COPD)患者稳定期支气管肺泡灌洗液(bronchoalveolar lavage fluid,BALF)中T细胞功能的影响.方法 流式细胞术测定吸烟的COPD患者稳定期BALF中CD3+、CD4+、CD8+T细胞和CD4+/CD8+水平.结果 与非COPD组相比,COPD患者稳定期BALF中CD3+、CD4+、CD4+/CD8+水平明显下降(P＜0.05),CD8+明显升高(P＜0.05);在COPD患者中,稳定期BALF中CD3+、CD4+、CD8+T细胞无明显差异(P＞0.05),但持续吸烟组CD4+/CD8+明显下降(P＜0.05).结论 COPD患者稳定期气道T细胞功能明显异常,其中持续吸烟的COPD患者气道CD4+/CD8+下降最为显著.     

慢性阻塞性肺疾病患者纤毛摆动时间的研究

目的 探讨慢性阻塞性肺疾病(COPD)患者纤毛摆动时间和肺功能的关系,及黏液纤毛清除功能(MCC)在COPD发病中的作用.方法 手术切除的支气管组织30例,分为COPD组、吸烟组及对照组,每组10例,光镜下观察活性纤毛细胞的百分率及纤毛摆动时间,并计数各个时间点的摆动纤毛细胞的百分率,收集临床资料包括吸烟指数、体质量指...     

烟草烟雾暴露大鼠中CD4白细胞介素-17+T细胞与CD+4Foxp3+调节性T细胞的变化及意义

目的 观察烟草烟雾暴露大鼠外周血与BALF中CD+4白细胞介素(IL)-17+T细胞(Th17细胞)与CD; Foxp3+调节性T细胞(Treg细胞)及相关因子的水平变化,探讨Th17和Treg细胞在烟草诱导气道炎症和COPD的发生与发展中的作用.方法 将40只健康清洁级雄性Wistar大鼠分为暴露12周组和24周组、对照12周组和24周组,每组10只.用烟熏法复制大鼠气道炎症的动物模型.收集BALF进行细胞学计数和分类,采用酶联免疫吸附法检测大鼠血清和BALF上清液中IL-17和IL-6水平,用流式细胞术检测Th17和Treg细胞比例,用实时荧光定量PCR法检测IL-17和Foxp3 mRNA的表达.多组间比较采用单因素方差分析,组内两两比较采用SNK法和GamesHowell法.结果 暴露12周组和24周组大鼠外周血IL-17浓度分别为(52.6±1.8) ng/L和(75.4±6.0) ng/L,BALF中IL-17浓度分别为(78.1 ±5.8) ng/L和(95.0±6.8)ng/L,均显著高于对照12周组[(40.0±3.2) ng/L和(54.5±4.6) ng/L]及24周组[(36.7±3.2) ng/L和(53.9±3.7) ng/L],暴露24周组与其他3组比较,差异均有统计学意义(均P＜0.05).暴露24周组大鼠外周血中IL-6浓度为(31.4±2.1)ng/L,显著高于对照24周组[(11.5±0.5)ng/L];暴露12周组和24周组大鼠BALF中IL-6浓度分别为(33.3 ±2.3)ng/L和(44.6±3.0)ng/L,显著高于对照12周组和24周组[(15.6±1.8)ng/L和(18.0±1.9) ng/L].暴露12周组和24周组大鼠外周血中Th17细胞比例分别为(1.81±0.19)％和(3.74±0.55)％,BALF中Th17细胞比例分别为(7.84±0.28)％和(8.01±0.39)％,均显著高于对照12周组[(0.97±0.08)％和(5.64±0.54)％]及24周组[(1.08±0.10)％和(5.95±0.48)％],暴露24周组与其他3组比较,差异均有统计学意义(均P＜0.05).暴露12周组和24周组大鼠BALF中Treg细胞比例分别为(8.81±0.49)％和(11.98±0.72)％,均显著高于对照12周组和24周组[(4.34±0.28)％和(5.21±0.42)％].暴露12周组和24周组大鼠外周血中IL-17 mRNA表达量分别为25.7±2.0和33.9±1.5,BALF中IL-17 mRNA表达量分别为22.2±1.8和34.7±4.2,均显著高于对照12周组(11.3±2.6和11.6 ±2.4)及24周组(11.1±2.0和13.5±3.4);暴露12周组和24周组大鼠BALF中Foxp3 mRNA表达量分别为24.4±2.7和30.3±2.7,显著高于对照12周组和24周组(12.7±2.7和14.6±3.8).暴露组大鼠BALF中Th17细胞与其BALF中细胞总数和巨噬细胞数呈正相关(r值分别为0.512和0.543,均P＜0.05).结论 烟草暴露可导致大鼠气道炎症模型的Th17细胞和Treg细胞及相关炎症因子水平升高,提示Treg细胞可能参与气道炎症的免疫调节,Th17细胞异常升高可能与大鼠气道炎症反应的发生及持续进展有关.     

人外周血树突状细胞体外稳定培养方法的建立及与磁珠法的比较

目的 建立一种稳定的人外周血树突状细胞(DCs)体外培养的方法,并与磁珠分选法进行比较.方法 通过密度梯度离心法分离出志愿者的外周血单个核细胞(PBMC),再分别应用磁珠分选法、贴壁法对PBMC进行培养,应用重组人集落刺激因子(rhGM-CSF)、重组人白细胞介素-4(rhIL-4)诱导获得DCs.倒置显微镜观察细胞形态变化,并分别在第3、5、6天用台盼蓝染色法进行细胞活力检测;经过1、2、5 h的贴壁培养后,应用流式细胞仪检测单核细胞表面CD14、CD1a、HLA-DR的表达以确定最佳贴壁时间;经人重组细胞因子诱导培养后,对所获得的细胞检测CD14、CD1a、CD86、CD83、HLA-DR的表达.采用同种混合淋巴细胞反应,评价DCs刺激T淋巴细胞增殖的能力.结果 经贴壁2 h后诱导培养的DCs形态较典型.磁珠分选法获得的DCs第5、6天细胞活力[(53.333±5.774)%、(38.333±7.638)%]明显低于第3天[(68.667±3.215)%,P均＜0.05];贴壁培养法获得的DCs第3、5、6天的细胞活力[(92.667±3.055)%、(94.000±1.000)%和(94.667±1.528)%]比较,差异无统计学意义(F=0.737,P＞0.05);贴壁培养法获得的DCs第3、5、6天细胞活力均高于磁珠分选法(t值分别为9.374、12.021、12.527,P均＜0.05).PBMC经磁珠分选前后CD14的阳性表达率分别为(32.457±12.351)%、(41.914±14.858)%,二者比较差异无统计学意义(t=1.295,P＞0.05).单核细胞表面CD14的阳性表达率在培养2 h时[(35.267±4.658)%]高于培养1、5 h时[(15.033±6.189)%、(21.233±4.895)%,P均＜0.05].培养第6天,DCs表面CD14的阳性表达率[(2.200±1.356)%]较第1天[(32.328±14.517)%]明显下降(t=5.467,P＜0.05),CD1a的阳性表达率[(43.371±16.250)%]较第1天[(12.300±6.223)%]显著升高(t=2.545,P＜0.05);而CD86、CD83、HLA-DR的阳性表达率[(16.857±5.686)%、(9.343±5.230)%、(72.800±17.881)%]与第1天[(12.550±16.758)%、(6.250±1.323)%、(64.671±15.588)%]比较,差异无统计学意义(t值分别为0.652、1.137、0.907,P均＞0.05).同种混合淋巴细胞反应,随着淋巴细胞的增多,增殖能力下降.磁珠分选法中,DCs与淋巴细胞的比例为1:50、1:100时,细胞增殖能力(1.502±0.055、1.507±0.029)较1:10时(1.859±0.049)降低(P均＜0.05);贴壁培养法中,DCs与淋巴细胞的比例为1:100时,细胞增殖能力(1.545±0.066)较1:10时(2.015±0.301)降低(P＜0.05).在DCs与淋巴细胞的比例相同时,两种方法得到的DCs在刺激T淋巴细胞方面的能力相近(P＞0.05).结论 与磁珠分选法比较,贴壁培养2 h后的人外周血PBMC再行诱导可获得形态与功能较优的DCs,且此法稳定、简便、经济,是一种适于基础、临床研究的DCs体外培养方法.