细胞凋亡对中性粒细胞功能的影响

目的：为了探讨中性粒细胞（ＰＭＮ）的自然凋亡和ＰＭＮ凋亡对细胞吞噬、趋化和活性氧（ＲＯＳ）生成等功能的影响。方法：采用光镜、电镜、电泳和流式细胞仪等研究手段探讨ＰＭＮ的自然凋亡;通过葡萄球菌法、滤膜法和化学发光的方法研究凋亡ＰＭＮ吞噬、趋化和ＲＯＳ生成的改变。结果：分离健康成人外周血ＰＭＮ,在体外进行１０％血清培养,随着培养时间的延长,ＰＭＮ凋亡率升高,同时ＰＭＮ的吞噬、趋化以及ＲＯＳ生成等功能都     

Th1型和Th2型细胞因子对人中性粒细胞吞噬结核杆菌活性的影响

目的 建立用流式细胞术检测人外周血中性粒细胞(PMNs)吞噬结核分枝杆菌(Mtb)的方法 ,并探讨Th1和Th2型细胞因子对PMNs吞噬Mtb活性的影响.方法 运用抗酸染色、激光共聚焦显微镜观察人PMNs吞噬Mtb,并用流式细胞术检测人PMNs对FTTC标记Mtb的吞噬活性.外周血预先分别与IL-2、IFN-γ、GM-CSF和IL-4等细胞因子孵育,再加FTTC标记Mtb后,用流式细胞术检测PMNs对Mtb吞噬率,并与对照组比较吞噬率的变化.结果 抗酸染色和激光共聚焦显微镜均能观测到人PMNs吞噬Mtb.用流式细胞术检测健康人外周血PMNs对Mtb的吞噬率在5 min时为47%,15～20 min达到平台期,为66%～72%.外周血预先加IL-2或IFN-γ作用后,PMNs对Mtb的吞噬率可分别增加76.7%和75.2%;而预先加IL-4作用后,吞噬率降低31.7%.结论 IL-2和IFN-γ对PMNs吞噬Mtb功能有增强作用,而IL-4有降低作用,表明Th1型和Th2型细胞因子参与调节PMNs抗结核杆菌感染的免疫作用.     

白细胞介素17对结核病患者中性粒细胞凋亡的影响

目的探讨白细胞介素-17(IL-17)对结核病患者外周血中性粒细胞(PMN)凋亡的影响以及可能涉及的信号通路。方法取结核病患者和健康人外周血PMN培养0~24 h,annexinⅤ染色后流式细胞术检测PMN凋亡率;或加不同浓度IL-17刺激培养24 h再检测PMN凋亡率;或用MAPK特异性抑制剂U0126预处理30 min后,再加IL-17培养24 h后检测PMN的凋亡。结果结核病患者和正常人PMN凋亡率均随培养时间延长而增加(P0.05),结核病患者PMN在0、6、12、24 h时的annexinⅤ+细胞分别为(4.49±1.39)%、(21.89±2.90)%、(39.96±4.15)%、(68.35±7.01)%,均分别高于正常人组的(2.65±0.75)%、(11.00±1.72)%、(25.84±3.90)%,(45.59±4.10)%(P0.05)。而经IL-17刺激后,结核病患者与正常人PMN的凋亡率,在IL-17 0.5μg/L组,分别为(59.81±7.19)%和(34.65±4.79)%;在IL-17 5μg/L组,分别为(51.62±6.91)%和(29.04±3.62)%;均低于未加IL-17对照组(68.35±7.01)%和(45.59±4.10)%(P0.05);而在IL-17 50μg/L组,凋亡率则分别达到(76.04±5.59)%和(53.24±4.62)%,明显高于对照组(P0.05)。预先用U0126处理,可大部分阻断IL-17对结核病患者和正常人PMN凋亡的抑制作用。结论结核病患者和正常人PMN凋亡率均随培养时间延长而增加,且结核病患者PMN凋亡率高于正常人。IL-17较低浓度延迟凋亡,较高浓度促进凋亡。IL-17抑制PMN凋亡的作用和ERK途径有关。     

Th1型和Th2型细胞因子对人中性粒细胞吞噬结核杆菌活性的影响

Objective To establish a flowcytometry method for detecting phagocysis of Mycobacterium tuberculosis(Mth) by the human peripheral blood neutrophils( PMNs), and to explore the effects of Thl and Th2 cytokine on phagocytotic activity of PMNs. Methods By using acid-fast staining the phagocytosis of Mtb by human neutrophil was observed by microscopy, and the phagocytosis of FTTC labeled Mtb by human neutrophils was detected under confocal microscope. The whole fresh peripheral blood of healthy adults was incubated with FTTC labeled Mtb and the phagocytosis were measured by flow cytometry. The altered phagocytosis of FTTC-Mtb by neutrophils pretreated with IL-2, IFN-γ, GM-CSF or IL-4 were measured. Results Human peripheral blood neutrophils activity of Mtb phagocytosis was observed by acid-fast staining and confocal microscope. The percentage of phagocytosis of Mtb was dependent on the time of incubation with Mtb. The percentages were 47% at 5 min and reached plateau about 66% ～72% at 15 min to 20 min.Pretreatment with different concentrations of IL-2 or IFN-γincreased the phagocytosis of Mtb by 76.7% and 75.2%, respectively; but pretreatment with IL-4 decreased the phagocytosis by 31.7%. Conclusion IL-2and IFN-γcan increase phagocytosis of Mth by neutrophils; while IL-4 can reduced neutrophil activity of phagocytosis of Mtb by human neutrophils, and demonstrate that Th1/Th2 type cytokins involve in regulating the acitvities of neutrophils anti-Mtb infection.     

Th1型和Th2型细胞因子对人中性粒细胞吞噬结核杆菌活性的影响

Objective To establish a flowcytometry method for detecting phagocysis of Mycobacterium tuberculosis(Mth) by the human peripheral blood neutrophils( PMNs), and to explore the effects of Thl and Th2 cytokine on phagocytotic activity of PMNs. Methods By using acid-fast staining the phagocytosis of Mtb by human neutrophil was observed by microscopy, and the phagocytosis of FTTC labeled Mtb by human neutrophils was detected under confocal microscope. The whole fresh peripheral blood of healthy adults was incubated with FTTC labeled Mtb and the phagocytosis were measured by flow cytometry. The altered phagocytosis of FTTC-Mtb by neutrophils pretreated with IL-2, IFN-γ, GM-CSF or IL-4 were measured. Results Human peripheral blood neutrophils activity of Mtb phagocytosis was observed by acid-fast staining and confocal microscope. The percentage of phagocytosis of Mtb was dependent on the time of incubation with Mtb. The percentages were 47% at 5 min and reached plateau about 66% ～72% at 15 min to 20 min.Pretreatment with different concentrations of IL-2 or IFN-γincreased the phagocytosis of Mtb by 76.7% and 75.2%, respectively; but pretreatment with IL-4 decreased the phagocytosis by 31.7%. Conclusion IL-2and IFN-γcan increase phagocytosis of Mth by neutrophils; while IL-4 can reduced neutrophil activity of phagocytosis of Mtb by human neutrophils, and demonstrate that Th1/Th2 type cytokins involve in regulating the acitvities of neutrophils anti-Mtb infection.     

pHGF对人中性粒细胞吞噬功能的影响

我们曾报道，从乳猪肝脏提取的促肝细胞生长因子（ｐＨＧＦ）能增强小鼠中性粒细胞的吞噬功能，本文进而用吞噬杀菌试验、化学发光技术和促凝血活性试验证明，ｐＨＧＦ也能显著增强人中性粒细胞的吞噬和杀菌功能。     

小鼠骨髓中性粒细胞与脾脏中性粒细胞的异质性比较北大核心CSCD

目的:探讨小鼠骨髓与脾脏中性粒细胞的异质性。方法:ImmGEN数据库检索获得小鼠骨髓和脾脏Ly6G~+CD11b~+中性粒细胞相关细胞因子的表达水平,流式细胞术分选获得骨髓和脾脏Ly6G~+CD11b~+中性粒细胞,LPS刺激6 h后采用实时定量PCR方法检测小鼠骨髓与脾脏中性粒细胞IL-1β、IL-6及TNF表达的变化,LPS刺激12 h后采用Annexin V和PI双染法检测中性粒细胞凋亡水平,使用FITC标记的大肠埃希菌检测两组中性粒细胞的细菌吞噬能力。结果:小鼠骨髓中性粒细胞高表达的细胞因子数量(11.36%,5/44)低于脾脏中性粒细胞(45.45%,20/44);LPS作用后,与对照组相比,小鼠骨髓与脾脏中性粒细胞的IL-1β、IL-6及TNF表达显著升高(P<0.05),而骨髓与脾脏中性粒细胞的凋亡水平差异无统计学意义;大肠埃希菌吞噬实验显示,骨髓中性粒细胞的吞噬阳性率显著高于脾脏中性粒细胞(P<0.05)。结论:小鼠骨髓中性粒细胞与脾脏中性粒细胞在炎症因子释放、吞噬能力两方面呈现一定的差异性,而自发凋亡水平具有一致性。     

中性粒细胞弹性蛋白酶和IL-8在大鼠哮喘中的作用及地塞米松调控

目的 观察中性粒细胞弹性蛋白酶（NE）和IL-8在大鼠哮喘中的作用及地塞米松（DM）对其功能的影响.方法 采用大鼠哮喘模型,随机分成哮喘组（A组）、正常对照组（B组）、地塞米松治疗组（C组）,对周围血内中性粒细胞（PMN）进行分离纯化,ELJSA法检测血PMN和支气管肺泡灌洗液（BALF）中NE和IL-8的表达水平.结果 A组血PMN和BALF中NE、IL-8表达水平均显著高于B组（P＜0.01）,C组均显著低于A组（P＜0.01）.C组血PMN中NE的表达水平显著高于B组（p＜0.05）,而BALF中则无显著性差异（P＞0.05）.C组血PMN和BALF中IL-8的表达水平与B组相比差异均无统计学意义（P＞0.05）.BALF和肺组织中PMN计数A组显著高于B组（P＜0.01）,肺组织中PMN计数C组显著高于A组（P＜0.01）,而两组BALF中PMN占细胞总数的百分比差异无统计学意义（P＞0.05）.BALF中PMN计数和IL-8呈显著正相关（P＜0.05）.结论 PMN、NE和IL-8参与了哮喘的炎症过程,PMN可能通过合成IL-8、NE起作用,其合成功能可以被地塞米松所抑制.IL-8与PMN在BALF中浸润密切相关.     

比较两种麻醉剂对胆道手术患者中性粒细胞凋亡、TNF-α、IL-6和IL-8的影响

探讨TNF α、IL 6、IL 8与异丙酚麻醉下胆道手术患者中性粒细胞凋亡的关系。ASAI～II级择期行胆道手术的非肿瘤患者 4 3例 ,随机分为异丙酚麻醉组 (P组 ,n =2 2 )及安氟醚麻醉组 (E组 ,n =2 1)。另选择健康成年人 16例作健康对照组。取手术前 (T1)、手术开始 (T2 )、手术开始后 3h (T3)、 2 4h (T4 )、 72h (T5 )外周静脉血 10ml,检测中性粒细胞凋亡及血浆细胞因子的水平。结果发现胆道手术患者术前中性粒细胞凋亡率明显低于健康对照组 ;同手术前比 ,T3、T4、T5时点中性粒细胞的凋亡率均明显降低 (P <0 0 1) ;而与E组比 ,P组分别在T3、T4时间点中性粒细胞凋亡率明显升高 (P <0 0 1)。胆道手术患者围术期血浆TNF α变化差异不明显 ;IL 6在手术创伤后升高 ,术后 1d达峰值 ,但在组间无明显的差异 ;围术期血浆IL 8的水平变化同IL 6 ,但与E组比 ,P组分别在T3、T4时间IL 8的血浆水平明显降低 (P <0 0 5、P <0 0 1)。本研究认为异丙酚麻醉与安氟醚麻醉相比 ,具有明显地减轻手术创伤后中性粒细胞凋亡的延迟作用 ,可能与异丙酚麻醉组创伤后细胞因子IL 8的释放较低有关     

GLS和IL-12偶联重组耻垢分枝杆菌免疫效果的观察

目的：研究携带编码人天然颗粒溶素（GLS）和小鼠IL-12基因质粒（pZM03）偶联重组耻垢分枝杆菌（ATCC607）经鼻黏膜免疫小鼠后,小鼠体内的免疫状况.方法：BALB/c小鼠36只,随机分为生理盐水、pZM03、ATCC607、卡介苗（BCG）、重组ATCC607（即携带pZM03的ATCC607）、灭活重组ATCC607组;采用滴鼻法免疫小鼠,BCG组0、14天各1次,其它组0、4、14天各1次,第0天免疫后4周处死小鼠,用ELISA检测血清中IFN-γ、IL-12、IgG2a、lL-4的分泌水平和淋巴细胞PPD诱导的IFN-γ分泌水平以及肺泡灌洗液特异性SIgA的水平,用MTS法检测免疫小鼠脾淋巴细胞增殖情况.结果：重组ATCC607血清中IFN-γ、IL-12水平与BCG组无明显差异但明显高于其他组（P＜0.05）,IgG2a水平重组ATCC607组高于BCG组和其他组（P＜0.05）,各组间IL-4水平差异无统计学意义.重组ATCC607、BCG、灭活重组ATCC607及ATCC607组用PPD均可诱导小鼠脾淋巴细胞增殖,各组间差异无显著意义,但与生理盐水和pZM03组间差异有显著意义（P＜0.05）.重组ATCC607组PPD诱导淋巴细胞IFN-γ明显高于其它组（P＜0.05）,与BCG组比较无显著差异.重组ATCC607等含菌组经鼻黏膜免疫可产生黏膜特异性SIgA.结论：重组ATCC607经鼻黏膜免疫小鼠后,机体特异性免疫特别是细胞免疫和黏膜免疫增强,为重组ATCC607治疗结核病的研究奠定了基础.     

Dysregulation in IL-12 secretion by neutrophils from HIV-infected patients

It is generally believed that neutrophils from HIV-infected patients are functionally competent, but several studies have shown impairment in neutrophil fungal killing and cytokine production. In this study we evaluated the ability of neutrophils from healthy donors and HIV-infected patients to produce IL-12 in response to stimulation with Candida albicans, lipopolysaccharide (LPS) and Cryptococcus neoformans (acapsular and encapsulated), with and without MoAb opsonization. Neutrophils from healthy donors secreted IL-12 in response to LPS or C. albicans but not in response to encapsulated or acapsular C. neoformans, regardless of MoAb opsonization. Surprisingly, neutrophils from HIV-infected patients demonstrated constitutive IL-12 production, although these cells were not responsive to LPS stimulation. The inability of MoAb to C. neoformans capsular polysaccharide to promote IL-12 production by neutrophils excludes phagocytosis and/or CD16 cross-linking in this process, and distinguishes neutrophils from monocytes. Our results provide additional evidence for cytokine dysregulation in neutrophils from HIV-infected patients. Furthermore, the IL-12 response of neutrophils and monocytes to CD16 stimulation appears to be different, suggesting differences in the role of these phagocytic cells during the inflammatory response.     

结核分枝杆菌Ag85B与IL-12基因真核共表达质粒的构建及表达

目的 :构建结核分枝杆菌Ag85B和鼠IL 12基因的共表达载体pBud85B IL12。方法 :将结核分枝杆菌Ag85B基因和鼠IL 12基因同时克隆入含多启动子的共表达载体pBudCE4 .1中 ,构建真核共表达质粒pBud85B IL12。以pBud85B IL12转染COS 7细胞 ,通过RT PCR及ELISA方法检测目的基因的表达。结果 :在COS 7细胞中同时可检测到Ag85B和IL12的表达。结论 :pBud85B IL12共表达质粒的成功构建 ,为对其免疫原性、免疫反应性及免疫保护作用的进一步研究奠定了基础     

Impacts of interleukin-12 on multiplication of Mycobacterium tuberculosis and Mycobacterium avium complex in mice

Objective: To evaluate the potential impacts of exogenous administration of murine recombinant interleukin-12 (IL-12) on multiplication of Mycobacterium tuberculosis and M. avium complex (MAC) in murine models.
Methods: Swiss or beige mice were infected intravenously with M. tuberculosis H37Rv or MAC respectively, and were treated by subcutaneous injection with various doses of IL-12, either alone or in combination with chemotherapy. Effectiveness of treatment was assessed by the enumeration of CFUs in the spleens and lungs, together with other indicators.
Results: Multiplication of M. tuberculosis was reduced by IL-12 in a dose-dependent manner if the treatment began at day 1, whereas no statistically significant suppression was observed if the treatment began at day 14. Combination with IL-12 did not enhance the bactericidal activity of antituberculosis chemotherapy. The growth curves of MAC in IL-12-treated mice were almost identical to those of untreated controls, indicating that IL-12 did not affect the multiplication of MAC in beige mice. In both experiments, the dosing of IL-12 approached levels of severe toxicity for the mouse strains used.
Conclusions: IL-12 had a positive affect on early multiplication of M. tuberculosis . It had no effect on early multiplication of M. avium complex.     

结核杆菌抗原激活MEK-ERK信号通路延迟中性粒细胞自发性凋亡的作用

探讨结核杆菌抗原(Mtb-Ag)对中性粒细胞自发性凋亡的影响。取健康成人外周血分离中性粒细胞,加Mtb-Ag培养24 h,或用Mek抑制剂PD98059预处理30 min,Annexin V染色流式细胞术观察细胞凋亡;并用Western blot印迹法检测Mek活化情况。与中性粒细胞体外培养24 h后自发凋亡(55%±6%)相比,加入Mtb-Ag(1.125 mg/ml)后,中性粒细胞凋亡率(31%±3%)明显降低;并且Mtb-Ag可诱导Mek的激活;而PD98059(50μmol/L)预处理可阻断Mtb-Ag的抑制凋亡作用。Mtb-Ag对中性粒细胞自发凋亡有抑制作用,这一作用涉及Mek-Erk途径。     

脂多糖和金黄色葡萄球菌对IL-23、IL-12表达的调控

目的：检测脂多糖（LPS）和金黄色葡萄球菌（SAC）对人外周血单个核细胞（PBMC）IL-23和IL-12各亚基表达的调节作用并探索其作用机制。方法：用LPS和SAC直接刺激人PBMC，或用CD14抗体或p38丝裂原活化蛋白激酶（MAPK）抑制剂Mastoparan预处理PBMC后再予LPS和SAC刺激，半定量RT-PCR方法检测IL-23p19、IL-12p35和IL-12p40亚基的基因表达变化。结果：人PBMC组成性表达p19和p35，不表达p40。LPS和SAC可上调各亚基的表达。LPS诱导的IL-23和IL-12各亚基表达均需通过CDl4；CDl4仅部分参与SAC诱导的IL-12p40和p35表达上调，而与p19上调无关。LPS和SAC诱导的IL-23和IL-12各亚基表达均需要p38MAPK通路。结论：LPS和SAC刺激下IL-23和IL-12亚基表达及信号传导通路既有相似之处又有不同点，为单独或同时调控这两种因子的表达提供线索。     

小鼠psIL-12质粒抗结核分枝杆菌感染的研究

目的观察、评价小鼠白细胞介素12真核表达质粒psIL-12对结核分枝杆菌感染小鼠细胞因子水平的影响及疗效.方法结核分枝杆菌H37Rv感染的C57BL/J6小鼠随机分成生理盐水、pcDNA3.1对照组,psIL-12治疗组,每组各6只.感染4周后分别予以生理盐水、pcDNA3.1、psIL-12,第1次治疗后8周处死检测器官活菌数,脏器重量指数(WI),脾淋巴细胞特异性IFN-γ、IL-4细胞因子分泌水平,并观察肺、脾组织病理改变情况.结果 psIL-12治疗组肺组织活菌数(每mg log10CFU/ml)为7.41±0.50,与对照组(8.15±0.37、8.19±0.29)比较显著降低(P＜0.05);脾组织荷菌量(每mg log10 CFU/ml)为5.31±0.21,与对照组(5.76±016、5.88±0.21)比较显著降低(P＜0.05).psIL-12治疗组脾脏重量指数为0.58±0.05,与对照组(1.02±0.08、1.10±0.02)比较显著降低(P＜0.05).脾淋巴细胞IFN-γ(pg/ml)为478.0±10.1,与对照组(134.5±15.7、125.1±8.2)比较显著升高(P＜0.05).各组间IL-4水平差异无显著性.对照组肺组织病理改变以变质、渗出为主,而psIL-12治疗组以增生改变为主.对照组与治疗组间比较脾脏病理改变不明显.结论 psIL-12真核表达质粒对小鼠结核分枝杆菌感染有一定的治疗作用,可能通过诱导促进IFN-γ产生,调节TH1/TH2应答类型,起到抗结核分枝杆菌感染的作用.