中试生产的结核DNA疫苗免疫效能研究

目的 以300 L发酵罐中试规模生产的冻干Mtb Ag85A质粒DNA疫苗为基础免疫小鼠,对该疫苗的免疫效能进行研究。 方法 将4~6周龄的BALB/c小鼠20只通过数字表法随机分为2组,一组免疫Mtb Ag85A质粒DNA疫苗（结核DNA疫苗免疫组）,另一组免疫PBS作为阴性对照（PBS对照组）;于免疫前采血,肌内注射免疫3次。在第3次免疫后3周摘眼球处死小鼠,收集血清,进行血清特异性抗体检测和细胞因子(IFN-γ、IL-4)检测;同时对每只小鼠取脾细胞进行特异性γ干扰素 (IFN-γ) 分泌检测和脾细胞CD4⁺、CD8⁺百分率检测。 结果 （1）免疫前后免疫组与对照组抗体水平吸光度A值均小于0.2。（2）结核DNA疫苗免疫组血清细胞因子IL-4水平［（146.2±34.3）pg/ml］低于PBS对照组［（177.7±28.1）pg/ml］,差异有统计学意义（t=2.244,P=0.038）,而IFN-γ水平［（129.6±159.0）pg/ml］虽然高于PBS对照组［（76.5±21.5）pg/ml］,但差异无统计学意义（t=－1.047,P=0.309）。（3）酶联免疫斑点检测结果表明结核DNA疫苗免疫组脾细胞特异性IFN-γ分泌水平（分泌IFN-γ细胞个数）（103.60±112.14）高于PBS对照组（5.78±5.83）,差异有统计学意义（t=36.538,P=0.018）。（4）结核DNA疫苗免疫组CD4⁺细胞百分率均值（21.57%）高于PBS对照组（12.17%）,差异有统计学意义（t=3.043,P=0.038）;CD8⁺细胞百分率均值（13.70%）与PBS对照组（10.57%）相比,差异无统计学意义（t=0.847,P=0.445）。 结论 Mtb Ag85A质粒DNA疫苗主要刺激机体Th1型细胞免疫,而对诱导机体产生Th2型细胞免疫应答没有显著的促进作用。     

结核分枝杆菌休眠相关抗原Rvl733cDNA疫苗的构建及免疫学特性研究

目的 构建结核分枝杆菌（Mtb）休眠相关抗原Rv1733c的真核表达载体,并评价其作为DNA疫苗的免疫学特性。 方法 利用限制性酶切的方法从本室前期保存的pMD-18T-Rv1733c质粒中构建Rv1733c的真核表达载体pcDNA-Rv1733c。将pcDNA-Rv1733c重组质粒稳定转染P815细胞,并用间接免疫荧光法检测Rv1733c的表达。采用数字表法随机将BALB/c小鼠分为3组,每组10只,即pcDNA-Rv1733c质粒DNA组、生理盐水组和BCG组。pcDNA-Rv1733c质粒DNA组和生理盐水组采用肌内注射方式免疫,间隔2周免疫1次,共免疫3次。BCG组采用皮下免疫一次。各组小鼠每2周采血,ELISA检测血清中特异性抗体水平和IgG2a/IgG1抗体亚类比率与比值。初次免疫8周后,MTS［3-(4,5-diethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-etrazolium,inner salt］（四氮唑蓝盐化合物）法检测小鼠脾淋巴细胞特异性增殖、ELISPOT检测脾淋巴细胞分泌IFN-γ的水平。流式细胞法检测脾淋巴细胞中CD4⁺和CD8⁺ T细胞所占比率;LDH法检测CTL（cytotoxic T lymphocytes;细胞毒性T淋巴细胞）活性。 结果 成功构建Rv1733c的真核表达载体pcDNA-Rv1733c。间接免疫荧光实验表明pcDNA-Rv1733c质粒稳定转染的P815细胞中能够稳定表达Rv1733c蛋白。pcDNA-Rv1733c质粒DNA免疫小鼠后能诱导小鼠产生特异性抗体,抗体亚类以IgG2a为主,随着免疫时间的延长,IgG2a/IgG1的比值趋于平衡;脾淋巴细胞增殖指数（2.00±0.36）高于BCG组（1.1±0.06）（t=3.096,P<0.05）;特异性分泌IFN-γ的脾淋巴细胞频数(41.48±5.30) SFC/10⁶高于生理盐水组（2.75±1.37）SFC/10⁶（t=4.752,P<0.05）;然而脾淋巴细胞中CD4⁺、CD8⁺ T细胞所占比率［分别为(18.15±2.30)%、(7.68±1.34)%］、CTL杀伤活性［(29.52±1.96)%］都与生理盐水组相当［(16.43±2.02)% (t=0.571,P>0.05)、(7.32±0.42)% (t=0.234,P>0.05)、（25.28±2.51）%（t=0.726,P>0.05）］。 结论 成功构建Rv1733c真核表达载体pcDNA-Rv1733c;并能够诱导小鼠机体产生特异性的体液和细胞免疫应答,提示用于结核病新型疫苗的研究具有一定的意义。     

复苏促生长因子结构域及其突变体重组蛋白诱导小鼠免疫应答的研究

目的 评价复苏促生长因子结构域蛋白（Rpfd）及其突变体蛋白（Rpfd1、Rpfd2）等3种重组蛋白的免疫效能。 方法 2011年11月至2012年2月间,分别以本实验前期制备的重组蛋白Rpfd（A组）、Rpfd1（A1组）、Rpfd2（A2组）分别于0、2、4周免疫无特定病原体（SPF）级BALB/c小鼠,每组14只,并分别以BCG（B组）和生理盐水（C组）同期免疫同种小鼠各14只作为对照。第5周,每组取7只小鼠摘眼球取血,ELISA法检测血清特异性抗体、血清IFN-γ和IL-2表达水平;第8周,每组剩余7只小鼠用1×10⁵ CFU结核分枝杆菌标准株H37Rv感染小鼠,第9周检测感染后小鼠血清细胞因子IFN-γ和IL-2水平。 结果 （1）抗体水平：①Rpfd抗原包被。A1组刺激小鼠产生抗体A₄₅₀的检测值（0.990±0.272）高于B组（0.631±0.180）（t=4.635,P<0.05）;A2组（1.470±0.455）高于B组（t=6.634,P<0.05）。②Rpfd1抗原包被。A组刺激小鼠产生抗体A₄₅₀的检测值（1.030±0.304）高于B组（0.573±0.004）（t=5.276,P<0.05）;A1组（1.368±0.171）高于B组（t=17.20,P<0.05）;A2组（2.766±0.245）高于B组（t=31.643,P<0.05）;③Rpfd2抗原包被。A1组刺激小鼠产生抗体A₄₅₀的检测值（1.055±0.202）高于B组（0.538±0.100）（t=8.009,P<0.05）;A2组（1.605±0.544）高于B组（t=8.192,P<0.05）。（2） IFN-γ水平：①感染前。A组刺激小鼠产生IFN-γ水平［（553.47±132.00）pg/ml］高于B组［（385.28±129.07）pg/ml］（t=3.150,P<0.05）;②感染后。A1蛋白组刺激小鼠产生IFN-γ水平［（492.41±211.74）pg/ml］高于B组［（335.36±207.72）pg/ml］（t=2.874,P<0.05）;A2组［（543.09±223.07）pg/ml］高于B组（t=3.15,P<0.05）。（3）IL-2水平：①感染前。A组刺激小鼠产生IL-2水平［（1490.05±215.35）pg/ml］高于B组［（718.70±269.29）pg/ml］（t=7.763,P<0.05）;A1组［（1738.91±358.40）pg/ml］高于B组（t=7.903,P<0.05）;A2组［（2270.74±193.40）pg/ml］高于B组（t=16.308,P<0.05）;②感染后。A组刺激小鼠产生IL-2水平［（806.81±306.39）pg/ml］高于B组［（335.26±176.81）pg/ml］（t=4.627,P<0.05）;A1组［（1373.22±143.75）pg/ml］高于B组（t=15.90,P<0.05）。 结论 Rpfd、Rpfd1、Rpfd2蛋白具有启动宿主体液免疫功能及增强宿主细胞免疫功能的双重作用,可能成为预防及治疗Mtb感染的免疫新策略。     

CD4＋T淋巴细胞对肺结核患者血清白蛋白的影响

目的 探讨外周血CD4⁺ T淋巴细胞对肺结核患者血清白蛋白的影响。 方法 对119例初治肺结核患者进行淋巴细胞亚群和血清白蛋白测定,探讨细胞免疫功能与血清白蛋白相关性;同时,根据其CD4⁺ T淋巴细胞亚群水平,分为免疫低下组62例和正常组57例,探讨细胞免疫功能低下对低蛋白血症的影响。 结果 （1）所有纳入患者外周血CD4⁺ T淋巴细胞［（516.6±266.1）×10⁹/L］与CD3⁺ T淋巴细胞［（841.6±398.8）×10⁹/L］、CD8⁺ T淋巴细胞［（261.0±142.6）×10⁹/L］、CD4⁺/CD8⁺ T淋巴细胞比值（2.33±1.40）呈正相关（分别为r=0.883,P=0.000;r=0.579,P=0.000;r=0.365,P=0.000）。（2）外周血CD4⁺T淋巴细胞与血清白蛋白呈正相关（r=0.116,P=0.033）。（3）在免疫低下组中,肺部病灶范围达4个肺野以上者43例（占69.4%）,明显高于正常组的22例（占38.6%）,差异有统计学意义（χ²=11.335,P=0.001）;免疫低下组治疗前血清白蛋白［（33.9±5.5）g/L］明显低于正常组［（36.1±5.7）g/L］（t=2.187,P=0.031）。 结论 在肺结核患者中CD4⁺ T淋巴细胞与治疗前血清白蛋白水平呈正相关。CD4⁺ T淋巴细胞低下肺结核患者,其病灶范围较广,容易并发低蛋白血症。     

肺结核患者疗程中外周血辅助性和调节性T细胞的动态研究

目的 探讨辅助性T细胞17（Th17）和CD4⁺CD25⁺CD127 ^low调节性T细胞与结核病的发病及抗结核治疗转归的关系。 方法 纳入对象包括32例活动性肺结核患者、25例Mtb潜伏感染者、45例健康对照者。流式细胞术检测外周血Th17细胞分泌细胞因子IL-17和CD4⁺CD25⁺CD127 ^low调节性T细胞表达强度。结果以x±s表示,所有数据均使用Prism 4.0统计软件进行分析,两组间比较采用非配对t检验,多组间的比较采用ANOVA方差分析,以P<0.05为差异有统计学意义。 结果 肺结核患者组治疗前IL-17表达为（3.25±1.68）%,明显低于健康对照组［（4.62±1.46）%］（F=6.633,P<0.0001）。比较活动性肺结核患者组IL-17表达在治疗前、治疗3个月［（4.17±2.27）%］、治疗6个月［（5.58±1.66）%］时的检测结果,发现治疗3个月时高于治疗前,但差异无统计学意义（F=12.244,P=0.057）;而治疗6个月时明显高于治疗前（F=12.244,P<0.0001）和治疗3个月时（F=12.244,P=0.004）。健康对照组CD4⁺CD25⁺CD127 ^low调节性T细胞表达为（4.97±1.60）%,与Mtb潜伏感染组［（5.00±1.08）%］比较,差异无统计学意义（F=11.986,P=0.937）;活动性肺结核患者组治疗前表达为（6.59±1.73）%,显著高于健康对照组及Mtb潜伏感染组(F=11.986,P<0.0001)。比较活动性肺结核患者组治疗前、治疗3个月［（8.28±2.04）%］、治疗6个月［（7.46±1.87）%］时的CD4⁺CD25⁺CD127 ^low调节性T细胞表达,发现治疗3个月时的表达明显高于治疗前（F=6.458,P=0.001）;治疗6个月时的表达低于治疗3个月时（F=6.458, P=0.085）,但差异无统计学意义;治疗6个月与治疗前的表达相比较,差异无统计学意义（F=6.458,P=0.068）;治疗6个月CD4⁺CD25⁺CD127 ^low调节性T细胞表达明显高于健康对照组（t=6.255,P<0.0001）。 结论 结核病患者外周血Th17细胞明显减少,经有效抗结核治疗后,Th17细胞逐渐增加,表明Th17细胞在抗结核免疫中起保护作用。结核病患者外周血CD4⁺CD25⁺CD127 ^low调节性T细胞明显增多,经抗结核治疗后CD4⁺CD25⁺CD127 ^low调节性T细胞逐渐减少,进一步说明CD4⁺CD25⁺CD127 ^low调节性T细胞在抗结核免疫中起抑制作用。     

结核分枝杆菌ESAT6-RpfE亚单位疫苗的构建与免疫原性研究

目的 构建Mtb ESAT6-RpfE（早期分泌抗原靶6-复活促进因子E）的原核表达载体,表达和纯化融合蛋白,并对其免疫原性进行研究。方法 分别以Mtb H37Rv及临床株的基因组为模板,PCR扩增esat6、rpfE基因,依次插入pET30a质粒并在大肠埃希菌BL21中表达纯化ESAT6-RpfE蛋白。采用完全随机的方法将C57BL/6小鼠分组,每组6只,共进行了两次实验,第一次实验分为ESAT6、RpfE、ESAT6 RpfE、PBS、BCG五组,佐剂为二甲基三十六烷基胺（dimethyl dioctyldecyl ammonium bromide,DDA）;第二次实验有ESAT6-RpfE、PBS、BCG三组,佐剂为DPG［DDA+poly(I:C)+明胶］。分别于0、3、6周皮下免疫C57BL/6小鼠。末次免疫8周后,用特异性抗原ESAT6及RpfE刺激,检测其分泌IFN-γ的水平;ELISA检测血清特异性抗体IgG2b、IgG1。结果 PCR扩增的esat6、rpfE基因序列与GenBank报道一致;融合蛋白主要以包涵体形式表达;亚单位疫苗免疫动物能够分泌RpfE特异性IFN-γ的脾淋巴细胞数量［(427.13±100.46)］显著高于PBS组(26.13±22.34;q=7.924,P＜0.001); 分泌ESAT6刺激特异性IFN-γ的脾淋巴细胞数量［(506.50±140.40)］明显高于PBS组(19.25±14.75;q=11.36,P＜0.001)和 BCG组(125.5±46.41;q=8.882, P＜0.001)。ESAT6-RpfE免疫组能诱导产生特异性抗体。结论 成功构建并表达ESAT6-RpfE融合蛋白。此融合蛋白可在C57BL/6小鼠中诱导抗原特异性的免疫应答,可作为新型结核亚单位疫苗的候选疫苗予以进一步研究。     

我国卡介苗保护力评价参考体系建立的探讨

目的 探讨我国结核病新疫苗评价用卡介苗参考体系对豚鼠的保护力。 方法 采用我国卡介苗（D₂ PB 302）菌株制备的疫苗,以1/10人用剂量（0.005 mg/0.2 ml）经皮下注射免疫豚鼠,对照组等体积注射生理盐水。共进行4个实验,免疫与感染间隔分别为：实验Ⅰ与Ⅱ免疫后进行Mtb感染的时间间隔为5周（其中实验Ⅱ为实验Ⅰ的重复实验,实验Ⅰ中BCG组和对照组均入组8只豚鼠;实验Ⅱ中BCG组入组6只豚鼠,对照组入组8只豚鼠）,实验Ⅲ为28周（BCG组入组5只豚鼠,对照组入组6只豚鼠）,实验Ⅳ为60周（BCG组和对照组均入组8只豚鼠）,感染剂量为每只豚鼠2×（10²～10³）CFU Mtb/ml,注射0.5 ml,感染途径为皮下注射,感染后6周,进行豚鼠解剖,观察豚鼠肝脾肺的病变程度并进行脾脏Mtb的分离,计算脾脏Mtb分离数和对数值。 结果（1） BCG免疫5周组,豚鼠的肝脾肺综合病变指数为10.0±3.8,较对照组（46.9±4.8）差异有显著统计学意义（t=-0.63, P<0.01）;脾脏Mtb分离数的对数值(lg)为0.49±0.49,较对照组（5.41±0.18）差异有显著统计学意义（t=-9.43, P<0.01）。（2）BCG免疫5周重复实验组,豚鼠的肝脾肺综合病变指数为6.7±2.1,较对照组（36.3±6.0）差异有显著统计学意义（t=-4.63, P<0.01）;脾脏Mtb分离数的对数值(lg)为0.38±0.38,较对照组（5.11±0.19）差异有显著统计学意义（t=-11.22, P<0.01）。（3）BCG免疫28周组,豚鼠的肝脾肺综合病变指数为10.0±4.5,较对照组（57.5±6.2）差异有显著统计学意义（t=-6.24, P<0.01）;脾脏Mtb分离数的对数值(lg)为2.16±0.93,较对照组（5.44±0.27）差异有统计学意义（t=-3.39, P<0.05）。（4）BCG免疫60周组,豚鼠的肝脾肺综合病变指数为14.4±4.8,较对照组（56.9±5.0）差异有显著统计学意义（t=-6.16, P<0.01）;脾脏Mtb分离数的对数值(lg)为1.46±0.69,较对照组（5.43±0.20）差异有显著统计学意义（t=-5.55, P<0.01）。 结论 卡介苗保护力评价参考体系可靠稳定,我国临床应用卡介苗在该体系呈现优良保护效果。     

结核亚单位疫苗AEC／BC—C02诱导小鼠长期的抗原特异性细胞应答

目的动态评价结核亚单位候选疫苗AEC/BC-C02在小鼠中的细胞免疫应答水平。方法6～8周龄SPF级BALB/c雌鼠48只,按数字表法随机分成两组,一组免疫AEC/BC-C02,另一组免疫PBS。末次免疫后第1、2、4、8周分别取两组小鼠（6只/组）分离脾淋巴细胞,ELISPOT检测分泌抗原特异性IFN-γ的T细胞频率;在第2、4、8周ELISA检测抗原特异性IFN-γ的分泌量;在第4、8周,Cell Counting Kit-8（CCK-8）法检测脾淋巴细胞增殖。结果(1)末次免疫后第1、2、4、8周,对疫苗组小鼠脾淋巴细胞,Ag85B特异性的IFN-γ斑点形成细胞（spot forming cells,SFC）分别为168.8±103.5、205.2±51.0、206.8±65.3和160.0±67.9,与PBS对照组的8.9±6.0、16.1±18.8、9.3±4.9和7.7±6.6比较,差异均有统计学意义（成组t检验,t值分别为3.779、8.525、7.424、5.473;P值均<0.01）;EC特异性的IFN-γ SFC分别为45.1±18.6、75.6±39.3、86.2±50.4和54.3±26.3,与PBS对照组的4.5±3.5、11.7±10.5、3.8±5.8和5.2±4.3比较,差异均有统计学意义（成组t检验,t值分别为5.258、3.850、3.977、4.521;P值均<0.01）。(2)末次免疫后第2、4、8周,对疫苗组小鼠脾淋巴细胞,Ag85B多肽刺激的IFN-γ分泌量分别是(1.27±0.13)ng/ml,(1.76±0.55)ng/ml和(1.44±0.44)ng/ml;EC多肽刺激的IFN-γ分泌量分别是(0.81±0.33)ng/ml,(0.81±0.69)ng/ml和(0.54±0.29)ng/ml,两者间差异有统计学意义（配对t检验,分别为：t=3.008,P<0.05;t=2.631,P<0.05;t=10.02,P<0.01）。(3) 末次免疫后第4、8周,Ag85B多肽对疫苗组小鼠脾淋巴细胞的刺激指数（SI）分别为1.756±0.339和1.936±0.287,均分别高于PBS组的1.287±0.0581和1.382±0.114,差异有统计学意义（成组t检验,分别为：t=3.030,P<0.05;t=4.387,P<0.01）;EC多肽对疫苗组SI分别为1.599±0.154和1.581±0.156,均分别高于PBS组的1.380±0.126和1.314±0.170,差异有统计学意义（成组t检验,t值分别为2.540、2.844;P值均<0.05）。结论新型结核亚单位疫苗AEC/BC-C02免疫小鼠可诱导长期稳定存在的抗原特异性记忆T细胞,为后期抗Mtb保护力研究提供药理学基础。     

结核分枝杆菌Rpf-DNA疫苗对Mtb感染小鼠免疫保护作用的研究

目的 评价Mtb复苏因子DNA疫苗（resuscitation-promoting factor DNA,Rpf-DNA）对Mtb感染宿主的免疫保护作用。 方法 构建复苏因子D(Rpf D-DNA)和复苏因子E(Rpf E-DNA)质粒疫苗,180只BALB/c小鼠（4周龄）分为6组,每组30只,分别用空质粒、Rpf D-DNA质粒、Rpf E-DNA质粒、BCG、生理盐水免疫,H37Rv标准株气溶胶感染。检测血清复苏因子（Rpf）抗体、IFN-γ;RpfD、E刺激淋巴细胞后进行淋巴细胞增殖、细胞杀伤实验;检测细胞上清IFN-γ、IL-12、IL-2;对肺组织进行病理学检测和细菌负荷（CFU）计数。 结果 （1）疫苗免疫组血清Rpf D、Rpf E抗体水平：Rpf D组0.32±0.1,Rpf E组0.39±0.1,BCG组0.02±0.01,空质粒组0.01±0.0,生理盐水组0.09±0.04,空白对照组0.03±0.01,RpfD组与RpfE组抗体水平与BCG组、空质粒组、生理盐水组、空白对照组比较差异有显著统计学意义,F值分别为Rpf D：45.6,43.2,45.1,45.7;Rpf E：51.6,53.6,51.0,52.2;P值均<0.01。血清IFN-γ：Rpf D组（43.9±24.8）pg/ml,Rpf E组（45.9±21.0）pg/ml,BCG组（21.0±11.0）pg/ml,空质粒组（7.9±4.9）pg/ml,生理盐水组（4.7±2.1）pg/ml,空白对照组（5.8±4.7）pg/ml,Rpf D、Rpf E质粒组与BCG组比较差异有统计学意义（F值分别为Rpf D：10.2;Rpf E：12.4;P值均<0.05）, 与空质粒组、生理盐水组、空白对照组比较差异有显著统计学意义,F值分别为Rpf D：15.6,17.8,17.3;Rpf E：17.5,21.1,20.7;P值均<0.01。（2）淋巴细胞增殖实验CCK-8：Rpf D组176.0±4.2,Rpf E组183.0±4.3,BCG组101.0±1.1,空质粒组100.0±6.8,生理盐水组104.0±8.4,空白对照组116.0±2.2,RpfD、RpfE质粒组与BCG组、空质粒组、生理盐水组、空白对照组比较差异有统计学意义,F值分别为Rpf D：9.5,10.1,10.0,9.0;Rpf E：11.2,12.9,11.7,10.3;P值均<0.05。细胞杀伤实验：Rpf D组32.0±3.2,Rpf E组30.0±4.2,BCG组16.0±5.9,空质粒组3.3±1.5,生理盐水组6.7±0.5,空白对照组7.3±3.5,Rpf D、Rpf E质粒组与BCG组、空质粒组、生理盐水组、空白对照组比较差异有统计学意义,F值分别为Rpf D：8.8,14.5,13.7,11.9;Rpf E：8.1,12.5,11.6,11.1;P值均<0.05。（3）Rpf D、Rpf E蛋白各自刺激淋巴细胞后上清细胞因子检测：IL-2：Rpf D组9.5±2.4,Rpf E组9.2±1.2,BCG组2.4±2.1,空质粒组1.2±0.3,生理盐水组1.8±1.0,空白对照组1.5±0.7,Rpf D、Rpf E质粒组与其他各组比较差异有统计学意义,F值分别为Rpf D：12.5,14.6,13.5,13.9;Rpf E：12.0,13.6,13.1,13.2;P值均<0.05。IFN-γ：Rpf D组22.2±5.7,Rpf E组28.7±14.4,BCG组16.1±10.1,空质粒组9.8±1.6,生理盐水组13.2±2.1,空白对照组15.7±2.9,RpfD、RpfE质粒组与其他各组比较差异有统计学意义,F值分别为Rpf D：7.8,12.6,8.7,8.3;Rpf E：11.3,16.4,14.7,14.2;P值均<0.05。 结论 实验表明复苏因子疫苗能诱导Mtb感染小鼠产生体液免疫和细胞免疫,有希望成为结核病候选疫苗之一。     

县（区）级结核病防治定点医院模式的实施效果分析

目的 分析江苏省定点医院在结核病防治新体系中的实施效果,为制定结核病防控策略提供科学依据。 方法 选择江苏省采用定点医院模式和CDC模式的县（市、区）各10个,收集这20个地区2012年1月至2012年12月期间发现登记的10 578例肺结核患者的诊断延迟、转诊追踪、系统管理、治疗结局、资料质量等数据,采用SPSS 17.0软件进行分析,探讨两种模式的运行效果。 结果 定点医院模式和CDC模式下的肺结核登记率［分别为55.60/10万（4768/8 575 823）和55.93/10万（5810/10 388 634）,χ²=0.091,P=0.763］、转诊追踪总体到位率［分别为99.1%（4939/4985）和99.2%（6064/6111）,χ²=0.780,P=0.3771］、从诊断至报告的时间［分别为（0.021±0.009）d和（0.025±0.010）d,t=0.419,P>0.05］、系统管理率［分别为99.0%（5048/5097）和98.7%（5645/5719）,χ²=2.65,P=0.103］、涂阴患者完成疗程率［分别为96.6%（3182/3295）和97.4%（3661/3760）,χ²=3.82,P＞0.05］的差异均无统计学意义。定点医院模式下的肺结核患者从发病到诊断的平均时间为（11.117±6.109）d,显著低于CDC模式的（15.399±6.866）d（t=12.42,P<0.001）,而且因症就诊比率定点医院模式与CDC模式［分别为52.0%（2479/4768）和33.8%（1966/5810）,χ²=352.468,P＜0.0001）、转诊率［分别为99.1%（4941/4985）和98.5%（6017/6111）,χ²=9.606,P=0.0019］、追踪到位率［分别为99.6%（1320/1325）和97.9%（1559/1593）,χ²=16.993,P=0.000 04］比较前者高于后者,差异有统计学意义。而定点医院模式与CDC模式比较,新涂阳治愈率［分别为89.8%（1172/1305）和94.7%（1293/1366）,χ²=22.03,P＜0.0001］、复治涂阳治愈率（分别为80.8%（274/339）和88.9%（335/377）,χ²=9.06,P＜0.01）、信息录入及时率［分别为98.9%（23 571/23 840）和99.0%（28 773/29 050）,χ²=4.088,P=0.043］和完整率［分别为98.8%（28 272/28 608）和99.4%（34 646/34 860）,χ²=60.113,P＜0.0001］均低于CDC模式,但均能够满足相关工作要求。 结论 江苏省县（区）级定点医院模式的实施效果较好,定点医院模式是符合我国医疗机构改革大方向的必然趋势,能适应不断发展的结核病防治工作需求,可以因地制宜地稳步推行。     

传统BCG初免IL-12联合Ag85A DNA疫苗加强序贯免疫小鼠效果观察

目的 探讨BCG初次免疫,IL-12联合结核分枝杆菌Ag85A DNA疫苗加强免疫对小鼠的免疫效果。方法 实验小鼠随机分为7组,即PBS阴性对照组、BCG组、pcAg85A组、BCG初免pcAg85A加强免疫组、BCG初免pcAg85A联合IL-12加强免疫组、BCG初免IL-12加强免疫组、以及BCG初免pcDNA3.1加强免疫组。按BCG初免,细胞因子IL-12联合结核分枝杆菌Ag85A DNA加强的免疫程序进行免疫实验,在末次免疫后的4、6、8周通过检测小鼠血清总IgG抗体、特异性淋巴细胞增殖,细胞因子的水平,观测对小鼠的免疫效果。结果 采用BCG初免,细胞因子IL-12联合结核分枝杆菌Ag85A DNA疫苗加强的免疫策略组的小鼠与其它免疫方式组相比,IgG明显升高(P<0.05)、特异性淋巴细胞明显增殖,加强免疫后IFN-γ水平、IL-2水平、IL-4水平BCG/ Ag85A+IL-12组在3个时间段分别为128.2±20.4、190.2±16.51、244.2±39.14;146.2±17.29、271.6±16.36、419.3±28.12;68.6±6.62、96.6±5.5、117.4±10.71均高于其它各组(P<0.05)。结论 采用BCG初免,细胞因子IL-12联合结核分枝杆菌Ag85A DNA疫苗加强的免疫能明显增强机体体液和细胞免疫,为进一步在动物体内进行保护性效应试验的研究提供了依据。     

结核复合抗原疫苗不同抗原组合对小鼠免疫功能的影响

目的 探讨在辅以卡介苗非甲基化的胞嘧啶鸟嘌呤二核苷酸重复序列(CpG)佐剂和铝佐剂的情况下,结核病复合抗原疫苗中抗原85b(Ag85b)、融合蛋白早期滤液蛋白10(CFP-10)和相对分子质量为6000的早期分泌抗原靶蛋白(ESAT-6)及热休克蛋白X(HspX)不同组合对小鼠免疫功能的影响.方法 将48只BALB/c小鼠按抗原组合分为8组:(1)A组:Ag85b+CFP-10/ESAT-6+HspX+佐剂,(2)B组:CFP-10/ESAT-6+HspX+佐剂,(3)C组:Ag85b+HspX+佐剂,(4)D组:Ag85b+CFP-10/ESAT-6+佐剂,(5)E组:Ag85b+佐剂,(6)F组:CFP-10/ESAT-6+佐剂,(7)G组:HspX+佐剂,(8)对照组:生理盐水.每组6只,皮下免疫,共免疫3次,间隔2周,末次免疫后1周处死小鼠.酶联免疫斑点实验和淋巴细胞增殖实验检测小鼠脾淋巴细胞分泌γ-干扰素和白细胞介素-4及抗原特异性淋巴细胞增殖情况.酶联免疫吸附实验检测小鼠血清中IgG、IgG1和IgG2a抗体滴度.结果 E组γ-干扰素的斑点数[中位数(四分位间距),122.8个(78.4～184.4个)]明显高于C组和对照组[14.3个(6.5～14.6个)和0.5个(0.5～1.3个)],差异均有统计学意义(u值均为0.0,均P＜0.01);D组白细胞介素-4的斑点数[173.5个(7 8.8～233.4个)和132.8个(50.3～159.4个)]明显多于对照组[0.5个(0.5～1.3个)和5.3个(2.9～6.5个)],差异有统计学意义(u值均为0.0,均P＜0.01);A、C、D和E组的刺激指数(2.42±0.50、2.18±0.37、2.86±0.51和2.70±0.15)明显高于对照组(1.11±0.13),差异有统计学意义(F值为20.96,均P＜0.01);HspX激发的抗原特异性IgG、IgG1和IgG2a抗体稀释倍数对数值(3.90～5.21)均明显高于Ag85b(3.30～4.51)和CFP-10/ESAT-6蛋白(3.10～4.05),差异均有统计学意义(F值为43.8～70.4,均P＜0.01).结论 在辅以佐剂的情况下,不同抗原组合对小鼠免疫功能的影响有差别,3种抗原同时免疫并未获得最好的免疫效果,提示抗原间的相互作用可能对免疫效果产生影响.

Abstract：

Objective To study the immune function of mice immunized by different combinations of antigen 85b ( Ag85b), fusion protein culture filtered protein 10 ( CFP-10), early secreted antigenic target 6 kDa protein (ESAT-6) and heat shock protein X (Hsp X) with combined adjuvants of Bacille CalmetteGuerin (BCG) CpG and aluminum.Methods According to antigen combinations, 48 BALB/c mice were divided into 8 groups: ( 1 ) group A: Ag85b + CFP-10/ESAT-6 + HspX + adjuvant; (2) group B: CFP-10/ESAT-6 + HspX + adjuvant; ( 3 ) group C: Ag85b + HspX + adjuvant; (4) group D: Ag85b + CFP-10/ESAT-6 + adjuvant; (5) group E: Ag85b + adjuvant; (6) group F: CFP-10/ESAT-6 +adjuvant; (7)group G: HspX + adjuvants; (8) control group: saline (6 mice per group).The mice were subcutaneously immunized 3 times.One week after the third subcutaneous immunization, spleens were collected for enzymelinked immunospot (ELISPOT) assay to detect IFN-γ and IL-4 secretion, and for the lymphocyte proliferation assay to observe antigen-specific lymphocyte proliferation.Serum samples were separated for enzyme-linked immunosorbent assay (ELISA) to detect the titers of antigen-specific IgG, IgG1 and IgG2a antibodies.Results The amount of IFN-γ spots in Group E [median ( quartile), 122.8 (78.4 - 184.4 )]was significantly more than that in group C [14.3 (6.5 - 14.6)] and the control group [0.5 (0.5 - 1.3)](u =0.0, P ＜ 0.01 ).The amount of IL-4 spots in Group D stimulated with Ag85b and CFP-10/ESAT-6 [173.5 (78.8 -233.4), 132.8 ( 50.3 - 159.4)] were significantly more than those in the control group [0.5 (0.5 - 1.3 ), 5.3 ( 2.9 - 6.5 )] ( u = 0.0, P ＜ 0.01 ).The level of stimulation index of lymphocyte proliferation in Group A, C, D, E(2.42 ±0.50, 2.18 ±0.37, 2.86 ±0.51, 2.70 ±0.15) was significantly higher than that of the control group ( 1.11 ± 0.13 ) ( F= 20.96, P ＜ 0.01 ).The level of antigen-specific IgG, IgG1 , IgG2a antibody titers induced by Hsp X [lg( antibody dilution degree), 3.90 -5.21] was significantly higher than those induced by Ag85b (3.30-4.51 ) and CFP-10/ESAT-6 (3.10 -4.05) ( F = 63.8 - 70.4, P ＜ 0.01 ) .Conclusions With the use of adjuvants, different antigen combinations showed different influences on the immune function in mice.A combination of 3 antigens did not elicit the best immune effect, suggesting that the interaction among antigens may affect their immunity.     

二甲基三十六烷基铵.卡介苗多糖核酸在结核亚单位疫苗中的佐剂效应

目的 探讨二甲基三十六烷基铵(DDA)、卡介苗多糖核酸(BCG-PSN)作为结核病融合蛋白疫苗佐剂的免疫效应.方法 采用热酚法制备BCG-PSN,与DDA联合作为融合蛋白AMM(Ag85B-MPT64190-198-Mth8/4)佐剂免疫小鼠,设卡介苗、生理盐水、弗氏不完全佐剂(IFA)及单独应用DDA或BCG-PSN一种佐剂的各组作为对照.应用ELISA和酶联免疫斑点法检测免疫小鼠的体液与细胞免疫反应.结果 分离培养疫苗免疫小鼠的脾脏淋巴细胞,用特异性抗原AgSSB刺激后,AMM+DDA+BCG-PSN疫苗组、AMM+DDA组和卡介苗组小鼠每1×106个脾淋巴细胞分泌γ-干扰素的细胞数分别为222±79、259±85和230±64,均明显高于AMM+PBS组(40±4)、AMM+IFA组(10 ±3)、AMM+BCG-PSN组(132±18)和生理盐水组(8±4),差异有统计学意义(t值为2.923～7.118,均P<0.05);与单用DDA组比较,添加DDA和BCG-PSN两种佐剂的疫苗组小鼠血清抗体滴度Ig2a/IgG1较高(0.125和0.025),但γ-干扰素分泌水平没有增高.结论 AMM+DDA+BCG-PSN结核病亚单位疫苗能够引起较强的Th1细胞免疫为主的免疫反应,DDA+BCG-PSN(尤其是DDA)具有增强结核病亚单位疫苗Th1细胞免疫应答的佐剂效应.     

Immunogenicity of a DNA Vaccine Encoding Ag85a-Tb10.4 Antigens from Mycobacterium Tuberculosis

Background: Tuberculosis is a life threatening disease that is partially prevented by BCG vaccine. Development of more effective vaccines is an urgent priority in TB control. Ag85a and Tb10.4 are the members of culture filter protein (CFP) of M. tuberculosis that have high immunogenicity. Objective: To analyze the immunogenicity of Ag85a-Tb10.4 DNA vaccine by enzyme-linked immunosorbent assay (ELISA). Methods: In this study a previously described plasmid DNA vaccine encoding Ag85a-Tb10.4 was used to examine its capability in the stimulation of immune responses in an animal model. Female BALB/c mice were vaccinated with 100 μg of purified recombinant vector intramuscularly 3 times at two-week intervals and the levels of five cytokines including IFN-γ, IL-12, IL-4, IL-10 and TGF-β were measured. Results: The levels of IFN-γ and IL-12 for the mice following immunization with Ag85A-Tb10.4 was significantly greater than that of the BCG and control group (p<0.05). However there was no significant difference in the levels of IL-4, IL-10 and TGF-β between groups. Conclusion: IFN-γ and IL-12 Th1 cytokines increased significantly in mice vaccinated with Ag85a-TB10.4 DNA vaccine in comparison to the control and BCG groups. Our results may serve as groundwork for further research into the prevention and treatment of tuberculosis.     

Efficacy of recombinant bacille Calmette-Guérin vaccine secreting interleukin-15/antigen 85B fusion protein in providing protection against Mycobacterium tuberculosis

Protection against Mycobacterium tuberculosis not only depends on CD4+ T helper type 1 (Th1) cells but, also, on CD8+ T cells. Interleukin (IL)-15 has an important function in the maintenance of memory CD8+ T cells. In the present study, we examined the efficacy of recombinant Mycobacterium bovis bacille Calmette-Guérin (rBCG) secreting fusion protein antigen (Ag) 85B murine IL-15 (rBCG-Ag85B-IL15) in providing protection against M. tuberculosis infection. The levels of major histocompatibility (MHC) class Ib (H2-M3)-binding TB2- or MHC class Ia (H-2Db)-binding MPT64-specific CD8+ T cells producing interferon (IFN)-gamma were significantly higher after immunization with rBCG-Ag85B-IL15 than after immunization with rBCG secreting Ag85B (rBCG-Ag85B). The levels of purified protein derivative- or Ag85B-specific CD4+ T cells producing IFN-gamma were also higher in mice immunized with rBCG-Ag85B-IL15 than in mice immunized with rBCG-Ag85B. Mice immunized with rBCG-Ag85B-IL15 exhibited CD8+ and CD4+ T cells responses that were stronger than those in mice immunized with rBCG-Ag85B, as well as robust protection in the lung against intratracheal challenge of M. tuberculosis. Thus, rBCG-Ag85B-IL15 vaccination capable of inducing efficient cell-mediated immunity might be used as an effective vaccine for tuberculosis.     

Novel vaccines against M. tuberculosis

CDC and ACET in U.S.A. reported that novel vaccines instead of BCG are required for the protection against infection of Mycobacterium tuberculosis worldwide. However, no novel vaccine for clinical use has not yet been developed in the world including U.S.A. and Europe. We have developed two novel tuberculosis (TB) vaccines; a DNA vaccine combination expressing mycobacterial heat shock protein 65 (HSP 65) and interleukin-12 (IL-12) by using the hemagglutinating virus of Japan (HVJ)-liposome (HSP 65 + IL-12/HVJ). A mouse IL-12 expression vector (mIL-12 DNA) encoding single-chain IL-12 proteins comorised of p40 and p35 subunits were constructed. In a mouse model, a single gene gun vaccination with the combination of HSP 65 DNA and mIL-12 DNA provided a remarkably high degree of protection against challenge with virulent Mycobacterium tuberculosis; bacterial numbers were 100 fold lower in the lungs compared to BCG-vaccinated mice. To explore the clinical use of the DNA vaccines, we evaluated HVJ-liposome encapsulated HAP 65 DNA and mIL-12 DNA (HSP 65 + mIL-12/ HVJ). The HVJ-liposome method improved the protective efficacy of the HSP 65 DNA vaccine compared to gene gun vaccination. This vaccine provide remarkable protective efficacy in mouse and guinea pig models, as compared to the current by available BCG vaccine. HSP 65 + IL-12/HVJ vaccine induced CD8+cytoxic T lymphocyte activity against HSP 65 antigen. Protective efficacy of this vaccine was associated with the emergence of IFN-gamma-secreting T cells and activation of proliferative T cells as well as CTL induction upon stimulation with the HSP 65 and antigens from M. tuberculosis. Furthermore, we extended our studies to a cynomolgus monkey model, which is currently the best animal model of human tuberculosis, to evaluate the HSP 65 + IL-12/HVJ vaccine. Vaccination with HSP 65 + IL-12/HVJ provided better protective efficacy as assessed by the Erythrocyte Sedimentation Rate, chest X-ray findings, and immune responses than BCG. Most importantly, HSP 65 + IL-12/HVJ resulted in an increased survival for over a year. This is the first report of successful DNA vaccination against M. tuberculosis in the monkey model. Novel TB vaccines using the monkey model will be discussed in this issue. The development of novel vaccines against tuberculosis was also studied in murine and cynomolgus monkey systems. Four distinct methods; DNA vaccination (1. plasmid, 2. adenovirus vector, 3. adenoassouated virus), 4. recombinant BCG, and 5. subunit (recombinant protein) were used for the development of novel vaccines. Genes (HSP 65 gene, IL-12 gene as well as Ag 85A-, 85B-, MPB51-gene) and IL-6 related genes (IL-6 gene + IL-6R gene +gp130 gene) were administered into the Balb/c mice infected (i.v. or intra-tracheal injection) with Mycobacterium tuberculosis (M. tuberculosis). Elimination of M. tuberculosis in lungs, liver, and spleen of these mice and survival were studied in these models. HSP 65 gene + IL-12 gene vaccination, or recombinant BCG (BA51 : Antigen 85B(-) + Antigen 85A(-) + MPB51-gene recombinant BCG) were more prophylactically efficient than parental BCG Tokyo vaccination. In contrast, IL-6 related genes vaccination using adenovirus vector showed therapeutic effect on M. tuberculosis infected mice. Cytotoxic T cells (CTL) activity against M. tuberculosis in the spleen cells from mice treated with IL-6 related genes vaccination were significantly augmented. Furthermore, NOD-SCID-PBL/hu mice treated with anti-IL-2 receptor beta-chain antibody provide an useful tool for analyzing in vivo human T cell immunity against tuberculosis. In conclusion, we demonstrate the development of a novel HVJ-liposome DNA vaccine encapsulating HSP 65 DNA plus IL-12 DNA. These results suggest that HSP 65 + IL-12/HVJ could be a promising candidate for a new tuberculosis DNA vaccine, which is superior to the currently available BCG vaccine. The goal of our study is to develop a new tuberculosis vaccine superior to BCG. To this aim, we believe that the protective efficacy and protective immune responses for vaccine candidates should be addressed in larger animals, such as nonhuman primates, before proceeding to human clinical trials. Although other DNA vaccine candidates that appear to protect against virulent M. tuberculosis in mice better than BCG have failed to provide better protection than BCG in guinea pigs against aerosol challenge of a low dose of virulent M. tuberculosis, some of them are being prepared to enter early human clinical trials. More recently, we evaluated the HSP 65 + hIL-12/HVJ vaccine in the cynomolgus monkey model, which is currently the best non-human primate animal model of human tuberculosis. Monkeys were subsequently challenged with virulent M. tuberculosis by the intra-tracheal route after the third vaccination. This challenge dose normally causes death from acute respiratory infection within 4-6 months. In this particular experiment, monkeys vaccinated with HSP 65 + hIL-12/HVJ induced HSP 65-specific T-cell proliferation and improvement of chest X-P findings, resulting in an increased survival for over a year, superior to BCG group. Thus, we are taking advantage of the availability of multiple animal models (mouse, guinea pig, and monkey) to accumulate essential data of the HVJ-liposome DNA vaccine, including the vaccine efficacy and safety, for up-coming Phase I clinical trials.     

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.     

表达Ag85B与ESAT6融合蛋白的基因疫苗的免疫学特性

目的测定表达Ag85B与ESAT6融合蛋白的两种重组质粒AZ-pcDNA3-EF和EZ-pcDNA3-AF在小鼠体内诱导的免疫应答及保护力。方法50只BALB/c小鼠随机分为5组(每组10只),将表达Ag85B与ESAT6融合蛋白的两种重组质粒AZ-pcDNA3-EF和EZ-pcDNA3-AF分别免疫小鼠3次,每次间隔2周,同时设卡介苗(BCG)免疫组、空载体质粒免疫组和生理盐水对照组。最后一次免疫结束后,每组取5只小鼠血清,酶联免疫吸附测定(ELISA)法检测特异性抗体的滴度,并分离小鼠的脾淋巴细胞,并在体外用结核分枝杆菌(MTB)培养滤液蛋白(cu lture filtrate prote in,CFP)刺激,测定脾淋巴细胞增殖指数和γ干扰素(IFN-γ)水平。用1 m l含1×105克隆形成单位(CFU)的MTB毒株H37Rv经尾静脉感染其余每组5只BALB/c小鼠,4周后计数脾脏细菌负荷数。结果质粒AZ-pcDNA3-EF和EZ-pcDNA3-AF免疫小鼠血清的特异性抗体滴度分别为1∶1 000和1∶1 500。其脾淋巴细胞刺激指数分别为2.2和2.4,而生理盐水对照组和空载体质粒免疫组的刺激指数只有0.9和1.1;脾淋巴细胞悬液中诱发的IFN-γ分别为(5.48±0.38)ng/m l和(5.76±0.51)ng/m l,显著高于生理盐水对照组和空载体质粒免疫组(P<0.05),但与BCG免疫组的(5.55±0.31)ng/m l比较差异无统计学意义。结核毒株攻击后,与空载体质粒免疫组相比,质粒AZ-pcDNA3-EF和EZ-pcDNA3-AF免疫的BALB/c小鼠其抗MTB在脾脏中增殖有显著作用,3组脾脏细菌负荷对数值(lg,CFU/g)分别为6.08±0.25、4.63±0.11、4.50±0.32,但不及BCG免疫组的4.09±0.27。结论表达Ag85B与ESAT6融合蛋白的基因疫苗在小鼠体内诱导的IFN-γ水平与BCG相当,其保护力有待进一步提高。