接种卡介苗对热休克蛋白65-MUC1抗原肽融合蛋白抑瘤作用的影响

目的：探讨接种卡介苗对热休克蛋白65-MUC1抗原肽融合蛋白（HSP65-MUC1）所激发的抑瘤作用的影响。方法：给经卡介苗免疫后产生HSP65抗体的小鼠注射HSP65-MUC1融合蛋白3次，然后给小鼠接种MUC1阳性的B16肿瘤细胞，观察HSP65-MUC1融合蛋白对MUC1阳性的B16肿瘤细胞在已产生HSP65抗体的小鼠体内生长的抑制作用。结果：HSP65-MUC1能够显著抑制MUC1阳性的B16肿瘤细胞在已产生HSP65抗体小鼠体内的生长，HSP65-MUC1组小鼠的肿瘤重量和体积显著地低于PBS组。结论：接种卡介苗并不影响HSP65-MUC1所激发的对MUC1阳性B16肿瘤细胞生长的抑制作用。     

HSP65-MUC1 VNTR2融合蛋白的表达、纯化及其抗肿瘤作用

目的：通过基因工程手段在大肠杆菌中表达HSP65-MUC1 VNTR：融合蛋白、鉴定和纯化，并研究其在动物体内的肿瘤生长抑制活性。方法：通过PCR获得结核分支杆菌HSP65基因，以重叠PCR获得人MUC1基因VNTR的2个重复序列，构建原核重组表达载体HSP65-MUC1 VNTR2-pET28a（＋）。在大肠杆菌BL21（DE3）中利用IPTG诱导表达；利用单克隆抗体（mAb）进行Western blot鉴定，以Q柱和凝胶过滤纯化获得纯化蛋白。通过建立转染人MUC1基因的B16黑色素瘤小鼠肿瘤模型，在C57BL／6小鼠体内研究HSP65-MUC1 VNTR，融合蛋白的肿瘤生长抑制活性。结果：获得的结核分支杆菌HSP65基因和人MUC1基因VNTR区的2个重复片段，经测序证明分别与GenBank中结核杆菌HSP65基因和人MUC1基因的VNTR完全相符；构建的原核表达载体HSP65-MUC1 VNTR2-pET28a（＋）可稳定的可溶性表达HSP65-MUC1VNTR2蛋白；经Q柱和凝胶过滤纯化，纯化的目的蛋白纯度达95％以上；利用鼠抗人MUC1mAb对表达蛋白进行验证，结果阳性；小鼠肿瘤预防免疫实验表明，实验组小鼠抑瘤率大于对照组，且具有明显性差异。结论：成功地利用原核表达系统实现了对HSP65-MUC1 VNTR2蛋白的可溶性表达，并对其体内预防实验进行了初步研究，证明该融合蛋白能明显抑制表达MUC1的肿瘤生长。为进一步评价其作为肿瘤疫苗可能性的研究打下了基础。     

CpG ODN增强乙型肝炎表面抗原免疫小鼠的抗体产生

目的：探讨合成含CpG基序的寡核苷酸（CpG ODN)对重组乙型肝炎表面抗原（rHBsAg)及乙型肝炎疫苗增强小鼠特异性抗体产生的效应。方法：采用非纯系（Km)及纯系（Balb/c)小鼠作为免疫对象，经后腱胫骨前肌免疫2次，ELISA法检测血清乙型肝炎表面抗体（抗－HBs)效价。结果：加CpG ODN组，其抗-HBs效价均较单独注射rHBsAg和疫苗组明显增高，持续时间长，且纯系鼠的抗体效价明显高于非纯系鼠。结论：CpG ODN对小鼠抗－HBs产生具有增强作用，具与疫苗中的铝佐剂有协同效应。     

MUC1基因疫苗对膀胱肿瘤抑制的免疫学实验研究

目的：研究人粘蛋白MUC1基因DNA疫苗诱导小鼠细胞毒性T细胞（CTL）及体液免疫应答的作用，为膀胱癌的疫苗治疗提供实验资料。方法：接种pcDNA3．1（＋）-MUC1质粒，通过ELISA法检测小鼠血清MUC1抗体生成和脾细胞产生IL-2和1FN-7的量，通过LDH释放法测定CTL对BIU-87细胞的杀伤活性。结果：接种pcDNA3．1（＋）-MUC1疫苗后，小鼠血清中抗MUC1抗体水平明显升高，与对照组相比有显著性差异（P〈0．01）。脾细胞产生的IL-2和IFN-7水平较对照组高（P〈0．01）。在效靶比为100：1、50：1、25：1、12．5：1时，MUC1基因疫苗免疫组小鼠CTL对BIU-87杀伤率（分别为58．4％、47．2％、35．7％、27．4％），较空载体pcDNA3．1（＋）组小鼠和灭菌生理盐水组小鼠（分别为11％、19．2％、9．5％、14％和16．5％、11．9％、8．6％、10．3％）均明显升高，且有显著性差异（P〈0．01）。结论：MUC1基因DNA疫苗能够诱导小鼠产生抗MUC1特异性抗体，诱导产生杀伤表达MUC1细胞的CTL，为MUC1基因疫苗用于膀胱肿瘤生物治疗提供了一定的实验依据。     

CpG ODN对rHBsAg免疫小鼠淋巴细胞增殖反应的影响

目的：进一步探讨以合成含CpG基序的寡核苷酸（CpG ODN）作为免疫增强剂与重组乙型肝炎表面抗原（rHBsAg）联合免疫小鼠,观察其淋巴细胞增殖反应效应.方法：经后腿胫骨前肌初次免疫BALB/c小鼠两周后加强免疫1次;MTT和3H-TdR掺入法分别检测免疫小鼠胸腺及脾脏淋巴细胞增殖反应.结果：加CpG ODN组与单纯注射抗原及疫苗组相比,淋巴细胞增殖反应显著增强.而且,除胸腺特异性增殖中的CpG加疫苗组和疫苗组外,其他各实验组与对照组相比,淋巴细胞增殖反应也都显著增强.结论：CpG ODN能够明显刺激小鼠胸腺及脾脏淋巴细胞增殖反应,显示出强而有效的免疫增强效应,有望成为一种新型的免疫佐剂.     

CpG DNA中ISS数量与免疫刺激效果间的关系初探

研究CpG免疫刺激DNA中免疫刺激序列（ISS）的数量与免疫刺激效果间的关系。方法：将3种CpGODN（分别含2,4,8个ISS）与甲型肝炎灭活疫苗混合,通过腹腔注射途径免疫小鼠,于免疫后4,6,8w采血,用全自动微粒子酶联免疫测定分析检测抗HAVIgG水平.结果：含2个ISS的CpG ODN具有较好的佐剂效应,能增强甲型肝炎灭活疫苗的免疫效果,含4个ISS的CpG ODN不具有免疫刺激作用,而含8个ISS的CpG ODN则抑制了甲型肝炎灭活疫苗所诱导的免疫反应,结论：适当长度的CpG ODN（含2个ISS）可作为有效的佐剂来增强疫苗的免疫效果,而当其中的ISS含量过多,则反而具有免疫抑制作用。     

人乳腺癌疫苗HSP65-HER2联用CpG684的抗肿瘤作用

目的:检测人乳腺癌融合蛋白疫苗HSP65-HER2联用CpG684的抗肿瘤效果。方法:C57BL/6小鼠分别皮下注射PBS,CpG684,HSP65-HER2和CpG684混合物,一周一次,共三次,随后给小鼠腹腔接种7.5×104个转染了pcDNA3-GFP-HER2质粒的B16肿瘤细胞(HER2+B16),监测各组小鼠的生存期至接种肿瘤后70天。结果:免疫了HSP65-HER2和CpG684的小鼠在接种肿瘤后70天时,仍有80%存活,而GpG684组的小鼠仅有10%存活,PBS组小鼠在接种肿瘤后36天后全部死亡。与PBS和CpG684对照组相比,免疫了HSP65-HER2和CpG684的小鼠的生存期显著延长(P<0.01)。结论:HSP65-HER2联用CpG684在体内发挥了强大的抗肿瘤活性,为进一步的临床研究和应用奠定了基础。     

一种B型CpG ODN对乙肝疫苗的免疫佐剂作用

目的 研究一种本室设计的B型CpG ODN(BW006)作为乙肝疫苗佐剂的可行性.方法 以琼脂糖凝胶电泳法对BW006的质量进行初步检定;H3-掺人法检测BW006对小鼠脾细胞的刺激作用;乙肝疫苗与BW006联合免疫Balb/c小鼠,并用ELISA法检测小鼠血清中的HBsAb水平.结果 BW006的纯度和完整性符合实验要求,并能显著的刺激小鼠脾细胞的增生,同乙肝疫莳联用后能够明显增强乙肝疫苗的免疫效果.结论 BW006对乙肝疫苗有较强的免疫佐剂作用.     

微波消融联合消融灶周边注射CpG ODN诱导抗小鼠肝癌免疫效应

目的:探讨消融灶周围注射CpG ODN诱导抗肿瘤免疫反应的效能。方法:建立C57BL/6J小鼠Hepa 1-6细胞双侧皮下肝癌模型,根据右侧皮下肝癌治疗的方法分为联合治疗组(微波消融联合消融灶周边注射CpG ODN治疗)、消融治疗组、CpG ODN治疗组和对照组。观察双侧皮下肝癌体积变化、小鼠生存时间。免疫组化检测右侧肿瘤组织热休克蛋白70(HSP70)表达和左侧肿瘤组织CD4、CD8T淋巴细胞的数量。ELISA法检测各组小鼠血清IL-12、IL-10含量。LDH释放法检测各组脾源性CTL杀伤活性。另选取单侧皮下肝癌小鼠,分别给予联合治疗和消融治疗,30天后在对侧皮下再次接种同种肝癌细胞,观察再接种肿瘤的成瘤率和肿瘤体积变化。结果:双侧皮下肝癌模型小鼠联合治疗组和消融治疗组右侧肿瘤体积体积显著小于CpG ODN治疗组和对照组(P<0.05)。联合治疗组中左侧未治疗的肿瘤体积在第10天后明显小于其他各组(P<0.05)。中位生存时间分别为:联合治疗组73天,消融治疗组60天,CpG ODN治疗组55天,对照组38天。联合治疗组左侧肿瘤组织内CD8T淋巴细胞数量为(25.17±5.46)/HP,显著高于其他3组(P<0.05)。联合治疗组和消融治疗组右侧肿瘤组织HSP70标记指数分别为(39.95±9.03)%和(33.90±7.98)%,明显高于CpG ODN治疗组和对照组(P<0.05)。联合治疗组小鼠血清IL-12含量显著高于其他各组(P<0.05),IL-10含量显著低于其他各组(P<0.05)。联合治疗组中脾CTL细胞对Hepa 1-6杀伤率显著高于其他各组(P<0.05)。单侧皮下肝癌小鼠联合治疗组再接种肿瘤细胞的成瘤率为58.33%,而消融治疗组的成瘤率为83.33%,且联合治疗组的再接种肿瘤体积在第20天后明显小于消融治疗组(P<0.05)。结论:微波消融能促使肿瘤细胞HSP70表达,微波消融联合消融灶周边注射CpG ODN能激活小鼠体内的CD8 T淋巴细胞,促使小鼠体内发生Th2/Th1免疫偏移,从而产生抗肿瘤的免疫效应,抑制肿瘤生长。     

CpG ODN增强乙肝疫苗在老年小鼠中的免疫应答

目的：探讨CpG ODN对老年小鼠的体液和细胞免疫应答的增强作用。方法：选用老年C57BL／6小鼠，将乙肝疫苗和10μg、20μg CpG ODN同时或单独肌注到小鼠体内，两周后以同样剂量加强免疫一次，再过3周后摘除眼球取血，用EILSA方法检测抗HBs16；抗体和IL-12；无菌取脾脏作HE染色，观察脾脏淋巴细胞变化。结果：10μg和20μg CpG ODN与疫苗同时注射组产生的抗体绝对量分别是单独注射疫苗组的3倍和4倍；产生的IL-12水平较对照组有明显升高，且20μg比10μCpG组产生的IL-12水平更高。光镜下各组的脾脏淋巴细胞的变化如下：正常老年鼠组脾脏淋巴细胞较正常青年鼠组明显稀少；老年鼠 10μgCpG组脾脏淋巴细胞较正常老年鼠组有了明显增加，且细胞核也明显增大；20μgCpG组增加的更加明显。结论：CpG ODN能增强乙肝疫苗在老年小鼠中的体液和细胞免疫应答。     

CpG Oligodeoxynucleotide Inhibits Cockroach-Induced Asthma via Induction of IFN-γ+ Th1 Cells or Foxp3+ Regulatory T Cells in the Lung

PurposeCpG oligodeoxynucleotide (CpG-ODN), a TLR9 agonist, activates innate immunity and induces Th1 response. Although the immune modulatory effect of CpG-ODN has been extensively studied, its function in cockroach extract-induced allergic asthma has not been studied. Here, we investigated the inhibitory function of CpG-ODN in cockroach extract-induced asthma in mice with different treatment schemes.MethodsScheme 1: BALB/C mice were intra-nasally co-administered by cockroach extract and CpG-ODN twice a week for 3 weeks; Scheme 2: The mice were intra-nasally pre-treated with CpG-ODN at day 0 and cockroach allergen challenge was performed from day 3 as in scheme 1. Scheme 3: Cockroach allergen challenge was performed as in scheme 1 and CpG-ODN was post-treated at day 21. Then, BAL cell count, flow cytometric analysis of alveolar macrophages, regulatory T cells, and lung tissue histology, Th1 and Th2 cytokines, serum IgE, cockroach specific IgE, IgG1/IgG2a ratio, and airway hyper-responsiveness were evaluated.ResultsMice with repeated intra-nasal exposure to CpG-ODN showed a dramatic decrease in eosinophilic inflammation, goblet cell hyperplasia, and airway hyper-responsiveness with reduction of IL-13, IL-5, and serum IgE, cockroach specific IgE and IgG1/IgG2a ratio. This inhibitory function might be related to the up-regulation of IL-10 and CD4⁺Foxp3⁺ regulatory T cells in the lung. Interestingly, one-time challenge of CpG-ODN either prior or posterior to cockroach extract exposure could modulate airway inflammation and hyper-responsiveness via increase of Th1 response.ConclusionsCollectively, our data suggest that CpG-ODN treatment modulates Th2 inflammation in the lung by induction of regulatory T cells or Th1 response in a cockroach-induced asthma model.     

三种类型CpG-ODN作为蛋白亚单位疫苗佐剂的比较研究

目的 比较A、B和C3种类型CpG-ODN在动物模型中的疫苗佐剂活性,为设计高活性的新型人用疫苗佐剂提供理论基础.方法 用不同CpG-ODN刺激体外培养的小鼠脾细胞,检测上清中的IFN-γ和IgM,从而确定3种类型的CpG-ODN.以基因工程乙肝表面抗原(HBsAg)为模型抗原、不同类型CpG-ODN为佐剂免疫BALB/c小鼠,用间接ELISA法检测被免动物血清中抗原特异性ISG、IgG1和IgG2a抗体.结果 小鼠免疫结果表明,与Al(OH),对照组相比,3种类型CpG-ODN均具有良好的疫苗佐剂活性,但B和C型CpG-ODN诱生的抗原特异性IgG和IgG2a抗体水平远高于A型CpG-ODN.虽然A型CpG-ODN也能够显著增强总抗体水平,但并不改变IgG1和IgG2a抗体亚型的比值.这与B和C型CpG-ODN能够极大地促进TH1类免疫应答并降低IgG1/IgG2a比值明显不同,提示不同类型CpG-ODN可能通过不同机制起到疫苗佐剂作用.结论 不同类型CpG-ODN具有不同疫苗佐剂活性,B和c型CpG.ODN在小鼠体内的体液免疫佐剂效果优于A型CpG.ODN.     

卡介苗作为抗肿瘤重组蛋白疫苗佐剂的研究

目的：探讨卡介苗（BCG）能否增强抗肿瘤疫苗HSP-MUC1的特异性抑瘤作用，从而将BCG开发为肿瘤疫苗的新型佐剂。方法：动物水平上，BCG和HSP-MUC1共免疫小鼠，观察BCG能否增强HSP-MUC1所激发的特异性抑瘤作用。细胞水平上对BCG发挥佐剂作用的机制进行探讨，将BCG和HSP-MUC1共刺激树突状细胞（DC），观察BCG能否协同HSP-MUC1刺激DC表面的CD86分子表达的上调；并对DC培养上清中IL-6、TNF-α的水平进行测定。结果：BCG＋HSP-MUC1组小鼠的肿瘤重量显著地低于HSP-MUC1组（P〈0.05）。细胞学试验显示，BCG能够显著增强HSP-MUC1对DC的激活作用，使DC表面的CD86分子显著上调。BCG＋HSP-MUC1组的DC培养上清中细胞因子IL-6、TNF-α的水平显著地高于HSP-MUC1组（P〈0.05）。结论：BCG能够显著地增强HSP-MUC1的抗肿瘤活性，具有肿瘤疫苗佐剂的良好功效。     

Induction of persistent autoimmune haemolytic anaemia in mice by combined use of rat erythrocyte preimmunization and chronic GVHR

Female F1 mice (BDF1: C57BL/6 x DBA/2, BCF1: C57BL/6 x C3H/HeN, BBF1: C57BL/6 x BALB/c) were preimmunized with rat erythrocytes and then received splenic T cells from parental female C57BL/6 mice injected with hydrocortisone acetate before killing. In BDF1 and BCF1 mice, direct Coombs' test (DCT) positivity persisted for more than 4 months in almost all mice. They also showed haematological sign of anaemia, and the survival of infused 51Cr-labelled parental C57BL/6 erythrocytes was reduced. In BBF1 mice, DCT positivity returned to negative within 2 months. Autoantibodies were eluted from DCT-positive erythrocytes and their specificities were examined. They were capable of binding equally well to erythrocytes from all strains of mice tested, including the donor mice. They also cross-reacted with rat erythrocytes but not with sheep, rabbit or human erythrocytes. Isotypes of the autoantibodies eluted were also examined. Interestingly, isotypes of the autoantibodies obtained from individual mice were confined to one or other subclass of IgG isotype: IgG1, IgG2a or IgG2b.     

Monoclonal antibodies to mammalian heat shock proteins impair mouse embryo development in vitro

Two-cell mouse embryos (B6D2F1) were cultured in the presence or absence of 100 microg/ml monoclonal antibodies specific for the mammalian 60 kDa (HSP60), 70 kDa (HSP70) and 90 kDa (HSP90) heat shock proteins. Embryo development was evaluated after 3, 5 and 7 days in culture by determining the number of blastocysts, hatched blastocysts and outgrown trophoblasts at the successive time points. At day 3, only 29% (22/75) of the embryos cultured with anti-HSP60 antibody developed to the blastocyst stage (P < 0.0001) as compared to 67% (31/46) of the embryos cultured with anti-HSP70, 72% (43/60) cultured with anti-HSP90, and 79% (49/62) in medium plus mouse IgG1. By day 5, hatched embryos were present in 28% (13/ 46) of the cultures containing anti-HSP70 (P < 0.0001), as opposed to 57% (34/60) containing anti-HSP90 and 73% (45/62) containing IgG1. At day 7, outgrown trophoblasts were observed in 9% (4/46) of cultures containing anti-HSP70 (P < 0.0001), 45% (27/60) containing anti-HSP90 (P < 0.01) and 66% (41/62) cultured in medium plus IgG1. Antibodies to different heat shock proteins exerted a detrimental effect on mouse embryo development at unique development stages. Immune sensitization to heat shock proteins may be a cause of reproductive failure.      

Vaccinations with T-helper type 1 directing adjuvants have different suppressive effects on the development of allergen-induced T-helper type 2 responses

BACKGROUND: Allergen-induced T-helper type 2 (Th2) responses can be inhibited with Th1 directing vaccines. However, studies comparing the efficacy of the different adjuvants have not been performed in detail. OBJECTIVE: For this reason we compare the effects of live Bacillus-Calmette-Guerin(BCG), heat-killed (hk)-BCG, CpG-ODN (oligodeoxynucleotide) or PPD on the development of allergen-induced Th2 responses in mice. METHODS: Ovalbumin (OVA)-specific allergic responses were induced in C57BL/6 mice by two intraperitoneally (i.p.) applications of OVA/alum followed by the intranasal challenge with OVA. The different Th1-inducing adjuvants were applied to the mice together with OVA/alum i.p. during the OVA-sensitization period and, subsequently, different parameters of allergic immune responses were evaluated. RESULTS: All the adjuvants were effective in inhibiting the development of allergen-induced airway eosinophilia, mucous production and, with the exception of PPD, also airway hyper-reactivity, when they were applied together with OVA/alum. However, allergen-specific IgG1 and IgE serum levels were only reduced in live BCG- and PPD-treated mice. Suppression of airway eosinophilia was not observed in IFN-gamma- or IL-12-deficient mice (hk-BCG, CpG-ODN and PPD). Interestingly, live BCG was still able to suppress allergen-induced Th2 responses in the absence of either IFN-gamma or IL-12. When mice vaccinated with the different adjuvants together with OVA/alum were subjected to a second period of OVA/alum immunization, only live and hk-BCG were able to efficiently suppress the development of airway inflammation. This effect could be adoptively transferred by splenic CD4(+) T cells. CONCLUSIONS: Taken together our data suggest that live BCG>hk-BCG>CpG-ODN >PPD are effective in suppressing allergen-induced Th2 responses. The degree of suppression and the component of the Th2 response affected (airway inflammation vs. the production of allergen-specific IgE and IgG1) were dependent upon the adjuvant used and how it was applied. Our results contribute to the design of novel vaccines protecting humans from developing allergic disorders.     

糖皮质激素对BXSB狼疮小鼠肾组织fractalkine/CX3CR1表达的影响

目的：研究BXSB狼疮小鼠肾组织趋化因子fractalkine及其受体CX3CR1的表达以及给予泼尼松治疗后的改变,探讨两者在狼疮肾炎发病机制中的可能作用。方法：12 周龄雄性BXSB狼疮小鼠随机分成泼尼松治疗组（n=6）和实验对照组（n=6）;另取同周龄雄性C57BL/6J小鼠6只作为正常对照组。正常对照组和实验对照组小鼠每天给予0.5 mL生理盐水灌胃;泼尼松治疗组小鼠每天给予0.18 mg/20 g BW的泼尼松溶于0.5 mL生理盐水灌胃。持续10周结束实验。应用逆转录-聚合酶链反应（RT-PCR）及Western印迹检测小鼠肾组织fractalkine和CX3CR1 mRNA和蛋白的表达,并检测小鼠实验室指标以及肾脏组织病理学的变化。结果： BXSB狼疮小鼠肾组织fractalkine以及CX3CR1 mRNA和蛋白表达均较C57BL/6J小鼠明显增高,而经过泼尼松治疗后的BXSB小鼠两者的表达均较未治疗组（实验对照组）明显下降,同时伴有血清免疫球蛋白G（IgG）、IgM、血清抗双链脱氧核糖核酸（dsDNA）抗体水平以及血尿素氮（BUN）、血肌酐（SCr）水平的明显改善,尿蛋白减少;肾小球内免疫复合物沉积和肾脏组织病理学改变亦显著减轻。结论： 实验结果提示fractalkine/CX3CR1可能参与了小鼠狼疮肾炎的发病机制,且糖皮质激素可能通过抑制肾脏fractalkine的表达而发挥其治疗效应。     

Antibody-mediated protection against Cryptococcus neoformans pulmonary infection is dependent on B cells

The pathogenesis of pulmonary Cryptococcus neoformans infection and the efficacy of passive immunoglobulin G1 (IgG1) administration were investigated in B-cell-deficient and C57BL/6J mice. C57BL/6J mice lived longer than B-cell-deficient mice after both intratracheal and intravenous infections. Administration of IgG1 prior to infection prolonged the survival of C57BL/6J mice but had no effect on the survival or numbers of CFU in the lungs of B-cell-deficient mice. C. neoformans infection in B-cell-deficient mice resulted in significantly higher levels of gamma interferon (IFN-gamma), monocyte chemoattractant protein-1 (MCP-1), and macrophage inflammatory protein-1alpha (MIP-1alpha) than in C57BL/6J mice. IgG1 administration reduced IFN-gamma and MCP-1 levels in C57BL/6J mice but not in B-cell-deficient mice. In addition, compared to its effect in C57BL/6J mice, C. neoformans infection in FcRgammaIII-deficient, athymic, and SCID mice significantly increased IFN-gamma and MCP-1 levels. IgG1 administration was associated with reduced IFN-gamma levels in C57BL/6J mice but not in FcRgammaIII-deficient, athymic, and SCID mice. These observations suggest that IgG1-mediated protection in this system is a consequence of alterations in the inflammatory response that translate into less damage to the host without directly reducing the fungal burden. For hosts with impaired immunities, the ineffectiveness of passive antibody (Ab) may reflect an inability to down-regulate inflammation and avoid self-damage. The results indicate an important role for B cells in host defense against C. neoformans infection and demonstrate a surprising dependence of Ab-mediated protection on B cells in this system.     

TGF-β1诱导的调节性T细胞体内输注延长小鼠皮肤移植物存活

目的 利用TGF-β1体外诱导naYve T细胞分化为凋节性T细胞(Treg),通过体内输注延长小鼠皮肤移植物存活时间,并研究其相关机制. 方法 根据诱导条件不同分为3组:对照组(加入IL-2培养的C57BL/6小鼠T细胞)、MLR组(即混合淋巴细胞反应组,经同种抗原刺激活化的C57BL/6小鼠T细胞)和TGF-β组(经同种抗原刺激活化的C57BL/6小鼠T细胞,同时加入5.0ng/ml TGF-β1诱导).利用FACS检测CD4+CD25+T细胞比例,并用RT-PCR检测Foxp3的表达水平.建立小鼠皮肤移植模型,并于第0、l、2和3天输注上述细胞,观察皮肤移植物存活时间.术后第9天,取部分受鼠行移植物病理检测,用FACS检测脾脏中THl、TH2和CD4+CD25+Treg比例,并用Alamar Blue法检测淋巴细胞增殖能力. 结果 TGF-β组中CD4+CD25+T细胞比例高于对照组和MLR组(P<0.05),且其高表达Foxp3.将培养的细胞输注给受鼠后发现,输注MLR组细胞的受鼠其移植物平均存活时间(mean survival time,MST)为(9.4±1.3)d,低于对照组(P<0.05);而输注TGF-13组细胞小鼠MST为(22.8±1.9)d,较对照组和MLR组明显延长(P<0.05).病理检测亦显示TGF-β组受鼠移植物结构完整,无明显淋巴细胞浸润.FACS结果显示TGF-β组小鼠体内TH1细胞(CD4+TIM-3+)比例低于对照组和MLR组(P0.05),但低于对照组(P<0.05);而且TGF-β组中CD4+CD25+Treg比例明显高于对照组和MLR组(P<0.05).用Alamar Blue法检测受鼠外周淋巴细胞增殖活性显示,TGF-β组受鼠淋巴细胞增殖能力被明显抑制,低于对照组(P<0.05). 结论 TGF-β1可诱导T细胞分化为具有抑制能力的Treg将诱导后的细胞进行过继输注可使小鼠体内CD4+CD25+Treg比例升高,同时抑制TH1和TH2细胞分化扩增,并使得外周淋巴细胞增殖能力减弱,从而延长小鼠皮肤移植物存活时间.