促甲状腺素受体在BALB/c小鼠主要组织和器官的分布及其意义

目的:研究正常BALB/c小鼠体内各主要组织、器官的促甲状腺素受体(thyrotrophin receptor,TSHR) mRNA的分布,为Graves病(GD)或／和甲状腺相关眼病(TAO)动物模型的制作提供病理学依据。方法:采用实时荧光定量PCR技术检测BALB/c小鼠甲状腺及腺外部分组织、器官中TSHR mRNA表达水平。结果:以甲状腺组织TSHR mRNA表达水平为标准1,呈相对高表达组织依次为肾上腺、胸腺、睾丸、卵巢及眼外肌,呈相对低表达的组织依次为肾脏、脑组织、腹壁脂肪、下颌下腺、肺脏、腹壁皮肤、眼眶脂肪、肝脏直至脾脏,胰腺、心脏及腹直肌中未见TSHR mRNA表达。结论:BALB/c小鼠体内,TSHR除了在甲状腺中表达外,还广泛存在于甲状腺外组织,且全身各处的TSHR作用机制可能不同。     

表达TSHR-A亚单位的腺病毒免疫BALB/c雌性小鼠建立甲亢模型

应用表达TSHR-A亚单位的重组腺病毒免疫BALB/c小鼠建立甲亢模型。分别用1010和108病毒颗粒/50μl PBS的腺病毒免疫6~8周雌性BALB/c小鼠,在完成3次免疫后通过检测血清T4、TRAb水平,甲状腺体积及甲状腺病理来鉴定动物模型。在所有接受免疫的小鼠中,甲亢的发生率为75%,表现为血清T4、TRAb增高,甲状腺体积增大,甲状腺病理表现符合甲亢。低免疫剂量组和高免疫剂量组小鼠血清T4较对照组均显著增高,两组间无显著性差异;低免疫剂量组和高免疫剂量组小鼠血清TRAb水平均显著高于对照组,前者血清TRAb水平低于后者。该模型具有成模率高,稳定性好的特点。低免疫剂量建立的动物模型更符合GD特点。     

BALB/c小鼠胸腺巨噬细胞不同亚群特性研究

目的研究小鼠胸腺巨噬细胞组成、分布、形态、功能。方法糖皮质激素诱导小鼠胸腺细胞凋亡,采用免疫荧光染色法,免疫组织化学和酸性磷酸酶(ACP)双重染色方法,发现小鼠胸腺巨噬细胞的不同表型。结果发现小鼠胸腺巨噬细胞存在四种表型:树状突起型细胞:可被Mac-2,F4/80单克隆抗体标记,数量最多,分布于整个胸腺实质且有吞噬作用;扁平型细胞:可被F4/80单克隆抗体标记,分布于皮质被膜下方;小卵圆型细胞:可被Mac-2单克隆抗体标记,分布于胸腺髓质和皮髓交界(CMR),此类细胞未见吞噬现象。ED1~+细胞:细胞形态不规则,主要分布于皮髓质交界区。以上不同的巨噬细胞亚群均可显示酸性磷酸酶活性。结论小鼠胸腺巨噬细胞存在不同亚群,表现在其组成、形态、分布、特点及功能差异等方面。     

幽门螺杆菌感染BALB/c小鼠模型的研究

随着对幽门螺杆菌 (Ｈ .ｐｙｌｏｒｉ)疫苗、致病机理、药物筛选等方面研究的深入 ,人们越来越多地需用动物模型。我们用含有ｃａｇＡ和ｖａｇＡ基因阳性的Ｈｐ株ｓｙｄ ｎｅｙｓｔｒａｉｎ1 (ＳＳ1 )感染ＢＡＬＢ ｃ小鼠 ,作Ｈｐ慢性感染小鼠的动物模型 ,试验用空肠弯曲菌琼脂基础培养基培养 ,微需氧条件下 37℃培养 3～ 5ｄ。ＳＰＦ级ＢＡＬＢ ｃ小鼠 40只 ,雌性 ,7～ 8周龄 ,重 17～ 2 0克 ,分实验组、对照组。实验组动物模型灌喂Ｈ .ｐｙｌｏｒｉ菌液 ,0 4ｍｌ 只 (每ｍｌ约含Ｈｐ 10 9条 ) ,连续 5次 ,10ｄ完成 ,对照组动物则不作相应…     

BCR-ABL-pIRES-SEA重组质粒在BALB/c小鼠肌肉表达分析

检测BCR-ABL-pIRES-SEA重组基因疫苗在小鼠肌肉中的表达。用已构建BCR-ABL-pIRES-SEA重组质粒免疫BALB/c小鼠一侧后肢骨骼肌,每两周一次,每次注射200μg,第七周取材用RT-PCR及免疫组织化学染色法检测目的基因在注射部位肌肉中转录和表达。结果发现:实验小鼠骨骼肌内检测到BCR-ABL和金黄色葡萄球菌肠毒素A(SEA)基因的转录,以及BCR-ABL蛋白的表达,证明所构建的BCR-ABL-pIRES-SEA重组质粒在小鼠体内可以有效地表达基因产物。     

BALB/c小鼠肥大细胞瘤/白血病模型的建立

目的研究建立BALB/c小鼠肥大细胞瘤/白血病模型。方法 BALB/c小鼠尾静脉注入小鼠肥大细胞瘤P815细胞,实验按每只小鼠接种细胞数量分为1×10^7/只组、5×10^6/只组、2.5×10^6/只组、1×10^6/只组、5×10^5/只组、1×105/只组和溶剂（RPMI1640培养液）对照组。观察小鼠的生存状态、体重及肝肺脾（重量、形态、病理）、染色体、血涂片、骨髓涂片、白细胞及血小板计数。结果注入P815细胞≥1×106/只的所有组小鼠均死亡,〈1×10^6/只的所有组生存良好,且死亡小鼠的生存时间与注入细胞数量呈负向依赖关系（P〈0.05）。死亡组小鼠体重较注射前相当或减轻、肝肺脾重较对照组及未死亡组显著增加（P〈0.05）;肝质地变硬,表面可见大量大小不一的白色结节突起,部分脾肺可见出血梗死灶,病理示肝脾有大量异形肥大细胞瘤细胞浸润;脾细胞染色体分析可见肥大细胞瘤细胞的非正常染色体核型;白细胞和血小板计数较对照组降低（P〈0.01）,血涂片可见少量肥大细胞瘤细胞,骨髓涂片示骨髓原始细胞比例增高。结论 P815细胞通过静脉注射能使BALB/c小鼠形成肥大细胞瘤/白血病,可作为肿瘤实验研究的一种模型构建方法。     

BALB/c小鼠巨细胞病毒性心肌炎模型的特征

目的：探讨BALB/c小鼠巨细胞病毒(MCMV)性心肌炎模型的特征。方法:60只4周龄BALB/c小鼠随机分成2组：实验组(36只，MCMV腹腔注射)和对照组(24只，3T3细胞裂解液腹腔注射)。在注射后3、7、14、21、28、35、42、49、56、63和70 d，记录心电图，测血清抗心肌β1受体抗体，分批处死小鼠行心肌病理、免疫组化和MCMV DNA检测。结果:实验组心肌炎累积发病25只(25/36，69.4%)，死亡4只(4/36，11.1%)；心肌炎急性期心肌呈现弥漫性炎性细胞浸润和灶性心肌细胞变性坏死，慢性期心肌间质散在炎性细胞浸润，急慢性期心肌炎病理积分均在2分或以下；免疫组化示急性期心肌IL-1β和TNF-α蛋白强阳性表达。实验组心律失常累计发生率达50.0%，可出现各种心律失常，急性期以窦性、房性心律失常和传导阻滞为主，慢性期以室性和房性心律失常为主。实验组3-7 d时心肌组织可检测到MCMV DNA片段，14-70 d时则不能检测到。实验组前5周血清抗β1受体抗体滴度为0，第6-10周明显升高；对照组前7周该抗体滴度为0，第8-10周轻微升高。结论:MCMV心肌炎并不严重，心肌细胞变性坏死具有局灶性和散在性特点，可出现各种心律失常；心肌炎早期病变和心律失常的发生可能与病毒感染直接损伤有关，慢性期病变和心律失常的发生则可能与抗β1受体抗体的作用有关。     

杨树花粉过敏BALB / c 小鼠动物模型的建立

目的:建立杨树花粉粗提物致敏激发的小鼠过敏模型。方法:将60 只BALB/ c 小鼠随机均分为正常组、卵清蛋白(OVA)阳性对照组和模型组,每隔5 d 经腹腔注射致敏1 次,共4 次,末次致敏后第5 天滴鼻激发。观察支气管肺泡灌洗液(BALF)中炎症细胞变化情况;PAS 染色观察气道黏液分泌情况;HE 染色观察鼻黏膜、肺炎症情况;免疫组化法检测肺组织中IL-4 及IFN-α阳性细胞平均光密度值;酶联免疫吸附试验检测血清中的总IgE 水平。结果:与正常组比较,模型组诱导鼻黏膜、肺组织出现明显的变应性炎症且气道黏液分泌明显增加;BALF 涂片显示明显的炎症细胞(嗜酸性粒细胞、中性粒细胞)增多;肺组织中IL-4 阳性细胞平均光密度值均高于正常组,IFN-α阳性细胞平均光密度值均低于正常组;血清中总IgE 高于正常组。结论:杨树花粉粗提物能够成功建立小鼠过敏模型。该模型的建立有利于过敏性疾病机制的研究。     

正常BALB/c小鼠鼻部解剖组织学特点的实验研究

目的　为研究人员提供正常BALB/c的鼻部组织结构特点,为有关的动物模型的研究提供解剖基础。 方法　正常BALB/c小鼠头部去掉外部软组织,分别进行矢状位显微镜下大体解剖成像和冠状位石蜡切片。苏木苏-伊红和高典酸希夫染色进一步确定鼻甲、鼻窦、鼻黏膜等鼻部显微组织形态特征。 结果 本研究发现正常BALB/c小鼠鼻部主要结构包括犁鼻器、嗅觉上皮、上下两个鼻甲、鼻咽、窦口鼻道复合体、上颌窦、以及筛迷路形成的前组筛窦和后组筛窦。而且上颌窦以黏膜下腺体显著增生为主要特点。但是正常小鼠鼻部可能没有额窦和蝶窦。 结论　本研究结果提示正常BALB/c小鼠鼻部和正常人的鼻部结构有显著的区别。了解正常BALB/c小鼠鼻部组织结构特点及其和人的差异对于开展小鼠变应性鼻窦和慢性鼻窦炎等动物模型的实验研究具有重要的指导意义。     

T细胞免疫BALB/c小鼠诱导调节性体液免疫应答的研究

目的 :研究T细胞免疫后正常小鼠的调节性体液免疫应答。方法 :用经γ射线照射的体外扩增的活化OVA特异T细胞克隆免疫BALB c小鼠 ,FACS法分析血清中抗T细胞抗体水平 ,免疫共沉淀法分析靶抗原。结果 :T细胞免疫能诱导BALB c小鼠产生抑制T细胞增殖的抗T细胞抗体 ,这种抗体不是多克隆活化的结果 ,其靶抗原可能是T细胞上 93、87、5 5和 4 5kD的蛋白。结论 :T细胞免疫能诱导正常小鼠产生调节性体液免疫应答     

分子识别理论在促甲状腺激素受体系统的应用

目的:观察促甲状腺激素受体(TSHR)分子上的特定片段-TR1(aa37-45)、TR2(aa353-366)和TR3(aa648-655)与其相应的反义肽- RT1(aa45-37)、 RT2(aa366-353) 和RT3(aa655-648)之间的选择性识别。方法:制备固相反义肽亲和层析柱-RT1-sepharose 4B, RT2-sepharose 4B和RT3-sepharose 4B,采用阶段洗脱法,观察相应天然肽- TR1、TR2 和TR3 在柱上的滞留行为。结果: TR1、TR2和 TR3与其相应的反义肽之间存在选择性识别,而反义肽RT1且能识别TSHR大分子; 此外, 牛促甲状腺激素(bTSH)蛋白还能被TR1识别。 结论:本实验证实了分子识别理论在促甲状腺激素受体系统的存在,它可能用于生物大分子蛋白质的分离、促甲状腺激素(TSH)与受体(TSHR)结合位点的测定,并用于实验性自身免疫性甲状腺炎大鼠的实验性治疗。     

大鼠脂肪细胞TSH受体机能研究

目的：研究TSH及Graves'TgC对大鼠脂肪细胞的作用。方法：获取大鼠游离的脂肪细胞。溶脱TSH受体。采用A蛋白尾析柱纯化Graves患者的IgG以脂肪细胞内cAMP浓度及释放到孵化液中甘油浓度作为脂肪分解指标,用cAMP及甘油试剂盒测定cAMP浓度及甘油浓度。结果：1mU／ml的TSll能增加大鼠雅高脂肪细胞的cAMP浓度及甘油的释放。脓苷引起cAMP及甘油的TSH剂量依赖曲线向右移。Gare’TgG抑制[125I]-TSH结合于大鼠脂肪细胞膜上ISH受体,并能刺激雅高脂肪细胞内cAMP的形成。结论：ISH受体功能性表达于脂肪细胞,其生物学效应是通过活化腺耷环化酶而实现的。     

Characterization of Bartonella henselae-specific immunity in BALB/c mice

BALB/c mice were inoculated with Bartonella henselae by both systemic and mucosal routes. Culture analysis of tissues from mice infected intraperitoneally with a high dose of B. henselae yielded positive results 24 hr after infection. However, culture analysis of blood taken between 6 hr and 7 days after infection from groups receiving live B. henselae were negative. Following intraperitoneal infection, B. henselae was detected by polymerase chain reaction in liver and mesenteric lymph nodes by 6 hr and up to 7 days after infection in liver, kidney and spleen tissue. Enzyme-linked immunosorbent assay (ELISA) of serum samples collected as early as 13 days after infection indicated humoral immune responses to B. henselae. Specific humoral responses remained through week 6. Analysis of faecal samples revealed induction of B. henselae-specific immunoglobulin A by day 28 after infection. In addition, B. henselae-specific cellular responses were indicated by a positive delayed-type hypersensitivity and a T helper 1 (Th1) (CD4+ T cell)-type cytokine response following in vitro stimulation of splenocytes. The significance and implications of these data in relation to B. henselae infections are discussed.     

Induction of thyroiditis in mice with thyrotropin receptor lacking serologically dominant regions

Grave's disease (GD) is characterized by pathogenic autoantibodies to the human thyrotropin receptor (hTSH-R), and is frequently associated with a lymphocytic infiltrate of the thyroid gland. In attempts to establish a murine model of GD, we and others have previously shown that immunization of mice with recombinant preparations of the hTSH-R ectodomain induces high titres of specific antibodies, which, however, are not pathogenic, nor is the response accompanied by the development of thyroiditis. Since earlier reports identified the serological immunodominant determinants within the N- and C-terminal regions of hTSH-R ectodomain, we reasoned that immunization of mice with truncated fragments of ectodomain lacking these dominant regions might result in skewing of the response to other determinants of the molecule, with consequent induction of immunopathological features present in GD. We show here that multiple challenge of BALB/c mice with an amino acid fragment of residues 43–282 generates antibodies directed at hTSH-R peptides 37–56, 157–176, 217–236 and 232–251. This reactivity pattern is distinct from that induced previously with the whole ectodomain of hTSH-R in BALB/c animals. Thyroid function remained unaffected in these mice, suggesting that pathogenic antibodies were not being induced. Interestingly, some animals developed lymphocytic infiltration of the thyroid gland, clearly indicating the presence of pathogenic T cell determinants within the 43–282 fragment. Challenge with the related fragment 43–316 produced the same pattern of serological response to the synthetic peptides as fragment 43–282, but was not accompanied by thyroiditis. The results demonstrate: (i) the presence of thyroiditogenic determinants within hTSH-R, and (ii) that these pathogenic determinants are likely to be cryptic, as their effect is exhibited only when the hierarchy of immunodominance within hTSH-R is drastically altered.     

Induction of hyperthyroidism in mice by intradermal immunization with DNA encoding the thyrotropin receptor

Intramuscular injection with plasmid DNA encoding the human thyrotropin receptor (TSHR) has been known to elicit symptoms of Graves' disease (GD) in outbred but not inbred mice. In this study, we have examined, firstly, whether intradermal (i.d.) injection of TSHR DNA can induce hyperthyroidism in BALB/c mice and, secondly, whether coinjection of TSHR- and cytokine-producing plasmids can influence the outcome of disease. Animals were i.d. challenged at 0, 3 and 6 weeks with TSHR DNA and the immune response was assessed at the end of the 8th or 10th week. In two experiments, a total of 10 (67%) of 15 mice developed TSHR-specific antibodies as assessed by flow cytometry. Of these, 4 (27%) mice had elevated thyroxine (TT4) levels and goitrous thyroids with activated follicular epithelial cells but no evidence of lymphocytic infiltration. At 10 weeks, thyroid-stimulating antibodies (TSAb) were detected in two out of the four hyperthyroid animals. Interestingly, in mice that received a coinjection of TSHR- and IL-2- or IL-4-producing plasmids, there was no production of TSAbs and no evidence of hyperthyroidism. On the other hand, coinjection of DNA plasmids encoding TSHR and IL-12 did not significantly enhance GD development since two out of seven animals became thyrotoxic, but had no goitre. These results demonstrate that i.d. delivery of human TSHR DNA can break tolerance and elicit GD in inbred mice. The data do not support the notion that TSAb production is Th2-dependent in murine GD but they also suggest that codelivery of TSHR and Th1-promoting IL-12 genes may not be sufficient to enhance disease incidence and/or severity in this model.     

Maternal immunization modulates the primary immune response to 2-phenyl-oxazolone in BALB/c mice

The development of the antibody repertoire in newborn mice is greatly influenced by idiotypic network interactions. It has been demonstrated that anti-idiotypic antibodies either directly injected or transferred from the mother may alter the repertoire for life. For an elucidation of the underlying mechanisms we have analyzed the primary immune response to 2-phenyl-5-oxazolone (phOx) coupled to chicken serum albumin (CSA) in BALB/c mice after complete disappearance of maternal antibodies which originated from different stages of affinity maturation. Depending on the serum titers of the mothers after primary (1° mo), secondary (2° mo) or tertiary (3° mo) immunization, maternal anti-phOx IgG persisted in F1 mice for up to 9 months. In addition, F1 mice born to 2° mo developed – even without immunization – an anti-phOx IgM titer which reached levels similar to an antigen-induced primary response. An enhancement of the early primary anti-phOx as well as anti-CSA response was seen in F1 mice born from 1° mo, whereas the response was delayed when born to 2° mo and 3° mo. The antibody titers in the latter group of mice remained at a lower level for 3 months. In contrast, mice of the F2 generation which received a smaller amount of the same collection of maternal antibodies as F1 mice from 3° mo exhibited a quite different primary response: (i) They showed an earlier onset in their anti-CSA response. (ii) Whereas normally a plateau in antibody titer was reached by the 4th weak after immunization, in 55 % of the F2 mice a prolonged increase of the anti-phOx and anti-CSA antibody titers was observed. At 12 weeks after antigenic challenge, titers reached plateau levels of 6 × 10⁵ which were never before seen in a primary phOx or CSA response. Thus, depending on its own immunological experience, the maternal immune system induces a state of memory in the offspring which results in a faster and/or enhanced immune response in the F1 and F3 generations.     

IL-2 preS DNA疫苗免疫正常小鼠和HBV转基因鼠的免疫应答研究

目的 探讨IL—2 preS DNA疫苗作为预防和治疗性疫苗的可行性及作用机理。方法 应用基因重组技术，构建人白细胞介素2(hIL-2)和前表面抗原(preS)的真核表达载体，将此重组载体用基因枪分别注射正常的BALB／c小鼠和HBV转基因小鼠，通过ELISA方法检测BALB／c小鼠和HBV转基因鼠的抗-preS2、HBsAg及抗-HBs抗体水平；荧光定量PCR方法检测HBV转基因鼠血清中HBV DNA拷贝数，并用免疫病理HE染色观察小鼠肝组织炎症活动度，同时检测肝功能指标。结果 ①基因枪注射真核表达质粒免疫正常小鼠后，100％小鼠能在第4、6周检测到抗preS1抗体，持续时间长达10周。②用IL-2 preS真核表达质粒基因枪肌肉注射方式优于正常肌肉注射和皮下注射的方式，且所需质粒量(10μg／只)仅为后者(100μg／只)的1／10。③在第4周高峰期检测IgG亚类，是诱导以TH1(IgG2a)细胞免疫为主的反应。④基因枪注射真核表达质粒(1μg／只)免疫转基因小鼠后，80％的小鼠产生了抗体，HBV DNA量下降，其中20％的小鼠HBsAg转阴。⑤HE肝组织染色显示：肝组织有明显的炎细胞浸润、肝细胞肿大、颗粒样变性、转氨酶升高。结论 IL-2 preS DNA疫苗能刺激小鼠机体产生体液和细胞免疫，可部分打破小鼠机体的免疫耐受，为新型乙肝疫苗和抗HBV持续性感染的特异性免疫治疗剂的设计和构建提供理论与实践依据。     

参芪扶正注射液对化疗后小鼠免疫功能的保护作用研究

目的:探讨参芪扶正注射液对化疗后小鼠免疫功能的保护作用。方法:用5-氟尿嘧啶(5-FU)50mg/(kg·d)×3天腹腔注射BALB/c小鼠,检测小鼠免疫功能。治疗组给予参芪扶正注射液40、80ml/(kg·d)分别腹腔注射给药,连续7天;对照组予生理盐水40ml/(kg·d)。于治疗第7天和第15天予免疫器官重量法检测胸腺、脾脏指数,用流式细胞仪(FACS)测定脾淋巴细胞中T细胞亚群、B淋巴细胞及脾淋巴细胞膜IL-2R(即活化T淋巴细胞CD3+CD25+)百分率;MTT法测ConA诱导的脾淋巴细胞增殖能力。结果:用5-FU化疗后小鼠免疫功能低下;应用参芪扶正注射液治疗后小鼠脾CD3+、CD3+CD4+CD8-、CD3+CD4+CD8-/CD3+CD4-CD8+(即CD4+/CD8+)、CD3+CD25+百分率及ConA诱导的脾淋巴细胞增殖能力均明显高于对照组(P<0·05),且能持续1周以上。结论:参芪扶正注射液可提高化疗后免疫功能低下小鼠的细胞免疫功能,且疗效较长。临床上可试用参芪扶正注射液对化疗患者的免疫功能进行保护,或对化疗后免疫功能低下患者进行治疗。