皮肤组织特异性表达hCTLA4-Ig转基因小鼠品系的建立

为研究皮肤特异性高效表达hCTLA4-Ig分子对移植皮肤存活及受体免疫功能的影响,充分实现hCTLA4-Ig分子在皮肤移植中的免疫调控功能,利用K14（角蛋白14）基因启动子,构建了hCTLA4-Ig分子皮肤组织特异性表达载体,并通过受精卵原核显微注射技术制备了K14／hCTLA4-Ig转基因小鼠并建立了品系。通过RT-PCR和Northern blot分析表明,hCTLA4-Ig在转基因小鼠体内呈皮肤组织特异性高效表达;以GAPDH基因的表达量为内部参照分析表明,hCTLA4-Ig的表达水平在不同世代之间以及转基因个体的不同生命时相点之间保持相对恒定,说明皮肤组织特异性稳定表达hCTLA4-Ig的转基因小鼠品系已被建立。     

不同手术方法对小鼠同种异基因移植皮片存活的比较

目的采用不同手术缝合方式观察小鼠移植皮片存活及生长情况,以建立一种简单易行、快速经济的小鼠同种异基因皮肤移植方法.方法 受体BALB/c小鼠随机分为3组(Z组、J组和T组),每组20只,通过背-背法进行供体C57BL/6的皮片移植,Z组、J组和T组分别采用四周缝合、对角缝合和粘贴固定的皮片缝合方法.每天监测小鼠体重变化及生长状态,观察26天,实验第3、7、15和26天观察各组受体移植皮片存活情况,其中皮片变硬、结痂脱落或坏死判定为移植排斥.第26天取小鼠皮肤移植组织以及肝脾组织进行病理检测.结果 各组小鼠移植物平均体重变化趋势类似,第6天达到最低水平,移植皮片病理检测结果显示,各组均见移植皮片真皮内淋巴细胞浸润,3组病理结果类似,肝脾病理检测发现淋巴细胞大量浸润,说明造模成功.实验26天后,Z组、J组和T组小鼠皮片分别有7、5和3只受体小鼠皮片仅观察到炎症反应.结论 3种手术方式建模均成功,但采用粘贴固定方式进行小鼠背部皮肤移植具有手术方式简单有效、对皮肤损伤小的优点,是一种较好的小鼠同种异基因皮肤移植手术方法.     

中医病证相符的糖尿病慢性皮肤溃疡大鼠造模方法初探

目的观察不同造模方法致糖尿病大鼠慢性皮肤溃疡创面形态及愈合时间的影响。方法 50只SD大鼠随机分为5组:皮肤缺损组(QS组:剪皮),糖尿病组(DM组:STZ+剪皮),糖尿病加金黄色葡萄球菌组(DMJJ组:STZ+剪皮+金葡菌),糖尿病加激素组(DMJS组:STZ+剪皮+激素注射),糖尿病加激素加异物组(DMYW组:STZ+剪皮+激素注射+异物埋置)。糖尿病模型稳定后每周测量血糖1次,每日称量体重、观察疮面情况、测量创面面积。12 d后处死,石蜡包埋肉芽组织观察其组织病理形态。结果 DMJJ组前5d愈合速度快于其余组(P0.01);DMYW组的愈合时间延长,DMYW组愈合率显著偏低,与其余组比较有统计学意义(P0.01)。造模12 d其余组愈合率无统计学差异,DMYW组愈合率显著低于其余组(P0.01)。结论注射激素大鼠表现出中医"阴证"证型特点,糖尿病加激素注射加异物埋置复合因素造模法能使大鼠创面表现出与临床相似的"阴证"证型特点。     

小鼠胚胎干细胞移植入成体大鼠脑内的区域特异性存活与分化

全能区域非特异性的胚胎干细胞是研究成体不同脑区控制干细胞分化能力的十分有力的工具。胚胎干细胞源性神经前体细胞移植入成体脑后可分化为功能性神经元,但是未分化的胚胎干细胞在成体脑内各个部位的存活、生长与分化的潜能差异尚不清楚。本文旨在探讨成体脑组织对胚胎干细胞的影响及胚胎干细胞在成体脑内的一系列行为。将少量转绿色荧光蛋白未分化的小鼠胚胎干细胞移植入成体大鼠脑内不同部位,分别于移植5、14和28d后处死大鼠,进行形态学观察及免疫组化定性,以了解未分化的小鼠胚胎干细胞在大鼠脑内不同区域的存活、生长与分化。结果发现未分化的小鼠胚胎干细胞可逐步整合入受体组织并向nestin阳性神经前体细胞分化。移植细胞及其后裔在海马生长最为旺盛,而在隔区最差（P〈0．01）;移植细胞分化为神经干细胞的效率也是在海马最高,而在隔区最低（P〈0．01）。提示只有部分脑区适合胚胎干细胞及其后裔生存,并提供促进其分化的有益环境。因此,由于位置特异的微环境因子及环境因素的存在,宿主组织特性对决定中枢神经系统疾病的细胞替代疗法策略是相当重要的。     

妊娠对于父源性皮肤移植存活时间的影响

母胎耐受机制的阐明,将为器官移植免疫耐受方案的研究提供重要启示.本研究旨在阐明妊娠状态对父系来源移植皮片的存活是否有保护作用.2月龄雌性C57BL/6小鼠和2～4月龄BALB/c雄性小鼠同笼受孕.采用流式细胞技术确定妊娠过程中调节性T细胞（Treg）比例的时间变化规律.以单向混合淋巴细胞反应（MLR）手段比较研究妊娠对于父系来源脾细胞刺激后产生的增殖反应的影响.通过同种异体小鼠全厚皮片移植模型,观察妊娠对于父系来源移植皮片的存活是否具有保护作用.并用分子生物学技术研究此种效能的可能机制.结果显示,C57BL/6小鼠妊娠过程中,Treg占CD4＋T细胞的比例从妊娠前的4.2%逐渐上升,受孕8天左右达到高峰值（6.8%）,此后开始下降并逐渐回复至基线水平.MLR结果表明,针对父系来源脾细胞的刺激,妊娠组较对照组呈现显著的低反应性,其平均刺激指数分别是7.8和13.6（P〈0.05）.定量PCR研究表明,血红素加氧酶-1和吲哚胺2,3双加氧酶mRNA在胎盘高表达,在脾脏低表达（P〈0.05）.父系来源的移植皮片的平均存活时间在妊娠组和非妊娠组分别是7.67和7.08天,无统计学差异（P〉0.05）.由此认为,在小鼠妊娠过程中,尽管出现了具有免疫抑制功能的Treg的比例增加,尽管有针对父系来源刺激细胞的较低的MLR反应性,但是单次妊娠对于父系来源的移植皮片的存活,在本研究条件下,未能显示具有统计学意义的保护作用.     

转BmK IT4基因烟草的抗虫性

用酶切方法从「ｐＢＳ－ＢｍＫ ＩＴ４质粒中获得ＢｍＫ ＩＴ４基因片段,并构建ＣａＭＶ ３５Ｓ启动子下的ＢｍＫ ＩＴ４基因表达质粒ｐＥ３－ＢｍＫ ＩＴ４,以根癌土壤杆菌（Ａｇｒｏｂａｃｔｅｒｉｕｍ ｔｒｍｅｆａｃｉｅｎｓ（Ｓｍｉｔｈ ｅｔ Ｔｏｗｎｓｅｎｄ）Ｃｏｎｎ）介导的叶盘法转化云烟（Ｎｉｃｏ－ｔｉａｎａ ｔａｂａｃｕｍ Ｌ．）Ｋ３２６叶片,获得４５株抗卡那霉素的再生植株。用这些再生植株进行抗虫     

一种简易小鼠尾部皮肤移植教学实验改良方法的建立

目的 为本校生物工程系实验动物学课程小鼠尾部皮肤移植实验课提供一种合适的操作方法.方法 分别用0.7%、1%、1.5%戊巴比妥钠和10%水合氯醛对小鼠进行麻醉试验,选出合适的麻醉方法;再进行小鼠尾部皮肤移植,对比玻璃套管法和创可贴法2种包扎方法的效果,以选出合适的手术方法.结果 0.7%和1%戊巴比妥钠麻醉持续时间过短,1.5%戊巴比妥钠和10%水合氯醛能维持足够手术操作所需的时间,但1.5%戊巴比妥钠易导致动物死亡,故采用10%水合氯醛麻醉小鼠较合适;玻璃套管法包扎的效果较创可贴法为好且更简便.结论 10%水合氯醛麻醉结合玻璃套管法包扎进行小鼠尾部皮肤移植是一种有效、简便、经济的小鼠尾部皮肤移植手术实验方法;此方法技术失败率低,适合学生实验课采用.     

激光照射、免疫抑制剂及二者配合应用对大鼠全胰十二指肠移植的影响Ⅰ大鼠糖尿病模型制备与全胰十二指肠移植术研究

用１％的Ａｌｌｏｘａｎ以５０ｍｇ／ｋｇ·Ｗ的剂量经尾静脉快速注射,成功地在健康雄性Ｗｉｓ－ｔａｒ大鼠制成了糖尿病模型。以健康大鼠为供体,成功地进行了模型大鼠全胰十二指肠移植术。为激光等抗移植排斥反应的深入研究打下了良好的基础。     

全蝎软膏对大鼠糖尿病皮肤溃疡创面愈合的效果评价

目的:探讨全蝎软膏治疗糖尿病皮肤创面愈合的作用及其机制。方法:采用SPF级SD大鼠构建糖尿病皮肤溃疡模型,将其随机平均分为对照组、模型组、全蝎软膏组和湿润烧伤膏组。造模后,分别在创面处涂抹全蝎软膏或湿润烧伤膏处理,空白组和模型组用PBS处理,观察和比较各组创面的愈合情况,并收集用药后3 d、7 d、10 d和14 d的创面肉芽组织进行HE染色,通过ELISA及荧光定量PCR检测肉芽组织内碱性成纤维细胞生长因子(bFGF)、血管性假血友病因子(v WF)、肿瘤坏死因子α(TNF-α)与Smad 4基因的表达。结果:成功构建大鼠糖尿病皮肤溃疡模型,成模率为96.15%。创伤后第3 d、7 d、10 d和14 d,各治疗组创面愈合率均显著高于模型组(P0.05),给药14 d时,全蝎软膏组创面愈合率已接近对照组水平。HE染色结果显示与模型组相比,全蝎软膏组中肉芽组织生长较快、毛细血管数量增多且组织纤维化程度较低。ELISA和荧光定量PCR检测结果显示与模型组和湿润烧伤膏组相比,全蝎软膏组肉芽组织内bFGF、v WF含量上调更显著(P0.05),而TNF-α含量与Smad 4 m RNA显著降低(P0.05)。结论:全蝎软膏可促进糖尿病皮肤溃疡创面肉芽组织的生长,可能与其抑制炎症反应并改善血管功能有关。     

转OsMAPK4基因烟草的抗旱性研究与遗传分析

以低温处理的水稻辽盐241植株叶片总RNA为模板, 用OsMAPK4基因特异引物通过RT-PCR扩增出OsMAPK4基因, 构建了由E12启动子调控的OsMAPK4基因植物表达载体pBME12。通过农杆菌介导法将OsMAPK4基因导入烟草, 筛选获得25株转基因植株。抗旱性研究结果表明, OsMAPK4基因的超量表达提高了T1代转基因植株的抗旱性。卡那霉素抗性在T1代转基因植株中的分离情况表明, 大多数转基因株系符合单基因遗传规律。     

GLP-1-IgG4-Fc融合蛋白的表达与鉴定

将合成的人胰高血糖素样肽-1(glucagon-like peptide-1,GLP-1)突变体基因与IgG4抗体的Fc部分进行融合获得GLP-1-IgG4-Fc片段,获得的基因片段与pXC17.4载体进行连接,用电转化方法将线性化质粒稳定转染CHO-K1细胞,通过Clone Pix 2筛选出高表达细胞株,产量达1.5g/L。收集培养上清并经Protein A和Source 30Q纯化,得到的GLP-1-IgG4-Fc融合蛋白,SDS-PAGE纯度高于95%,高效液相色谱(high performance liquid chromatography,HPLC)纯度和毛细管区域凝胶电泳(capillary zone electrophoresis,CZE)纯度均不低于80%,尺寸排阻层析(size-exclusion chromatography,SEC)纯度高于99%。经质谱和肽图谱测定,分子量与理论值一致,肽图谱序列与对照品高度一致。生物学活性分析表明,GLP-1-IgG4-Fc融合蛋白具有促进表达有GLP-1受体的HEK293细胞分泌环磷酸腺苷(cyclic adenosine monophosphate,c AMP)的活性,并且该活性与对照品高度相似。     

Expression of a soluble form of CTLA4 on macrophage and its biological activity

INTRODUCTI0NItisrecentlyreportedthatB7andCD28/CTLA4pathwayplaysacriticalroleintheactivationofT-ce1l[1-3],aswellasintheonsetofautoimmunitybothinhumanandmurinem0delofaut0immunity[4-7].B7moleculesareexpressedonavarietyofcelltypes,includingdentriticcells,Bcells,T-cellsandmacrophages[8-1l].CTLA4Ig,asolubleform0fCTLA4,cou1db10ckB7andCD28/CTLA4pathwayandresultsintheinhibitationofT-cellactivationandautoimmuneresponse[12-19].ThemacrophagicMm1ce1llinewasregardedasag0odmodelforstudyingmacr…     

Influence of local and systemic CTLA4Ig gene transfer on corneal allograft survival

BACKGROUND: To analyse the effects of local (ex vivo) or systemic (in vivo) administration of adenovirus type 5 encoding CTLA4Ig (AdCTLA4Ig) on its influence to prolong corneal allograft survival and to study the underlying mechanisms. METHODS: A MHC class I/II mismatched rat corneal transplant model was used. Recipients were randomly assigned to receive ex vivo gene-modified corneas expressing either CTLA4Ig, CTLA4Ig/IL-10 or a single intraperitoneal (i.p.) injection (1.0 x 10(9) or 1.0 x 10(10) infectious particles) of AdCTLA4Ig 1 day before transplantation and graft survival was analysed. The immunoregulatory effect of this treatment was examined by analysing intra-graft cytokine mRNA expression pattern at day 12 post-transplant. The anti-adenovirus immunity also was investigated. RESULTS: Ex vivo gene transfer resulted in a modest but significant prolongation of graft survival (p = 0.0036 compared to no treatment). In contrast, systemic gene therapy (1.0 x 10(9) or 1.0 x 10(10) infectious particles) significantly prolonged graft survival (p = 0.0007 and 0.0001, respectively, compared to no treatment). Systemic (1.0 x 10(10) infectious particles) therapy resulted in frequent indefinite survival of allogeneic grafts which was not observed in the other therapeutic regimens. Moreover, systemic therapy prevented the intra-graft accumulation and activation of T cells and resulted in a reduced mRNA expression of both TH1 and TH2 cytokines. The generation of anti-adenovirus antibodies was also efficiently inhibited. CONCLUSIONS: CTLA4Ig gene therapy is a successful strategy for the prevention of allogeneic graft rejection in corneal transplantation. Our work has further elucidated the mechanisms of corneal allograft rejection which may lead to novel therapeutic strategies.     

Enhancement of immunotherapeutic effects of HPV16E7 on cervical cancer by fusion with CTLA4 extracellular region

Cervical cancer is caused by infection by high-risk human papillomavirus (HPV), especially HPV16. Limitations in current treatments of cervical cancers call for the development of new and improved immunotherapies. This study aims at investigating the efficacy of a novel vaccine consisting of modified HPV 16E7 fused with human cytotoxic T-lymphocyte antigen 4 (CTLA4). The regions in HPV16 E7 gene associated with its transformation and CTL-enhanced response were modified; the resultant HPV16mE7 was fused with extracellular region of CTLA4 to generate HPVml6E7-eCTLA4 fusion protein. Binding of this fusion protein to B7 molecules expressed on antigen presenting-cells (APCs) was demonstrated. C57BL/6 (H-2^b) mice immunized with low dose of the fusion protein (10 μg) produced higher titer antibody and stronger specific CTL response, and expressed higher levels of IFN-γ and IL-12, compared with those immunized with HPVml6E7 only or admixture of HPVml6E7 and CTLA4, or PBS; and were protected from lethal dose tumor challenge. Tumor growth was retarded and survival prolonged in mouse models with the fusion protein treatment. Our results demonstrate that fusion of HPV16 E7 with eCTLA4 targeting APCs resulted in enhanced immunity, and that this fusion protein may be useful for improving the efficacy of immunotherapeutic treatments of cervical cancer and other HPV16 infection-associated tumors.     

人轮状病毒NSP4蛋白的表达及其致小鼠腹泻作用的初步研究

研究人轮状病毒非结构蛋白NSP4在轮状病毒致病性中的作用.分离得到我国人轮状病毒97SZ8株,以谷胱甘肽S-转移酶融合蛋白的形式在大肠杆菌BL-21中表达NSP4蛋白C端86-175氨基酸并用GlutathioneSepharoseTM 4B亲和纯化.将纯化蛋白分别以0.4nmol和1.5nmol的剂量腹腔注射新生Balb/C乳鼠,记录腹泻发生和体重变化情况.当注射0.4nmol GST-NSP4T重组蛋白时,有1只小鼠发生一过性腹泻(1/6),给予1.5nmol重组蛋白时,实验组所有乳鼠都先后出现了腹泻,存在一定的剂量依赖性.本研究初步在新生小鼠建立了一种人轮状病毒腹泻动物模型,该模型有望在人轮状病毒的致腹泻机理、治疗和预防研究中发挥重要作用.     

食物供应量对转红色荧光蛋白基因唐鱼 生存和生长的影响

选取雄性杂合子转红色荧光蛋白基因唐鱼(Tanichthys albonubes)(简称:转基因唐鱼)与雌性非转基因唐鱼交配产卵,出膜7d后,在水温(25.0 ±2.0)℃条件下,选择健康仔鱼进行饱食(食物供应量高)、半饥饿(食物供应量中)和饥俄(食物供应量低)3种处理,结果显示:(1)出膜后7～72 d的唐鱼饱食组与半...     

人轮状病毒NSP4蛋白的表达及其致小鼠腹泻作用的初步研究

研究人轮状病毒非结构蛋白NSP4在轮状病毒致病性中的作用。分离得到我国人轮状病毒97SZ8株,以谷胱甘肽S-转移酶融合蛋白的形式在大肠杆菌BL-21中表达NSN蛋白C端86-175氨基酸并用G1utathione SepharoseTM 4B亲和纯化。将纯化蛋白分别以0．4nmol和1．5nmol的剂量腹腔注射新生Balb／C乳鼠,记录腹泻发生和体重变化情况。当注射0．4nmol GST-NSP4重组蛋白时,有1只小鼠发生-过性腹泻(1／6),给予1．5nmol重组蛋白时,实验组所有乳鼠都先后出现了腹泻,存在一定的剂量依赖性。本研究初步在新生小鼠建立了一种人轮状病毒腹泻动物模型,该模型有望在人轮状病毒的致腹泻机理、治疗和预防研究中发挥重要作用。     

Effects of Metformin on Tissue Oxidative and Dicarbonyl Stress in Transgenic Spontaneously Hypertensive Rats Expressing Human C-Reactive Protein

Inflammation and oxidative and dicarbonyl stress play important roles in the pathogenesis of type 2 diabetes. Metformin is the first-line drug of choice for the treatment of type 2 diabetes because it effectively suppresses gluconeogenesis in the liver. However, its “pleiotropic” effects remain controversial. In the current study, we tested the effects of metformin on inflammation, oxidative and dicarbonyl stress in an animal model of inflammation and metabolic syndrome, using spontaneously hypertensive rats that transgenically express human C-reactive protein (SHR-CRP). We treated 8-month-old male transgenic SHR-CRP rats with metformin (5 mg/kg/day) mixed as part of a standard diet for 4 weeks. A corresponding untreated control group of male transgenic SHR-CRP rats were fed a standard diet without metformin. In a similar fashion, we studied a group of nontransgenic SHR treated with metformin and an untreated group of nontransgenic SHR controls. In each group, we studied 6 animals. Parameters of glucose and lipid metabolism and oxidative and dicarbonyl stress were measured using standard methods. Gene expression profiles were determined using Affymetrix GeneChip Arrays. Statistical significance was evaluated by two-way ANOVA. In the SHR-CRP transgenic strain, we found that metformin treatment decreased circulating levels of inflammatory response marker IL-6, TNFα and MCP-1 while levels of human CRP remained unchanged. Metformin significantly reduced oxidative stress (levels of conjugated dienes and TBARS) and dicarbonyl stress (levels of methylglyoxal) in left ventricles, but not in kidneys. No significant effects of metformin on oxidative and dicarbonyl stress were observed in SHR controls. In addition, metformin treatment reduced adipose tissue lipolysis associated with human CRP. Possible molecular mechanisms of metformin action–studied by gene expression profiling in the liver–revealed deregulated genes from inflammatory and insulin signaling, AMP-activated protein kinase (AMPK) signaling and gluconeogenesis pathways. It can be concluded that in the presence of high levels of human CRP, metformin protects against inflammation and oxidative and dicarbonyl stress in the heart, but not in the kidney. Accordingly, these cardioprotective effects of metformin might be especially effective in diabetic patients with high levels of CRP.