新制抗癌方对AFB1染毒的HBV转基因小鼠肝癌前病变组织PCNA、P21ras和NF-KB P65表达的影响

我们已从形态学角度观察了新制抗癌方对AFB1染毒的HBV转基因小鼠肝癌前病变的阻断作用[1].本研究旨在运用免疫组化法从分子生物学角度探讨其作用机理,为新制抗癌方的进一步研究奠定部分实验依据.

优化反向PCR法对hTF转基因小鼠整合位点旁侧序列分析

自1982年世界上首次通过转基因动物技术获得"巨鼠"[1]以来,转基因动物的研究发展迅速.随着多种转基因动物的成功获得,研究者设想利用动物表达外源基因,以动物个体作为一个反应器来生产有价值的蛋白质.

氧化苦参碱对HBV转基因鼠HBV抗原表达的抑制作用

目的:研究氧化苦参碱对HBV转基因鼠表达HBsAg,HBeAg,HBcAg的抑制作用,探讨氧化苦参碱抗HBV的机制.方法:用显微注射法建立HBV转基因鼠动物模型,并进行HBV整合和表达鉴定.获得的HBV复制型转基因鼠随机分成3组,分别用生理盐水(9只),氧化苦参碱50 mg/kg(8只)和100 mg/kg(9只)腹腔注射,1次/d,30 d后处死,取肝组织定量测定HBsAg和HBeAg的量,免疫组织化学观察肝组织表达HBsAg和HBcAg.结果:HBV转基因鼠经氧化苦参碱治疗后,与生理盐水组相比,肝脏组织HBsAg的量有所降低,但统计学无显著差异(F=1.29,P＞0.05);而HBV转基因鼠经氧化苦参碱高和低剂量组治疗后,肝脏组织HBeAg量明显降低(F=9.09,P＜0.01),但在氧化苦参碱高和低剂量组之间无显著性差异(F=1.58,P＞0.05).肝脏组织免疫组化HBsAg和HBcAg检测结果显示生理盐水组,氧化苦参碱低和高剂量组肝组织内HBsAg阳性细胞数变化不明显,统计学无显著性差异(x2=1.61,P＞0.05).氧化苦参碱高剂量组肝组织内HBcAg阳性细胞数明显低于生理盐水组(x2=4.73,P＜0.05),而生理盐水组与氧化苦参碱低剂量组,氧化苦参碱低和高剂量组之间均无显著性差异.结论:氧化苦参碱对HBV转基因鼠HBV的表达有抑制作用.

HBV转基因小鼠免疫耐受机制的实验研究

目的:观察乙型肝炎病毒(HBV)转基因小鼠(Tg鼠)树突状细胞(DC)抗原递呈功能,T淋巴细胞(TC)免疫活化状态和肝细胞(HC)的应答能力,探讨慢性HBV感染免疫耐受形成的机制.方法:用流式细胞术,氚一胸腺嘧啶核苷(3H-TdR)掺入法,ELISA和免疫组织化学方法,分别测Tg鼠脾DC,TC和HC表面分子,HBsAg刺激淋巴细胞增生反应以及TC产生的细胞因子含量.结果:Tg鼠DC表面MHC-Ⅱ类分子和CDS0表达低下,DC诱导脾淋巴细胞对HBsAg刺激的增生反应减弱,TC分泌的mIFN-γ,mIL-2,mIL-6,mIL-10均显著减少,HC膜MHC-I类分子表达量也降低.结论:Tg鼠DC的HBV抗原递呈功能低下,TC免疫活化受到抑制,HC的应答能力减弱,其中DC功能低下起关键作用.

A novel coronavirus, severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV), has been identified as the causal agent of SARS. Spike (S) protein is a major structural glycoprotein of the SARS virus and a potential target for SARS-specific cell-mediated immune responses. A panel of S protein-derived peptides was tested for their binding affinity to HLA-A * 0201 molecules. Peptides with high affinity for HLA-A * 0201 were then assessed for their capacity to elicit specific immune responses mediated by cytotoxic T lymphocytes (CTLs) both in vivo, in HLA-A2. 1/Kb transgenic mice, and in vitro, from peripheral blood lymphocytes (PBLs) harvested from healthy HLA-A2.1 + donors. SARS-CoV protein-derived peptide-1 (SSp-1 RLNEVAKNL), induced peptide-specific CTLs both in vivo (transgenic mice) and in vitro (human PBLs), which specifically released interferon-gamma (IFN-gamma) upon stimulation with SSp-1-pulsed autologous dendritic cells (DCs) or T2 cells. SSp-1-specific CTLs also lysed major histocompatibility complex (MHC)-matched tumor cell lines engineered to express S proteins. HLA-A * 0201-SSp-1 tetramer staining revealed the presence of significant populations of SSp-1-specific CTLs in SSp-1-induced CD8+ T cells. We propose that the newly identified epitope SSp-1 will help in the characterization of virus control mechanisms and immunopathology in SARS-CoV infection, and may be relevant to the development of immunotherapeutic approaches for SARS.

Emerging evidence has indicated that BRCC 36-mediated K63-linked ubiquitination modification was involved in diverse cellular functions , including endocytosis , apoptosis and DNA damage repair .We previously showed that activation of cGMP/PKG pathway con-tributed to the binding of BRCC36 and the pro-fibrotic factor Smad3.The current study tested the hypothesis that BRCC 36 functions as a negative regulator of transforming growth factor-beta ( TGF-β)/Smad3 pathway and participates in cardiac remodeling .In isolated adult mouse cardiac fibroblasts , we have demonstrated that TGF-β1 treatment significantly increased the expression of BRCC 36.Over-expression BRCC36 suppressed TGF-β1-induced Smad3 phosphorylation, nuclear translocation, extracellular matrix molecular expres-sion and cell proliferation .On the contrary, silencing BRCC36 by transfection of adenovirus-carrying BRCC36 shRNA potentiated to
enhance the pro-fibrotic effect of TGF-β.In vivo, under chronic pressure overload condition-induced by transverse aortic constriction , myocardial pro-survival protein Bcl-2 and Mcl-1 expression were significantly decreased and the pro-apoptosis protein Puma was in-creased.However, the cardiac-specific over-expression of BRCC36 significantly increased myocardial Bcl-2 and Mcl-1 and inhibited Puma expression .Interestingly , we also found that sustained pressure overload resulted in a significant myocardial DNA injury in wild type mice, which was characterized by the increase of γH2AX level.However, cardiac-specific BRCC36 over-expression significantly decreased the level of γH2AX in the pressure overloaded heart in the transgenic mice , while effectively enhanced myocardial RAD 51 expression, a marker of DNA damage repair.Furthermore, BRCC36 over-expression effectively attenuated TAC-induced cardiac fibro-sis and remodeling in the transgenic mice , compared with the wild type mice .Collectively , the results have suggested that BRCC 36 ef-fectively protected heart against chronic pressure overload-induced cardiac remodeling though antagonizing TGF-β/Smad3 pathway and enhancing myocardial DNA injury repair response .

CMV-hFasL转基因小鼠容易被小剂量的链尿菌素诱导至糖尿病

AIM: To investigate the role of Fas-FasL pathway in the pathogenesis of streptozotocin (STZ)-induced type I diabetes mellitus. METHODS: Low dose injections of STZ were used to induce type I diabetes mellitus in the CMV-hFasL transgenic mice. Blood glucose concentration was measured with Glucotrand Plus blood glucose test strips. Expression of hFasL was detected by RT-PCR and Western blotting. The severity of insulitis was determined by histologicalexamination. Expressions of IL- 1 β and TNF-α mRNA in the pancreas were detected by semi-quantitative RT-PCR analysis. Fas expression in apoptotic RIN-5F cells was also confirmed by RT-PCR in vitro. RESULTS: hFasL was expressed in the islets of CMV-hFasL transgenic mice. The transgenic mice were sensitive to diabetic induction than the control WT mice. IL-1 β and TNF-α expressions in the pancreas of CMV-hFasL transgenic mice were far more than that in WT mice. We also found STZ and IL-1β could both induce higher expression of Fas in RIN-5F. The combining of Fas-FasL could lead to the apoptosis of β cells in the CMV-hFasL transgenic mice. CONCLUSION: Fas-FasL interaction plays a significant role in the pathogenic mechanism of type I diabetes mellitus.