重组腺相关病毒-阳离子脂质体载体的构建及评价

目的探讨重组腺病毒相关病毒（rAAV）-阳离子脂质体（LyoVec）复合载体的构建方法及其转染率评价。方法构建rAAv～GFP，rAAv—LyoVec复合载体及rAAV—GFP—LyoVec。倒置荧光显微镜观察，测定rAAV—GFP，LyoVec—GFP及rAAV—GFP-LyroVec对定量C6细胞的转染率，筛选出高转染率载体。结果rAAV—GFP组24、48、72和96h转染率分别为16％、23％、21％和9％；LyoVec-GFP组为28．5％、33％、32％和11％；rAAV—GFP—Lyovec组为49％、59％、64％和20％。rAAV-GFP—LyoVec纽分别在24、48、72和96h的转染率显著高于rAAV—GFP组（P〈0．01）及LyoVec—GFP组（P〈0．01）。结论新构建rAAV—LyoVec复合载体基因的转染效率远远高于rAAV或LyoVec独立转染。     

Bacterial ghosts (BGs)—Advanced antigen and drug delivery system

Bacterial ghosts (BGs) are empty bacterial envelopes of Gram-negative bacteria produced by controlled expression of cloned gene E, forming a lysis tunnel structure within the envelope of the living bacteria. BGs are devoid of cytoplasmic content and possess all bacterial bio-adhesive surface properties in their original state while not posing any infectious threat. BGs are ideally suited as an advanced drug delivery system (ADDS) for toxic substances in tumor therapy. The inner space of BGs can be loaded with either single components or combinations of peptides, drugs or DNA which provides an opportunity to design new types of (polyvalent) drug delivery vehicles. Uptake of BGs loaded with Doxorubicin (Dox) by CaCo2 cells led to effective Dox release from endo-lysosomal compartments and accumulation in the nucleus. Viability and proliferative capacity of the cells were significantly decreased (2–3 orders of magnitude) after internalization of Dox loaded BGs as compared to cells incubated with free Dox. The same effect was observed with leukemia cells. Melanoma cells also revealed a high capability to internalize BGs. These results indicate that BGs are able to target a range of types of cancer. BGs have also been investigated as DNA delivery vectors. Studies show DNA loaded BGs are efficiently phagocytosed and internalized by both professional APCs and tumor cells with up to 82% of cells expressing the plasmid-encoded reporter gene. Our studies with BGs as an ADDS system contribute (i) to optimize drug delivery for the treatment of cancer; (ii) define specific conditions for selection and preparation of BG formulations; (iii) and provide a background for the clinical application of BGs in cancer therapy.     

HBV前C/C基因与绿色荧光蛋白基因融合表达载体的构建及在HEK293细胞中的表达

目的 :构建含HBV前C C基因与绿色荧光蛋白基因的融合表达载体 ,并在真核细胞中表达。方法 :用PCR法扩增含 1.3倍HBVayw型全基因组真核表达载体pHBV1.3的前C C基因片断 ,应用含绿色荧光蛋白基因的真核表达质粒构建融合表达载体 ,采用脂质体介导方法将其转染到HEK2 93细胞 ,并用荧光显微镜及ELISA法检测融合蛋白的表达。结果 :经PCR及酶切鉴定 ,证实成功构建了含HBV前C C基因的真核表达重组体pEGFP -HB VC ,显微镜下观察及ELISA法证实在HEK2 93细胞中表达了相应融合蛋白。结论 :构建的重组表达载体能在真核细胞中表达目的蛋白与GFP的双功能融合蛋白 ,适合于针对HBV前C C区的相关研究。     

Preservation and redirection of HPV16E7-specific T cell receptors for immunotherapy of cervical cancer

Human papilloma virus (HPV) type 16 infections of the genital tract are associated with the development of cervical cancer (CxCa) in women. HPV16-derived oncoproteins E6 and E7 are expressed constitutively in these lesions and might therefore be attractive candidates for T-cell-mediated adoptive immunotherapy. However, the low precursor frequency of HPV16E7-specific T cells in patients and healthy donors hampers routine isolation of these cells for adoptive transfer. To overcome this problem, we have isolated T cell receptor (TCR) genes from four different HPV16E7-specific healthy donor and patient-derived human cytotoxic T lymphocyte (CTL) clones. We examined whether genetic engineering of peripheral blood-derived CD8+ T cells in order to express HPV16E711-20-specific TCRs is feasible for adoptive transfer purposes. Reporter cells (Jurkat/MA) carrying a transgenic TCR were shown to bind relevant but not irrelevant tetramers. Moreover, these TCR-transgenic Jurkat/MA cells showed reactivity towards relevant target cells, indicating proper functional activity of the TCRs isolated from already available T cell clones. We next introduced an HPV16E711-20-specific TCR into blood-derived, CD8+ recipient T cells. Transgenic CTL clones stained positive for tetramers presenting the relevant HPV16E711-20 epitope and biological activity of the TCR in transduced CTL was confirmed by lytic activity and by interferon (IFN)-gamma secretion upon antigen-specific stimulation. Importantly, we show recognition of the endogenously processed and HLA-A2 presented HPV16E711-20 CTL epitope by A9-TCR-transgenic T cells. Collectively, our data indicate that HPV16E7 TCR gene transfer is feasible as an alternative strategy to generate human HPV16E7-specific T cells for the treatment of patients suffering from cervical cancer and other HPV16-induced malignancies.     

Engulfment of mast cell secretory granules on skin inflammation boosts dendritic cell migration and priming efficiency

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Ras homolog gene family H (RhoH) deficiency induces psoriasis-like chronic dermatitis by promoting TH17 cell polarization

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Mast cell corticotropin-releasing factor subtype 2 suppresses mast cell degranulation and limits the severity of anaphylaxis and stress-induced intestinal permeability

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Isolation of a peptide inhibitor of human rhinovirus

Cell culture-based transdominant genetic techniques provide new methods for discovering peptide/RNA modulators of cellular pathways. We applied this technology to isolate a peptide inhibitor of human rhinovirus. A green fluorescent protein (GFP)-scaffolded library of cDNA fragments was expressed in HeLa cells from a retroviral vector and screened for inhibitors of rhinovirus-mediated cell killing. A DNA clone, I421, increased cell survival in an HRV14 challenge assay from less than 0.5% to greater than 60%. It encodes a 53-amino-acid C-terminal extension of the GFP scaffold. Particular subclones of Hela cells expressing I421 (exemplified by I421dp3) show a delay in virus production and a 50-fold decrease in viral RNA levels at 6-8 h postinfection. HRV2, HRV14, and HRV16 show a dramatic decrease in plaque-forming ability on I421dp3 while Coxsackievirus B3 showed a small reduction. Levels of ICAM-1, the receptor for the main rhinovirus serotype, are not altered in I421dp3.     

谷氨酸脱羧酶67-绿色荧光蛋白基因敲入小鼠三叉神经尾侧亚核内GFP阳性神经元的分布及与小白蛋白的共存

目的观察谷氨酸脱羧酶67-绿色荧光蛋白（GAD67-GFP）基因敲入小鼠三叉神经尾侧亚核（Vc）浅层内，表达GFP的GABA能神经元的分布及其与小白蛋白（PV）的共存。方法分别运用原位分子杂交与免疫组织化学相结合；GFP与神经元标记物——神经元核蛋白（NeuN）或PV免疫荧光染色相结合的双重标记方法，在光学显微镜和激光共聚焦显微镜下进行观察。结果1．Vc浅层内90％以上的GFP阳性神经元同时表达GAD67 mRNA，而几乎所有表达GAD67 mRNA的阳性神经元都呈GFP阳性；2．GFP阳性神经元主要分布于Ｖc的Ⅰ－Ⅱ层内，细胞较小，尤其在Ⅱ层内可见大量密集分布的GFP阳性细胞和突起。GFP阳性神经元分别占Ⅰ、Ⅱ层内NeuN阳性神经元总数的19．4％和24．3％；3．GFP／PV双标神经元主要分布于Ｖc的Ⅰ－Ⅱ层，这些双标神经元大约占PV阳性神经元的62．4％，占GFP阳性神经元的12．8％。结论在Ｖc表达GFP的GABA能神经元主要密集分布于与外周伤害性信息传递关系密切的板层内，且大部分PV样阳性神经元属于GABA能神经元。     

NMDA receptor mediated dendritic plasticity in cortical cultures after oxygen-glucose deprivation

Dendrites and spines undergo dynamic changes in physiological and pathological conditions. Dendritic outgrowth has been observed in surviving neurons months after ischemia, which is associated with the functional compensation. It remains unclear how dendrites in surviving neurons are altered shortly after ischemia, which might reveal the mechanisms underlying neuronal survival. Using primary cortical cultures, we monitored the dendritic changes in individual neurons after oxygen-glucose deprivation (OGD). Two to four hours of OGD induced approximately 30–50% cell death in 24 h. However, the total dendritic length in surviving neurons was significantly increased after OGD with a peak at 6 h after re-oxygenation. The increase of dendritic length after OGD was mainly due to the sprouting rather than the extension of the dendrites. The dendritic outgrowth after 2 h of OGD was greater than that after 4 h of OGD. Application of NMDA receptor blocker MK-801 abolished OGD-induced dendritic outgrowth, whereas application of AMPA receptor antagonist CNQX had no significant effects. These results demonstrate a NMDA receptor-dependent dendritic plasticity shortly after OGD, which provides insights into the early response of surviving neurons after ischemia.