N-亚甲基磷酸化壳聚糖基因纳米粒子的体外细胞毒性及基因转染实验研究

目的 考察N-亚甲基磷酸化壳聚糖( NMPCS)基因纳米粒子的体外细胞毒性及基因转染效率.方法 采用均相反应法制备了NMPCS,用复凝聚法制备了NMPCS/DNA纳米粒子;通过MTT实验考察了NMPCS及其与DNA复合物对HeLa细胞的细胞毒性,以荧光索酶质粒为报告基因考察了NMPCS及NMPCS-CaZ+载体介导的体外基因转染效率.结果 NMPCS及其与DNA的复合物在体外表现出很小的细胞毒性,远远低于同等浓度时聚乙烯亚胺(PEI)的毒性.通过对壳聚糖进行亚甲基磷酸化修饰后,可大幅提高载体的基因转染效率.结论 N-亚甲基磷酸化壳聚糖有望成为一种新型、安全、高效的非病毒基因载体.     

光谱法研究亚甲基绿(MG)、亚甲基蓝(MB)与DNA的识别作用及纳米材料对识别作用的影响

通过可见光谱法研究了亚甲基绿（MG）、亚甲基蓝（MB）与CT-DNA的相互作用，并且研究了纳米材料胶体金对该识别作用的影响。研究结果表明，MG、MB与DNA相互作用方式是不相同的，尽管它们在化学结构上只存在很小的差异。MG是以插入方式与DNA作用的，而MB与DNA作用存在沟槽和插入两种方式，并以沟槽方式为主。此外，通过在胶体金溶液中的研究表明，胶体金可以明显地提高小分子化合物与DNA作用检测的灵敏度。     

甲基硝基亚硝胍处理的vero细胞中磷酸化蛋白的差异显示

目的：初步了解哺乳类细胞非定标性突变形成是否与以蛋白质磷酸化级联反应为主要形式的细胞信号传导通路有关;方法：采用［３２Ｐ］体内预标记及凝胶双向电泳方法差异显示甲基硝基亚硝胍（ＭＮＮＧ）处理组和对照组磷酸化蛋白,蛋白质免疫印迹杂交试验初步鉴定差异显示蛋白斑点性质;结果：在处理组发现两个与对照组不同的蛋白斑点,分子量分别在６８×１０３和４３×１０３附近,与抗酪氨酸磷酸化蛋白抗体未发生阳性反应;结论：根据差异显示蛋白斑点的分子量和印迹杂交试验结果,初步认为所见蛋白斑点可能为丝／苏氨酸磷酸化蛋白,且可能为丝裂原激活的蛋白激酶（ＭＡＰＫ）成员     

低浓度N-三甲基壳聚糖对依托泊苷经肠黏膜透过性的影响

背景：N-三甲基壳聚糖作为透皮吸收促进剂,可以影响口服药物在体内经肠黏膜的透过性。 目的：考察低浓度N-三甲基壳聚糖对依托泊苷经肠黏膜透过性的影响。 方法：使用体外扩散池法评价依托泊苷经肠黏膜经时吸收方向和分泌方向的透过量和透过系数,并测定10%N-三甲基壳聚糖对依托泊苷经肠黏膜透过性的影响。依托泊苷在接受室中的浓度用高效液相色谱法测定。 结果与结论：10%N-三甲基壳聚糖具有增加依托泊苷经吸收方向的透过性,减少经分泌方向的透过性。说明低浓度的N-三甲基壳聚糖可用于改善受P-糖蛋白介导药物的吸收,有望提高此类药物的口服生物利用度。关键词：依托泊苷;P-糖蛋白; N-三甲基壳聚糖;扩散池;透过性 doi:10.3969/j.issn.1673-8225.2012.16.012     

壳聚糖-质粒DNA层层组装及携载DNA量的评价方法

目的 壳聚糖与质粒DNA可以层层组装形成多层膜,可用于金属表面载基因涂层.本研究采用表面等离子共振(SPR)技术,实时检测金属表面与壳聚糖、壳聚糖与质粒DNA(pDNA)的相互作用,并分析壳聚糖携载质粒DNA的能力以及壳聚糖-质粒DNA多层膜在液流作用下的稳定性.方法 在11-巯基十一羧酸处理过的裸金芯片上自组装不同浓度、不同相对分子量的壳聚糖(50～400 ku)和质粒DNA分子.结果 层层组装方法中壳聚糖对质粒的携载量与相对分子量和浓度密切相关,即高浓度、高分子量的壳聚糖可以形成更加厚实的多层膜,并可以携载更多的质粒DNA,自组装形成的多层膜还具有耐液流冲刷性,物理性质稳定.结论 采用壳聚糖与治疗基因的多层自组装可以在血管支架上携载质粒DNA,为探索载基因血管支架涂层的构建提供了新的方法和手段.     

N-乙酰化壳聚糖膜的制备和性质研究

以乙酸和甲醇为介质，利用壳聚糖和乙酸酐制备出N-乙酰化壳聚糖膜。对膜的亲水性、吸水性、结晶性、透光性、渗透性以及对血清蛋白的吸附性和与兔角膜上皮细胞的生物相容性进行了研究。结果表明N-乙酰化壳聚糖膜有一定的亲水性、吸水性、结晶性，有很好的透光性和渗透性，对血清蛋白有一定的吸附能力，以该膜为载体培养兔角膜上皮细胞实验结果表明膜与兔角膜上皮细胞具有很好的生物相容性。     

载基因壳聚糖纳米粒的制作优化和表征

目的 改良和优化载基因壳聚糖纳米微粒制作方法.方法 制备具有水溶性的磷酸化壳聚糖(pCS),再将pCS与甲胎蛋白基因的探针按不同比例浓度混合制作纳米粒.测量纳米粒径及电位变化,以及改变溶液pH值对包封率的影响.应用拉曼光谱分析纳米粒荧光强度变化.结果 改良制作纳米粒的方法更简单,粒径(144.6±6.8)nm与常规方法制作纳米粒粒径(153.4±18.9)mn差异无统计学意义(P＞0.05).通过优化条件,pCS与基因探针摩尔浓度比例为2∶1时最理想,改良法制作纳米粒径为(102.6±12.0)nm,zeda电位为(1.45±1.75)mV,包封率为(87.6±3.5)%.纳米材料的表征分析显示pCS与探针可结合形成纳米颗粒,并且包封基因探针.结论 优化微量法制作载基因壳聚糖纳米粒的方法可行和简单,pCS可包封基因探针.     

DNA的甲基化修饰与DNA甲基转移酶

组织特异的DNA甲基化谱是哺乳动物基因组的显特征。DNA的甲基化修饰参与基因表达调控、发育调节、基因组印迹和X染色体灭活等诸多重要生物学过程。大多数印迹基因可以调节胚胎的生长和发育。印迹功能的紊乱将导致发育异常及死胎。基因组范围内的广泛的去甲基化及随后发生的重新甲基化,可能使胚胎消除其亲本特异的甲基化谱,从而进入政党发育程序。DNA甲基化异常与肿瘤等疾病有关。肿瘤细胞的总体甲基化水平比正常细胞低,但是伴有某些CpG岛甲基化程度增高。抑菌基因启动子的异常甲基化和癌基因的去甲基化均影响肿瘤发生发展过程。哺乳动物DNA甲基化谱的建立和维持需要维持DNA甲基化的甲基转移酶和DNA重新甲基转移酶。     

DNA的甲基化修饰与DNA甲基转移酶

组织特异的 DNA甲基化谱是哺乳动物基因组的显著特征。DNA的甲基化修饰参与基因表达调控、发育调节、基因组印迹和 X染色体灭活等诸多重要生物学过程。大多数印迹基因可以调节胚胎的生长和发育。印迹功能的紊乱将导致发育异常及死胎。基因组范围内的广泛的去甲基化及随后发生的重新甲基化 ,可能使胚胎消除其亲本特异的甲基化谱 ,从而进入正常发育程序。DNA甲基化异常与肿瘤等疾病有关。肿瘤细胞的总体甲基化水平比正常细胞低 ,但是伴有某些 Cp G岛甲基化程度增高。抑癌基因启动子的异常甲基化和癌基因的去甲基化均影响肿瘤发生发展过程。哺乳动物 DNA甲基化谱的建立和维持需要维持 DNA甲基化的甲基转移酶和 DNA重新甲基转移酶。     

载基因壳聚糖纳米粒的制作优化和表征

Objective To improve the preparation method of chitosan nanoparticle for gene probe.Methods The water soluble phosphonic chitosan (pCS) was synthesized, then mixed with gene probe of alpha fetoprotein with different molar concentrations to synthesize nanoparticles. The size and zeta potential of the nanoparticles were determined. The pH in gene solution was modulated and the rate of envelopment of pCS for the gene was examined. Fluorescence intensity of nanoparticles was analyzed by laser Raman spectroscopy. Results The modified method of nanoparticles was simpler, and the size of nanoparticle synthesized by modified method was (144.6±6.8) nm, comparable to the size as synthesized by conventional The size, zeta potential and rate of chitosan combined gene of nanoparticle produced by modified method were ( 102.6± 12.0) nm, ( 1.45 ± 1.75 ) mV, and (87.6 ± 3.5 )% respectively. The Raman spectra showed that the pCS could combine and envelop the gene probe. Conclusion The modified method for synthesizing chitosan nanoparticle is simple and feasible and pCS can envelop the gene probe.     

表面等离子共振检测金属表面不同相对分子质量的壳聚糖对质粒DNA的保护作用

目的研究采用表面等离子共振（SPR）技术，检测不同相对分子质量壳聚糖对质粒DNA（pDNA）的保护作用，以防止核酸酶（DNaseⅠ）的降解。方法用具有羧基化葡聚糖表面的CM5芯片固定在Biacore3000生物大分子相互作用系统中制成壳聚糖芯片，采用SPR检测在核酸酶存在下，不同相对分子质量壳聚糖对pDNA的保护作用。结果在人体温度（37℃）下，浓度5U／μ1的DNaseⅠ在lmin内即可降解没有保护的质粒DNA，而相对分子质量为200000以上的壳聚糖对质粒具有良好的保护作用。结论表面等离子共振技术可以作为体外研究金属表面高分子对于质粒DNA保护作用的一种新方法。     

Immunostimulatory effects of plasmid DNA and synthetic oligodeoxynucleotides

Nucleic acid immunization is a new vaccination technology. DNA vaccines do not only carry the genetic information for the antigen of interest but also deliver an adjuvant effect due to the presence of immunostimulatory sequences within the plasmid backbone. It is generally assumed that the adjuvant properties of plasmid DNA are equal to those described for oligodeoxynucleotides (ODN) containing immunostimulatory CpG motifs. To challenge this hypothesis we have carried out a series of experiments comparing the ability of single- and double-stranded ODN containing CpG motifs to induce the activation of mouse spleen cells. Moreover, we compared the immunostimulatory properties of plasmids that were modified by the addition of two to four CpG motifs. Our results establish that plasmid DNA express their adjuvanticity as either double or single strands, and no differences were observed between modified and unmodified plasmids. On the other hand, the strongest stimulatory ODN sequences lost their adjuvant properties when administered as double-strand DNA. Furthermore, the profile of cytokines induced on spleen cells by plasmid DNA and ODN is different. Strikingly, plasmid DNA induces a moderate synthesis of IL-6 and a strong synthesis of IFN-gamma, whereas stimulation with ODN showed an inverse profile with a higher increase in the synthesis of IL-6 but a moderate increase in IFN-gamma. Finally, in vivo studies were consistent with the results obtained in vitro. Mice immunized with modified or unmodified plasmids encoding the glycoprotein D of HSV showed similar levels of cellular and humoral immune responses.     

Topical application of plasmid DNA to mouse and human skin

Gene expression following direct injection of naked plasmid DNA into the skin has been demonstrated in the past. Topical application of plasmid DNA represents an attractive route of gene delivery. If successful, it would have great prospects in skin gene therapy since it is painless and easy to apply. In this study, we analyzed the expression of plasmid DNA in vivo and in vitro following topical application of plasmid DNA in various liposomal spray formulations. Therefore, different concentrations of plasmid DNA expressing enhanced green fluorescent protein (pEGFP-N1) were sprayed onto mouse or human skin once daily for three consecutive days and compared with direct injection. Gene expression was assessed 24 h after the final topical application of various liposomal DNA formulations. The results showed that EGFP mRNA and protein were detectable by RT-PCR and Western blot, respectively. However, epicutaneously applied EGFP plasmid DNA did not lead to microscopically detectable EGFP protein, when assessed by confocal laser microscopy or fluorescence-activated cell sorting in contrast to about 4% of fluorescent keratinocytes following intradermal injection. In an in vivo mouse model, the application of pEGFP-N1 DNA led to the generation of GFP-specific antibodies. These results indicate that topical spray application of pEGFP-N1 liposomal DNA formulations is a suitable method for plasmid DNA delivery to the skin, yielding limited gene expression. This spray method may thus be useful for DNA vaccination. To increase its attractiveness for skin gene therapy, the improvement of topical formulations with enhanced DNA absorption is desirable.     

Enhancement of plasmid DNA immunogenicity with linear polyethylenimine

The utility of plasmid DNA as an immunogen has been limited by its weak immunogenicity. In the present study, we evaluated the ability of a family of linear polyethylenimine (PEI) polymers, complexed to plasmid DNA, to augment DNA expression in vivo and to enhance antigen‐specific adaptive immune responses. We showed that four of five structurally different PEIs that we evaluated increased in vivo DNA expression 20‐ to 400‐fold, and enhanced DNA‐induced epitope‐specific CD8+ T‐cell responses 10‐ to 25‐fold in BALB/c and C57BL/6J mice respectively, when delivered intravenously. Functional studies of the PEI‐DNA‐induced CD8+ T‐cell responses demonstrated that formulation of DNA with PEI was associated with increased numbers of cells secreting type I cytokines. In addition, PEI‐DNA complexes improved antigen‐specific T_H1‐helper cell and humoral responses. Most importantly, the PEI‐DNA complexes elicited memory cellular responses, capable of rapid expansion and accelerated clearance of a lethal dose of recombinant Listeria monocytogenes. Lastly, we identified physical properties of PEI‐DNA complexes that are associated with enhanced DNA‐elicited immunogenicity. These findings demonstrate that PEI polymers can play an important role in the development of DNA‐based vaccines in the setting of infectious disease prevention and cancer therapy.     

丙型肝炎病毒核心蛋白DNA免疫初步研究

目的　观察HCV核心蛋白基因的DNA免疫效果。方法　将HCV核心蛋白基因插入真核表达载体pcDNA3.1( ) ,构建重组质粒pcDNA3.1 c。在证明该重组质粒可在哺乳动物COS7细胞中表达的基础上 ,用重组质粒 10 0 μg免疫小鼠 ,同时设立空白质粒组和PBS组两组对照 ,初次免疫后 4周、8周各进行一次加强免疫。小鼠体液免疫反应和T淋巴细胞增殖检测分别采用间接免疫荧光法和MTT法。结果　pcDNA3.1 c可在COS7细胞内表达HCV核心抗原 ,接种于Balb c小鼠能有效诱导体液和细胞免疫应答。结论　重组质粒pcDNA3.1 c对于丙型肝炎防治具有潜在价值     

Heterokaryotic transmission of senescence plasmid DNA in Neurospora

Summary Heterokaryotic transmission is one of the major techniques for the study of cytoplasmic inheritance and here we have applied it to the senescence-determining plasmids kalilo (Hawaiian) and maranhar (Indian). We have shown that kalilo-based senescence is effectively transmitted by cytoplasmic contact, both in N. crassa and in N. intermedia. In the first place, the heterokaryons themselves are senescent, confirming the suppressivity of the senescence phenotype in mixtures of normal and senescent cytoplasms. Second, senescence is found in new nuclear associations, as shown by analysis of conidial isolates and meiocytes stemming from the heterokaryons. In addition, the free plasmid AR-kalDNA, and its form that is inserted into mtDNA, (mtIS-kalDNA), are both transmitted to new nuclear associations. In a transient fusion between senescent N. intermedia and nonsenescent N. crassa cells, AR-kalDNA was transmitted to N. crassa and mtIS-kalDNA was transmitted to N. crassa mtDNA. A cryptic mitochondrial plasmid, not associated with senescence, was also transmitted very efficiently to N. crassa mitochondria. In mixed kalilo/maranhar fusions, both plasmids coexisted, approximately equally, in the heterokaryons themselves, and in conidial isolates. However, in sexual derivatives, AR-marDNA was in an excess and AR-kalDNA was sometimes absent. The efficient heterokaryotic transmission of these elements suggests that this is one of their natural modes of spread in populations.     

不同剂量结核病DNA疫苗及细胞因子联合免疫的研究

目的：评价结核分枝杆菌MPT64（简称M64）和ESAT6（简称E6）DNA疫苗不同剂量联合免疫、与细胞因子联合免疫的保护效力，以及Ag85A（简称85A）和Ag5B（简称85B）DNA疫苗的保护效力。方法：将C57BL／6小鼠随机分为14组免疫：①生理盐水；②载体pVAX1 100μg；③卡介苗；④M64100μg＋E6100μg；⑤M6450μg＋E650μg；⑥M6475μg＋E625μg；⑦M6425μg＋E675μg；⑧M6425μg＋E625μg；⑨M64100μg＋IFN-γ 100μg；⑩E6100μg＋IFN-γ100μg；（11）M64100μg＋IL-12 100μg；（12）E6100μg＋IL-12 100μg；（13）85A100μg；逼（14）5B100μg。用ELISA法检测血清抗体，结核分枝杆菌通过尾静脉攻击小鼠，动态观察小鼠体重变化；攻击4周后，取肺、肝和脾观察病理改变、称重量、做菌落计数。结果：不同剂量的M64和E6DNA联合免疫、85A和85BDNA分别免疫均能诱导小鼠产生特异性抗体，但M64或E6与IFN-γ或IL-12质粒DNA共同免疫后特异性抗体水平与对照组无显著性差异；各组血清抗体亚型IgG2a均无显著升高，IgG2b均显著高于IgGI。第2、3A、8、9、11、12组在感染后4周体重明显增加，而第1、14、5、6组体重下降；第3、11、9、7组的肺菌落指数较少。第10、13组肺肉眼未见明显病变；第2～4、7、9～13组肺组织主要为不同程度的增殖性反应，第1、6、8、14组肺组织为增殖性反应与渗出性反应并存，第5组为渗出性反应。结论：DNA免疫的量需100μg才能诱导产生强的保护效力；M64和E6DNA各100μg混合免疫、85ADNA疫苗的保护效力与卡介苗相当；b164和E6DNA与IFN-γ或IL-12质粒DNA共同免疫后，明显增强其保护效力。     

含恶性疟原虫Pf70基因片段表达载体DNA的免疫学特性

目的 研究恶性疟原虫Pf70 DNA片段作为DNA疫苗候选抗原基因的可能性。方法 应用PCR方法从含有恶性疟原虫Pf70基因DNA片段的质粒pGEX-Pf70中扩增出包含Pf70 DNA片段的长度为957bP的PCR产物。将其插人带有乙肝表面抗原基因的真核表达载体pCMV-S中，构建出重组质粒pCMV-S-pf70。用提纯的重组质粒pCMV-S-Pf70作为DNA免疫制剂，以质粒pCMV-S DNA和经过谷胱甘肽亲和层析法纯化的融合蛋白GST-Pf70作为对照，免疫昆明种小白鼠。每隔14d加强免疫1次，加强免疫2次后第7天采小鼠全血，用流式细胞仪检测CD8 T淋巴细胞和CD4 T淋巴细胞的水平，以检测体液免疫和细胞免疫状况。结果 接种了重组质粒pCMV-S-Pf70小鼠CD8 T淋巴细胞的绝对量增加，其百分比含量增加了39％；CD4 T淋巴细胞的相对含量保持恒定，绝对量稍有增加。用ELISA法分别检测经重组质粒pCMV-S-Pf70免疫的小鼠和经融合蛋白GST-Pf70免疫的小鼠血清中抗Pt70蛋白质的抗体滴度，发现前者抗体滴度约为1：800，远远低于后者的滴度(1：5000)。研究结果证明，重组质粒pCMV-S-Pf70 DNA可以诱导小鼠产生很强的细胞免疫和相对于蛋白质免疫而言较弱的体液免疫；Pt70蛋白质可以诱导小鼠产生很强的体液免疫。结论 恶性疟原虫红内期Pf70 DNA片段是有前景的红内期DNA疫苗候选抗原基因片段。     

氯化锂离心纯化质粒DNA方法的建立及其效果评价

本文建立了一种采用氯化锂离心纯化质粒ＤＮＡ的方法。该法不需要特殊的仪器设备和昂贵的试剂材料，操作简便易行。用此法对ｐＵＣ和ｐＧＥＭ等多种系统的质粒进行了提取纯化。结果表明：纯化的质粒ＤＮＡ无ＲＮＡ、蛋白质和染色质ＤＮＡ污染，其得率与氯化铯超离心法相近；酶切效果良好；可直接用于哺乳动物细胞的基因转移并获得有效表达。