白细胞介素2脂质体的制备及其抗肿瘤作用的研究

采用逆相蒸发法制备稳定的白细胞介素2（IL－2）大单层脂质体，对其包封率、稳定性及活性进行了测定。建立C57BL／6小鼠荷瘤动物模型，通过给荷瘤小鼠腹腔注射空白脂质体、单纯IL-2及IL-2脂质体来比较其在肿瘤生长中的抑制作用，结果3组抑癌率分别为4．26％、34．04％和54.60％。空白脂质体组与单纯对照组相比尤显著性差异（P＞0．05），脂质体-IL-2组与单纯IL-2组之间存在显著性差异（P＜0．05），抑瘤率提高了20.6％。     

姜黄素-胡椒碱共载脂质体的制备及其体外抗肿瘤活性评价

以姜黄素和胡椒碱的包封率为指标,通过单因素试验和Box-Behnken设计结合响应面法优化姜黄素-胡椒碱共载脂质体的处方参数。结果显示,脂质体采用薄膜水化法制备,优化参数为：卵磷脂与胆固醇的质量比为5.16,成膜溶剂体积为20 mL、成膜温度为40℃、水化溶剂体积为20 mL、水化温度为60℃。所得优化脂质体的粒径为(95.5±1.8)nm,ζ电位为(–0.3±0.1)mV,姜黄素和胡椒碱的包封率为(97.46±0.36)%和(96.74±1.09)%,二者的载药量为(1.56±0.09)%和(0.07±0.01)%。然后,采用四甲基偶氮唑盐(MTT)比色法检测制品对人肺癌细胞(A549细胞)增殖的抑制作用。结果显示,与姜黄素原料药及其脂质体相比,姜黄素-胡椒碱共载脂质体对A549细胞增殖的抑制作用更强。Transwell迁移试验结果显示,与姜黄素原料药及其脂质体相比,姜黄素-胡椒碱共载脂质体作用下的细胞迁移数目明显减少。     

血清中VEGF，VEGFR1与VEGFR2在肺癌中的表达及其相互关系

目的本研究为探讨肺癌患者血清及组织中VEGF,VEGFR1,VEGFR2的表达在肺癌发生发展中的变化,为肺癌的诊断、发病机制提供生物学指标。方法选择山西省肿瘤医院胸外科2008年1月到2008年10月收治的初治原发性肺癌患者65例作为病例组,术后均经组织病理学证实。按照频数匹配的方法选择本院健康体检中20例正常人作为健康对照组。采集外周静脉血,用EHSA法检测病例组外周血中VEGF,VEGFR1,VEGFR2含量,并与健康对照组比较。采用SPSS12．0软件包进行统计学分析VEGF,VEGFR1和VEGFR2与患者病理类型、临床分期、性别、年龄之间的关联性及其三者之间的相关性。结果①肺癌患者血清VEGF、VEGFR：表达与健康对照组比较差异有显著性（均P〈0．01）;VEG—FR,表达在两组之间比较差异无显著性（P〉0．05）;②血清VEGF、VEGFR1、VEGFR2表达在性别,年龄及临床分期之间比较差异均无显著性（均P〉0．05）。结论①肺癌血清中VEGF,VEGFR2的表达在腺癌,鳞癌及小细胞癌患者中明显高于正常对照,而VEGFR1的表达与对照组比较无显著性差异;②患者肺癌血清中VEGF,VEGFR1,VEGFR2的表达在性别,年龄,临床分期中差异均无显著性。     

重组CEA和IL-2抗肿瘤基因疫苗的构建及其抗肿瘤作用研究

目的成功构建重细CEA、CEA／IL-2抗肿瘤基因疫苗,并检测其激活的淋巴细胞对肿瘤细胞的杀伤作用。方法将CEA、CEA／IL-2基因导入真核表达质粒pcDNA3．1,并转染入DC细胞,通过转染DC细胞活化淋巴细胞,将活化的淋巴细胞与人CEA＋肝癌细胞共培养,检测其对肿瘤细胞的杀伤作用。结果重组CEA、CEA／IL-2组均检测出较强的抗肿瘤效应,且重组CEA／IL-2组与重组CEA组差异具有统计学意义（P〈0．05）。结论联合CEA／IL-2基因疫苗具有抗肿瘤作用,且其抗肿瘤作用明显优于单基因CEA基因疫苗,证实了重组肿瘤基因疫苗对肿瘤细胞的杀伤作用。     

苦参碱脂质体的制备及抗肿瘤活性

目的:探讨增强苦参碱抗肿瘤作用方法.方法:用熔融法制备苦参碱脂质体;制备小鼠活体肿瘤模型,对比观察苦参碱及苦参碱脂质体抗肿瘤作用.结果:熔融法制备的脂质体外观圆整,粒径分布均匀,包封率理想,稳定性良好.苦参碱及苦参碱脂质体均有明显的抗小鼠S-180、H-22、EAC肿瘤的作用,且苦参碱脂质体作用大大强于苦参碱.结论:将苦参碱制备成脂质体剂型能显著增强苦参碱抗肿瘤作用,具有广阔的应用前景.     

冻融人外周血树突状细胞基因疫苗体外抗肿瘤活性研究

目的观察EB病毒潜伏膜蛋白重组腺病毒(rAd-EBV-LMP2)转染的冻融人外周血树突状细胞(DC)诱导的特异性细胞毒性T淋巴细胞(CTL)抗肿瘤活性。方法人外周血来源的单核细胞经细胞因子扩增培养为成熟DC,液氮冻存;rAd-LMP2重组腺病毒转染冻融DC制备疫苗。动态观察细胞形态学特征,四甲基偶氮唑蓝(MTT)法检测DC疫苗刺激同种T淋巴细胞增殖活性,以及诱导的CTL对肿瘤细胞的杀伤活性。结果rAd-LMP2转染的冻融DC疫苗具有形态迥异、多突起的典型形态学特征,其刺激同种T淋巴细胞的增殖能力明显高于对照组(P<0.01),能诱导高效的CTL活性(P<0.01),与新鲜DC疫苗差异无统计学意义(P>0.05)。结论rAd-LMP2转染冻融DC疫苗在体外能激发较强的EBV-LMP2特异性的功能性CTL,为EBV相关的鼻咽癌(NPC)的免疫治疗提供策略。     

槐定碱阳离子脂质体的制备及体外抗肿瘤活性的研究

目的制备槐定碱阳离子脂质体,并探讨其对肿瘤细胞的抑制作用。方法采用主动载药法制备槐定碱阳离子脂质体,并对其进行表征研究,采用MTS方法考察槐定碱阳离子脂质体对3种肿瘤细胞的抑制作用。结果制备得到的槐定碱阳离子脂质体呈类圆形,表面光滑,其平均粒径和聚分散指数分别为242.2 nm和0.180,表面电荷为+32.5 m V,其包封率和载药量分别为88.62%和5.97%。槐定碱阳离子脂质体对3种肿瘤细胞的IC50值均明显高于槐定碱,而空白阳离子脂质体对细胞并无明显的抑制作用。结论采用阳离子脂质体作为槐定碱的载体有利于将药物透过细胞膜,提高抗肿瘤作用,值得进行深入的系统研究。     

阿魏酸脂质体的制备及其体外抗氧化活性评价

采用醋酸钙梯度法制备阿魏酸脂质体，考察不同因素对包封率的影响。制备的脂质体包封率可达到(80.2±5.2)%，粒径约150 nm。空白脂质体和载药脂质体的zeta 电位分别为(13.14±1.67)mV和(4.12±0.05)mV。冷冻蚀刻电镜观察结果为单室脂质体。建立了U937细胞株氧化损伤模型，采用MTT法、荧光显微镜及光学显微镜考察了细胞活度、线粒体膜电位及细胞的形态学变化，评价阿魏酸脂质体对U937细胞株氧化损伤的保护作用。结果表明，阿魏酸脂质体组体外抗氧化活性强于阿魏酸溶液。     

藤黄酸脂质体的制备及抗肿瘤活性研究

目的 制备藤黄酸脂质体（GA Lip）,优化其处方并进行表征,考察GA Lip对4T1乳腺癌的抗肿瘤活性。方法 采用薄膜分散法制备GA Lip,通过高效液相色谱（HPLC）法对藤黄酸（GA）含量测定进行方法学考察;以粒径和多分散系数为主要指标优化GA Lip的药脂比和投药量;测定GA Lip包封率;采用透射扫描电镜（TEM）观察其形貌;采用四甲基偶氮唑盐比色（MTT）法检测GA及GA Lip对4T1细胞的增殖抑制活性;建立4T1荷瘤小鼠模型,对比游离GA与GA Lip的体内抗肿瘤效果。结果 优化后的GA Lip药脂比为1∶5,投药量为8 mg,包封率为（89.15±0.12）%,呈大小均一的椭圆形;GA及GA Lip对4T1细胞48 h的IC50分别为0.635μmol/L和0.294μmol/L;与游离GA组比较,GA Lip组小鼠肿瘤体积及重量更小。结论 优化后的GA Lip稳定性较好,包封率高,与游离GA相比,GA Lip抗肿瘤活性更好,药效作用有所增强。     

Enhanced synergistic anti‐Lewis lung carcinoma effect of a DNA vaccine harboring a MUC1‐VEGFR2 fusion gene used with GM‐CSF as an adjuvant

In order to achieve a synergistic effect on anti‐tumour and anti‐angiogenesis activity, we designed and constructed a DNA vaccine that expresses MUC1and VEGFR2 in the same reading frame. The aim of this study was to investigate the anti‐tumour activity of this DNA vaccine. Furthermore, we also investigated the enhanced synergistic anti‐Lewis lung carcinoma effect of this DNA vaccine by using GM‐CSF as an adjuvant. A series of DNA plasmids encoding MUC1, VEGFR2, GM‐CSF, and their conjugates were constructed and injected into mice intramuscularly (i.m.) followed by an electric pulse. The humoral and cellular immune responses after immunization were detected by enzyme‐linked immunosorbent assay (ELISA) and enzyme‐linked immunospot (ELISPOT), respectively. To evaluate the anti‐tumour efficacy of these plasmids, murine models with MUC1‐expressing tumours were generated. After injection into the tumour‐bearing mouse model, the plasmid carrying the fusion gene of MUC1 and VEGFR2 showed stronger inhibition of tumour growth than the plasmid expressing MUC1 or VEGFR2 alone, which indicated that MUC1 and VEGFR2 could exert a synergistic anti‐tumour effect. Furthermore, mice vaccinated with the combination of the GM‐CSF expressing plasmid and the plasmid carrying the fusion gene of MUC1 and VEGFR2 showed an increased inhibition in the growth of MUC1‐expressing tumours and prolonged mouse survival. These observations emphasize the potential of the synergistic anti‐tumour and anti‐angiogenesis strategy used in DNA vaccines, and the potential of the GM‐CSF gene as an adjuvant for DNA vaccines, which could represent a promising approach for tumour immunotherapy.     

A bivalent cyclic RGD–siRNA conjugate enhances the antitumor effect of apatinib via co-inhibiting VEGFR2 in non-small cell lung cancer xenografts

The vascular endothelial growth factor receptor 2 (VEGFR2) is considered to be a pivotal target for anti-tumor therapy against angiogenesis of non-small cell lung cancer (NSCLC). However, effective and low-toxicity targeted therapies to inhibit VEGFR2 are still lacking. Here, biRGD–siVEGFR2 conjugate comprising murine VEGFR2 siRNA and [cyclo(Arg-Gly-Asp-D-Phe-Lys)-Ahx]₂-Glu-PEG-MAL (biRGD) peptide which selectively binds to integrin αvβ3 receptors expressing on neovascularization endothelial cell was synthesized. The anti-tumor activity and renal toxicity of biRGD–siVEGFR2 or its combination therapy with low-dose apatinib were investigated on NSCLC xenografts. The immunogenicity of biRGD–siVEGFR2 was also evaluated in C57BL/6J mice. In vivo, intravenously injected biRGD–siVEGFR2 substantially inhibited NSCLC growth with a marked reduction of vessels and a down-regulation of VEGFR2 in tumor tissue. Furthermore, biRGD–siVEGFR2 in combination with low-dose apatinib achieved powerful anti-tumor effect with less nephrotoxicity compared with the regular dose of apatinib. Besides, no obvious immunogenicity of biRGD–siVEGFR2 was found. These findings demonstrate that biRGD–siVEGFR2 conjugate can be used as a new candidate for the treatment of NSCLC and its combination therapy with apatinib may also provide a novel strategy for cancer treatment in clinic.     

载碱性成纤维细胞生长因子质粒纳米粒的制备及体外转染活性研究

目的:制备壳聚糖载bFGF基因纳米粒,并对其体外性质及转染成纤维细胞效率进行考察。方法:以复凝集法制备载绿色荧光蛋白(EGFP)与人碱性成纤维细胞生长因子(bFGF)融合蛋白EGFP-bFGF质粒(pEGFP/bFGF)的壳聚糖纳米粒CS-pEGFP/bFGF,透射电镜、纳米粒度电位仪测定纳米粒形态、粒径和表面电位,凝胶阻滞实验分析质粒与壳聚糖结合情况,细胞增殖实验考察载bFGF质粒转染后对细胞增殖的影响,荧光分光光度计及荧光显微镜观测纳米粒转染成纤维细胞后细胞对EGFP的表达情况。结果:壳聚糖纳米粒可将质粒pEGFP/bFGF成功转入成纤维细胞中并能有效降低转染产生的细胞毒性,细胞自分泌的bFGF能促进成纤维细胞的增殖。结论:载bFGF纳米粒具有提高转染效率、降低毒性及促进成纤维细胞增殖的作用。     

In vitro and in vivo evaluations of PLGA microsphere vaccine formulations containing pDNA coexpressing Hepatitis B surface antigen and Interleukin-2

Purpose. DNA-based vaccines encoding viral antigens have been shown to elicit immune responses in animal models. In this study, a plasmid DNA (pDNA) coexpressing the middle envelope protein of hepatitis B virus (HBV) and Interleukin-2 (IL-2) was incorporated into Poly (D,L-lactic-co-glycolic acid) (PLGA) microspheres and three different formulations were investigated for their potential as a vaccine delivery system.

Methods. Emulsion solvent evaporation methods of water-in-oil-in-water (w/o/w) and oil-in-water (o/w) were used to generate three different formulations in which PLGA microspheres contained pDNA either encapsulated within or adsorbed onto the microspheres.

Results. In vaccine formulation A2, prepared using the (w/o/w) method, pDNA was encapsulated within the microspheres. The other two formulations (B2 and B2a) were prepared using the (o/w) method and B2 contained pDNAs encapsulated within the microspheres while B2a contained pDNAs adsorbed onto the microspheres. pDNA loading efficiencies of A2, B2 and B2a were determined to be 15%, 25% and 45%, respectively. In vitro release of pDNAs from microspheres was evaluated for a 45-day period with no conformational changes and A2 displayed slower release than that of the B2 and B2a. When mice were immunized from anterior tibialis muscle using A2, B2 and B2a formulations containing 100 µg pDNA, antibody responses were detected for 6 months in mice sera.

Conclusions. Although all PLGA microsphere formulations containing pDNA elicited antibody responses by the end of the 6th month, the antibody titers were found to be higher with B2 and B2a formulations in comparison to A2 formulation and the naked pDNA in saline.     

汉滩病毒核酸疫苗滴鼻免疫小鼠表达动力学研究

目的 构建汉滩病毒DNA疫苗,考查小鼠鼻内接种DNA疫苗后的基因组织定位及表达动力学,为进一步研究汉滩病毒DNA疫苗粘膜免疫的疗效作前期准备。方法 采用PCR法从PJSA1175-S质粒扩增汉滩病毒的S基因片段后装入PcDNA3．1B(-)载体,并以此候选DNA疫苗滴鼻免疫BALB／c小鼠,在不同时间、不同部位取其组织抽提基因组DNA和组织总RNA,用PCR及RT-PCR检测质粒的分布和表达动力学。结果 构建了汉滩病毒DNA疫苗PcDNA3．1B-S1.3。免疫接种该疫苗第1天后在小鼠鼻咽、肺、肾、肝、脾、小肠等组织中均有质粒分布;第1周内上述组织中均可检测到特异mRNA,第2周起小肠、肝及肾中特异mRNA消失。结论成功构建了PcD-NA3．1B-S1.3DNA疫苗,构建的疫苗滴鼻免疫后在小鼠体内得到广泛的分布及表达。     

胰岛素对RF/6A细胞VEGF受体表达的影响

目的:探讨胰岛素对视网膜血管内皮细胞RF/6A血管内皮细胞生长因子受体(VEGFR)的表达及血管新生的影响.方法:分别用0、1和100 nmol/L的人胰岛素处理RF/6A细胞,采用MTS实验、细胞迁移实验、管腔形成实验分别检测胰岛素对RF/6A细胞增殖、迁移、管腔形成等生物学功能,RT-PCR、Western blot技术检测VEGFR的表达、磷酸化活性.结果:胰岛素可促进RF/6A的增殖、迁移、管腔形成(P＜0.05或P＜0.01),促进VEGFR2 mRNA的表达和VEGFR2蛋白的磷酸化(P＜0.05或P＜0.01).而对VEGFR1 mRNA表达的影响差异无统计学意义(P＞0.05).结论:胰岛素可经VEGFR2信号促进RF/6A细胞血管新生,而VEGFR1信号可能不参与胰岛素诱导的RF/6A细胞血管新生.     

Anlotinib improves bile duct ligature-induced liver fibrosis in rats via antiangiogenesis regulated by VEGFR2/mTOR pathway

Cholestasis is a main clinical feature of biliary atresia (BA), which leads to liver fibrosis (LF). The focus of BA treatment is preventing and slowing the progress of LF. This study reports the improvement effect of anlotinib on common bile duct ligature (BDL)-induced LF in young rats. The BDL young rats were treated with anlotinib and the serum levels of aspartate aminotransferase, alanine aminotransferase, albumin, and total bilirubin were determined. Histological staining was performed and pathological changes in liver tissue were observed. The expression levels of α-SMA, collagen I, CD31, TGF-β1, phospho-VEGFR2, phospho-4E/BP1, and phospho-S6K1 were determined. The results showed that anlotinib significantly improved the liver function and histopathological injury of BDL rats, inhibited the deposition of collagen and hepatocyte apoptosis, and downregulated the protein expression of α-SMA and collagen I. Furthermore, anlotinib treatment significantly inhibited microvascular formation in the liver and downregulated the expression level of phospho-VEGFR2, thereby suggesting that the antifibrosis effect of anlotinib may be achieved by antiangiogenesis. In addition, anlotinib downregulated the expression of phospho-S6K1 and upregulated the expression of phospho-4E/BP1, two downstream proteins of the mammalian target of rapamycin (mTOR) pathway. MHY1485, an agonist of mTOR, significantly reversed the inhibitory effect of anlotinib on angiogenesis and LF but did not influence the effect of anlotinib on the downregulation of phospho-VEGFR2 expression. Together, the above-mentioned results suggest that the effect of anlotinib on BDL-induced LF involves at least antiangiogenesis regulated by the VEGFR2/mTOR signaling pathway.     

非小细胞肺癌中LKB1和VEGFR2的表达及临床意义

目的:探讨LKB1和血管内皮生长因子受体-2(VEGFR2)在非小细胞肺癌中的表达情况及临床意义。方法:选取不同临床分型及进展程度的非小细胞肺癌78例,分别取材自癌组织及癌周围正常组织,应用免疫组化方法检测并分析组织中LKB1和VEGFR2的表达情况。结果:LKB1蛋白和VEGFR2在肺癌组织及癌旁正常肺组织中均有表达。在正常肺组织LKB1高于肺癌组织,VEGFR2低于肺癌组织;在肺癌组织LKB1在高分化组织和无淋巴转移组高于中、低分化和有淋巴转移组,而VEGFR2高分化组和无淋巴转移组低于中、低分化组,亦低于有淋巴转移组,差异均有统计学意义(P<0.05或P<0.01)。结论:LKB1蛋白和VEGFR2在非小细胞肺癌中的表达呈相反趋势,LKB1的低表达、VEGFR2的高表达可能与非小细胞肺癌的肿瘤细胞分化及淋巴结转移有关。     

In vitro transfection of plasmid DNA by amine derivatives of gelatin accompanied with ultrasound irradiation

Purpose. The purpose of this study is to examine the ultrasound (US)-enhanced gene expression by the complexes of a plasmid DNA with gelatin derivatives of aminization. Methods. Gelatin derivatives with different introduced extents of ethylenediamine (Ed), spermidine (Sd), and spermine (Sm) were prepared with a water-soluble carbodiimide. The molecular size and zeta potential of the gelatin derivatives before and after complexation with the plasmid DNA were examined. After incubation with the complexes with or without US exposure, the DNA expression of rat gastric mucosal cells was measured to evaluate the effect of the type of gelatin derivatives on their gene expression. The cell uptake of the complexes, the cell viability, and the buffering effect of gelatin derivatives were examined. Results. The apparent molecular size and zeta potential of gelatin derivatives became larger as their aminization extent increased although the Sm gelatin derivative of higher aminization showed a larger value than other corresponding derivatives. Irrespective of the type of gelatin derivatives, the apparent molecular size of plasmid DNA was reduced by increasing the gelatin-DNA mixing ratio to attain a saturated value of about 150 nm. The condensed gelatin-DNA complexes showed the zeta potential of 10-15 mV. The cells incubated with the complex exhibited significantly stronger luciferase activities than free plasmid DNA, and the activity was further enhanced by US irradiation. The enhancement was significant for the Sm derivative compared with the corresponding Ed and Sd derivatives. The amount of plasmid DNA internalized into the cells was significantly increased by the complexation with every gelatin derivative, whereas US irradiation did not significantly increase the DNA internalization. US irradiation had no effect on the viability of cells incubated with every gelatin derivative-plasmid DNA complex, although the viability was decreased by the complex incubation. The buffering capacity of Sm derivative was higher than that of Ed and Sd derivatives and comparable with that of polyethylene amine. Conclusion. Among amine derivatives of gelatin, the Sm derivative enabled the plasmid DNA to induce the US-enhanced gene expression of cells in vitro most effectively because of the superior buffering effect.