反应核酸药物与抗肿瘤研究

现在各国正在利用反义技术发展一类由核酸组成的新型药物,即反义核酸药物。我们合成了５种血管内皮生长因子（ＶＥＧＦ）的反义寡聚脱氧核苷酸。测定了它们在Ｓ１８０肿瘤细胞中对ＶＥＧＦ表达的抑制作用,还测定了它们在鼠角膜肿瘤模型上抑制由肿瘤引起的新生血管形成和肿瘤的生长。结果表明,部分硫代的或发夹结构修饰的ＯＤＮｓ均可有效抑制ＶＥＧＦ的表达、肿瘤的生长和新生血管的形成。线性的ＯＤＮ效果不好。两种作用于不同位     

脱氧核苷酸钠溶液电渗析法脱盐

<正> 核酸是生物体中传递遗传信息的一类非常重要的生物大分子。它们是由碱基、戊糖、磷酸所组成的核苷酸单体缩聚而成。根据所含戊糖的种类不同,核酸又分为核糖核酸(RNA)和脱氧核糖核酸(DNA)两大类。核酸在生物遗传变异、生长繁殖、分化发育等方面都起着决定性作用,核酸的研究对于肿瘤的发生和治疗也有重要意义。目前,核酸的研究已成为生物化学和分子生物学中一个十分活跃的领域。本文研究的脱氧核苷酸钠是由脱氧核糖核酸酶介而成的四种核苷酸单体钠盐的混合物(即脱氧腺嘌呤核苷酸钠、脱氧乌嘌呤核苷酸钠、脱氧胞嘧啶核苷酸     

以VEGF受体为靶向的融合毒素的研究

肿瘤的快速生长主要依赖于新生血管的形成。血管内皮生长因子(VEGF)在刺激血管内皮细胞生长和诱导血管生成方面起重要作用。肿瘤组织因血管含量丰富,VEGF特异性受体的数目远高于邻近正常组织,因此可以将血管内皮生长因子和某些细胞毒素构建为融合毒素,特异性定位于肿瘤部位,通过抑制血管的增生达到抑制肿瘤的目的。国外自二十世纪九十年代后期开展这方面研究,国内尚少见此类报道。本文就此类融合毒素的设计及临床研究等方面的进展作一综述。     

抗肿瘤新生血管生成抑制剂临床研究概况

肿瘤的生长和转移与血管有着密切的关系,抑制肿瘤血管生成可以调节肿瘤的生长。近年来,抑制肿瘤新生血管的药物已成为药学领域的研究热点,一些新生血管抑制剂已经进入临床试验阶段,本文对抗肿瘤血管生成的理论进行了概述,并总结了新生血管生成抑制剂的临床研究情况。     

血管内皮生长因子反义基因真核表达载体的构建及其对乳腺癌细胞的生长抑制作用

目的构建反义血管内皮生长因子(VEGF165)基因真核表达载体,分析该基因对乳腺癌细胞的生长抑制作用。方法将人VEGF165cDNA反向克隆至pcDNA3真核表达载体中,构建VEGF165反义基因的真核表达载体,转染人乳腺癌细胞MCF-7,观察转染前后MCF-7细胞的VEGF165表达及细胞生长周期。结果所构建的VEGF165反义基因真核表达载体转染MCF-7细胞后,VEGF165表达下降,细胞生存率下降,G1期细胞数量增加,S期细胞数量减少,细胞增殖能力降低。结论成功构建了VEGF165反义基因表达载体,该基因对乳腺癌细胞的生长具有明显的抑制作用。     

3种免疫调节性寡聚脱氧核苷酸佐剂体外免疫活性的比较

目的比较用于乙型肝炎疫苗的3种寡聚脱氧核苷酸(CpG-oligodeoxynueleotide,CpG ODN)佐剂体外的免疫活性。方法分别将CpG ODN 684(沃森公司)、CpG ODN 1018(Dynavax公司)和CpG ODN 7909(Coley公司)序列体外刺激免疫人外周血单个核细胞(peripheral blood mononuclear cell,PBMC)和人树突状细胞系GEN,采用氚-胸腺嘧啶核苷(3H-TdR)渗入法检测人PBMC增殖活力,ELISA法检测GEN细胞中IL-6和IFNα水平。结果 CpG ODN 7909、CpG ODN 1018和CpG ODN 684诱导人PBMC的增殖水平明显高于未刺激的对照组,差异有统计学意义(P0.000 1),其中CpG ODN 684和CpG ODN 7909序列诱导人PBMC的增殖水平相似,略高于CpG ODN 1018序列;3种CpG ODN在体外对免疫细胞均具有明显的活化作用,其中CpG ODN 684和CpG ODN 7909序列体外活化免疫细胞产生的IL-6和IFNα水平相似,但高于CpG ODN 1018序列。结论沃森公司CpG ODN 684序列体外活化免疫细胞功能与Coley公司CpG ODN 7909序列相似,略优于Dynavax公司CpG ODN 1018序列。     

CpG寡聚脱氧核苷酸佐剂对狂犬病疫苗免疫效果的影响

目的探讨CpG寡聚脱氧核苷酸(CpG oligodeoxynucleotides,CpG ODN)对狂犬病疫苗(rabies vaccine,RV)免疫效果的影响。方法以市售狂犬病疫苗为抗原,分别以低、中、高剂量的CpG ODN(1.25、5、20μg/剂)及其与氢氧化铝[(Al(OH)_3]联合的复合物作为疫苗佐剂,按Essen程序经腿部肌肉免疫BALB/c小鼠。免疫前2 d及首次免疫后分别于第6、8、10、14、35和42天采血,分离血清,通过快速荧光灶抑制试验(rapid fluorescent focus inhibition test,RFFIT)检测血清中抗狂犬病病毒中和抗体效价;并经ELISPOT法分析首次免疫第6和14天小鼠脾细胞分泌IFNγ的情况。结果低、中剂量的CpG ODN及其与Al(OH)_3联合使用均可使小鼠在首次免疫后第8天产生具有保护水平的中和抗体,第10天阳转率均达100%;在首次免疫后第35及42天,小鼠产生抗狂犬病病毒中和抗体水平均明显升高(P0.05);在首次免疫后第6及14天,低、中剂量的CpG ODN可明显提高小鼠脾细胞的IFNγ分泌水平(P0.05)。结论 CpG ODN对RV具有免疫增效作用,可能成为一种新型的RV佐剂。     

薰衣草精油和亚洲薄荷精油促毛发生长作用的研究

通过体外细胞实验和脱发小鼠模型,研究了薰衣草精油与亚洲薄荷精油对毛发生长的促进作用。通过细胞实验考察两种精油对人永生角质形成细胞血管内皮生长因子(VEGF)和角质形成细胞生长因子(KGF)分泌的影响;通过小鼠模型考察两种精油对新生毛发生长长度、脱毛区皮肤组织学变化、毛囊数目、真皮厚度、皮肤VEGF和KGF表达的影响。结果表明,两种精油均能促进毛发伸长,增加脱毛区皮肤毛囊数目和真皮层厚度,延长毛发处于生长期的时间,但二者的作用机制不同;薰衣草精油促进KGF的分泌,亚洲薄荷精油则促进VEGF的分泌,动物实验结果与细胞实验结果相符。说明薰衣草精油和亚洲薄荷精油均有促毛发生长的功效。     

CpG-ODN对乙肝核酸疫苗及重组疫苗免疫效果的影响

目的研究CpG寡聚脱氧核苷酸(CpGODN)对乙肝核酸疫苗及重组(CHO细胞)疫苗免疫效果的影响。方法核酸疫苗实验组用CpGODN与乙肝核酸疫苗联合免疫BALBc小鼠,阳性对照组注射核酸疫苗,阴性对照组注射质粒pVAX1;重组疫苗分4个实验组,用CpGODN与不同剂量的重组疫苗配伍后分别免疫BALBc小鼠,阳性对照组注射重组疫苗,阴性对照组注射生理盐水。应用ELISA检测小鼠血清中的抗HBs水平。结果CpG与乙肝核酸疫苗组抗体阳转率比阳性对照组提高3倍,抗体水平提高2倍。CpG与重组疫苗组的阳转率和阳转时间均高于或早于CpG与核酸疫苗实验组。加入CpGODN后,即使重组疫苗用量减少3倍,诱导抗体水平和阳转率均无明显改变。结论CpGODN可提高乙肝核酸疫苗和重组疫苗的免疫效果,并使抗体产生的时间提前。     

抗肿瘤血管新生的靶向药物研究进展

抗肿瘤研究一直以来都是医学界的研究热点,近年来随着医学研究的不断发展,抗肿瘤血管新生成为一种新型的靶向肿瘤治疗方法,这种方法具有诸多优势,所以迅速引起了业内外的广泛关注。就肿瘤血管生成机制及抗肿瘤血管新生的靶向药物研究进展进行了综述。     

On the in vitro and in vivo properties of four locked nucleic acid nucleotides incorporated into an anti-H-Ras antisense oligonucleotide

Locked nucleic acid (beta-D-LNA) monomers are conformationally restricted nucleotides bearing a methylene 2'-O, 4'-C linkage that have an unprecedented high affinity for matching DNA or RNA. In this study, we compared the in vitro and in vivo properties of four different LNAs, beta-D-amino LNA (amino-LNA), beta-D-thio LNA (thio-LNA), beta-D-LNA (LNA), and its stereoisomer alpha-L-LNA in an antisense oligonucleotide (ODN). A well-known antisense ODN design against H-Ras was modified at the 5'- and 3'-ends with the different LNA analogues (LNA-DNA-LNA gapmer design). The resulting gapmers were tested in cancer-cell cultures and in a nude-mouse model bearing prostate tumor xenografts. The efficacy in target knockdown, the biodistribution, and the ability to inhibit tumor growth were measured. All anti H-Ras ODNs were very efficient in H-Ras mRNA knockdown in vitro, reaching maximum effect at concentrations below 5 nM. Moreover, the anti-H-Ras ODN containing alpha-L-LNA had clearly the highest efficacy in H-Ras knockdown. All LNA types displayed a great stability in serum. ODNs containing amino-LNA showed an increased uptake by heart, liver, and lungs as compared to the other LNA types. Both alpha-L-LNA and LNA gapmer ODNs had a high efficacy of tumor-growth inhibition and were nontoxic at the tested dosages. Remarkably, in vivo tumor-growth inhibition could be observed at dosages as low as 0.5 mg kg(-1) per day. These results indicate that alpha-L-LNA is a very promising member of the family of LNA analogues in antisense applications.     

Enhanced Inhibition of Prostate Tumor Growth by Dual Targeting the Androgen Receptor and the Regulatory Subunit Type Iα of Protein Kinase A in Vivo

Progression to castration resistance is a major problem in the treatment of advanced prostate cancer and is likely to be driven by activation of several molecular pathways, including androgen receptor (AR) and cyclic AMP-dependent protein kinase A (PKA). In this study, we examined the therapeutic efficacy of a combined inhibition of the AR and the regulatory subunit type Iα (RIα) of protein kinase A with second generation antisense oligonucleotides (ODNs) in androgen-sensitive LNCaP and castration-resistant LNCaPabl tumors in vivo. We found that targeting the AR alone inhibited LNCaP, as well as LNCaPabl tumors. Combined inhibition resulted in an improved response over single targeting and even a complete tumor remission in LNCaPabl. Western blot analysis revealed that both ODNs were effective in reducing their target proteins when administered alone or in combination. In addition, treatment with the ODNs was associated with an induction of apoptosis. Our data suggest that dual targeting of the AR and PKARIα is more effective in inhibiting LNCaP and LNCaPabl tumor growth than single treatment and may give a treatment benefit, especially in castration-resistant prostate cancers.     

小剂量长春新碱和恩度联合用药对横纹肌肉瘤皮下移植瘤的抑制作用

目的研究小剂量长春新碱(Vincristine,VCR)和恩度(Endostar,Endo)联合用药对横纹肌肉瘤(Rhabdomyosarcoma,RMS)BALB/c裸鼠皮下移植瘤的生长抑制作用,并探讨其机制。方法经BALB/c裸鼠右侧腋部皮下注射RMS细胞PLA-802悬液,建立RMS的皮下移植瘤动物模型;接种RMS后第8天,将选模成功的裸鼠随机分为对照组(注射生理盐水)、VCR组、Endo组和联合组,注射部位均为腹腔,注射体积均为0.2 ml/(只.日),每3 d称重1次,并测量肿瘤的长短径,计算肿瘤体积,绘制肿瘤生长曲线,2周后处死裸鼠,称量瘤重,并计算抑瘤率;RT-PCR和Western blot法检测移植瘤细胞中血管内皮生长因子(Vascular endothelial growth factor,VEGF)基因mRNA的转录水平和蛋白的表达水平;免疫组化法检测VEGF和CD31的分布和表达。结果裸鼠接种PLA-802细胞第8天,RMS裸鼠模型复制成功,裸鼠成瘤率为84%(42/50);联合组裸鼠体重和移植瘤的体积、重量均明显小于对照组、VCR组和Endo组(P<0.01);VCR组、Endo组和联合组抑瘤率分别为31.41%、20.75%和48.41%;与对照组相比,VCR组、Endo组和联合组裸鼠移植瘤细胞中VEGF基因mRNA的转录水平和蛋白表达水平均显著下降,且以联合组下降最明显(P<0.05);免疫组化结果显示,联合组VEGF和CD31的表达水平明显低于其他3组。结论小剂量VCR和Endo对RMS的皮下移植瘤的生长具有明显的抑制作用,而联合用药能起到增效作用,其机制可能是通过抑制肿瘤的血管生成而发挥其抑制作用。     

Vascular Endothelial Growth Factor,a Key Modulator of the Anti-Tumor Immune Response

During tumor growth, angiogenesis is required to ensure oxygen and nutrient transport to the tumor. Vascular endothelial growth factor (VEGF) is the major inducer of angiogenesis and appears to be a key modulator of the anti-tumor immune response. Indeed, VEGF modulates innate and adaptive immune responses through direct interactions and indirectly by modulating protein expressions on endothelial cells or vascular permeability. The inhibition of the VEGF signaling pathway is clinically approved for the treatment of several cancers. Therapies targeting VEGF can modulate the tumor vasculature and the immune response. In this review, we discuss the roles of VEGF in the anti-tumor immune response. In addition, we summarize therapeutic strategies based on its inhibition, and their clinical approval.     

Generation of Potent Anti-Vascular Endothelial Growth Factor Neutralizing Antibodies from Mouse Phage Display Library for Cancer Therapy

Vascular endothelial growth factor (VEGF) is an important stimulator for angiogenesis in solid tumors. Blocking VEGF activity is an effective therapeutic strategy to inhibit tumor growth and metastasis. Avastin, a humanized monoclonal antibody recognizes VEGF, has been approved by the US Food and Drug Administration. To generate potential VEGF-recognizing antibodies with better tumor regression ability than that of Avastin, we have designed a systematic antibody selection plan. From mice immunized with recombinant human VEGF, we generated three phage display libraries, scFv-M13KO7, Fab-M13KO7, and scFv-Hyperphage, in single-chain Fv (scFv) or Fab format, displayed using either M13KO7 helper phage or Hyperphage. Solid-phase and solution-phase selection strategies were then applied to each library, generating six panning combinations. A total of sixty-four antibodies recognizing VEGF were obtained. Based on the results of epitope mapping, binding affinity, and biological functions in tumor inhibition, eight antibodies were chosen to examine their abilities in tumor regression in a mouse xenograft model using human COLO 205 cancer cells. Three of them showed improvement in the inhibition of tumor growth (328%–347% tumor growth ratio (% of Day 0 tumor volume) on Day 21 vs. 435% with Avastin). This finding suggests a potential use of these three antibodies for VEGF-targeted therapy.     

Myricanol Induces Apoptotic Cell Death and Anti-Tumor Activity in Non-Small Cell Lung Carcinoma in Vivo

This study explored the inhibiting effect and mechanism of myricanol on lung adenocarcinoma A549 xenografts in nude mice. Forty nude mice with subcutaneous A549 xenografts were randomly divided into five groups: high-dose myricanol (40 mg/kg body weight) group; middle-dose myricanol (20 mg/kg body weight) group; low-dose myricanol (10 mg/kg body weight) group; polyethylene glycol 400 vehicle group (1 mL/kg); and tumor model group. Nude mice were sacrificed after 14 days of treatment and the tumor inhibition rate (TIR, %) was then calculated. The relative mRNA expression levels of Bax, Bcl-2, VEGF, HIF-1α, and survivin in the tumor tissues were determined by real-time PCR. TUNEL assay was applied to determine cellular apoptosis, while IHC test was performed to detect the protein expression levels of Bax, Bcl-2, VEGF, HIF-1α, and survivin. The TIR of the three myricanol-treated groups ranged from 14.9% to 38.5%. The IHC results showed that the protein expression of Bcl-2, VEGF, HIF-1α, and survivin were consistently downregulated, whereas that of Bax was upregulated after myricanol treatment. Myricanol also significantly upregulated the mRNA expression of Bax and downregulated that of Bcl-2, VEGF, HIF-1α, and survivin in a dose-dependent manner (p < 0.05 to 0.001). These results are consistent with those of IHC. The TUNEL assay results indicated that apoptotic-positive cells significantly increased in the myricanol-treated tumor tissues compared with the cells of the vehicle control group (p < 0.01 to 0.001). These data suggest that myricanol could significantly decelerate tumor growth in vivo by inducing apoptosis.     

Smart polyion complex micelles for targeted intracellular delivery of PEGylated antisense oligonucleotides containing acid-labile linkages

A novel pH-sensitive and targetable antisense ODN delivery system based on multimolecular assembly into polyion complex (PIC) micelles of poly(L-lysine) (PLL) and a lactosylated poly(ethylene glycol)-antisense ODN conjugate (Lac-PEG-ODN) containing an acid-labile linkage (beta-propionate) between the PEG and ODN segments has been developed. The PIC micelles thus prepared had clustered lactose moieties on their peripheries and achieved a significant antisense effect against luciferase gene expression in HuH-7 cells (hepatoma cells), far more efficiently than that produced by the nonmicelle systems (ODN and Lac-PEG-ODN) alone, as well as by the lactose-free PIC micelle. In line with this pronounced antisense effect, the lactosylated PIC micelles showed better uptake than the lactose-free PIC micelles into HuH-7 cells; this suggested the involvement of an asialoglycoprotein (ASGP) receptor-mediated endocytosis process. Furthermore, a significant decrease in the antisense effect (27 % inhibition) was observed for a lactosylated PIC micelle without an acid-labile linkage (thiomaleimide linkage); this suggested the release of the active (free) antisense ODN molecules into the cellular interior in response to the pH decrease in the endosomal compartment is a key process in the antisense effect. Use of branched poly(ethylenimine) (B-PEI) instead of the PLL for PIC micellization led to a substantial decrease in the antisense effect, probably due to the buffer effect of the B-PEI in the endosome compartment, preventing the cleavage of the acid-labile linkage in the conjugate. The approach reported here is expected to be useful for the construction of smart intracellular delivery systems for antisense ODNs with therapeutic value.     

ELISA法间接检测血管内皮生长因子抑制剂的相对结合力

目的采用ELISA法间接检测血管内皮生长因子抑制剂(VEGF Trap)的相对结合力。方法以固定量的VEGF165与VEGF Trap结合,通过双抗体夹心ELISA法检测未结合的、游离的VEGF165,进而求得VEGFTrap的半数抑制浓度(IC50),计算其与反应体系中参比品的IC50之比,即为VEGFTrap的相对结合力。结果同一批次的3支VEGF Trap样品分别经3次测定,相对结合力分别为(100.86±9.43)%、(88.46±13.94)%和(82.66±9.60)%,均值为(90.66±12.60)%,符合该制品质量标准的要求。3次试验的变异系数分别为9.35%、15.76%和11.61%。结论 ELISA法精密性良好,结果客观,影响因素少,可用于VEGF Trap相对亲和力的常规检测。