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Numéro de publicationCN1434054 A
Type de publicationDemande
Numéro de demandeCN 03115489
Date de publication6 août 2003
Date de dépôt21 févr. 2003
Date de priorité21 févr. 2003
Autre référence de publicationCN1176937C
Numéro de publication03115489.1, CN 03115489, CN 1434054 A, CN 1434054A, CN-A-1434054, CN03115489, CN03115489.1, CN1434054 A, CN1434054A
Inventeurs白春学, 张新, 张敏, 陈杰
Déposant复旦大学附属中山医院
Exporter la citationBiBTeX, EndNote, RefMan
Liens externes:  SIPO, Espacenet
Dowble-stranded RNA and use thereof
CN 1434054 A
Résumé
The present invention relates to a double-stranded RNA and its application for inhibiting mammalian tumor cell. Said invention adopts the RNA interference technique to introduce the externally-synthetic double-stranded RNA into mammalian tumor cell by means of combination of non-viral carrier and/or viral carrier, and can inhibit the expression of epidermal growth factor receptor so as to change NSCLC cell biological character and raise the sensitivity of mammalian tumor cell to radiation therapy and chemical therapy. Said invented double-strand RNA can be used for preparing antitumor biological preparation and antitumor medicine to make direct injection in tumor body in travenous medication for effectively inhibiting tumor growth.
Revendications(7)  Langue du texte original : Chinois
1.一种双链RNA,其特征是具有如下序列的21-nt双链RNA。 1. A double-stranded RNA, wherein the 21-nt double-stranded RNA has the following sequence. GGAGCUGCCCAUGAGAAAUdTdT……siRNAAUUUCUCAUGGGCAGCUCCdTdT……siRNA complement GGAGCUGCCCAUGAGAAAUdTdT ...... siRNAAUUUCUCAUGGGCAGCUCCdTdT ...... siRNA complement
2.按权利要求1所述的双链RNA,其特征是所述的双链RNA是体外合成。 According to claim 2. wherein the double-stranded RNA 1, characterized in that said double-stranded RNA is synthesized in vitro.
3.按权利要求1和2所述的双链RNA,其特征是所述双链RNA与非病毒载体和/或病毒载体结合导入肿瘤细胞,制备抗肿瘤生物制剂和抗肿瘤药物。 3. according to claim 1 and wherein the double-stranded RNA 2, wherein said double-stranded RNA and non-viral vectors and / or viral vector in combination introduced into tumor cells, preparation of anti-tumor biological agents and antineoplastic agents.
4.按权利要求3所述的双链RNA,其特征是所述非病毒载体是阳离子脂质体。 According to claim 4. wherein the double-stranded RNA 3, wherein said non-viral vector is a cationic liposome.
5.按权利要求4所述的双链RNA,其特征是所述阳离子脂质体是Lipofecta-mineTM2000。 According to claim 5. wherein the double-stranded RNA 4, wherein said cationic liposome is Lipofecta-mineTM2000.
6.按权利要求3所述的双链RNA,其特征是所述肿瘤细胞是哺乳动物肿瘤细胞。 According to claim 6. wherein the double-stranded RNA 3, wherein said tumor cell is a mammalian tumor cell.
7.按权利要求3所述的双链RNA,其特征是所述肿瘤细胞是非小细胞肺癌细胞。 According to claim 7. wherein the double-stranded RNA 3, characterized in that the tumor cells are non-small cell lung cancer cells.
Description  Langue du texte original : Chinois
一种双链RNA及其用途 Double-stranded RNA and its use

技术领域 FIELD

本发明属生物技术领域,涉及一种双链RNA及其抗肿瘤用途。 The present invention belongs to the field of biotechnology, to a double-stranded RNA and anti-tumor purposes. 具体涉及一种21-nt双链RNA及其在抑制非小细胞肺癌细胞株表皮生长因子受体表达的用途。 In particular to a 21-nt double-stranded RNA and its inhibition of non-small cell lung cancer cell lines epidermal growth factor receptor expression purposes.

目前,抗肿瘤药物的研制主要针对治疗靶标的高通量药物筛选和针对治疗靶标的理性化药物设计。 At present, the development of anticancer drugs for the treatment of major targets for high-throughput drug screening and rational drug design for the treatment targets. 近年来受到广泛重视的反义核酸技术是后者的一个重要代表,通过设计和靶标蛋白基因的反义核酸,导入到细胞中,对靶标蛋白的表达进行抑制,达到杀死和治疗肿瘤的目的。 Widespread attention in recent years, antisense technology is an important representative of the latter, by designing and antisense nucleic acid target gene introduced into cells expressing the target protein to suppress, to kill, and the purpose of treatment of cancer . 反义核酸由于其作用靶点序列清楚、易设计制备,被认为是极具潜力的肿瘤治疗药物,但在实际应用中,存在抑制效率低,专一性差,较大的毒性以及用药量较大和成本高等缺陷。 Antisense nucleic acid target sequence because of its role clearly designed and easy preparation, is considered to be of great potential cancer therapy, but in practice, there is a low inhibition efficiency, poor specificity, toxicity and dosage larger and larger high cost defects.

RNA干扰(RNA interference,RNAi)是由双链RNA(double-stranded RNA,dsRNA)引发的序列依赖性的转录后基因沉默机制(posttranscriptional genesilencing,PTGS)。 RNA interference (RNA interference, RNAi) is a post by double-stranded RNA (double-stranded RNA, dsRNA) triggered sequence-dependent transcriptional gene silencing mechanism (posttranscriptional genesilencing, PTGS). 在哺乳动物细胞中,21-23nt dsRNA进入细胞后,能以其为模板,降解与之序列相应的mRNA,从而达到专一性抑制特定基因的蛋白生成。 In mammalian cells, 21-23nt dsRNA into the cell, can be used as templates, with the degradation of the corresponding mRNA sequence, so as to achieve specific inhibition of protein to generate a specific gene. RNAi是真核生物中普遍存在的抵抗病毒入侵、抑制转座子活动、调控基因表达的监控机制。 RNAi is ubiquitous in eukaryotes resistance to viruses, transposon activity inhibition, regulation of gene expression monitoring mechanism. 在药物开发方面,RNAi除了具有和反义核酸相同的优点外,还具有针对靶点抑制的专一性高,用量少,药物毒性小的特点。 In drug development, RNAi and antisense nucleic acids in addition to the same advantages, it also has a target for inhibition of high specificity, with less drug toxicity characteristics. 目前将RNAi技术用于抗病毒(如HIV病毒)和细胞信号传递系统的研究方兴未艾,但用于肿瘤治疗未见报道。 The technology is currently used antiviral RNAi (eg HIV virus) studies and cell signaling systems in the ascendant, but has not been reported for tumor therapy.

本发明的进一步目的是提供新的抗肿瘤生物制剂和抗肿瘤药物。 A further object of the present invention is to provide new antitumor biological agents and antineoplastic agents.

本发明选定哺乳动物肿瘤细胞表皮生长因子受体(EGFR)作为肿瘤治疗靶点,采用RNA干扰(RNAi)技术,体外合成与EGFR cDNA序列互补的21nt双链RNA,与非病毒载体和/或病毒载体结合导入哺乳动物肿瘤细胞,抑制表皮生长因子受体的表达,从而改变NSCLC细胞生物学特性,增加放化疗敏感性。 The present invention is selected mammalian tumor epidermal growth factor receptor (EGFR) as a target for cancer therapy, use of RNA interference (RNAi) 21nt double-stranded RNA technology, synthesized in vitro with EGFR cDNA sequence complementary to, and non-viral vectors, and / or viral vectors introduced into mammalian tumor cell binding, inhibition of epidermal growth factor receptor expression, thereby changing the biological characteristics of NSCLC cells, increased sensitivity to chemotherapy.

本发明所述哺乳动物肿瘤细胞表皮生长因子受体(epidermal growthfactor receptor,EGFR)是一分子量为170KD的跨膜糖蛋白,可调控细胞周期,调节细胞增生、存活、粘附、迁移和分化,促进损伤修复。 The present invention is a mammalian tumor cell epidermal growth factor receptor (epidermal growthfactor receptor, EGFR) is a transmembrane glycoprotein having a molecular weight of 170KD, can regulate the cell cycle, regulate cell proliferation, survival, adhesion, migration and differentiation and promote damage repair. EGFR在包括非小细胞肺癌(non-small-cell-lung cancer,NSCLC)在内的多种上皮源性肿瘤中过度表达,与肿瘤增生、转移、放化疗敏感性下降关系密切。 EGFR in a variety of epithelial tumors including non-small cell lung cancer (non-small-cell-lung cancer, NSCLC), including over-expression, and tumor proliferation, metastasis, decreased sensitivity to put close to chemotherapy.

本发明按下述方法和步骤进行,1、测定EGFR受体(1)选用EGFR阳性的细胞株A549、SPC-A-1,通过免疫组化法和免疫荧光法对EGFR定性、定位。 The present invention is carried out according to the following methods and procedures, a determination EGFR receptor (1) selection of cell line A549 EGFR-positive, SPC-A-1, by immunohistochemistry and immunofluorescence characterization of EGFR, location.

免疫组化设低倍镜、高倍镜阴性对照和阳性对照。 Magnification, high magnification negative and positive controls immunohistochemistry set low. 免疫组化结果阳性对照在细胞膜,细胞浆和细胞核显示棕色颗粒,阴性对照未见特异性着色。 Immunohistochemistry positive control in the cell membrane, cytoplasm and nucleus show brown particles, the negative control no specific coloration.

上述方法所用一抗为鼠抗人EGFR单克隆抗体,由中科院细胞生物所提供,二抗为SABC标记的兔抗鼠IgG。 The method described above is used a mouse anti-human anti-EGFR monoclonal antibodies, biological cells provided by the Chinese Academy of Sciences, rabbit anti-mouse IgG secondary antibody for SABC tag.

所述细胞株由中科院上海细胞生物学研究所细胞库提供。 The cells were provided by the Institute of Cell Biology, Shanghai Institute of Cell Bank.

免疫荧光法设阴性和阳性对照荧光照片。 Immunofluorescence based fluorescence photo negative and positive controls. 试验结果同免疫组化染色。 Test results with immunohistochemical staining. 所述二抗采用FITC标记的兔抗鼠IgG。 The secondary antibody using rabbit anti-mouse IgG FITC-labeled.

(2).测定受体数量采用流式细胞仪测定受体数量,结果为A549:%gated:78.58%;mean:54.51,SPC-A-1:%gated:98.89%mean:103.79,H69:%gated:0.67%mean:67.54。 (2) the number of receptors was measured using flow cytometry receptor number, the result is A549:% gated:. 78.58%; mean: 54.51, SPC-A-1:% gated: 98.89% mean: 103.79, H69:% gated: 0.67% mean: 67.54.

2、筛选EGFR高表达细胞株取细胞株A549,SPC-A-1,ECV,HFL,H7402,BEL7402、SKOV3和H69进行筛选,结果表明A549,SPC-A-1为EGFR高表达细胞株,ECV、HFL、H7402、BEL7402、SKOV3细胞株EGFR表达较低。 2, EGFR overexpression cell line screening cell lines take A549, SPC-A-1, ECV, HFL, H7402, BEL7402, SKOV3 and H69 filter, the results show A549, SPC-A-1 cell line for EGFR expression, ECV , HFL, H7402, BEL7402, low EGFR expression in SKOV3 cell lines. H69细胞株无EGFR表达。 H69 cell line without EGFR expression. 设H69细胞株为阴性对照。 H69 cells were set up as a negative control.

所述细胞株由中科院上海细胞生物学研究所细胞库提供。 The cells were provided by the Institute of Cell Biology, Shanghai Institute of Cell Bank.

3、按下述原则设计ssRNA,1)EGFR的cDNA启动子AUG下游75碱基处,2)找到第一个AA二聚体,3)记录AA下游19个核苷酸(siRNA-EGFR),其核苷酸序列为:5'AAGGAGCUGCCCAUGAGAAAU……mRNA4)计算嘌呤和嘧啶的百分比(G/C),G/C比值为30%至70%,50%最佳。 3, according to the following principles designed ssRNA, 1) EGFR promoter of cDNA downstream AUG 75 bases, 2) find the first AA dimer, 3) AA record 19 nucleotides downstream (siRNA-EGFR), nucleotide sequence: 5'AAGGAGCUGCCCAUGAGAAAU ...... mRNA4) calculating the percentage of purines and pyrimidines (G / C), G / C ratio of 30 to 70%, 50% preferred.

4、按已知的体外化学合成方法合成siRNA-EGFR,其核苷酸序列为:GGAGCUGCCCAUGAGAAAUdTdT……siRNAAUUUCUCAUGGGCAGCUCCdTdT……siRNA complement5、拟和(annealing)siRNA双链1)独立分装上述化学合成的RNA单链,用无RNase水稀释,2)正义、反义RNA混合,加入拟和缓冲液, 4, according to the known chemical synthetic methods in vitro synthesized siRNA-EGFR, which nucleotide sequences: GGAGCUGCCCAUGAGAAAUdTdT ...... siRNAAUUUCUCAUGGGCAGCUCCdTdT ...... siRNA complement5, fitting (annealing) siRNA duplexes 1) independent dispensing the chemical synthesis of RNA single chain, diluted with RNase-free water, 2) sense and antisense RNA were mixed, and the proposed buffer

3)90℃孵育,离心,收集管底液体。 Incubate 3) 90 ℃, centrifugation, collect the liquid bottom of the tube.

4)37℃孵育1小时。 4) 37 ℃ for 1 hour.

5)拟和的双链RNA的终浓度为20uM。 5) intends to double-stranded RNA and a final concentration of 20uM. 形成的双链具有如下序列。 Double-stranded form has the following sequence. GGAGCUGCCCAUGAGAAAUdTdT……siRNAAUUUCUCAUGGGCAGCUCCdTdT……siRNA complement6、sRNA-unrelated的设计、合成以及拟和同dsRNA-EGFR。 GGAGCUGCCCAUGAGAAAUdTdT ...... siRNAAUUUCUCAUGGGCAGCUCCdTdT ...... siRNA complement6, sRNA-unrelated design, synthesis and fitting with dsRNA-EGFR. 序列为:5'-GAACUUCAGGGUCAGCUUGCCUU-3'——dsRNA-unrelated5'-GGCAAGCUGACCCUGAAGUUCUU-3'——unrelated completement7、15%PAGE-SDS电泳鉴定拟和的双链,拟和完全,双链片段大小正确。 Sequence: 5'-GAACUUCAGGGUCAGCUUGCCUU-3 '- dsRNA-unrelated5'-GGCAAGCUGACCCUGAAGUUCUU-3' - unrelated completement7,15% PAGE-SDS electrophoresis and identified the proposed double-stranded, and fully intends to double-stranded fragments of the correct size.

8、筛选阳离子脂质体取阳离子脂质体TKO,Oligofectamine,LipofectamineTM2000进行筛选,采用高转导效率的LipofectamineTM2000用于dsRNA的转导。 8, screening cationic liposomes take cationic liposomes TKO, Oligofectamine, LipofectamineTM2000 screening, high transduction efficiency LipofectamineTM2000 for dsRNA transduction.

9、RNA干扰技术用于肿瘤细胞:1)制备贴壁细胞,2)制备dsRNA-Lipofectamine复合物,3)流式细胞仪检测EGFR受体数目。 9, RNA interference technique for tumor cells: 1) Preparation of adherent cells, 2) Preparation of dsRNA-Lipofectamine complex, 3) the number of flow cytometry EGFR receptor.

实验结果证实,A549细胞株EGFR表达降低了73.23%,SPC-A-1细胞株受体表达降低了76.01%。 Experimental results show that, EGFR expression in A549 cells decreased 73.23%, receptor expression in SPC-A-1 cell line decreased 76.01%. 本发明将体外合成的dsRNA借助阳离子脂质体导入哺乳动物肿瘤细胞,能抑制表皮生长因子受体的表达,从而改变NSCLC细胞生物学特性,增加哺乳动物肿瘤细胞对放化疗的敏感性,获得肯定的RNAi效果。 The present invention is synthesized in vitro by means of cationic liposomes dsRNA introduced into mammalian tumor cells, can inhibit the expression of epidermal growth factor receptor, thereby altering the biological characteristics of NSCLC cells, mammalian tumor cells increased the sensitivity to radiotherapy and chemotherapy, obtaining a positive The RNAi effect.

本发明双链RNA可与非病毒载体和/或病毒载体结合,制备新的抗肿瘤生物制剂和抗肿瘤药物,行瘤体内直接注射或静脉给药有效抑制肿瘤生长。 Double-stranded RNA of the present invention may be combined with non-viral vectors and / or viral vectors, the preparation of novel antitumor biological agents and anticancer drugs, the line direct intratumoral injection or intravenous administration effectively inhibit tumor growth.

其中1:分子量标准,2:正义RNA-EGFR,3:反义RNA-EGFR, Where 1: molecular weight standards, 2: Justice RNA-EGFR, 3: antisense RNA-EGFR,

4:双链RNA-EGFR,5:非特异双链RNA图2是RNAi对SPC-A-1细胞EGFR的抑制作用。 4: double-stranded RNA-EGFR, 5: non-specific double-stranded RNA of FIG. 2 is a RNAi SPC-A-1 cells EGFR inhibition.

按已知体外化学合成方法合成siRNA-EGFR,其序列为:5'AAGGAGCUGCCCAUGAGAAAU……mRNA,GGAGCUGCCCAUGAGAAAUdTdT……siRNA,长度:21,分子量:6732.9(g/mole),ODU260:31.9(ug/ODU260),数量:50nmol/管×2管,AUUUCUCAUGGGCAGCUCCdTdT……siRNA complement长度:21,分子量:6583.7(g/mole),ODU260:33.4(ug/ODU260),数量:50nmol/管×2管,上述合成的每条RNA单链独立分装,RNase灭活的水稀释成50uM,正义RNA30ul与反义RNA30ul混合,加入15ul 5×拟和缓冲液,终体积为75ul,90℃孵育1分钟,离心15秒,收集管底液体,37℃孵育1小时,dsRNA双链终浓度为20uM。 By known chemical synthetic methods in vitro synthesized siRNA-EGFR, whose sequence is: 5'AAGGAGCUGCCCAUGAGAAAU ...... mRNA, GGAGCUGCCCAUGAGAAAUdTdT ...... siRNA, length: 21, molecular weight: 6732.9 (g / mole), ODU260: 31.9 (ug / ODU260), Quantity: 50nmol / tube × 2 管, AUUUCUCAUGGGCAGCUCCdTdT ...... siRNA complement Length: 21, molecular weight: 6583.7 (g / mole), ODU260: 33.4 (ug / ODU260), Quantity: 50nmol / tube × 2 tubes, each of the above synthetic single-stranded RNA independent aliquots diluted RNase inactivation of water into 50uM, justice RNA30ul mixed with antisense RNA30ul, adding 15ul 5 × buffer proposed and final volume of the incubation 75ul, 90 ℃ 1 minutes, and centrifuged for 15 seconds, the collection tube Bottom liquid, 37 ℃ incubated for 1 hour, dsRNA duplexes final concentration of 20uM. 拟和缓冲液的组成为:100mMKOAc,30mMHEPES-KOHpH7.4,2mM MgOAc。 And buffer composition intended for: 100mMKOAc, 30mMHEPES-KOHpH7.4,2mM MgOAc. 经15%PAGE-SDS电泳,结果表明拟和的双链拟和完全,双链片段大小正确。 By 15% PAGE-SDS electrophoresis, and the results show that the proposed double-stranded and fully intends to double-stranded fragments of the correct size.

实施例2 RNA干扰技术用于非小细胞肺癌细胞制备贴壁细胞:转染前一天接种A549或SPC-A-1细胞于12孔培养板,使得次日转染的时候细胞密度达40-50%(0.5-2×105细胞/孔/12孔培养板),更换培养液(DMEM+10%FBS),体积1ml。 EXAMPLE 2 RNA interference technique used in the preparation of non-small cell lung cancer cells adherent cells: the day before transfection inoculation A549 or SPC-A-1 cells in 12-well culture plate, making the cells transfected the next day, when the density of 40-50 % (0.5-2 × 105 cells / well / 12 well plate), replacement of culture medium (DMEM + 10% FBS), the volume of 1ml.

制备dsRNA-Lipofectamine复合物:1)将dsRNA稀释于100ul无血清DMEM,混匀。 Preparation of dsRNA-Lipofectamine complexes: 1) The dsRNA was diluted in 100ul of serum-free DMEM, and mix.

2)Lipofectamine使用前混匀,然后4ul Lipofectamine与100ul无血清DMEM混匀。 2) Lipofectamine mix before use, then 4ul Lipofectamine and 100ul serum free DMEM mix. 室温孵育5min。 Incubated at room temperature for 5min.

3)将dsRNA稀释液与Lipofectamine稀释液混匀,总体积200ul,室温孵育20min,室温保存。 3) The dsRNA dilution with Lipofectamine dilution mixing, the total volume of 200ul, incubated at room temperature for 20min, stored at room temperature.

4)加入12孔培养板,混匀,培养箱内孵育24-48小时。 4) was added to 12-well culture plate, mix, incubate incubator for 24-48 hours.

流式细胞仪检测受体数目,结果为:A549细胞株EGFR表达降低了73.23%(dsRNA-EGFR16ul/lipofectamine2000 4ul/12孔培养板)。 Flow cytometry detection of receptor number, the result is: EGFR expressing A549 cells reduced 73.23% (dsRNA-EGFR16ul / lipofectamine2000 4ul / 12 well plate). SPC-A-1细胞株受体表达降低了76.01%(dsRNA-EGFR16 ul/lipofectamine 2000 4ul/12孔培养板)。 Receptor expression in SPC-A-1 cell line decreased 76.01% (dsRNA-EGFR16 ul / lipofectamine 2000 4ul / 12-well plates).

结果证实,本发明dsRNA能抑制表皮生长因子受体的表达,改变NSCLC细胞生物学特性,增加哺乳动物肿瘤细胞对放化疗的敏感性,获得肯定的RNAi效果。 The results confirmed that, dsRNA present invention can inhibit the epidermal growth factor receptor expression, alter the biological properties of NSCLC cells, increasing the sensitivity of tumor cells in mammals chemotherapy, received a positive RNAi effect.

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Classifications
Classification internationaleC07H21/02, A61P35/00, A61K31/7088, A61K48/00
Événements juridiques
DateCodeÉvénementDescription
6 août 2003C06Publication
22 oct. 2003C10Request of examination as to substance
24 nov. 2004C14Granted
11 mai 2011C17Cessation of patent right