CN101974529A - 含自由三磷酸基团的TGF-β特异性siRNA及其应用 - Google Patents
含自由三磷酸基团的TGF-β特异性siRNA及其应用 Download PDFInfo
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Abstract
本发明属于RNA干扰技术领域，公开了含自由三磷酸基团的TGF-β特异性siRNA及其应用。该含自由三磷酸基团的TGF-β特异性siRNA的反义链和正义链序列5’端均为鸟嘌呤核苷，该鸟嘌呤核苷的戊糖3’位点上修饰有一个自由三磷酸基团。本发明siRNA结合了TGF-β特异性基因沉默作用机制和诱导真核细胞抗病毒的固有免疫作用机制，不仅能抑制介导肿瘤免疫逃逸的重要分子TGF-β，还能有效激活机体的抗肿瘤免疫，能显著性地提高治疗肿瘤的效果。有效地解决了传统siRNA(OH-RNA)仅有单一基因沉默功能，而治疗肿瘤疗效不佳的问题。本发明ppp-TGF-β可在制备治疗肿瘤特别是胰腺癌的药物中应用。
Description
技术领域
本发明属于RNA干扰技术领域，涉及siRNA及其应用，具体涉及含自由三磷酸基团的TGF-β特异性siRNA及其应用。
背景技术
由于早期的转移、高度耐受化疗和放疗等原因，使胰腺癌的预后极差。在胰腺癌的进程中，生长转化因子β(TGF-β)的异常表达起到了关键性的作用。高表达的TGF-β能促进肿瘤的生长、侵润、转移及血管生成，另外，肿瘤来源的TGF-β具有很强的抑制机体免疫的作用，能帮助肿瘤建立免疫逃逸机制。如何抑制肿瘤介导的免疫抑制，是今后治疗胰腺癌的一大挑战。针对这个与肿瘤生长、浸润、转移及免疫逃逸相关的重要分子，目前最有效的方法之一是使用小干扰核糖核酸技术(siRNA)直接沉默，以阻断其促进肿瘤生长的通路。这种含21-23个碱基对的特异双链RNA序列，其反义链能靶向识别这些分子相应的信使RNA(mRNA)，并在相关酶的协同作用下，剪切mRNA从而干预TGF-β的蛋白合成。但前期临床肿瘤治疗效果并不理想，原因可能与其作用靶点单一、不能有效激活机体对肿瘤的免疫监视/杀伤功能、缺乏直接促进肿瘤细胞凋亡作用等有关。
发明内容
本发明的目的是针对现有技术的上述不足，提供含自由三磷酸基团的TGF-β特异性siRNA，该ppp-siRNA分子同时具备TGF-β特异性基因沉默、激活机体免疫应答和直接促进肿瘤细胞凋亡的三重功能，从而能很好的抑制肿瘤。
本发明的另一目的是提供该siRNA的应用。
本发明的技术方案如下：
含自由三磷酸基团的TGF-β特异性siRNA(简称为ppp-TGF-β)，其反义链和正义链序列5’端均为一鸟嘌呤核苷，该鸟嘌呤核苷戊糖3’位点上修饰有一个自由三磷酸基团。
所述的TGF-β特异性siRNA序列选自：ppp-TGF-β1，其正义链序列为SEQ IDNO.1，反义链序列为SEQ ID NO.2；ppp-TGF-β2，其正义链序列为SEQ ID NO.3，反义链序列为SEQ ID NO.4。
本发明所述的含自由三磷酸基团的TGF-β特异性siRNA在制备治疗肿瘤的药物中的应用。
本发明所述的含自由三磷酸基团的TGF-β特异性siRNA在制备治疗胰腺癌的药物中的应用。
本发明的有益效果：
发明人通过大量实验设计并筛选出具有良好基因沉默功能的小鼠和人TGF-β特异性siRNA，通过设计5’→3’端依次含有TGF-β特异性siRNA的eDNA序列、胞嘧啶核苷及T7RNA聚合酶启动子序列的DNA模板，在体外利用T7RNA聚合酶转录合成得到5’端含自由三磷酸基团的TGF-β特异性siRNA(以下简称ppp-TGF-β)。该ppp-TGF-β通过如下三种机制抑制肿瘤：
(1)本发明ppp-TGF-β能有效地特异性沉默TGF-β基因，抑制TGF-β蛋白的表达。
(2)本发明ppp-TGF-β能被细胞胞浆内的特异性识别受体-视磺酸诱导基因-I(RIG-I，一种真核细胞胞浆螺旋酶，参与机体抗病毒的固有免疫)识别，并激活其下游I-型干扰素信号转导通路，能诱导细胞产生IFN-β和干扰素诱导蛋白10(IP-10)、显著提高肿瘤细胞免疫递呈分子MHC-I的表达(能提高机体免疫系统对肿瘤细胞的识别、攻击)，从而激活细胞及机体对肿瘤的免疫应答。
(3)本发明ppp-TGF-β还能激活内源性凋亡通路中caspase-9而促进肿瘤细胞凋亡。
本发明将TGF-β特异性基因沉默作用机制和诱导真核细胞抗病毒的固有免疫作用机制，引入到抗肿瘤的治疗中，不仅能抑制介导肿瘤免疫逃逸的重要分子TGF-β，还能有效激活机体的抗肿瘤免疫，能显著性地提高治疗肿瘤的效果。有效地解决了传统siRNA(OH-RNA)仅有单一基因沉默功能，而治疗肿瘤疗效不佳的问题。故本发明ppp-TGF-β可在制备治疗肿瘤特别是胰腺癌的药物中应用。
附图说明
图1实施例2中定量RT-PCR检测ppp-TGF-β特异性基因沉默及免疫诱导功能结果图。
其中，图1A为ppp-TGF-β体外转染人胰腺癌细胞，对TGF-βmRNA表达量的影响；图1B为ppp-TGF-β体外转染人胰腺癌细胞，对I-型干扰素mRNA的表达量的影响；图1C为ppp-TGF-β体外转染鼠胰腺癌细胞，对TGF-βmRNA表达量的影响；图1D为ppp-TGF-β体外转染人胰腺癌细胞，对I-型干扰素mRNA的表达量的影响；图1E为鼠ppp-TGF-β经静脉注射胰腺癌小鼠，对其肿瘤实体中TGF-β的mRNA表达水平的影响；图1F为鼠ppp-TGF-β经静脉注射胰腺癌小鼠，对其肿瘤实体中I-型干扰素的mRNA表达水平的影响；图1G为鼠ppp-TGF-β经静脉注射胰腺癌小鼠，对其肿瘤实体中干扰素诱导蛋白(IP-10)的mRNA表达水平的影响。
图2实施例3中ELISA检测ppp-TGF-β基因沉默及免疫诱导功能结果图。
其中图2A～2B依次为ppp-TGF-β体外转染小鼠胰腺癌细胞，24h后对TGF-β蛋白水平和干扰素诱导蛋白水平的影响；图2C～图2F为鼠ppp-TGF-β治疗的荷瘤小鼠，对血浆TGF-β水平的抑制情况；图2G～2I为鼠ppp-TGF-β静脉注射荷瘤小鼠6小时后，对血浆IFN-α、IP-10和TNF-α蛋白水平的影响。
图3ppp-TGF-β转染胰腺癌细胞，肿瘤细胞表面I-型抗原呈递分子表达情况。
其中见图3A为人ppp-TGF-β转染不同人胰腺癌细胞，肿瘤细胞表面I-型抗原呈递分子表达情况；图3B为鼠ppp-TGF-β转染鼠胰腺癌细胞，肿瘤细胞表面I-型抗原呈递分子表达情况。
图4ppp-TGF-β静脉注射小鼠后12小时，B淋巴细胞、CD4、CD8淋巴细胞、NK细胞及NKT细胞的活化情况。
图5ppp-TGF-β处理鼠及人胰腺癌细胞后48小时，细胞凋亡情况。
图6实施例4中ppp-TGF-β转染人及鼠的胰腺癌细胞48小时后，活化caspase-9在细胞中表达情况。
图7ppp-TGF-β转染小鼠胰腺癌细胞后48小时，肿瘤细胞活性情况。
图8ppp-TGF-β转染人胰腺癌细胞后48小时，肿瘤细胞活性情况。
图9实施例6中ppp-TGF-β转染小鼠胰腺癌细胞后24小时，胞内活化的caspase-9荧光免疫染色照片。
图10ppp-TGF-β治疗原位胰腺癌小鼠组，肿瘤TUNEL染色结果。
图11小鼠生存曲线图。
本发明各幅附图横坐标：A均为空白组，即未给予RNA治疗组；B均为OH-RNA组，即给予无意义siRNA治疗组；C均为ppp-RNA组，即给予含自由三磷酸基团的无意义siRNA治疗组；D均为OH-TGF-β组，即给予TGF-β特异性siRNA治疗组；E均为ppp-TGF-β组，即给予5’端含自由三磷酸基团的TGF-β特异性siRNA治疗组。
具体实施方式
实施例1 本发明ppp-TGF-β的合成
1.1人及小鼠TGF-β特异小干扰RNA的设计和筛选：
根据PUBMED基因库中，人及小鼠的TGF-βmRNA完整编码序列，使用siRNA设计软件(Dharmacon RNAi Technologies)，筛选出数条相应匹配的反义短序列，各含19核糖核苷。在体外合成相应的siRNA(Metabion，Germany)，并于每条单链3’端链接2个游离尿嘧啶核苷，用作体外筛选及抑瘤实验的OH-TGF-β组。
将合成的siRNA序列(OH-TGF-β)，在体外用脂质体(Lipofectamine 2000)按0.5微克/微升在OptiMEM培养基中混合包被，体外转染胰腺癌细胞，在不同的时间24h、48h及72h提取细胞RNA，用定量RT-PCR方法检测TGF-β1的mRNA表达量，根据对TGF-β的抑制作用效果而选择抑制效果最佳的siRNA，从而得到两条TGF-β特异性siRNA。其中一条为人TGF-β特异性siRNA，其正义链序列为SEQ ID NO.5，反义链序列为SEQID NO.6；另一条为鼠TGF-β特异性siRNA，其正义链序列为SEQ ID NO.7，反义链序列为SEQ ID NO.8。
同时，设计并合成对人及小鼠均无意义siRNA，其正义链序列为SEQ ID NO.9，反义链序列为SEQ ID NO.10，该序列通过与PUBMED基因库中相关mRNA进行匹配分析，证实无任何干扰作用。合成时为增强其稳定性，于每条单链3’端链接2个游离尿嘧啶核苷。该3’端链接2个UU的无意义siRNA，在考察抑瘤效果时作为OH-RNA对照。
1.2合成ppp-TGF-β的DNA模板设计：
根据1.1筛选得到的序列，在相应的正义链或反义链的cDNA序列的3’端依次链接一个胞嘧啶核苷和一段T7RNA聚合酶启动子序列SEQ ID NO.11，作为编码相应的ppp-TGF-β正义链或反义链的DNA模板，序列见表1，送交基因公司(Metabion，Germany)合成。其中胞嘧啶的加入是为了增强转录效率，T7RNA聚合酶启动子序列的加入是使T7RNA聚合酶能特异识别并启动从5’端向3’端的转录，从而得到在5’端核糖核苷的戊糖3’位点上修饰有一自由三磷酸基团转录的产物，即ppp-TGF-β。
表1.在体外利用T7RNA聚合酶合成ppp-TGF-β的DNA模板
| 类别 | 序列 |
| 无意义siRNA正义链DNA模板 | SEQ IDNO.12 |
| 无意义siRNA反义链DNA模板 | SEQ IDNO.13 |
| 含自由三磷酸基团的人TGF-β特异性siRNA正义链DNA模板 | SEQ IDNO.14 |
| 含自由三磷酸基团的人TGF-β特异性siRNA反义链DNA模板 | SEQ IDNO.15 |
| 含自由三磷酸基团的鼠TGF-β特异性siRNA正义链DNA模板 | SEQ IDNO.16 |
| 含自由三磷酸基团的鼠TGF-β特异性siRNA反义链DNA模板 | SEQ IDNO.17 |
1.3DNA模板双链的合成：
A.引物与模板链杂交：体外合成识别T7RNA聚合酶启动子序列的引物：SEQ IDNO.18(Metabion，Germany)。于PCR反应微管中，加入10微升杂交缓冲液、2微升引物、2微升相应的DNA模板，于70℃孵育5分钟，再于25℃孵育约25分钟。
B.在上述PCR反应管中，加入5微升不含RNA酶的纯水、2微升Klenow缓冲液、2微升dNTP混合液(2.5mM)和1微升Exo-Minus Klenow DNA多聚酶(20单位/微升，Fermentas)，置于37℃孵育30分钟，以合成双链DNA。PCR反应管再于70℃孵育5分钟，以灭活剩余Exo-Minus Klenow DNA多聚酶。
1.4ppp-TGF-β体外转录：
自由三磷酸基团siRNA合成的原理：T7RNA聚合酶能特异识别DNA模板链上的T7RNA聚合酶启动子序列，并启动从5’端向3’端(即从T7RNA聚合酶启动子端向另一端)的转录，并在RNA的转录过程中，于新合成的RNA序列5’端的首个核糖核苷戊糖3’位点上，修饰一个不被其它分子包被的自由三磷酸基团。
合成过程与反应体系：在灭菌PCR反应管中，加入6微升不含RNA酶的无菌纯水、2微升10xT7RxN缓冲液、8微升NTP混合物、2微升双链DNA模板和2微升T7RNA聚合酶(MegashortskriptTMT7kit，Ambion)，在37℃过夜，合成5’端含三磷酸基团TGF-βsiRNA的正义链或反义链。再将合成的正义链和反义链混合，置于37℃过夜，杂交形成稳定双链(即为ppp-TGF-β)。其中人ppp-TGF-β(即ppp-TGF-β1)其正义链序列为SEQID NO.1，反义链序列为SEQ ID NO.2；鼠ppp-TGF-β(即ppp-TGF-β2)，其正义链序列为SEQ ID NO.3，反义链序列为SEQ ID NO.4。
利用上述方法，转录合成5’端含自由三磷酸基团的无意义siRNA，正义链序列为SEQID NO.19，反义链序列为SEQ ID NO.20，在考察抑瘤效果时作为ppp-RNA对照。
1.5ppp-TGF-β的提纯：
在1.4合成产物中，加入2微升Turbo-DNA酶并孵育30分钟去除残存的DNA链，再加入30微升乙酸铵灭活DNA酶，将合成产物转移到1.5毫升反应管，加入115微升不含RNA酶的纯水，再加入300微升酚：氯仿混合物混合均匀，于12000G室温下离心5分钟，将上层苯酚(相)吸出，转移到新的1.5毫升EP管中，再加入300微升氯仿，混合均匀后，于12000G室温下离心5分钟，吸出上层苯酚(相)，转移至新的EP管。向苯酚/RNA混合液中加入600微升100％乙醇，吹打均匀，将混合物置于-20℃过夜，使RNA沉淀析出，再于14000G 4℃离心30分钟，取出EP管立即置于冰上，弃去上清液，将所得的RNA沉淀溶于50微升不含RNA酶纯水中。进一步可以将RNA加入到Mini QuickSpin Columns(Roche)过柱，离心进行提纯，最后进行RNA定量及纯度测定。
实施例2 定量RT-PCR检测ppp-TGF-β基因沉默及免疫诱导功能：
细胞、小鼠的转染：人胰腺癌细胞株PANC-1，MIAPaCa-2(来自美国ATCC)，PaTu8988t(DSMZ，德国微生物菌种保藏中心)、BxPC-3(中国科学院上海生科院细胞资源中心)、IMIM PC-1(由德国Marburg大学Patrick Michl博士赠送，其分离及体外培养方法由ViláMR等详细描述[1]。Panc02细胞株是由甲基胆蒽诱导的小鼠胰腺腺癌(德国慕尼黑大学)，其诱导及培养方法首先由Corbett等人公开[2]。
2.1体外转染：肿瘤细胞株按3x105每孔种植在6孔细胞培养板中。将RNA用Lipofectamine 2000(Invitrogen)按厂家说明书方法包被，然后分别溶于OptiMEM培养基(Gibco BRL)，终浓度为0.5微克/毫升，用于体外转染细胞。转染细胞18或24小时后用胰酶消化细胞，离心收集细胞(以下体外转染细胞同)。实验共有为五组，A组为空白组，即空白Lipofectamine 2000，B组为Lipofectamine 2000包被的OH-RNA，C组为Lipofectamine 2000包被的ppp-RNA，D组为Lipofectamine 2000包被的OH-ppp-TGF-β，E组为Lipofectamine 2000包被的ppp-TGF-β，以下实验的分组标记同本实验。
2.2体内实验，2x105鼠Panc02细胞株经开腹手术注射至6周龄雌性小鼠C57B1/6(Harlan-Winkelmann，每只平均体重约25克)胰腺包膜下，12天后，将50微克RNA按说明书的方法包被于in vivo-JetPEI试剂(Peqlab)，并溶于200微升5％葡萄糖溶液中静脉注射小鼠，48小时后取出肿瘤组织。实验分组情况同2.1体外转染实验。
mRNA的提取与定量：将肿瘤细胞和组织在液氮中速冻后匀浆，用RNeasy试剂盒(Qiagen，Invitrogen)按说明书提供的方法提取细胞总mRNA，然后用DNA合成试剂盒(New England BioLabs，Germany)，按说明书提供的方法转录成cDNA，最后，定量RT-PCR采用LC 480Probes Master试剂盒在Light Cycler 480定量RT-PCR(Roche)仪上检测相关指标.引物根据the Universal Probes library(Roche)设计并由Metabion(Martinsried，Germany)合成。每样本拷贝数均以HPRT拷贝数为参考。
结果显示：含自由三磷酸基团的人TGF-β特异性siRNA(简称人ppp-TGF-β)体外转染人胰腺癌PANC-1细胞，其TGF-βmRNA的表达量显著降低(见图1A)，人ppp-TGF-β转染不同的人胰腺癌细胞，I-型干扰素mRNA表达水平均较OH-TGF-β显著增高(见图1B)；含自由三磷酸基团的小鼠TGF-β特异性siRNA(简称鼠ppp-TGF-β)体外转染小鼠胰腺癌细胞Panc02，其TGF-βmRNA的表达量显著降低(见图1C)，鼠ppp-TGF-β转染小鼠胰腺癌细胞Panc02，其I-型干扰素mRNA表达量较OH-TGF-β显著增高(见图1D)，鼠ppp-TGF-β经静脉注射胰腺癌小鼠，其肿瘤实体中TGF-β的mRNA表达水平较对照组显著降低，I-型干扰素的mRNA表达水平和干扰素诱导蛋白mRNA水平较OH-TGF-β显著增高(见图1E～G)。
实施例3 酶联免疫吸附法(ELISA)检测ppp-TGF-β基因沉默及免疫诱导功能
小鼠胰腺癌肿瘤细胞Panc02或实验小鼠按实施例2所述方法转染RNA或接受RNA治疗后，在不同时间点收集细胞培养上清、小鼠的血清或肿瘤实体的匀浆上清液，用酶联免疫吸附检测试剂盒，按厂家说明书提供的方法分别检测IFN-α(PBL Interferon source)、IP-10(R&D Systems)、TGF-β(eBiosciences)和TNF-α(BD Biosciences)。结果显示：ppp-TGF-β体外转染小鼠胰腺癌细胞Panc02，24小时后TGF-β蛋白水平显著降低(见图2A)，干扰素诱导蛋白水平较OH-TGF-β显著增高(见图2B)；鼠ppp-TGF-β治疗的荷瘤小鼠，其血浆TGF-β水平在治疗后14天及25天后，仍被显著抑制(见图2C～F)；鼠ppp-TGF-β静脉注射荷瘤小鼠6小时后，血浆IFN-α、IP-10和TNF-α蛋白水平较OH-TGF-β显著增高(见图2G～I)。
实施例4 流式细胞分析法(FACS)检测ppp-TGF-β免疫诱导及促进肿瘤细胞凋亡的功能
人及小鼠胰腺癌细胞表面I-型抗原递呈分子检测抗体分别为抗人HLA-A，B，C (cloneG46-2.6，BD Pharmingen)或抗鼠H-2Kb(clone AF6-88.5，BD Pharmingen)，在流式细胞分析仪(BD)上检测RNA处理后肿瘤细胞的表达。结果显示人ppp-TGF-β转染不同人胰腺癌细胞，肿瘤细胞表面I-型抗原呈递分子表达均较OH-TGF-β处理后显著增高(见图3A)，ppp-TGF-β转染小鼠胰腺癌细胞Panc02后，I-型抗原呈递分子表达较OH-TGF-β增高约5倍(见图3B)。
淋巴细胞活化实验：小鼠在接受RNA治疗后12小时，取出小鼠脾脏，通过40μm细胞滤网分离单细胞悬液，红细胞则用BD PharmLyseTM裂解液(BD Bioscience)去除。再将脾脏细胞用CD69(clone H1.2F3，Caltag Laboratories)单克隆抗体作为细胞活化的阳性标记，结合其它小鼠单克隆抗体，检测B细胞(CD3-CD19+)、辅助性T细胞(CD3+CD4+)、CD8+性T细胞(CD3+CD8+)、NK细胞(CD3-NK1.1+)、NKT(CD3+NK1.1+)中活化水平，抗体均购自BD Biosciences。结合抗体后，细胞在流式细胞分析仪上检测。结果用FlowJo软件进行分析(版本8.5.3，Tree Star Inc.，Ashland，Oregon)。结果显示ppp-TGF-β静脉注射小鼠后12小时，活化的B淋巴细胞、CD4、CD8淋巴细胞、NK细胞及NKT细胞数较OH-TGF-β显著增高(见图4)。
细胞凋亡检查：经RNA处理后48小时的肿瘤细胞，用Annexin V-APC(Invitrogen)和Propidium iodide(PI，Sigma)进行荧光染色，再于流式细胞分析仪检测细胞凋亡。结果显示：ppp-TGF-β处理鼠及人胰腺癌细胞后48小时，Annexin-V阳性的凋亡细胞数较OH-TGF-β显著增高(见图5)。
肿瘤细胞Caspase-9活化检测：肿瘤细胞经RNA转染后48小时，细胞用Caspase-9FLICA试剂盒(ImmunoChemistry，Biomol)进行免疫荧光染色，活化的Caspase-9用流式细胞分析仪检测。结果显示：ppp-TGF-β转染人及鼠的胰腺癌细胞48小时后，活化的caspase-9阳性细胞较OH-TGF-β显著增高(见图6)。
实施例5 XTT法检测肿瘤细胞活性
ppp-TGF-β转染细胞后，采用XTT法(Sigma-Aldrich)检测细胞活性。细胞以4x103每孔种植于96孔板使贴壁，用2微克/毫升RNA浓度转染细胞，48小时后，将培养基更换为含20％XTT无酚红培养基，孵育4小时后在450纳米波长下检测吸光度。结果显示：ppp-TGF-β转染小鼠胰腺癌细胞后48小时，细胞活性较OH-TGF-β组显著降低，并呈剂量依赖趋势(见图7)；ppp-TGF-β转染人胰腺癌细胞PANC-148小时，肿瘤细胞活性较OH-TGF-β显著降低(见图8)。
实施例6 免疫荧光法检测ppp-TGF-β体内促肿瘤凋亡作用及诱导肿瘤凋亡机制
体外实验：小鼠肿瘤细胞经RNA按实施例2的方法处理后24小时，细胞用caspase-9FLICA试剂盒(ImmunoChemistry，Biomol)按照厂家提供的操作方法进行荧光免疫染色，最后细胞膜用Alexa
647标记的choleratoxin subunit B(Invitrogen)染色5分钟，PBS洗后在共聚焦荧光显微镜下观察照相(Leica TCS SP5)。结果显示：ppp-TGF-β转染小鼠胰腺癌细胞后24小时，显示胞内caspase-9活化(照片所呈现的亮点即为活化的)caspase-9，对照及OH-TGF-βcaspase-9染色阴性，说明未活化(见图9)。
体内实验：按实施例2所述方法处理后的小鼠肿瘤实体进行冰冻切片，常规固定后，用原位杂交细胞死亡检测试剂盒(TUNEL，Roche)，按厂家提供说明书操作，然后用含DAPI的荧光上样液
(Vector Laboratories)盖片，用共聚焦荧光显微镜观察照相(Leica TCS SP5)，见图10。结果显示：ppp-TGF-β治疗原位胰腺癌小鼠组，肿瘤TUNEL染色阳性(图中呈现亮点)，说明肿瘤细胞凋亡；PBS对照组及OH-TGF-β则为阴性，说明肿瘤细胞未凋亡。
实施例7 原位胰腺癌小鼠试验检测ppp-TGF-β抗肿瘤的作用
6周龄雌性小鼠C57B1/6(Harlan-Winkelmann)，每只平均体重约25克，全身麻醉后，经开腹手术暴露小鼠胰腺，将2x105胰腺癌细胞悬于20微升PBS中注射至胰腺包膜下，回置胰腺，关腹，小鼠保暖至苏醒活动。于第10天予以尾部静脉注射RNA，每周两次共三周，每天观察小鼠生存情况，当小鼠出现不能进食、运动等明显垂危征兆时处死小鼠。每组小鼠数目为9-11只，至第100天时终止实验。以上操作均根据当地政府伦理道德条例规定进行。绘制小鼠生存曲线图(图19)，由图可知：ppp-TGF-β组小鼠较之其他四组平均生存时间显著延长，说明本发明ppp-TGF-β抗胰腺癌肿瘤的作用优越于其他四组。
参考文献
1.ViláMR，Lloreta J，Schüssler MH，Berrozpe G，Welt S，Real FX.New pancreas cancers celllines that represent distinct stages of ductal differentiation.Lab Invest.1995Apr；72(4)：395-404.2.Corbett TH，Roberts BJ，Leopold WR，Peckham JC，Wilkoff LJ，Griswold DP Jr，Schabel FMJr.Induction and chemotherapeutic response of two transplantable ductal adenocarcinomas ofthe pancreas in C57BL/6mice.Cancer Res.1984；44：717-726.
Claims (4)
1.含自由三磷酸基团的TGF-β特异性siRNA，其特征在于该TGF-β特异性siRNA的反义链和正义链序列5’端均为鸟嘌呤核苷，该鸟嘌呤核苷的戊糖3’位点上修饰有一个自由三磷酸基团。
2.根据权利要求1所述的含自由三磷酸基团的TGF-β特异性siRNA，其特征在于所述的TGF-β特异性siRNA序列选自：ppp-TGF-β1，其正义链序列为SEQ ID NO.1，反义链序列为SEQ ID NO.2；或ppp-TGF-β2，其正义链序列为SEQ ID NO.3，反义链序列为SEQ IDNO.4。
3.权利要求1所述的含自由三磷酸基团的TGF-β特异性siRNA在制备治疗肿瘤的药物中的应用。
4.权利要求1所述的含自由三磷酸基团的TGF-β特异性siRNA在制备治疗胰腺癌的药物中的应用。
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