CN112067805B - 一种β-1,4-半乳糖基转移酶-IsiRNA在克服肝癌化疗耐药药物中的应用 - Google Patents

一种β-1,4-半乳糖基转移酶-IsiRNA在克服肝癌化疗耐药药物中的应用 Download PDF

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CN112067805B
CN112067805B CN202010862247.XA CN202010862247A CN112067805B CN 112067805 B CN112067805 B CN 112067805B CN 202010862247 A CN202010862247 A CN 202010862247A CN 112067805 B CN112067805 B CN 112067805B
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吴圆圆
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Abstract

本发明公开了一种β‑1,4‑半乳糖基转移酶‑I siRNA在克服肝癌化疗耐药药物中的应用，用阿霉素处理肝癌细胞Huh‑7和HepG2，通过PCR检测β‑1,4‑半乳糖基转移酶‑I糖基化和基因表达水平变化；倒置显微镜观察发现阿霉素诱导肝癌细胞形态变化；检测β‑1,4‑半乳糖基转移酶‑I，MDRI表达：β‑1,4‑半乳糖基转移酶‑I的特异性siRNA构建和转染；观察β‑1,4‑半乳糖基转移酶‑I对EMT相关分子的影响；划痕实验检测β‑1,4‑半乳糖基转移酶‑I对肝癌细胞迁移能力的影响：阿霉素诱导的肝癌细胞huh‑7、hepG2，观察细胞迁移能力，观察β‑1,4‑半乳糖基转移酶‑I对肝癌细胞侵袭能力的影响。

Description

一种β-1,4-半乳糖基转移酶-IsiRNA在克服肝癌化疗耐药药 物中的应用
技术领域
本发明属于医药领域，具体涉及一种β-1,4-半乳糖基转移酶-IsiRNA在克服肝癌化疗耐药药物中的应用。
背景技术
原发性肝癌是全世界范围内常见的威胁人类生命的疾病之一，是全球第五大高发和第三致死性的恶性肿瘤，每年增加超过56万新发病例并引起超过50万例病人死亡。目前肝癌的治疗方案包括手术切除、肝脏移植、射频消融、肝动脉化疗栓塞和索拉菲尼。尽管手术是治疗肝癌的首选，然而，绝大多数肝癌患者都是在中晚期发现，而此时肿瘤已不能被手术切除而被迫采取姑息治疗。需要注意的是，虽然化疗在很多其他类型肿瘤病人中都得到非常广泛的应用，对于肝癌病人却没有明显的效果。由于肝癌具有内在的化疗药物抵抗的特性，导致肝癌患者的化疗药物的用量往往很大。因此，不管是全身性的化疗或者局部动脉内给药，往往对患者产生较大的毒副作用，却不能有效的抑制肿瘤生长和延长病人的生存期。
EMT概念的首次提出源于首次提出源于 1982 年学者在三维胶原凝胶中培养的晶状体上皮细胞出现间质细胞样形态。作为一个动态变化的过程， EMT表现为上皮细胞失去分化特征，细胞与细胞间的紧密连接以及细胞极性消失，并获得间质细胞特性，包括迁移性增加、 侵袭性增加，耐受凋亡能力的增加。在多种细胞外刺激以及转录因子的调控作用下，E-钙黏连蛋白等细胞粘附分子的表达下调， 同时波形蛋白等间质细胞表型分子的表达上调，最终导致 EMT 的发生。一系列的研究表明，EMT的发生在促进上皮源性肿瘤细胞间质表型发生的同时，细胞对化疗药物的敏感性也发生显著的变化。在多种耐药细胞株的构建过程中， 均发现细胞形态发生间质性的改变。2006 年Yang等发现对奥沙利铂耐药的结直肠细胞形态发生改变，出现极性丧失、细胞间连接消失以及伪足出现的改变， E-钙黏连蛋白以及波形蛋白等上皮与间质细胞标志物的表达改变。2009 年Rho等发现，耐吉非替尼的肿瘤细胞在对药物敏感性降低的同时，细胞的侵袭性能以及运动能力明显增加。2009 年Black等学者在细胞水平干预 E-钙黏连蛋白表达后，细胞对西妥昔单抗的敏感性明显降低。上述研究结果表明某些上皮源性的肿瘤在经历 EMT过程中，细胞形态、表面标志物以及运动侵袭能力发生改变，使细胞对化疗药物的敏感性亦产生显著的改变。EMT与肿瘤细胞对化疗药物的敏感性以及耐药现象的发生是密切相关的。但目前肿瘤特别是普遍对化疗耐药的肝癌其内在耐药的分子机制尚不清楚。
研究证明糖蛋白糖基化的改变是一个恶性肿瘤的现象，而与细胞的正常生长无关。糖蛋白的糖基化需要糖基转移酶的参与，因此肝癌细胞糖蛋白中糖链结构的改变必然与转移酶的活性改变有关。β-1,4-半乳糖基转移酶-I（β-1,4-galactosyltransferase-I,β-1,4-GalT-I）属于β-1,4-半乳糖基转移酶家族，是该家族七个已经确认的成员中最早被成功克隆的。它是一种既能定位于高尔基体参与细胞糖基化又能定位于细胞膜表面介导细胞识别的II型跨膜糖蛋白。其一个基因通过不同的起始位点能转录两种长度的mRNA，分别编码长型β-1,4-GalT-I（含399个氨基酸）和短型β-1,4-GalT-I（含386个氨基酸）。长型β-1,4-GalT-I和短型β-1,4-GalT-I都包含三部分结构：相对短的氨基端胞浆结构域，跨膜信号序列结构域和较大的羧基端胞内或胞外催化结构域，但由于转录翻译起始位点的不同，长型β-1,4-GalT-I比短型β-1,4-GalT-I在胞内氨基端多附加了13个氨基酸的延伸序列，该序列使得长型β-1,4-GalT-I能够从高尔基体定位到细胞膜表面，并表现出不同的生物学功能。通常认为高尔基体上的长型和短型β-1,4-GalT-I能将半乳糖苷基团从UDP-半乳糖苷上转移到N-多糖复合物的末端N-乙酰氨基葡萄糖或葡萄糖上。除此之外，β-1,4-GalT-I还以可溶性的形式存在于许多体液中，包括血清、脑脊液、玻璃体液、羊水、初乳和牛奶中，而这些体液中的β-1,4-GalT-I将首先被纯化成为乳糖合成复合物的一部分。除了传统定位于高尔基复合物上和存在于体液中的β-1,4-GalT-I以外，细胞膜表面的长型β-1,4-GalT-I可作为一种细胞间识别分子，与相邻细胞膜或细胞外基质中糖复合物糖链末端N-乙酰氨基葡萄糖或半乳糖基结合，参与神经细胞轴突延伸、肿瘤细胞转移扩散、卵细胞受精反应、胚胎细胞生长发育、乳腺组织形态构建、表皮细胞增殖凋亡等许多生物学过程，并在细胞与细胞间及细胞与细胞外基质间发挥重要的细胞粘附作用。β-1,4-GalT-I的表达在多种肿瘤中增加，并和肿瘤的恶性程度相关。改变β-1,4-半乳糖基转移酶-I的表达影响黑色素瘤和肺癌细胞的侵袭能力。有研究发现β-1,4-半乳糖基转移酶-I在人耐药白血病细胞株中表达增加，提示β-1,4-半乳糖基转移酶-I在人白血病细胞株的多药耐药过程中发挥重要作用。但是关于β-1,4-半乳糖基转移酶-I能否调控肝癌耐药细胞EMT的发生从而影响其耐药国内外文献尚未报道。我们实验研究结果阿霉素能诱导肝癌细胞Huh-7形态改变，且在此过程中β-1,4-半乳糖基转移酶-I的表达上调，故推测β-1,4-半乳糖基转移酶-I可能参与肝癌细胞的化疗抵抗过程。免疫荧光共聚焦检测发现红色荧光β-1,4-半乳糖基转移酶-I与绿色荧光MDR1（耐药相关分子）存在共定位，提示β-1,4-半乳糖基转移酶-I可能通过与MDR1相互作用参与肝癌耐药发生。在此基础上利用RNA干扰技术下调β-1,4-半乳糖基转移酶-I的表达，其迁移和侵袭增加的现象被抑制，提示下调β-1,4-半乳糖基转移酶-I能抑制肝癌耐药过程中增强的肿瘤细胞恶性行为学现象。
由于肝癌具有内在的化疗药物抵抗的特性，导致肝癌患者的化疗药物的用量往往很大。因此，不管是全身性的化疗或者局部动脉内给药，往往对患者产生较大的毒副作用，却不能有效的抑制肿瘤生长和延长病人的生存期。肿瘤细胞的耐药性包括原发性耐药和获得性耐药，多次化疗诱导肿瘤细胞产生的获得性耐药更为常见，且肿瘤细胞对一种化疗药物耐药后对其他抗肿瘤药物也有耐药性，故而克服恶性肿瘤的耐药性成为迫切需要解决的问题。上皮源性的肿瘤在经历 EMT 过程中， 细胞形态、 表面标志物以及运动侵袭能力发生改变， 使细胞对化疗药物的敏感性亦产生显著的改变。EMT与肿瘤细胞对化疗药物的敏感性以及耐药现象的发生是密切相关的。但目前肿瘤特别是普遍对化疗耐药的肝癌其内在耐药的分子机制尚不清楚。我们以β-1,4-半乳糖基转移酶-I在肝细胞肝癌耐药发生发展中的调控机制为核心研究内容，以β-1,4-半乳糖基转移酶-I对EMT过程的调控作用为主要切入点分析β-1,4-半乳糖基转移酶-I在肝癌耐药过程中所发挥的作用。 利用干扰技术下调β-1,4-半乳糖基转移酶-I的表达，从而逆转化疗药物诱导的肝癌细胞EMT的改变，增加肝癌细胞对化疗药物的敏感性，为临床肝癌的治疗提供新的思路。
发明内容
解决的技术问题：针对上述在耐药的分子机制尚不清楚等技术问题，本发明目的之一为提供一种β-1,4-半乳糖基转移酶-IsiRNA在克服肝癌化疗耐药药物中的应用。
技术方案
一种β-1,4-半乳糖基转移酶-IsiRNA在克服肝癌化疗耐药药物中的应用。
进一步的，步骤为：
第一步：用阿霉素 doxorubicin处理肝癌细胞Huh-7和HepG2，通过lectin blot和realtime PCR检测doxorubicin不同浓度（0.25uM及1uM）处理肝癌细胞Huh-7、HepG2后，β-1,4-半乳糖基转移酶-I糖基化和基因表达水平变化；
第二步：倒置显微镜观察发现阿霉素诱导肝癌细胞形态变化；
第三步，检测β-1,4-半乳糖基转移酶-I，MDRI表达：Realtime PCR技术检测耐药过程中β-1,4-半乳糖基转移酶-I，MDRI表达变化; 双抗染色后在激光共聚焦显微镜下观察β-1,4-半乳糖基转移酶-I和MDRI共定位情况；
第四步：β-1,4-半乳糖基转移酶-I的特异性siRNA构建和转染；
第五步，观察β-1,4-半乳糖基转移酶-I对EMT相关分子的影响：阿霉素诱导肝癌细胞Huh-7、HepG2及siRNA转染细胞组，western blot检测E-Cadherin，Vimentin及MDR1表达变化；
第六步，划痕实验检测β-1,4-半乳糖基转移酶-I对肝癌细胞迁移能力的影响：阿霉素诱导的肝癌细胞huh-7、hepG2，观察细胞迁移能力，在此基础上利用siRNA下调β-1,4-半乳糖基转移酶-I的表达，观察下调β-1,4-半乳糖基转移酶-I的表达对细胞迁移的影响；
第七步，观察β-1,4-半乳糖基转移酶-I对肝癌细胞侵袭能力的影响：阿霉素诱导的肝癌细胞huh-7、hepG2，transwell检测肝癌细胞侵袭能力，而干扰β-1,4-半乳糖基转移酶-I的表达后观察侵袭能力的变化，研究下调β-1,4-半乳糖基转移酶-I是否能抑制肝癌耐药过程中增强的肿瘤细胞侵袭行为。
进一步的，所述第四步中β-1,4-半乳糖基转移酶-I的特异性siRNA载体的构建和转染具体实验方案为：由吉玛基因公司合成针对β-1,4-半乳糖基转移酶-I的特异性siRNA序列，具体序列：sense（正义链）：CCAUGCUGAUUGAGUUUAATT， antisense（反义链）:UUAAACUCAAUCAGCAUGGTT, 将生长50%-60%的Huh-7和HepG2细胞培养液倒出，用PBS冲洗两遍，加入lml不含双抗的无血清培养基，放入培养箱中培养，将生长50%-60%的Huh-7和HepG2细胞培养液倒出，用PBS冲洗两遍，加入lml不含双抗的无血清培养基，放入培养箱中培养，用250μl双无DMEM稀释siRNA 4μl，混匀; 用250μl双无DMEM稀释脂质体8μl，混匀，室温5min，不超过10min；将上述SiRNA和脂质体液体混匀，室温孵育20min，6孔板加入混合液体，轻轻摇晃6孔板使其中的液体混合均匀，放入培养箱终培养，6-8h后将液体倒出，加入正常培养基培养。
技术效果：利用干扰技术下调β-1,4-半乳糖基转移酶-I的表达，从而逆转化疗药物诱导的肝癌细胞EMT的改变，增加肝癌细胞对化疗药物的敏感性，为临床肝癌的治疗提供新的思路。阿霉素能诱导肝癌细胞Huh-7形态改变，且在此过程中β-1,4-半乳糖基转移酶-I的表达上调，故推测β-1,4-半乳糖基转移酶-I可能参与肝癌细胞的化疗耐药过程。免疫荧光共聚焦检测发现红色荧光β-1,4-半乳糖基转移酶-I与绿色荧光MDR1（耐药相关分子）存在共定位，提示β-1,4-半乳糖基转移酶-I可通过与MDR1相互作用参与肝癌耐药发生。利用RNA干扰技术下调β-1,4-半乳糖基转移酶-I的表达，其迁移和侵袭增加的现象被抑制，提示下调β-1,4-半乳糖基转移酶-I能抑制肝癌耐药过程中增强的肿瘤细胞恶性行为学现象。肝癌的机制研究及相关药物开发对提高我国国民素质和身体健康具有举足轻重的意义。近年来，肿瘤的生物靶向治疗作为新兴的毒副作用小、靶向性强、效果优异的治疗手段，成为肿瘤治疗药物开发的热点领域。目前国外拥有一批年销售额高达数十亿美元的生物靶向肿瘤药物应用于临床，而我国在相关药物开发上存在明显的不足和滞后。因此，该发明如能进行应用开发，将会具有良好的应用前景和市场竞争力，对改善我国民生和社会具有良好的促进作用。
附图说明
图1为本申请实施例1阿霉素诱导肝癌细胞Huh-7、HepG2 β-1,4-半乳糖基转移酶-I糖基化和基因表达变化图；
图2为本申请实施例1中ADM处理48h Huh-7、HepG2形态变化图。
图3为本申请实施例1中免疫荧光激光共聚焦技术检测β-1,4-半乳糖基转移酶-I与MDR1在阿霉素诱导的肝癌细胞表面共定位情况图。
图4为本申请实施例1中β-1,4-半乳糖基转移酶-I对EMT相关分子的影响图。
图5为本申请实施例1中β-1,4-半乳糖基转移酶-I对肝癌细胞迁移能力的影响图。
图6为本申请实施例1中β-1,4-半乳糖基转移酶-I对肝癌细胞侵袭能力的影响图。
具体实施方式
以下通过实施例说明本发明的具体步骤，但不受实施例限制。
在本发明中所使用的术语，除非另有说明，一般具有本领域普通技术人员通常理解的含义。
在下面结合具体实施例并参照数据进一步详细描述本发明。应理解，这些实施例只是为了举例说明本发明，而非以任何方式限制本发明的范围。
在以下实施例中，未详细描述的各种过程和方法是本领域中公知的常规方法。
实施例1
一种β-1,4-半乳糖基转移酶-IsiRNA在克服肝癌化疗耐药药物中的应用，步骤为：
第一步：β-1,4-半乳糖基转移酶-I在肝癌细胞Huh-7、HepG2阿霉素化疗抵抗过程中糖基化及基因表达增加：通过筛选出0.25、1μM的阿霉素作用于肝癌细胞Huh-7、HepG2，分别采用凝集素印记技术lectin blot及实时定量PCR技术 Realtime PCR检测β-1,4-半乳糖基转移酶-I糖基化和基因表达水平，结果发现β-1,4-半乳糖基转移酶-I在阿霉素化疗抵抗过程中糖基化及基因表达明显增加，提示β-1,4-半乳糖基转移酶-I可能参与肝癌细胞Huh-7、HepG2阿霉素耐药发生。
如图1 所示，阿霉素诱导肝癌细胞Huh-7、HepG2 β-1,4-半乳糖基转移酶-I糖基化和基因表达变化。A图不同浓度(0.25、1μM)阿霉素作用于Huh-7、HepG2细胞，应用lectinblot检测β-1,4-半乳糖基转移酶-I糖基化水平。B图realtime PCR检测β1,4GT mRNA水平变化。
第二步：倒置显微镜观察发现阿霉素诱导肝癌细胞形态变化，阿霉素ADM诱导48h，Huh-7、HepG2形态由上皮样转变为间质细胞样的形态。
如图2 所示ADM处理48h Huh-7、HepG2形态变化。ADM处理后肝癌细胞由上皮细胞形态向间质细胞形态改变。
第三步：检测β-1,4-半乳糖基转移酶-I，MDRI表达： Realtime PCR技术检测耐药过程中β-1,4-半乳糖基转移酶-I，MDRI表达变化; 双抗染色后在激光共聚焦显微镜下观察β-1,4-半乳糖基转移酶-I和MDRI共定位情况。β-1,4-半乳糖基转移酶-I与耐药相关分子MDR1共定位：阿霉素诱导肝癌细胞Huh-7、HepG2，免疫荧光共聚焦检测发现红色荧光β-1,4-半乳糖基转移酶-I与绿色荧光MDR1存在共定位，提示β-1,4-半乳糖基转移酶-I可能通过与MDR1相互作用参与肝癌耐药发生。
如图3所示，β-1,4-半乳糖基转移酶-I与MDR1 相互作用。免疫荧光激光共聚焦技术检测β-1,4-半乳糖基转移酶-I（红色）与MDR1（绿色）在阿霉素诱导的肝癌细胞表面共定位情况。
第四步：β-1,4-半乳糖基转移酶-I的特异性siRNA构建和转染；由吉玛基因公司合成针对β-1,4-半乳糖基转移酶-I的特异性siRNA序列，具体序列：sense（正义链）：CCAUGCUGAUUGAGUUUAATT， antisense（反义链）:UUAAACUCAAUCAGCAUGGTT,将细胞分为三组，即空白组、空载体组、转染组；将生长50%-60%的Huh-7和HepG2细胞培养液倒出，用PBS冲洗两遍，加入lml不含双抗的无血清培养基，放入培养箱中培养，用250μl双无DMEM稀释siRNA 4μl，混匀; 用250μl双无DMEM稀释脂质体8μl，混匀，室温5min，不超过10min；将上述SiRNA和脂质体液体混匀，室温孵育20min，6孔板加入混合液体，轻轻摇晃6孔板使其中的液体混合均匀，放入培养箱终培养，6-8h后将液体倒出，加入正常培养基培养。
第五步：观察β-1,4-半乳糖基转移酶-I对EMT相关分子的影响：阿霉素诱导肝癌细胞Huh-7、HepG2及siRNA转染细胞组，western blot检测E-Cadherin，Vimentin及MDR1表达变化。阿霉素诱导肝癌细胞Huh-7、HepG2，E-Cadherin表达减少，Vimentin表达增加，MDR1表达增加，而干扰β-1,4-半乳糖基转移酶-I表达能逆转上述现象，提示β-1,4-半乳糖基转移酶-I在EMT过程中发挥重要作用。
如图4所示，β-1,4-半乳糖基转移酶-I对EMT相关分子的影响。
第六步：划痕实验检测β-1,4-半乳糖基转移酶-I对肝癌细胞迁移能力的影响：阿霉素诱导的肝癌细胞huh-7、hepG2，观察细胞迁移能力，在此基础上利用siRNA下调β-1,4-半乳糖基转移酶-I的表达，观察下调β-1,4-半乳糖基转移酶-I的表达对细胞迁移的影响。
如图5所示，阿霉素诱导的肝癌细胞huh-7、hepG2的迁移细胞明显增多，在此基础上利用siRNA下调β-1,4-半乳糖基转移酶-I的表达，其迁移增加的现象被抑制，提示下调β-1,4-半乳糖基转移酶-I能抑制肝癌耐药过程中增强的肿瘤细胞迁移现象。
第七步：观察β-1,4-半乳糖基转移酶-I对肝癌细胞侵袭能力的影响。阿霉素诱导的肝癌细胞huh-7、hepG2，transwell检测肝癌细胞侵袭能力，而干扰β-1,4-半乳糖基转移酶-I的表达后观察侵袭能力的变化，研究下调β-1,4-半乳糖基转移酶-I是否能抑制肝癌耐药过程中增强的肿瘤细胞侵袭行为。
如图6所示，β-1,4-半乳糖基转移酶-I对肝癌细胞侵袭能力的影响。阿霉素诱导的肝癌细胞huh-7、hepG2的迁移细胞明显增多，而干扰β-1,4-半乳糖基转移酶-I的表达该现象则被抑制，提示下调β-1,4-半乳糖基转移酶-I能抑制肝癌耐药过程中增强的肿瘤细胞侵袭行为。
序列表
<110> 南通大学
<120> 一种β-1,4-半乳糖基转移酶-IsiRNA在克服肝癌化疗耐药药物中的应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA/RNA
<213> 人工序列(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 1
ccaugcugau ugaguuuaat t 21
<210> 2
<211> 21
<212> DNA/RNA
<213> 人工序列(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 2
uuaaacucaa ucagcauggt t 21

Claims (1)

1.一种非疾病诊断治疗目的的β-1,4-半乳糖基转移酶-IsiRNA在克服肝癌化疗耐药药物中的应用，其特征在于步骤为：
第一步：用阿霉素 doxorubicin处理肝癌细胞Huh-7和HepG2，通过lectin blot和realtime PCR检测doxorubicin 0.25uM及1uM浓度处理肝癌细胞Huh-7、HepG2后，β-1,4-半乳糖基转移酶-I糖基化和基因表达水平变化；
第二步：倒置显微镜观察发现阿霉素诱导肝癌细胞形态变化；
第三步，检测β-1,4-半乳糖基转移酶-I，MDRI表达：Realtime PCR技术检测耐药过程中β-1,4-半乳糖基转移酶-I，MDRI表达变化; 双抗染色后在激光共聚焦显微镜下观察β-1,4-半乳糖基转移酶-I和MDRI共定位情况；
第四步：β-1,4-半乳糖基转移酶-I的特异性siRNA载体的构建和转染；
第五步，观察β-1,4-半乳糖基转移酶-I对EMT相关分子的影响：阿霉素诱导肝癌细胞Huh-7、HepG2及siRNA转染细胞组，western blot检测E-Cadherin，Vimentin及MDR1表达变化；
第六步，划痕实验检测β-1,4-半乳糖基转移酶-I对肝癌细胞迁移能力的影响：阿霉素诱导的肝癌细胞huh-7、hepG2，观察细胞迁移能力，在此基础上利用siRNA下调β-1,4-半乳糖基转移酶-I的表达，观察下调β-1,4-半乳糖基转移酶-I的表达对细胞迁移的影响；
第七步，观察β-1,4-半乳糖基转移酶-I对肝癌细胞侵袭能力的影响：阿霉素诱导的肝癌细胞huh-7、hepG2，transwell检测肝癌细胞侵袭能力，而干扰β-1,4-半乳糖基转移酶-I的表达后观察侵袭能力的变化，研究下调β-1,4-半乳糖基转移酶-I是否能抑制肝癌耐药过程中增强的肿瘤细胞侵袭行为；阿霉素诱导的肝癌细胞huh-7、hepG2的迁移细胞明显增多，而干扰β-1,4-半乳糖基转移酶-I的表达该现象则被抑制，提示下调β-1,4-半乳糖基转移酶-I能抑制肝癌耐药过程中增强的肿瘤细胞侵袭行为；
所述第四步中β-1,4-半乳糖基转移酶-I的特异性siRNA构建和转染具体实验方案为：由吉玛基因公司合成针对β-1,4-半乳糖基转移酶-I的特异性siRNA序列，具体序列：sense（正义链）：CCAUGCUGAUUGAGUUUAATT， antisense（反义链）:UUAAACUCAAUCAGCAUGGTT, 将生长50%-60%的Huh-7和HepG2细胞培养液倒出，用PBS冲洗两遍，加入lml不含双抗的无血清培养基，放入培养箱中培养，用250μl双无DMEM稀释siRNA 4μl，混匀; 用250μl双无DMEM稀释脂质体8μl，混匀，室温5min，不超过10min；将上述siRNA和脂质体液体混匀，室温孵育20min，6孔板加入混合液体，轻轻摇晃6孔板使其中的液体混合均匀，放入培养箱终培养，6-8h后将液体倒出，加入正常培养基培养。
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