药学学报

No.	Name		Sequence 5ʹ→3ʹ	(Calcd./Measured) molecular weight
1	siPLK1A	AS	UAU UUA AGG AGG GUG AUC UTT	6 716.1/6 718.1
SS	AGA UCA CCC UCC UUA AAU ATT	6 562.0/6 563.5
2	siPLK1A1	AS	UsAsU UUA AGG AGG GUG AUC UsTsT	6 994.6/6 996.0
SS	AsGsA UCA CCCUCC UUA AAU AsTsT	6 840.6/6 842.3
3	siPLK1A2	AS	UsAsU UUA AGGAGG GUG AUC UsTsT	6 970.6/6 972.0
SS	AsGsA UCA CCCUCC UUA AAU AsTsT	6 840.6/6 842.3
4	siPLK1A3	AS	UsAsUUUA AGG AGG GUG AUCUsTsT	6 934.5/6 935.7
SS	AsGsA UCA CCCUCC UUA AAU AsTsT	6 840.6/6 842.3
5	siPLK1B	AS	UUG AUC CGG AGG UAG GUC UTT	6 706.1/6 708.8
SS	AGA CCU ACC UCC GGA UCA UTT	6 678.1/6 679.5
6	siPLK1B1	AS	UsUsG AUC CGG AGG UAG GUC UsTsT	6 985.6/6 986.9
SS	AsGsA CCU ACCUCC GGA UCA AsTsT	6 894.7/6 896.2
7	siPLK1B2	AS	UsUsG AUC CGG AGG UAG GUC UsTsT	6 961.6/6 963.0
SS	AsGsA CCU ACCUCC GGA UCA AsTsT	6 894.7/6 896.2
8	siPLK1B3	AS	UsUsGAUC CGG AGG UAG GUCUsTsT	6 925.4/6 896.2
SS	AsGsA CCU ACCUCC GGA UCA AsTsT	6 894.7/6 896.2
9	siKSP	AS	AGU UAG UUU AGA UUC UGG ATT	6 712.9/6 713.2
SS	UCG AGA AUC UAA ACU AAC UTT	6 222.1/6 622.1
10	siKSP1	AS	AsGsU UAG UUU AGA UUC UCG AsTsT	6 915.6/6 917.3
SS	UsCsG AGAAUCUAA ACU AAC UsTsT	6 904.7/6 905.6

No.	Name		Sequence 5ʹ→3ʹ	(Calcd./Measured) molecular weight
1	siPLK1A	AS	UAU UUA AGG AGG GUG AUC UTT	6 716.1/6 718.1
SS	AGA UCA CCC UCC UUA AAU ATT	6 562.0/6 563.5
2	siPLK1A1	AS	UsAsU UUA AGG AGG GUG AUC UsTsT	6 994.6/6 996.0
SS	AsGsA UCA CCCUCC UUA AAU AsTsT	6 840.6/6 842.3
3	siPLK1A2	AS	UsAsU UUA AGGAGG GUG AUC UsTsT	6 970.6/6 972.0
SS	AsGsA UCA CCCUCC UUA AAU AsTsT	6 840.6/6 842.3
4	siPLK1A3	AS	UsAsUUUA AGG AGG GUG AUCUsTsT	6 934.5/6 935.7
SS	AsGsA UCA CCCUCC UUA AAU AsTsT	6 840.6/6 842.3
5	siPLK1B	AS	UUG AUC CGG AGG UAG GUC UTT	6 706.1/6 708.8
SS	AGA CCU ACC UCC GGA UCA UTT	6 678.1/6 679.5
6	siPLK1B1	AS	UsUsG AUC CGG AGG UAG GUC UsTsT	6 985.6/6 986.9
SS	AsGsA CCU ACCUCC GGA UCA AsTsT	6 894.7/6 896.2
7	siPLK1B2	AS	UsUsG AUC CGG AGG UAG GUC UsTsT	6 961.6/6 963.0
SS	AsGsA CCU ACCUCC GGA UCA AsTsT	6 894.7/6 896.2
8	siPLK1B3	AS	UsUsGAUC CGG AGG UAG GUCUsTsT	6 925.4/6 896.2
SS	AsGsA CCU ACCUCC GGA UCA AsTsT	6 894.7/6 896.2
9	siKSP	AS	AGU UAG UUU AGA UUC UGG ATT	6 712.9/6 713.2
SS	UCG AGA AUC UAA ACU AAC UTT	6 222.1/6 622.1
10	siKSP1	AS	AsGsU UAG UUU AGA UUC UCG AsTsT	6 915.6/6 917.3
SS	UsCsG AGAAUCUAA ACU AAC UsTsT	6 904.7/6 905.6

Name	Sequence
PLK1 gene forward	ATCACCTGCCTGACCATTCCACCAAGG
PLK1 gene reverse	AATTGCGGAAATATTTAAGGAGGGTGATCT
KSP forward	CTGAACAGTGGGTATCTTCCTTA
KSP reverse	GATGGCTCTTGACTTAGAGGTTC
β-Actin forward	CCAACCGCGAGAAGATGA
β-Actin reverse	CCAGAGGCGTACAGGGATAG

Name	Sequence
PLK1 gene forward	ATCACCTGCCTGACCATTCCACCAAGG
PLK1 gene reverse	AATTGCGGAAATATTTAAGGAGGGTGATCT
KSP forward	CTGAACAGTGGGTATCTTCCTTA
KSP reverse	GATGGCTCTTGACTTAGAGGTTC
β-Actin forward	CCAACCGCGAGAAGATGA
β-Actin reverse	CCAGAGGCGTACAGGGATAG

全2ʹ-F/OMe-siRNA的设计合成及其新型混合脂材纳米制剂抗肝癌活性评价

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高宇睛 ¹ , 王玺贤 ² , 潘宇飞 ² , 汪全鑫 ² , 朱月洁 ² , 潘德林 ¹^,^* , 关注 ² , 杨振军 ²^,^*

药学学报 | 研究论文 2023,58(6): 1634-1640

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药学学报 |研究论文 2023 , 58 (6) : 1634 -1640

全2ʹ-F/OMe-siRNA的设计合成及其新型混合脂材纳米制剂抗肝癌活性评价

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高宇睛¹, 王玺贤², 潘宇飞², 汪全鑫², 朱月洁², 潘德林¹^,^*, 关注², 杨振军²^,^*

作者信息

1.成都中医药大学药学院, 四川成都 611137

2.北京大学药学院/天然药物及仿生药物国家重点实验室, 北京 100191

通讯作者:

*杨振军, Tel: 86-10-82805781, E-mail: yangzj@bjmu.edu.cn;

潘德林, Tel: 13547460729, E-mail: linfeng_0130@163.com

Synthesis and anti-HCC activity of full 2ʹ-F/OMe-siRNA encapsulated with neutral cytidinyl/cationic lipid

Yu-jing GAO¹, Xi-xian WANG², Yu-fei PAN², Quan-xin WANG², Yue-jie ZHU², De-lin PAN¹^,^*, Zhu GUAN², Zhen-jun YANG²^,^*

Affiliations

1. School of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu 611137, China

2. State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China

出版时间: 2023-06-12 doi: 10.16438/j.0513-4870.2022-1345

文章导航

摘要

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本文设计合成了多条全2ʹ-F/OMe修饰的抗肝癌siRNA, 并在细胞水平进行活性评价, 探究2ʹ-F/OMe修饰的位置差异对siRNA活性的影响。使用K&A DNA/RNA H-8合成仪合成全2ʹ-F/OMe修饰siRNA, 利用中性胞苷脂材DNCA混合阳离子脂材CLD包载递送siRNA入胞。通过RT-qPCR实验检测Huh-7和HepG2细胞的靶基因沉默活性, CCK-8实验检测Huh-7和HepG2细胞增殖活力, RNA结合蛋白免疫沉淀及RT-qPCR实验检测siRNA载入Ago2蛋白的情况, 流式细胞术检测药物摄取和细胞凋亡, Western blot实验检测PLK1蛋白的表达。部分全2ʹ-F/OMe修饰siRNA增强了与Ago2蛋白的结合, 进而增强了基因沉默活性及肝癌细胞增殖抑制活性。此外, 多数siRNA修饰物制剂的Huh-7细胞摄取率提高, 更大幅度下调PLK1蛋白, 诱导更多Huh-7细胞凋亡, 其中siPLK1A3最优。以上结果为进一步研究siRNA修饰模式及研发抗肝癌新型制剂提供了工作基础。

关键词

小干扰RNA / 全2ʹ-修饰 / 抗肝癌 / Ago2 / DNCA/CLD

Abstract

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A variety of full 2ʹ-F/OMe-modified siRNAs were designed and synthesized, and the activity against hepatocellular carcinoma Huh-7 and HepG2 cells was evaluated. K&A DNA/RNA H-8 synthesizer was used to synthesize siRNAs, and neutral cytidinyl lipid DNCA mixed with cationic lipid CLD were used to transfect siRNA. By RT-qPCR and CCK-8 assay, the target gene silence and the proliferation of Huh-7 and HepG2 cells were detected. The siRNAs loading into Ago2 protein was detected by RNA-binding protein immunoprecipitation. Drug uptake and cell apoptosis were detected by flow cytometry, and the expression of PLK1 protein was detected by Western blot. Partial full 2ʹ-F/OMe modified siRNAs, especial siPLK1A3, increased the uptake of Huh-7 cells, enhanced their binding to Ago2 and gene silencing activity, down-regulated PLK1 protein, as well as induced more Huh-7 cell apoptosis and proliferation inhibition activity. It provides important data for the development of novel siRNA modification patterns and anti-HCC formulations.

Key words

siRNA / full 2ʹ-modification / anti-HCC / Ago2 / DNCA/CLD

引用本文

高宇睛, 王玺贤, 潘宇飞, 汪全鑫, 朱月洁, 潘德林, 关注, 杨振军. 全2ʹ-F/OMe-siRNA的设计合成及其新型混合脂材纳米制剂抗肝癌活性评价. 药学学报, 2023 , 58 (6) : 1634 -1640 . DOI: 10.16438/j.0513-4870.2022-1345

Yu-jing GAO, Xi-xian WANG, Yu-fei PAN, Quan-xin WANG, Yue-jie ZHU, De-lin PAN, Zhu GUAN, Zhen-jun YANG. Synthesis and anti-HCC activity of full 2ʹ-F/OMe-siRNA encapsulated with neutral cytidinyl/cationic lipid[J]. Acta Pharmaceutica Sinica, 2023 , 58 (6) : 1634 -1640 . DOI: 10.16438/j.0513-4870.2022-1345

正文

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肝癌是全球第六大常见癌症, 也是癌症相关死亡的第三大原因。临床上已有6种肝癌的全身性治疗方法, 主要是小分子化疗药物和单抗, 仅能一定程度上改善患者的预后, 严重的毒副作用及耐药性问题尚未得到有效解决^{[1, 2]}。探索肝癌发生和进展的潜在机制, 并开发靶向药物的治疗方案至关重要。

近年来, 小干扰RNA (siRNA) 正成为一种有潜力的抗肝癌疗法。TKM-080301是一种阳离子混合脂材SNALP递送的siRNA药物, 其靶标为Polo样激酶1 (PLK1), 已完成临床Ⅱ期试验^[3]。PLK1是一种广泛存在于真核细胞中高度保守的丝氨酸/苏氨酸激酶, 参与多种有丝分裂进程^[4], 在胶质母细胞瘤、乳腺癌、直肠癌、胃癌、肾癌及肝癌等中过表达^[5-8], 与癌症患者的预后不良有关^[9-11]。PLK1促进肝癌发展中的细胞生长, 是肝癌最可能的治疗靶点^[12]。ALN-VSP02是靶向血管内皮生长因子(VEGF) 和驱动蛋白纺锤体蛋白(KSP) 的双靶点siRNA药物, 已完成Ⅰ期临床试验^[13]。KSP在有丝分裂早期染色单体分离和双极纺锤体的形成中起重要作用, 其在正常细胞中低表达, 但在喉癌、肺癌、前列腺癌、神经胶质瘤、膀胱癌及肝细胞癌等中过表达^[14-17], 与肝癌预后不良有关^[18], 是抗肝癌治疗的良好分子靶标。

TKM-080301及ALN-VSP02均为部分位点2ʹ-OMe的siRNA, 易被血清核酸酶降解^[19]。2′-F与2′-OH大小和性质很相似, 用于修饰反义链切割位点也有较高的适应性。2′-OMe和2′-F修饰能够显著提高siRNA双链的结合亲和力、核酸酶抗性和靶基因沉默活性, 与2ʹ-F所处的位置和数量密切相关^{[20, 21]}。较为成功的siRNA使用全2ʹ-F和2ʹ-OMe交替修饰的ESC策略, 已经形成一些具有指导意义的修饰策略, 但其普适性有限^{[22, 23]}。截至2022年初, 已有五款FDA批准上市的siRNA药物, 除第一款外, 其余四款均为全2ʹ-修饰。在高胆固醇血症(inclisiran) 等治疗中取得的成功证实了全2ʹ-修饰siRNA药物具有很重要的发展潜力^{[24, 25]}。

Advanced ESC修饰策略是在siRNA反义链的2/6/14/16位和正义链的7/9/10/11位使用2ʹ-F修饰, 其他位置使用2ʹ-OMe修饰, 同时在反义链的两端以及正义链的5ʹ-端使用磷硫代(PS)^[26]。Lumasiran反义链8/9位由2ʹ-OMe修饰换成了2ʹ-F修饰^[26]。Inclisiran相对Advanced ESC的修饰不同, 为反义链4/5/8/10/18位由2ʹ-OMe换成了2ʹ-F修饰, 正义链10位由2ʹ-F换成了2ʹ-OMe修饰, 正义链11位用胸苷替代了尿苷(图 1)。对siRNA某个位点进行化学修饰会影响其相邻位点、整体构象及同互补链的结合^[27]。

本项全2ʹ-F/2ʹ-OMe修饰siRNA研究, 选择有抗肝癌活性的siPLK1A、siPLK1B和siKSP^{[28, 29]}。参考Alnylam公司的advanced ESC及其上市药物lumasiran和inclisiran的修饰策略, 设计合成了siPLK1A1/2/3、siPLK1B1/2/3; 参考advanced ESC修饰模式, 设计合成了siKSP1 (表 1)。本课题组前期利用自主知识产权的中性胞苷脂材DNCA和阳离子脂材CLD包载各类核酸药物, 实现了siRNA、反义核酸、mRNA及环二鸟苷酸的静脉注射及核酸适配体的瘤旁注射给药, 体内外药效非常显著, 制剂安全^[30-32]。继续使用DNCA/CLD转染上述修饰的siRNA, 三者摩尔比为21∶31.5∶1^{[30, 33]}, 评价其靶基因沉默等情况, 期望对全2ʹ-F/2ʹ-OMe修饰方案提供一些有意义的补充, 为进一步体内药效学研究奠定基础。

结果

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1 抗肝癌siRNA及其全2΄-F/OMe修饰物对靶基因沉默及细胞增殖抑制活性

RT-qPCR实验检测各序列转染细胞24 h后胞内靶mRNA的相对表达水平, 给药浓度为5 nmol·L^-1 (图 2A), CCK-8试剂检测抗肝癌siRNA各序列的细胞增殖抑制活性, 给药剂量为25 nmol·L^-1 (图 2B), 对于siPLK1A和siKSP, 全2ʹ-修饰后靶基因沉默活性及细胞增殖抑制能力均有所提升, 3种修饰模式在同一条序列上表现出相同的规律。而siPLKB1/2/3靶基因沉默活性及细胞增殖抑制活性则差于siPLKB, 可能由于全2ʹ-修饰后影响siRNA与靶标mRNA的互补配对、局部构象以及与Ago2相互作用, 从而影响其活性, 表明序列差异对修饰模式的筛选优化也具有一定影响; DNCA/CLD (Mix) 转染siPLK1A及siPLK1A3并以商用转染试剂Lipo 2000为对照, 考察对Huh-7细胞PLK1 mRNA的沉默活性及细胞增殖抑制作用。Mix显著优于Lipo 2000包载siRNA活性(图 2C、D); 进一步考察不同浓度siPLK1A及siPLK1A3对PLK1 mRNA的沉默活性及阴性对照(NC) 对Huh-7细胞增殖抑制活力。结果显示, siPLK1A3与siPLK1A相比EC₅₀、IC₅₀明显降低(图 2E、F)。因此, siPLK1A3更有效沉默PLK1基因表达, 对Huh-7细胞增殖抑制作用更佳。NC组对Huh-7细胞没有细胞毒性, 表明该制剂具有良好的细胞安全性。

2 siPLK1A及其修饰物与Ago-2蛋白免疫共沉淀(RIP)

RIP实验结果如图 3所示, 给药浓度为5 nmol·L^-1的siRNA/Mix转染Huh-7细胞, 与siPLK1A相比, Ago2蛋白富集结合了1.3倍的siPLK1A3。显然全2ʹ-F/OMe修饰的siRNA增强其与Ago2蛋白的结合能力, 是部分修饰物如siPLK1A3靶基因沉默活性提高的原因。

3 siPLK1A及其修饰物新型制剂的细胞摄取

利用Cy5.5标记的siPLK1A及siPLK1A3转染Huh-7细胞, 给药量为25 nmol·L^-1, 通过流式细胞术检测Huh-7的Cy5.5荧光情况(4 h)。结果如图 4所示, 与裸给组相比, siPLK1A及siPLK1A3制剂均显著被Huh-7细胞摄取, 后者具有更宽的单吸收峰, 显著提高了细胞摄取率。

4 siPLK1A及其修饰物对PLK1蛋白表达的影响

通过Western blot考察给药后Huh-7细胞内PLK1蛋白表达情况, 给药剂量为25 nmol·L^-1。结果如图 5所示, NC组与Blank组相比PLK1蛋白表达量无明显变化, 而siPLK1A及siPLK1A3显著抑制PLK1蛋白表达, siPLK1A下调了63% PLK1蛋白, siPLK1A3下调了69% PLK1蛋白。

5 siPLK1A及其修饰物对细胞凋亡的影响

利用Annexin V-FITC/PI双染法检测siPLK1A及siPLK1A3对细胞凋亡的影响, 给药剂量为25 nmol·L^-1, 结果如图 6所示, NC组对Huh-7细胞的凋亡无明显影响, 而siPLK1A能诱导Huh-7细胞的凋亡(18%), siPLK1A3能诱导更多Huh-7的细胞凋亡(25%)。

讨论

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肝癌治疗的一线及二线药物存在明显毒副作用及易产生耐药性等问题。siRNA可以在哺乳动物细胞中发挥特异性RNAi作用, 能在体内介导基因沉默, 尤其在局部组织中。全2ʹ-修饰可以有效抑制其免疫反应, 提高酶稳定性和靶基因沉默作用, 降低脱靶效应产生的毒性问题。本研究发现全2ʹ-F/OMe修饰的siPLK1A活性提高是源于基因沉默复合物中siRNA的负载量增加, 由此确定了可显著提高siRNA靶基因沉默活性的最佳2ʹ-F/OMe修饰模式。总的来说, 修饰后的siPLK1A和siKSP基因沉默活性和肝癌细胞增殖抑制活性均有所提升, 其中siPLK1A3和siKSP1活性显著上升, 可以作为体内抗肝癌研究的候选序列。而siPLK1B在修饰后多数情况下基因沉默活性有所下降, 其中siPLK1B3相对保持, 可作为体内抗肝癌研究的备选序列。

实验部分

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未修饰的siRNA及引物订购自生工生物工程(上海) 股份有限公司。siRNA合成使用K&A DNA/RNA H-8合成仪(德国, K&A LaborgeraeteGbR), 纯化siRNA使用GILSON液相系统(157 UV-VIS、306 PUMP、806, 811D) (美国, Gilson)、XBridge^TM OST C18 OBD^TM色谱柱(美国, Waters) 和HiPrep^TM 26/10 Desalting凝胶柱(美国, GE), GenOpti、DMEM高糖培养基、PBS系迈晨科技(北京) 有限公司产品, FBS购自美国Gibco公司, Cell Counting Kit-8购自日本同仁化学, RIP试剂盒系广州伯信生物科技有限公司产品, 总RNA提取试剂盒购自美国Promega公司, 凋亡试剂盒系美国BD公司产品, BCA试剂盒购自北京索莱宝科技有限公司, PLK1、Ago2抗体购自英国Abcam公司, 多功能酶标仪(美国, Molecular Devices), 贝克曼的流式细胞仪(美国, BECKMAN COULTER), Real-time PCR仪(德国, Agilent Technologies), eBLOT的Western blot化学发光蛋白印迹成像仪(中国, 易孛特)。

1 siRNA的合成及纯化

全2ʹ-修饰的siRNA使用固相合成法在K&A DNA/RNA H-8合成仪中自动合成。合成使用CPG固相载体, 使用2ʹ-F或2ʹ-OMe修饰的RNA亚磷酰胺单体、三氯乙酸的二氯甲烷溶液(3%)、5-乙硫基四氮唑的乙腈溶液(0.25 mol·L^-1)、CAP-A (10%Ac₂O和10%吡啶的乙腈溶液)、CAP-B (10% N-甲基咪唑的乙腈溶液)、氧化剂(0.02 mol·L^-1 I₂/2% H₂O/20%吡啶的四氢呋喃溶液) 及二苯乙酰基二硫化物的吡啶乙腈(50%) 溶液。

固相合成结束时保留最后一个5ʹ-端DMT保护基, 使用浓氨水脱下5′-DMT-AS/SS, 使用半制备HPLC系统和C18反相色谱柱纯化(洗脱方法: ACN/0.05 mol·L^-1 TEAB; 0 min-15% ACN, 22 min-40% ACN), 使用0.05 mol·L^-1 TCA水溶液脱除DMT保护基, 随后使用0.1 mol·L^-1 NaHCO₃水溶液中和过量TCA, 再使用Desalting凝胶柱除盐, 得到纯品进行ESI-LC/MS确证, 随后退火AS/SS得到相应的siRNA修饰物。

2 脂质复合物的制备和转染

siRNA溶于无酶水, DNCA、CLD溶于无水乙醇。加入一半总体积的GenOpti, 依次加入siRNA、DNCA、CLD溶液至液面下, 补加GenOpti至额定体积, 50 ℃超声10 min。

3 生物活性检测

人肝癌细胞Huh-7/HepG2使用含10%胎牛血清(FBS) 的DMEM高糖培养基在37 ℃、5% CO₂的恒温培养箱中培养。

3.1 细胞增殖抑制活性实验(CCK-8)

将Huh-7和HepG2细胞以每孔1×10⁴密度铺至96孔板, 孵箱培养24 h时转染siRNA制剂。48 h后, 真空泵吸弃孔内原有液体, 每孔加入100 μL含10% CCK-8工作液的培养基, 孵育显色30 min后, 使用酶标仪检测450 nm处吸光度值。细胞生存率(V) = (RA-RE)/(RB-RE)×100% (RA、RB、RE分别代表实验组、对照组及溶剂底色组的吸光度值)。

3.2 RNA结合蛋白免疫沉淀实验(RIP)

转染24 h后收集细胞, 加入裂解缓冲液, 细胞裂解液中加入Ago2抗体, 放至垂直混匀器4 ℃孵育16 h。平衡好A/G磁珠加入细胞裂解液中, 垂直混匀器4 ℃孵育1 h。裂解缓冲液洗涤磁珠两次以去除未结合的抗体, 55 ℃孵育1 h洗脱RNA。RT-qPCR实验定量与Ago2蛋白结合siRNA的量。

3.3 基因沉默活性实验(RT-qPCR)

将Huh-7和HepG2细胞以每孔5×10⁴密度铺至12孔板, 孵箱培养24 h后进行转染, 细胞转染24 h后进行RT-qPCR实验, 用Trizol裂解细胞并提取总RNA, 提取的RNA溶于无酶水, 按逆转录试剂盒操作得到cDNA。然后进行实时定量PCR, 获得各个样品组的Ct值。RT-qPCR实验涉及的引物序列如表 2所示。

3.4 细胞摄取测定

将Huh-7细胞以每孔7×10⁵个细胞的密度接种到12孔板中并生长24 h。用Cy5.5标记的siPLK1A和siPLK1A3纳米复合物处理细胞4 h后, 通过胰蛋白酶消化收集细胞并用预冷的PBS洗涤。流式细胞仪测定摄取情况。

3.5 Western blot实验检测蛋白表达

将Huh-7细胞以每孔7×10⁵个细胞的密度接种到12孔板中并生长24 h后进行转染。继续培养24 h后, RIPA裂解液裂解细胞。按照BCA蛋白定量试剂盒说明书操作定量, 在SDS-PAGE凝胶中每孔上样15 μg含loading buffer且已煮沸变性的蛋白样品, 180 V电泳分离20 min。利用iBlot 2转膜仪设置20 V转膜7 min, 用封闭液常温浸泡PVDF膜封闭过夜, 次日洗膜3次, 依次以一抗、二抗孵育, 最后滴加ECL显色液显色, 在蛋白印迹成像仪中拍照检测。

3.6 细胞凋亡测定

将Huh-7细胞以每孔7×10⁵个细胞的密度接种到12孔板中并生长24 h后进行转染, 继续培养24 h, 用0.25%胰蛋白消化细胞, 预冷的PBS洗涤2次, 重悬于100 μL 1×结合缓冲液中。向每个样品中加入5 μL FITC和5 μL PI, 将细胞在室温下避光孵育20 min。然后通过流式细胞仪测定染色样品。

3.7 统计学方法

所有分析均使用GraphPad Prism 8.0软件, 所有数据都是至少3个独立重复实验的平均值, 误差线代表标准偏差。两组独立数据使用单因素t检验, 多组独立数据使用单因素方差分析, P < 0.05认为有统计学意义。

作者贡献: 高宇睛完成了生化、细胞实验及数据分析处理和论文撰写; 王玺贤完成了修饰序列设计合成及论文修改; 潘宇飞、汪全鑫完成了部分修饰序列siRNA的合成; 朱月洁、潘德林和关注参与了指导了实验数据分析和讨论; 杨振军负责实验设计、结果讨论及论文修改定稿。

利益冲突: 所有作者均声明不存在利益冲突。

基金

收起

浙江大学(余杭) 基础医学创新研究院(校合-2019-KYY-A07043-0018)
国家自然科学基金资助项目(21778006)

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2023年第58卷第6期

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文章信息

doi: 10.16438/j.0513-4870.2022-1345

接收时间：2022-12-07
首发时间：2025-11-21
出版时间：2023-06-12

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文章信息

作者

出版历史

收稿日期：2022-12-07
修回日期：2023-01-13

基金

浙江大学(余杭) 基础医学创新研究院(校合-2019-KYY-A07043-0018)

国家自然科学基金资助项目(21778006)

作者信息

1.成都中医药大学药学院, 四川成都 611137

2.北京大学药学院/天然药物及仿生药物国家重点实验室, 北京 100191

通讯作者:

*杨振军, Tel: 86-10-82805781, E-mail: yangzj@bjmu.edu.cn;

潘德林, Tel: 13547460729, E-mail: linfeng_0130@163.com

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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No.	Name		Sequence 5ʹ→3ʹ	(Calcd./Measured) molecular weight
1	siPLK1A	AS	UAU UUA AGG AGG GUG AUC UTT	6 716.1/6 718.1
SS	AGA UCA CCC UCC UUA AAU ATT	6 562.0/6 563.5
2	siPLK1A1	AS	UsAsU UUA AGG AGG GUG AUC UsTsT	6 994.6/6 996.0
SS	AsGsA UCA CCCUCC UUA AAU AsTsT	6 840.6/6 842.3
3	siPLK1A2	AS	UsAsU UUA AGGAGG GUG AUC UsTsT	6 970.6/6 972.0
SS	AsGsA UCA CCCUCC UUA AAU AsTsT	6 840.6/6 842.3
4	siPLK1A3	AS	UsAsUUUA AGG AGG GUG AUCUsTsT	6 934.5/6 935.7
SS	AsGsA UCA CCCUCC UUA AAU AsTsT	6 840.6/6 842.3
5	siPLK1B	AS	UUG AUC CGG AGG UAG GUC UTT	6 706.1/6 708.8
SS	AGA CCU ACC UCC GGA UCA UTT	6 678.1/6 679.5
6	siPLK1B1	AS	UsUsG AUC CGG AGG UAG GUC UsTsT	6 985.6/6 986.9
SS	AsGsA CCU ACCUCC GGA UCA AsTsT	6 894.7/6 896.2
7	siPLK1B2	AS	UsUsG AUC CGG AGG UAG GUC UsTsT	6 961.6/6 963.0
SS	AsGsA CCU ACCUCC GGA UCA AsTsT	6 894.7/6 896.2
8	siPLK1B3	AS	UsUsGAUC CGG AGG UAG GUCUsTsT	6 925.4/6 896.2
SS	AsGsA CCU ACCUCC GGA UCA AsTsT	6 894.7/6 896.2
9	siKSP	AS	AGU UAG UUU AGA UUC UGG ATT	6 712.9/6 713.2
SS	UCG AGA AUC UAA ACU AAC UTT	6 222.1/6 622.1
10	siKSP1	AS	AsGsU UAG UUU AGA UUC UCG AsTsT	6 915.6/6 917.3
SS	UsCsG AGAAUCUAA ACU AAC UsTsT	6 904.7/6 905.6

No.

Name

Sequence 5ʹ→3ʹ

(Calcd./Measured) molecular weight

siPLK1A

UAU UUA AGG AGG GUG AUC UTT

6 716.1/6 718.1

AGA UCA CCC UCC UUA AAU ATT

6 562.0/6 563.5

siPLK1A1

UsAsU UUA AGG AGG GUG AUC UsTsT

6 994.6/6 996.0

AsGsA UCA CCCUCC UUA AAU AsTsT

6 840.6/6 842.3

siPLK1A2

UsAsU UUA AGGAGG GUG AUC UsTsT

6 970.6/6 972.0

AsGsA UCA CCCUCC UUA AAU AsTsT

6 840.6/6 842.3

siPLK1A3

UsAsUUUA AGG AGG GUG AUCUsTsT

6 934.5/6 935.7

AsGsA UCA CCCUCC UUA AAU AsTsT

6 840.6/6 842.3

siPLK1B

UUG AUC CGG AGG UAG GUC UTT

6 706.1/6 708.8

AGA CCU ACC UCC GGA UCA UTT

6 678.1/6 679.5

siPLK1B1

UsUsG AUC CGG AGG UAG GUC UsTsT

6 985.6/6 986.9

AsGsA CCU ACCUCC GGA UCA AsTsT

6 894.7/6 896.2

siPLK1B2

UsUsG AUC CGG AGG UAG GUC UsTsT

6 961.6/6 963.0

AsGsA CCU ACCUCC GGA UCA AsTsT

6 894.7/6 896.2

siPLK1B3

UsUsGAUC CGG AGG UAG GUCUsTsT

6 925.4/6 896.2

AsGsA CCU ACCUCC GGA UCA AsTsT

6 894.7/6 896.2

siKSP

AGU UAG UUU AGA UUC UGG ATT

6 712.9/6 713.2

UCG AGA AUC UAA ACU AAC UTT

6 222.1/6 622.1

siKSP1

AsGsU UAG UUU AGA UUC UCG AsTsT

6 915.6/6 917.3

UsCsG AGAAUCUAA ACU AAC UsTsT

6 904.7/6 905.6

Name	Sequence
PLK1 gene forward	ATCACCTGCCTGACCATTCCACCAAGG
PLK1 gene reverse	AATTGCGGAAATATTTAAGGAGGGTGATCT
KSP forward	CTGAACAGTGGGTATCTTCCTTA
KSP reverse	GATGGCTCTTGACTTAGAGGTTC
β-Actin forward	CCAACCGCGAGAAGATGA
β-Actin reverse	CCAGAGGCGTACAGGGATAG

Name

Sequence

PLK1 gene forward

ATCACCTGCCTGACCATTCCACCAAGG

PLK1 gene reverse

AATTGCGGAAATATTTAAGGAGGGTGATCT

KSP forward

CTGAACAGTGGGTATCTTCCTTA

KSP reverse

GATGGCTCTTGACTTAGAGGTTC

β-Actin forward

CCAACCGCGAGAAGATGA

β-Actin reverse

CCAGAGGCGTACAGGGATAG