脂质偶联siRNA：提升哺乳动物细胞基因抑制效果的新策略（上）

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Abstract 摘要

Nucleic acid conjugates are promising drugs for treating gene-related diseases. Conjugating specific units like lipids, cell-penetrating peptides, polymers, antibodies, and aptamers either at the 3′- or 5′-termini of a siRNA duplex molecule has resulted in a plethora of siRNA bioconjugates with improved stabilities in bloodstream and better pharmacokinetic values than unmodified siRNAs. In this sense, lipid-siRNA conjugates have attracted a remarkable interest for their potential value in facilitating cellular uptake. In this chapter, we describe a series of protocols involving the synthesis of siRNA oligonucleotides carrying either neutral or cationic lipids at the 3′- and 5′-termini. The resulting lipid-siRNA conjugates are aimed to be used as exogenous effectors for inhibiting gene expression by RNA interference. A protocol for the formulation of lipid siRNA using sonication in the presence of serum is described yielding interesting transfection properties for cell culture without the use of transfecting agents.

核酸偶联物有望成为治疗基因相关疾病的药物。将脂质、细胞穿透肽、聚合物、抗体和适体等特异性单元与 siRNA 双链分子的 3' 或 5' 末端结合，产生大量 siRNA 生物偶联物，这些偶联物具有比未修饰 siRNA 更高的血液稳定性和更好的药代动力学参数。从这个意义上说，脂质-siRNA 偶联物因其促进细胞摄取的潜在价值而备受关注。本章描述了一系列涉及合成携带中性或阳离子脂质的 3' 和 5' 末端 siRNA 寡核苷酸的方案。所得的脂质-siRNA 缀合物旨在作为外源效应物，通过 RNA 干扰抑制基因表达。此外，本章还介绍了在含血清的环境下，通过超声处理制备脂质 siRNA 的方法，这种方法无需使用转染试剂就能为细胞培养提供有效的转染特性。

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1 Introduction 引言

Nucleic acids represent a class of biomolecules that contain a great number of biological properties. Since Zamecknik and Stephenson reported in 1978 the use of a 13-mer antisense oligonucleotide as a tool for blocking the replication process of the RNA Rous sarcoma virus, new horizons and novel strategies have been opened up in the development of oligonucleotides as promising drug therapeutics. In fact, the first DNA oligonucleotide (Fomivirsen) approved by the Food and Drug Administration (FDA) for clinical use was effective in 1998. Since then it has come a long way making significant strides in the field of nucleic acid therapeutics with the approval of eight more antisense oligonucleotides (e.g., Pegaptanib, Mipomersen, Eteplirsen, Defibrotide, Nusinersen, and Inotersen) and two RNA interference-based therapeutics (e.g., Patisiran and Givosiran), the latter being accepted for human use in 2018 and 2019, respectively. This success has allowed nucleic acids to be launched to several advanced clinical trials and becoming a promising alternative to traditional therapy when using small molecule drugs to combat gene-related diseases.

核酸是一类具有众多生物学特性的生物分子。自 1978 年 Zamecknik 和 Stephenson 首次报道利用 13 聚反义寡核苷酸阻断 RNA Rous 肉瘤病毒的复制以来，寡核苷酸作为药物治疗工具的开发开辟了新的前景和策略。实际上，第一个获得美国食品药品监督管理局（FDA）批准用于临床的 DNA 寡核苷酸药物 Fomivirsen 于 1998 年投入使用。自那以来，核酸疗法领域取得了显著进展，八种反义寡核苷酸（如 Pegaptanib、Mipomersen、Eteplirsen、Defibrotide、Nusinersen 和 Inotersen）和两种基于 RNA 干扰的治疗药物（如 Patisiran 和 Givosiran）相继获批，后者分别在 2018 年和 2019 年获准上市。这一成就使得核酸疗法进入多个高级临床试验阶段，并成为小分子药物治疗基因相关疾病的传统疗法的有力替代方案。

RNA interference (RNAi) is a powerful process, discovered by Fire and Mello, in which a 21–25 base pair double-stranded small interference RNA (siRNA) is able to trigger the RNA-induced silencing complex (RISC) and interact specifically with a messenger RNA (mRNA). In this regard, this process degrades such mRNA inhibiting translation step by blocking protein production. Interestingly, this mechanism of action is catalytic making RNAi to be superior and more efficient in inhibiting specific target proteins. Despite this silencing activity, siRNA-based therapeutics must cope with many challenges including stability in bloodstream and low transfection efficiencies, among others. While formulation strategies have been developed to avoid degradation and therefore enhance cellular internalization, decoration of such nano-formulations with targeting ligands has also improved effectiveness and selectivity of siRNA molecules in vivo. Some comprehensive reviews have been recently published on this topic.

RNA 干扰（RNAi）是由 Fire 和 Mello 发现的一种强效机制，通过 21-25 碱基对的双链小干扰 RNA（siRNA）触发 RNA 诱导的沉默复合物（RISC），并与信使 RNA（mRNA）特异性结合，从而降解 mRNA，阻断蛋白质的翻译过程。这种催化性机制使得 RNAi 在抑制特定目标蛋白质方面更为高效。尽管如此，siRNA 疗法仍面临许多挑战，如在血液中的稳定性和低转染效率等。为了解决这些问题，已经开发了多种配方策略以防止降解并提高细胞内化。此外，通过在纳米配方上装饰靶向配体，进一步提高了 siRNA 分子的有效性和选择性。最近，关于这一主题已有多篇综合性综述发表。

It is well known that also modifying chemically certain positions of natural nitrogenous bases and sugar rings has remarkably improved stability without compromising original siRNA activities. On the other hand, conjugating either 3′- or 5′-end positions with bioactive molecules like lipids, cell-penetrating peptides, polymers, and/or antibodies has enabled the preparation of more stable siRNA conjugates when compared to their siRNA complex counterparts. In this regard, the use of chemical strategies has resulted in synthesizing siRNA bioconjugates with improved stabilities, potency, targetability as well as exhibiting better siRNA fate when administered in vivo. The development of various chemical strategies to modify siRNA backbone with lipids, cell-penetrating peptides, aptamers, and polymers has been reviewed in many revision articles.

众所周知，通过化学修饰天然氮碱基和糖环的特定位置可以显著提高 siRNA 的稳定性，同时不会影响其原有活性。另一方面，将生物活性分子如脂质、细胞穿透肽、高分子和/或抗体连接到 siRNA 的 3′或 5′末端，能够制备出比传统 siRNA 复合物更稳定的 siRNA 共轭物。在这方面，化学策略的应用使得合成的 siRNA 生物共轭物具有更高的稳定性、效力和靶向性，并在体内给药时表现出更好的效果。许多综述文章已经回顾了通过脂质、细胞穿透肽、适配体和高分子修饰 siRNA 骨架的各种化学策略的发展。

The presence of hydrophobic pendent groups has made RNA oligonucleotide chains to increase in lipophilicity and thus turn into more drug-like molecules to induce a specific activity in vitro and in vivo. Several chemical strategies have been successfully developed to modify siRNA backbones with lipid moieties either at 5′- or 3′-termini of the passenger strand including both stable and cleavable linkages. For instance, a direct conjugation involving a lipid residue and siRNA was reported by Kubo et al. in 2013. Such chemical combination required a series of siRNAs which were modified properly with an amino modifier and a N-hydroxysuccinimide ester derivative that was attached to the corresponding lipid chains. This protocol allowed the authors to introduce covalently hydrophobic residues into a series of siRNAs at the 5′-end in the presence of N,N-diisopropylethylamine (DIEA) and a mixture of isopropanol:water (1:1).

由于引入了疏水的侧链基团，RNA 寡核苷酸链变得更具亲脂性，因此更像药物分子，能够在体内外引发特定的活性。目前已经开发出多种化学策略，通过在正义链的 5'或 3'端附加稳定或可裂解的脂质部分来修饰 siRNA 骨架。例如，Kubo 等人在 2013 年报道了一种直接结合脂质残基和 siRNA 的方法。该方法需要使用适当修饰了氨基和连接了脂质链的 N-羟基琥珀酰亚胺酯衍生物的一系列 siRNAs。在 N,N-二异丙基乙胺（DIEA）和异丙醇：水（1:1）的混合物存在下，这种方法使得能够在一系列 siRNAs 的 5'端共价引入疏水残基。

Introducing lipid moieties at the 3′-termini of siRNAs requires additional synthetic efforts based on derivatizing Controlled Pore Glass (CPG) solid supports with small molecule linkers. These units containing hydrophobic residues have been further modified with specific acid-labile protecting groups like trityl (Tr), 4-monomethoxy trityl (MMTr), or 4,4′-dimethoxy trityl (DMTr) giving rise to lipid-modified CPG solid supports that have been used in conjunction with automatic oligonucleotide synthesizers. In this regard, many examples have been described for the chemical conjugations of cholesterol and other lipids (e.g., α-tocopherol, lithocolic-oleyl, docosanoic acid, docosahexaenoic acid, and eicosapentaenoic acid) with siRNA oligonucleotides showing improved silencing activities and specific accumulations in liver but also other tissues like skeletal muscle and heart when injected intravenously (Fig. 1). While internalization mechanisms of some siRNA conjugates remain unclear, tissue uptake and cellular internalization efficacies are driven not only by the hydrophobic nature of the lipid pendent groups but also the effective binding of such conjugates to serum proteins such as high-density and low-density lipoproteins (HDL and LDL, respectively) as well as albumin.

在 siRNA 的 3'端引入脂质部分需要额外的合成步骤，基于使用小分子连接物修饰的控制孔玻璃（CPG）固体支持。这些含有疏水残基的单元进一步用酸敏保护基如三苯甲基（Tr）、4-单甲氧基三苯甲基（MMTr）或 4,4'-二甲氧基三苯甲基（DMTr）修饰，从而产生脂质修饰的 CPG 固体支持，它们已经与自动寡核苷酸合成器一起使用。在这方面，已经有许多将胆固醇和其他脂质（如α-生育酚、石胆酸-油酸、二十二酸、二十二碳六烯酸和二十碳五烯酸）与 siRNA 寡核苷酸进行化学结合的例子，这些结合显示出改进的沉默活性，并且在肝脏和其他组织如骨骼肌和心脏中有特异性积累，特别是在静脉注射时（图 1）。尽管一些 siRNA 结合物的内化机制尚不清楚，但组织摄取和细胞内化效率不仅由脂质侧链基团的疏水性驱动，还由这些结合物与高密度和低密度脂蛋白（分别为 HDL 和 LDL）以及白蛋白的有效结合驱动。

Fig. 1 Scheme of some of the chemical structures of lipids that have been incorporated in siRNA

In an effort to find additional strategies addressed to enhance the efficiency of siRNAs in transfection processes, our research group has studied the effect of several lipid modifications introduced either at the 3′- or 5′-termini of a siRNA passenger strand taking TNF-α as a target protein model (Fig. 2). TNF-α was selected because overexpression of this gene is found associated to many diseases such as cancer, autoimmune diseases: rheumatoid arthritis, ankylosing spondylitis, Crohn’s disease, psoriasis, refractory asthma. In order to connect the lipid moiety to the RNA molecules, we selected glycerol as a small linker to prepare a series of siRNA conjugates containing lipid pendent groups in the form of ether lipids both neutral and cationic at the 3′-end. On the other hand, other neutral and cationic lipids were also used to modify siRNA passenger strand at the 5′-termini by using the well-known phosphoramidite chemistry strategy.

为了进一步增强 siRNA 在转染过程中的效率，我们的研究团队以 TNF-α为靶标蛋白模型（如图 2 所示），研究了在 siRNA 乘客链的 3′或 5′末端引入不同脂质修饰的效果。选择 TNF-α作为研究对象是因为该基因的过度表达与许多疾病有关，包括癌症和多种自身免疫性疾病：类风湿性关节炎、强直性脊柱炎、克罗恩病、银屑病和顽固性哮喘。为了将脂质基团与 RNA 分子连接，我们选用了甘油作为小分子链接剂，制备了一系列在 3′末端含有中性和阳离子醚脂的 siRNA 缀合物。同时，也通过广为人知的磷酰胺化学策略，将其他中性和阳离子脂质用于修饰 siRNA 乘客链的 5′末端。

Fig. 2 Schematic representation of some glycerol-based lipids described for the preparation of lipid-siRNAs modifying the RNA passenger strand

Herein, we describe specific experimental protocols that have been carried out in the field of siRNA delivery with the aim to increase both its potency and transfection efficiency. Particularly, we describe a protocol to synthesize a series of siRNA conjugates carrying neutral and cationic lipids at the 3′- and 5′-termini modifying chemically the siRNA guide strand (Fig. 2 and Table 1). In addition, a formulation method used for increasing the binding capacity of unmodified and chemically modified siRNAs through a process namely “pre-binding” is described. This protocol that we named as “f-siRNA” protocol is based on a short sonication of lipid-siRNA conjugates in the presence of serum with the aim to facilitate their binding to serum proteins such as albumin, HDL, and LDL. The ultrasound treatment carried out before adding the siRNA conjugates to cell culture showed a clear enhancement of cellular uptake and therefore resulted in an enhancement of the silencing activities promoted by such siRNA conjugates in vitro when compared with non-ultrasound-treated oligonucleotides.

本文中，我们介绍了一些在 siRNA 递送领域中执行的实验方案，目的是提升其效力和转染效率。特别是，我们详细描述了通过化学修饰 siRNA 引导链，在其 3′和 5′末端引入中性和阳离子脂质的合成过程（如图 2 和表 1 所示）。此外，我们还介绍了一种称为“预结合”的配方方法，用于增强未修饰和化学修饰 siRNA 的结合能力。我们将此方案命名为“f-siRNA”方案，其基于对脂质-siRNA 缀合物进行短时间的超声处理，以促进它们与血清蛋白如白蛋白、高密度脂蛋白（HDL）和低密度脂蛋白（LDL）的结合。实验结果显示，在将 siRNA 缀合物加入细胞培养之前进行超声处理，与未处理的寡核苷酸相比，显著提高了细胞摄取效率，从而在体外增强了这些 siRNA 缀合物的基因沉默效果。

Table 1 Lipid-siRNA conjugates carrying glycerol-based long linear alkyl chains.

Structures for Chol, lipidC14, lipidC18, and lipid_C14N are shown in Fig. 2. Underlined residues indicate the position of 2′-O-methyl-RNA residues

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2 Materials

Use deionized pure water with a resistivity of 18.2 MΩ to dissolve both RNA strands (passenger and guide) and prepare buffer solutions (see Note 1). Reagents were used as received without being further purified.

使用电阻率为 18.2 MΩ的去离子纯水来溶解 RNA 链（包括载链和导链）并配制缓冲溶液（详见 注释 1）。所有试剂在未经进一步纯化的情况下直接使用。

2.1 RNA Oligonucleotide Synthesis RNA 寡核苷酸的合成

Perform the protocols described below in a DNA synthesizer (Applied Biosystems 3400) using two different scales: LV200 and 1 μmol (see Note 2). Use solvents and ancillary reagents with DNA synthesis grade (see Note 3).

在 DNA 合成仪（Applied Biosystems 3400）上进行以下合成步骤，实验规模有 LV200 和 1 微摩尔两种（详见 注释 2）。使用符合 DNA 合成级别的溶剂和辅助试剂（详见 注释 3）。

1. A computer equipped with the 3400 DNA Synthesizer software. 配备 3400 DNA 合成仪软件的计算机

2. A DNA synthesizer (e.g., Applied Biosystems 3400). DNA 合成仪（例如，Applied Biosystems 3400）

3. Unmodified DNA controlled-Pore Glass (CPG) solid support (3′-dT-CPG) (see Note 4). 未修饰的 DNA 控制孔玻璃（CPG）固体载体（3′-dT-CPG）（详见 注释 4）

4. Unmodified RNA CPG solid supports (Bz-A-RNA, Ac-C-RNA, Ac-G-RNA and U-RNA) (see Note 5). 未修饰的 RNA CPG 固体载体（Bz-A-RNA, Ac-C-RNA, Ac-G-RNA 和 U-RNA）（详见 注释 5）

5. RNA 2-cyanoethyl (CE) phosphoramidites (Bz-A-CE, Ac-C-CE, dmf-G-CE and U-CE phosphoramidites) (see Note 5). RNA 2-氰乙基（CE）磷酰胺（Bz-A-CE, Ac-C-CE, dmf-G-CE 和 U-CE 磷酰胺）（详见 注释 5）

6. RNA phosphoramidites modified at their 2′-position of the sugar with OMe (2′-OMe-A-CE, 2′-OMe-Ac-C-CE, 2′-OMe-dmf-G-CE and 2′-OMe-U-CE phosphoramidites) (see Note 6). 2′-位经 OMe 修饰的 RNA 磷酰胺（2′-OMe-A-CE, 2′-OMe-Ac-C-CE, 2′-OMe-dmf-G-CE 和 2′-OMe-U-CE 磷酰胺）（详见 注释 6）

7. Anhydrous acetonitrile. 无水乙腈

8. 3% Trichloroacetic acid (TCA) in dichloromethane. 3%三氯乙酸（TCA）在二氯甲烷中的溶液

9. Anhydrous dichloromethane. 无水二氯甲烷

10. 0.4 M 1H-tetrazol in acetonitrile. 0.4 M 1H-四氮唑在乙腈中的溶液

11. Capping A: Acetic anhydride/pyridine/tetrahydrofurane (1:1:8). 封端试剂 A：乙酸酐/吡啶/四氢呋喃（1:1:8）

12. Capping B: 10% N-methylimidazole in tetrahydrofurane. 封端试剂 B：10% N-甲基咪唑在四氢呋喃中的溶液

13. 0.01 M Iodine in tetrahydrofurane/pyridine/water (7:2:1) (see Note 7). 0.01 M 碘在四氢呋喃/吡啶/水（7:2:1）中的溶液（详见 注释 7）

14. Concentrated ammonia (32%) in water. 水溶液中的浓氨水（32%）

15. Triethylamine trihydrofluoride, 98% (TREAT-HF). 三乙胺三氟化物，纯度 98%（TREAT-HF）

16. Triethylamine. 三乙胺

17. N-Methylpyrrolidone, 97%. N-甲基吡咯烷酮，纯度 97%

18. Isopropoxytrimethylsilane, 98%. 异丙氧基三甲基硅烷，纯度 98%

19. Annealing buffer: 10 mM Tris-Base [Tris(hydroxymethyl)aminomethane], 50 mM NaCl, 1 mM EDTA (ethylenediaminetetraacetic acid), pH 8.0. 退火缓冲液：10 mM Tris-碱 [Tris（羟甲基）氨基甲烷]，50 mM NaCl，1 mM EDTA（乙二胺四乙酸），pH 8.0

20. 3 M Sodium acetate solution, pH 5.2. 3 M 醋酸钠溶液，pH 5.2

21. Ethanol. 乙醇

22. A centrifuge 5424 R instrument. 5424 R 型离心机

23. Argon or nitrogen sources. 氩气或氮气源

24. 2 mL Glass screw vials. 2 毫升玻璃螺旋瓶

25. Dry bath tube heating block. 干浴加热块

26. Speed Vac evaporator. 旋转蒸发仪

2.2 High-Performance Liquid Chromatography (HPLC) 高效液相色谱法

Detach RNA single strand oligonucleotides from CPG solid supports once RNA synthesis has been completed (see Subheading 3.1). Analyze and purify by reverse-phase HPLC (RP-HPLC) in order to obtain the corresponding RNA oligonucleotide strands (guide or passenger) with high purity.

在 RNA 合成完成后，将 RNA 单链寡核苷酸从 CPG 固体载体中分离出来（详见小标题 3.1）。通过反相 HPLC（RP-HPLC）进行分析和纯化，以获得高纯度的 RNA 寡核苷酸链（导链或载链）

1. Analytical and/or semi-preparative RP-HPLCs are both equipped with a Waters 2695 Separation Module and a Waters 2998 Photodiode Array Detector. 分析型或半制备型 RP-HPLC，配有 Waters 2695 分离模块和 Waters 2998 光二极管阵列检测器

2. A computer equipped with Empower™ 3 chromatography data software. 配有 Empower™ 3 色谱数据软件的计算机

3. A HPLC reverse-phase column C18, with a size pore of 2.5 μm, and 4.6 × 50 mm column (see Note 8). HPLC 反相 C18 柱，孔径为 2.5 微米，柱长 4.6 × 50 毫米（详见 注释 8）

4. Gas-tight syringe.气密注射器

5. 2 mL Conical plastic tubes (see Note 9). 2 毫升锥形塑料管（详见 注释 9）

6. 1 M Aqueous triethylammonium acetate (TEAA): acetic acid, triethylamine, water, pH 7.01 (stock solution). 1 M 三乙胺醋酸盐（TEAA）水溶液：由醋酸、三乙胺和水配置，pH 7.01（储备溶液）

7. Buffered solution A (1 L): 5% acetonitrile, 0.1 M aqueous TEAA, pH 7.01. 缓冲液 A（1 升）：5%乙腈，0.1 M TEAA 水溶液，pH 7.01

8. Buffered solution B (1 L): 70% acetonitrile, 0.1 M aqueous TEEA, pH 7.01. 缓冲液 B（1 升）：70%乙腈，0.1 M TEAA 水溶液，pH 7.01

2.3 Desalting RNA Oligonucleotide Solutions RNA 寡核苷酸溶液的脱盐

1. Disposable NAP DNA/RNA size-exclusion purification columns (NAP-5 or NAP-10 columns) (see Note 10). 一次性 NAP DNA/RNA 尺寸排阻纯化柱（NAP-5 或 NAP-10 柱）（详见 注释 10）

2. Sephadex G-25. 葡聚糖凝胶 G-25

3. RNAse-free water (DEPC-water) (see Note 1). 无 RNA 酶水（DEPC 水）（详见 注释 1）

4. Eyedropper. 滴管

5. 2 mL Conical plastic tubes (see Note 9). 2 毫升锥形塑料管（详见 注释 9）

2.4 Ultraviolet-Visible (UV) Spectroscopy 紫外-可见光 (UV) 光谱分析

1. A computer equipped with the Spectra Manager™ II software. 配有 Spectra Manager™ II 软件的计算机

2. A Jasco V-650 UV-VIS spectrophotometer equipped with a thermoregulated cell holder. 配有温控池座的 Jasco V-650 紫外-可见分光光度计

3. A 1 mL-UV quartz Hellma absorption cuvette with PTFE stoppers. 1 毫升的 UV 石英 Hellma 吸收比色皿，配有 PTFE 塞子

4. RNAse-free water (DEPC-water) (see Note 1). 无 RNA 酶水（DEPC 水）（详见 注释 1）

5. Eyedropper. 滴管

6. 2 mL Conical plastic tubes (see Note 9). 2 毫升锥形塑料管（详见 注释 9）

2.5 Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry 基质辅助激光解吸/电离飞行时间 (MALDI-TOF) 质谱分析

Identify and characterize the correct mass of RNA oligonucleotides carrying lipid pendent groups using a MALDI-TOF mass spectrometry .

使用 MALDI-TOF 质谱法来识别和表征带有脂质侧链的 RNA 寡核苷酸的准确质量

1. Perseptive Voyager-DE™RP spectrometer (Applied Biosystems) in negative mode equipped with nitrogen laser at 337 nm with a 3 ns-pulse. 使用Perseptive Voyager-DE™RP 质谱仪（Applied Biosystems），负离子模式，配有 337 纳米波长的氮激光器，脉冲时间为 3 纳秒

2. A computer equipped with Data Explorer™ software. 配有 Data Explorer™软件的计算机

3. 2,4,6-Trihydroxyacetophenone (THAP): 10 mg/mL in a mixture of acetonitrile:water (1:1). 2,4,6-三羟基苯乙酮（THAP）：浓度为 10 mg/mL 的乙腈和水（1:1）混合溶液

4. Ammonium citrate (CA): 50 mg/mL aqueous solution (see Note 11). 柠檬酸铵（CA）：浓度为 50 mg/mL 的水溶液（详见 注释 11）

5. RNAse-free water (DEPC-water) (see Note 1). 无 RNA 酶水（DEPC 水）（详见 注释 1）

2.6 Cell Culture Models 细胞培养模型

1. Immortal human cervical cancer HeLa cells or 4T1 murine breast cancer cells. 永生化人宫颈癌 HeLa 细胞或 4T1 小鼠乳腺癌细胞

2. Dulbecco’s Modified Eagle Medium (DMEM) with Glutamax supplement (500 mL). 含 Glutamax 补充剂的杜氏改良鹰培养基（DMEM）（500 毫升）

3. Opti-MEM reduced serum medium (500 mL). Opti-MEM 低血清培养基（500 毫升）

4. 1× Phosphate-buffered saline solution (PBS) (500 mL). 1×磷酸盐缓冲盐溶液（PBS）（500 毫升）

5. Fetal bovine serum (FBS) (500 mL). 胎牛血清（FBS）（500 毫升）

6. Penicillin and streptomycin (100 U/mL and 100 mg/mL, respectively). 青霉素和链霉素（分别为 100 U/mL 和 100 mg/mL）

7. Lipofectamine™ 2000 transfection reagent (0.75 mL, ThermoFischer Scientific). Lipofectamine™ 2000 转染试剂（0.75 毫升，ThermoFischer Scientific）

2.7 Enzyme-Linked Immunosorbent Assay (ELISA) 酶联免疫吸附测定 (ELISA)

Detect and quantify the presence of ligands or antigens by carrying out the ELISA assay. To do so, use the Murin TNF-α Instant Elisa (Bender MedSystems) instead of the conventional one to detect quantitatively TNF-α from the cell culture supernatant following the manufacturer’s instructions.

通过进行酶联免疫吸附测定（ELISA）来检测和定量配体或抗原的存在。使用 Murin TNF-α Instant Elisa（Bender MedSystems）代替传统方法，根据制造商的说明，从细胞培养上清液中定量检测 TNF-α。

1. A pre-coated 96-well microplate. 预涂层 96 孔微孔板

2. A biotinylated detection antibody.生物素化的检测抗体

3. A SAV-HRP conjugate, generally an antibody or streptavidin. SAV-HRP 缀合物，通常是抗体或链霉亲和素

4. Diluent and wash buffers. 稀释液和洗涤缓冲液

5. A substrate solution. 底物溶液

6. Lysis buffer 10×.裂解缓冲液 10×