WO2019228108A1 - 用于提高细胞转染效率的试剂组合物 - Google Patents

用于提高细胞转染效率的试剂组合物 Download PDF

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WO2019228108A1
WO2019228108A1 PCT/CN2019/084394 CN2019084394W WO2019228108A1 WO 2019228108 A1 WO2019228108 A1 WO 2019228108A1 CN 2019084394 W CN2019084394 W CN 2019084394W WO 2019228108 A1 WO2019228108 A1 WO 2019228108A1
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cells
transfection
actinomycin
solution
medium
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陈骐
傅雅娟
郑立群
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福建师范大学
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the invention belongs to the field of biotechnology, and particularly relates to a reagent composition for improving the efficiency of cell transfection.
  • Gene therapy is a new research field in contemporary medicine and biology. It introduces normal genes or therapeutic DNA sequences into target cells in a certain way to correct gene defects or play a therapeutic role, thereby achieving the purpose of treating diseases.
  • scientists have been studying effective ways to deliver plasmid DNA.
  • conventional exogenous expression systems are mainly divided into two types: one is the method of using viral vectors.
  • the three mainstream viral vectors include lentiviral vectors, retroviral viral vectors, and adenovirus (AAV) vectors. These methods are the mainstream choice for clinical trials such as gene therapy.
  • Viral vectors such as AAV vectors have the characteristics of high transfection rate and continuous and stable expression of foreign genes, but they need to be integrated into the host cell's chromosome to enable the continuous expression of foreign genes, so they have the risk of causing cancer and teratogenicity.
  • the disadvantage of poor safety has become the main reason for limiting the widespread use of viral vectors.
  • the other is the use of non-viral vectors.
  • This kind of non-viral vector is highly safe and easy to prepare. In terms of effects and development prospects, it is more advantageous than viral vectors in gene therapy.
  • the introduction efficiency and targeting of this type of vector are low.
  • Most host cells are highly resistant to foreign DNA, and the expression time of foreign genes is short after they are transferred into the host cells. The foreign genes are quickly shut down after transient expression. All have severely restricted the application and development of such carriers.
  • Non-viral vectors include liposomes, polymer vectors, nanogene transporters, and the like.
  • the general transfection methods are mostly liposome and electroporation methods.
  • the surface of the cationic liposome is positively charged, and the DNA molecule is encapsulated into a DNA-liposome complex by electrostatic interaction with the phosphate of the nucleic acid. It can be adsorbed on the negatively charged cell membrane and enter through the membrane fusion or endocytosis. Cells in vivo. Sometimes DNA is also transferred into cells through the direct penetration of small holes in the cell membrane.
  • the electroporation method or electroporation method uses a high-intensity electric field to instantly increase the permeability of the cell membrane, thereby sending foreign molecules, such as DNA, mRNA, proteins, and sugars, into the cytoplasm of the host cell.
  • the methods currently used for genetic modification of primary T cells are mainly performed by viral infection and electroporation. Viral methods often require higher virus titers when infecting primary cells and are generally less effective. For T cells, additional cytokines, such as IL-2 and IL-7, are needed for stimulation in order to have a certain viral infection efficiency.
  • the electroporation method has advantages in terms of ease of operation and experimental cycle compared to viral infection of primary cells. However, the popularity of this method is limited by many factors such as electrotransformation efficiency, cell survival rate, and differences in experimental equipment. In addition, primary cells are highly resistant to a variety of transfection methods, making the means for efficient gene editing of cells relatively scarce.
  • This article introduces a set of reagent compositions to inhibit the cellular immune pathways to improve the expression efficiency of the genes carried by the vector in the target cells, so as to be used in the clinical application of gene therapy.
  • the purpose of the present invention is to provide a reagent composition for improving the transfection efficiency of cells.
  • the reagent composition and a complex containing DNA and a transfection reagent or a lentivirus packaged with a target vector are separately added to the cell culture solution to improve the cell Transfection efficiency.
  • the reagent mixture is easy to operate, highly reproducible, and widely used. It has been successfully used in cell lines such as L929, BJ and mouse primary mouse fibroblasts, primary lung fibroblasts, T lymphocytes and other primary cells. .
  • the present invention adopts the following technical solutions:
  • a reagent composition for improving cell transfection efficiency comprises: a compound BX795, a compound (Rusolitinib) Ruxolitinib, a compound Tofacitinib, and a compound Actinomycin D ).
  • the reagent composition is composed of two or more substances of compound BX795 0.01-10 ⁇ M, compound Ruxolitinib 0.1-100 ⁇ M, compound Tofacitinib 0.1-100 ⁇ M, or compound Actinomycin D 0.1-100 nM.
  • the combination and preparation method of the reagent composition include the following:
  • Ruxolitinib solution 5 mg Ruxolitinib was added to 0.3260 ml of DMSO to obtain a 50 mM Ruxolitinib solution;
  • Tofacitinib solution 10 mg Tofacitinib Citrate was added to 0.3964 ml DMSO to obtain a 50 mM Tofacitinib Citrate solution;
  • the reagent concentration is as follows: BX795 is 0.01-10 ⁇ M, Ruxolitinib 0.11-100 ⁇ M, Tofacitinib is 0.1-100 ⁇ M, Actinomycin D 0.1-100 nM.
  • the method of the present invention is particularly suitable for the transfection of adherent cell lines, primary mouse fibroblasts, and primary mouse lung fibroblasts, and is suitable for a variety of cell lines that are difficult to transfect or have low transfection efficiency. .
  • the method of the present invention is applicable to various transfection methods, such as PEI transfection method, Lipofectamine lipid system transfection method, electrotransfection method, etc.
  • the method of the present invention has low biological toxicity, is stable in serum, has higher transfection efficiency, and is easy to operate as a gene transfection auxiliary reagent.
  • the reagent is formulated in the form of a mixture, and its transfection and expression effect is far superior to that of a single-component compound.
  • FIG. 1 GFP fluorescence effect of L929 cells transfected with each component.
  • Figure 2 The effect of mixed solution A on cell efficiency of L929 cells transfected by liposome transfection was detected by immunoblotting.
  • Fig. 3 Fluorescent effect diagram of the effect of a pair of mixed drugs on the transfection efficiency of L929 cells.
  • Figure 4 Fluorescence effect diagram of the effect of mixing three drugs on the transfection efficiency of L929 cells.
  • Figure 5 Fluorescence effect of mixed solution A on lung fibroblasts of mice transfected with liposome transfection and flow cytometric analysis.
  • Figure 6 Fluorescent effect of mixed solution A on lentiviral infection of human primary T cells.
  • BX795 solution 10 mg of BX795 (purchased from Selleck) was added to 1.6907 ml of DMSO to obtain a 10 mM BX795 mother liquor;
  • Ruxolitinib solution 5 mg of Ruxolitinib (purchased from APEXBIO) was added to 0.3260 ml of DMSO to obtain 50 mM Ruxolitinib solution;
  • Tofacitinib Citrate solution 10 mg Tofacitinib Citrate (purchased from APEXBIO) was added to 0.3964 ml of DMSO to obtain 50 mM Tofacitinib Citrate solution
  • the final concentration range of the compound BX795 on the cells may be 0.01-10 ⁇ M;
  • the final concentration of the compound Ruxolitinib on the cells may range from 0.1 to 100 ⁇ M;
  • the final concentration range of the compound Tofacitinib Citrate acting on the cells may be 0.1-100 ⁇ M;
  • the final concentration of the compound Actinomycin D on cells can range from 0.1 to 100 nM;
  • An endotoxin-removing plasmid extraction kit (purchased from Qiagen) was used to prepare an expression plasmid with the desired gene for transfection (the plasmid used in the present invention is an expression vector pEGFP-N1 containing a green fluorescent protein reporter gene, which is derived from Clontech Company), see the manufacturer's instructions for the method.
  • the plasmid was dissolved in 200 ⁇ l of ddH 2 O buffer, and its concentration was measured by a UV spectrophotometer.
  • Example 1 The effect of mixed solution A on cell transfection efficiency was verified in L929 cells
  • the test method is as follows:
  • liposome Lipofectamine 2000 was used to transfect L929 cells.
  • L929 cells were seeded at a suitable concentration in a 6-well plate and cultured in an incubator containing 5% CO 2 and 37 ° C for 12 hours. When the cells were completely adherent, the transfection could begin to reach 60-90%; before transfection; Change the medium for 1 hour. Replace the old medium with 1 ml of fresh DMEM complete medium containing 10% FBS. Add Dmix Complete A to the DMEM complete medium. The ratio of the mixture to the medium is 1: 1000.
  • the final concentration in the medium was 0.25 ⁇ M BX795, 5 ⁇ M Ruxolitinib, 5 ⁇ M Tofacitinib Citrate, 5 nM actinomycin D.
  • Prepare the transfection mixture In a 1.5 ml round-bottom sterile tube, mix 40 ⁇ l of Opti-MEM solution and 2 ⁇ l of Lipofectamin 2000 (Thermo Fisher), shake in a vortex shaker, and let stand for 5 minutes; add 2 ⁇ g pEGFP-N1 plasmid, gently shake and mix, and let stand for 20 minutes; add the DNA-Lipofecatamine mixture evenly to L929 cells, and gently shake while adding; after 12 hours, aspirate the medium and DNA Precipitate, wash twice with 1x PBS solution, and then replace with fresh medium; the medium needs to contain mixed solution A.
  • the test results are as follows: the untreated L929 cells are set as the first group, the single-component pretreated L929 cells are set as the second group of control groups, and the mixed solution A pretreated L929 cells are set as the experimental group.
  • the pEGFP-N1 plasmid was transfected into the control group and the experimental group, and the results are shown in FIG. 1.
  • the results in Figure 1 show that under bright field conditions, whether in the control group or the experimental group, the cells adhere to the wall. Although the cell proliferation of the experimental group is slightly weaker than that of the control group, its cell state is good and there are fewer dead cells; in dark field conditions The expression of GFP in the cells was observed below.
  • the fluorescence number of cells in the experimental group of the pretreated mixed solution A was higher than that of the two control groups, and the fluorescence intensity was strong. Staining efficiency and GFP expression efficiency, the results are far better than single-component pretreated or untreated cells.
  • the expression of GFP protein was then detected by immunoblot experiments ( Figure 2). The results showed that when the internal parameters were consistent (indicating that the number of cells was approximately the same), the expression level of GFP protein in the experimental group was much higher than that in the two control groups, which proved that Pretreatment of mixed solution A during infection can effectively promote the transfection and expression of GFP plasmid in L929 cells.
  • the test method is as follows:
  • the solvent DMSO was added to DMEM complete medium (1 ⁇ l DMSO / ml DMEM medium); the experimental group was L929 cells with two or two mixed drugs added to DMEM complete medium: BX795 + Ruxolitinib, BX795 + Tofacitinib Citrate, BX795 + Actinomycin D, Ruxolitinib + Tofacitinib Citrate, Ruxolitinib + Actinomycin D, Tofacitinib Citrate + Actinomycin D.
  • L929 cells were seeded in a 6-well plate and cultured in an incubator containing 5% CO 2 and 37 ° C for 12 hours. When the cells were completely adherent, and the cells reached 60-90%, transfection was started. Change the solution 1 hour before transfection.
  • test results are as follows: As shown in Figure 3, the transfection efficiency of GFP expressing L929 pre-treated with drug pairing is higher than that of the control group, which proves that drug pairing can effectively promote GFP plasmid in L929 cells during transfection. Transfection and expression.
  • the test method is as follows:
  • any three drug mixtures of BX795 (final concentration 0.25 ⁇ M), Ruxolitinib (final concentration 5 ⁇ M), Tofacitinib Citrate (final concentration 2.5 ⁇ M), and Actinomycin D (final concentration 5 nM) were divided into 4 in proportion.
  • L929 cells were seeded at a suitable concentration in a 6-well plate and cultured in an incubator containing 5% CO 2 and 37 ° C for 12 hours. When the cells were completely adherent, the transfection could begin to reach 60-90%; before transfection; Change the medium for 1 hour, and replace the old medium with 1 ml of fresh DMEM complete medium containing 10% FBS; add the above four groups of mixed solutions to the DMEM complete medium, the ratio of the mixed solution to the medium is 1: 1000; Prepare the transfection mixture: in a 1.5 ml round-bottom sterile tube, mix 40 ⁇ l of Opti-MEM solution and 2 ⁇ l of Lipofectamine 2000 (Thermo Fisher), shake in a vortex shaker, and let stand for 5 minutes; Add 2 ⁇ g of pEGFP-N1, gently shake and mix, and let stand for 20 minutes; add the DNA-Lipofectamine mixture evenly to L929
  • test results are as follows: pEGFP-N1 plasmid was transfected into the control group and the experimental group. The results are shown in Figure 4. 0.25 ⁇ M BX795, 5 ⁇ M Ruxolitinib, 2.5 ⁇ M Tofacitinib Citrate and 10 nM Actinomycin D are mixed with each other to pretreat L929 cells. The effect is similar to mixing two pairs. Compared with untreated L929 cells, the expression of GFP is greatly improved. The concentration can greatly exert the combined effect of drugs, and more effectively promote the transfection and expression of GFP plasmid in L929 cells.
  • the test method is as follows:
  • mice primary lung fibroblasts As an example, the effects of drug mixing on the transfection efficiency of primary adherent cells were examined. The experiment was divided into two groups. The control group was DMEM complete medium. DMSO / ml DMEM) mouse lung fibroblasts. The experimental group consisted of mouse lung fibroblasts with mixed solution A in DMEM complete medium. The final concentration of mixed solution A in the medium is 5 ⁇ M BX795, 5 ⁇ M Ruxolitinib, 5 ⁇ M Tofacitinib Citrate, 5 nM Actinomycin D.
  • lipofectin 2000 was used to transfect mouse lung fibroblasts.
  • the mouse lung fibroblast cells were seeded in a 6-well plate at an appropriate concentration, and cultured in an incubator containing 5% CO 2 and 37 ° C. for 12 hours.
  • the transfection began to reach 60-90%; Change the medium 1 hour before transfection, replace the old medium with 1 ml of fresh DMEM complete medium containing 10% FBS; add the above-mentioned mixed solution A to the DMEM complete medium, and the ratio of the mixed solution to the medium is 1: 1000 ;
  • the ratio of mixed solution A is 5 ⁇ M BX795, 5 ⁇ M Ruxolitinib, 2.5 ⁇ M Tofacitinib Citrate, and 10 nM Actinomycin D.
  • the pEGFP-N1 plasmid was transfected into the control group and the experimental group, and the results are shown in FIG. 5.
  • the results showed that when pEGFP-N1 plasmid was used to transfect mouse lung fibroblasts, the transfection efficiency was extremely low.
  • the number of successfully transfected cells in a field of view was single digits.
  • the cells of mixed solution A were pretreated and expressed GFP after transfection. The number of cells is greatly increased. The results indicate that mixed solution A can improve the transfection efficiency of primary adherent cells, such as mouse lung fibroblasts, and promote the expression of foreign genes in cells.
  • the test method is as follows:
  • lentivirus was used to infect human primary T cells.
  • the specific experimental methods are as follows:
  • PBMC Peripheral blood mononuclear cells
  • human peripheral blood was drawn, diluted 1: 1 with PBS, and the diluted mixed liquid was added to Ficoll with a density of 1.077, the volume ratio was 2: 1, centrifuged at 1000 rpm, and 18 ° C for 30 minutes.
  • the turbid layer at the interface was aspirated, washed with twice the volume of PBS, centrifuged at 2000 rpm for 5 minutes, and the supernatant was discarded, and repeated-3 times.
  • the resulting cells were PBMC.
  • PBMC culture resuspend in 1640 medium containing 20% FBS 2.5 ⁇ g / ml CD3, 0.5 ⁇ g / ml CD28 1000 U / ml IL-2, 10 ng / ml IL-7 (1 can be added for every 5-10 ml blood) ml medium) was placed in a 6-well plate (or other adherent plate), and the cell suspension was collected after incubation at 37 ° C with 5% CO 2 for 2 hours. At this time, most of the macrophages had been removed. The suspension in the 6-well plate was transferred to an unattached culture flask and cultured for 4 days, and 1640 medium containing 10% FBS and 1000 U IL-2 was added depending on the cell density. During microscopy, the cells should show clumping.
  • lentivirus An endotoxin-removing plasmid extraction kit (purchased from Qiagen) was used to prepare an expression plasmid with the desired gene (the plasmid used in the present invention is an expression vector containing a green fluorescent protein reporter gene) pEGFP-N1, the plasmid required for virus assembly is psp2AX, VSVG); 293T cells were cultured in 10 cm plates using DMEM containing 10% FBS. When the cells grow to 70% -80%, lentiviral packaging can begin.
  • the total amount of plasmid was 20 ⁇ g, and it was dissolved in 250 ⁇ l Opti-MEM solution.
  • 60 ⁇ g PEI was dispensed into tubes and dissolved in Opti-MEM solution.
  • the configured solution was mixed and shaken vigorously for 15 seconds. After standing for 15 minutes, the solution was dropped evenly into the petri dish.
  • the solution was changed after 14 hours, and the supernatant was collected at intervals of 24 hours and 48 hours, which is the virus stock solution.
  • the collected virus stock was centrifuged at 2000 rpm for 5 minutes to remove larger cell debris.
  • the supernatant was filtered through a 0.45 ⁇ M filter membrane, and further concentrated by ultracentrifugation.
  • the obtained virus solution was stored in a refrigerator at -80 ° C.
  • T-cell infection preparation of plates required for infection: the day before the infection, the non-adherently treated 12-well plate was coated with Retronectin (TAKARA, 100T-A). Dilute Retronectin to 20 ⁇ g / ml with PBS, add 1 ml to each well, and overnight at 4 ° C. Before use, aspirate the liquid in the well, add PBS containing 2% BSA, and leave it at room temperature for 30 minutes. Aspirate the PBMC cultured in the flask, centrifuge and count. At the time of infection, 2 ⁇ 10 6 cells, 1640 medium containing 500 U / ml IL-2, 20% FBS, and virus solution were added to each well to a final volume of 2 ml. After culturing for about 5 days, supplement 1640 medium containing 500 U / ml IL-2 and 20% FBS according to the growth conditions. After 4 days, obvious fluorescence can be observed under a fluorescence microscope.
  • Retronectin TAKARA, 100T-A
  • human T cells can show partial fluorescence after being infected with lentivirus, indicating that the lentivirus successfully infected human T cells.
  • the human T cells pretreated with the mixed solution A were infected with lentivirus, and their fluorescence was relatively low.
  • the control group was significantly enhanced, indicating that mixed solution A had a significant enhancement effect on the T-cell lentivirus infection and the efficiency of foreign gene expression.

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Abstract

一种用于提高细胞转染效率的试剂组合物，所述组合物由BX795 0.01-10μM、鲁索利替尼0.1-100μM、托法替布0.1-100μM、放线菌素D 0.1-100 nM中的两种或两种以上物质组成。将这些试剂混合液与含有DNA和转染试剂的复合物或包装有目的载体的慢病毒分别加入细胞培养液中，以提高细胞的转染效率。

Description

用于提高细胞转染效率的试剂组合物 技术领域
本发明属于生物技术领域，具体涉及用于提高细胞转染效率的试剂组合物。
 背景技术
基因治疗是当代医学和生物学的一个新的研究领域，其通过一定方式将正常基因或有治疗作用的DNA序列导入靶细胞以纠正基因的缺陷或发挥治疗作用, 从而达到治疗疾病的目的。为了达到理想的治疗效果，科学家一直在研究传送质粒DNA的有效途径。目前常规的外源表达***主要分为两类：一类是采用病毒载体的方式，主流的三种病毒载体包括慢病毒（lentiviral）载体、逆转录（retroviral）病毒载体以及腺病毒（AAV）载体，这些方法是基因治疗等临床试验的主流选择。病毒载体如AAV载体具有高转染率、持续稳定表达外源基因的特点，但其需整合到宿主细胞的染色体中才能够使外源基因得以持续表达, 因此具有致癌、致畸的危险。安全性较差这一缺陷已成为限制病毒载体广泛应用的主要原因。另一类是采用非病毒载体的方式。这类非病毒载体安全性高，易于制备，在基因治疗中，从效果和发展前景上看，较病毒载体更具有优越性。但该类载体的导入效率和靶向性较低，较大部分宿主细胞对外源DNA抵抗性强，且外源基因转入到宿主细胞后表达时间短，短暂表达后外源基因快速关闭，这些都严重制约了该类载体的应用与发展。非病毒载体包括脂质体、高分子载体、纳米基因转运体等。
 技术问题
对于现有的普通细胞株或原代细胞，一般的转染方法多采用脂质体法和电穿孔的方法。阳离子脂质体表面带正电荷，与核酸的磷酸根通过静电作用将DNA分子包裹成DNA-脂质体复合物，能吸附在表面带负电荷的细胞膜，通过膜的融合或细胞内吞作用进入细胞体内。有时也通过细胞膜上小孔的直接渗透作用将DNA传递进入细胞。电穿孔法或电转法，是通过高强度的电场作用，瞬时提高细胞膜的通透性，从而将外源分子，如DNA、mRNA、蛋白、糖类等送入宿主细胞的胞浆内。但是对于很多细胞株或原代细胞，外源DNA通过非病毒载体方法进入细胞后并未能完全转录翻译成目的蛋白，表达效率较低，常常达不到实验要求。而通过电击法和病毒法进行转染的效率也不高，且实验操作过程麻烦。如何提高外源DNA在细胞内的表达成为了一个技术难题。在临床治疗方面，治疗造血***遗传功能障碍的疾病，如联合免疫缺陷（SCID），或治疗癌症的新型手段，如嵌合抗原受体T细胞免疫疗法（CAR-T）都有赖于一个高效转染T细胞的手段。现今用于原代T细胞进行基因改造的方法主要通过病毒感染和电穿孔的方式进行。病毒法对原代细胞进行感染时，往往需要较高的病毒滴度且通常效果不甚理想。对于T细胞而言，还需要额外的细胞因子，如IL-2和IL-7进行刺激，才能具备一定的病毒感染效率。而电穿孔的方法在操作简易度和实验周期上相比病毒法感染原代细胞更具优势。然而，该法的普及受制于电转效率、细胞存活率、实验仪器差异等诸多因素。加之原代细胞对于多种转染方法都具有较强的抵抗性，使得关于细胞进行高效基因编辑的手段较为匮乏。
本文介绍了一组试剂组合物通过对细胞免疫通路的抑制，以提高载体所运载的基因在目标细胞中的表达效率，从而用于基因治疗的临床应用。
 技术解决方案
本发明的目的在于提供用于提高细胞转染效率的试剂组合物，试剂组合物与含有DNA和转染试剂的复合物或包装有目的载体的慢病毒分别加入细胞培养液中，以提高细胞的转染效率。该试剂混合物操作简便、可重复性强，使用范围大，已成功运用于L929、BJ等细胞系和小鼠原代小鼠成纤维细胞、原代肺纤维细胞、T淋巴细胞等原代细胞中。
为实现上述目的，本发明采用如下技术方案：
用于提高细胞转染效率的试剂组合物，所述试剂组合物包含：化合物BX795、化合物（鲁索利替尼）Ruxolitinib、化合物托法替布（Tofacitinib）、化合物放线菌素D（Actinomycin D）。
其中所述试剂组合物由化合物BX795 0.01-10 μM、化合物Ruxolitinib 0.1-100μM、化合物Tofacitinib 0.1-100 μM或者化合物Actinomycin D 0.1-100 nM中的2种或2种以上物质组成。
试剂组合物的组合及制备方法，包括如下：
1）配制BX795溶液：将10 mg BX795加入1.6907 ml DMSO中，得到10 mM BX795母液；
2）配制 Ruxolitinib溶液：将5 mg Ruxolitinib加入0.3260 ml DMSO中，得到50 mM  Ruxolitinib溶液；
3）配制Tofacitinib溶液：将10 mg Tofacitinib Citrate加入0.3964 ml DMSO中，得到50 mM Tofacitinib Citrate溶液；
4）配制Actinomycin D溶液：将1 mg Actinomycin D加入79.65 μl DMSO中，得到10 mM Actinomycin D溶液；
5）将BX795、Ruxolitinib、Tofacitinib或者Actinomycin D中的任意2种化合物组合配制成特定终浓度的试剂混合液。
6）将BX795、Ruxolitinib、Tofacitinib或者Actinomycin D中的任意3种化合物组合配制成特定终浓度的试剂混合液。
7）将BX795、Ruxolitinib、Tofacitinib、和Actinomycin D中的4种化合物组合，配制成特定终浓度的试剂混合液，即混合液A。
8）所述试剂浓度如下：BX795 为0.01-10 μM、Ruxolitinib 为0.11-100 μM、Tofacitinib为 0.1-100 μM、Actinomycin D 0.1-100 nM。
 有益效果
（1）本发明的方法尤其适用于各贴壁细胞系，原代小鼠成纤维细胞，原代小鼠肺纤维细胞的转染，适用于多种难转染或转染效率低下的细胞系。
（2）本发明的方法适用于多种转染方式，如PEI转染法、Lipofectamine类脂质体系转染法、电转法等
（3）本发明的方法生物毒性小，在血清中稳定，转染效率更高并且操作简单的基因转染辅助试剂。
（4）本发明采用混合物形式配制试剂，其提高转染和表达效果远远优于单一成分的化合物。
 附图说明
图1添加各组分后L929细胞转染GFP荧光效果图。
图2免疫印迹法检测混合液A对脂质体转染法转染L929细胞的细胞效率影响。
图3两两混合药物对L929细胞的转染效率的影响的荧光效果图。
图4三种药物混合对L929细胞的转染效率的影响的荧光效果图。
图5混合液A对脂质体转染法转染小鼠肺纤维细胞的荧光效果图及流式细胞分析图。
图6混合液A对慢病毒法感染人原代T细胞的荧光效果图。
 本发明的实施方式
制备该混合液的步骤如下：
I. 试剂的配制
1. 母液的配置
1）配制BX795溶液：将10 mg BX795（购于Selleck公司）加入1.6907 ml DMSO中，得到10 mM BX795母液；
2） 配制 Ruxolitinib溶液：将5 mg Ruxolitinib（购于APEXBIO公司）加入0.3260 ml DMSO中，得到50 mM Ruxolitinib溶液；
3）配制Tofacitinib Citrate溶液：将10 mg Tofacitinib Citrate（购于APEXBIO公司）加入0.3964 ml DMSO中，得到50 mM Tofacitinib Citrate溶液
4） 配制Actinomycin D溶液：将1 mg Actinomycin D（购于Selleck公司）加入79.65 μl DMSO中，得到10 mM Actinomycin D溶液
2. 试剂混合液的配制
1）在1 ml DMSO中将上述 BX795、Ruxolitinib、Tofacitinib Citrate、和Actinomycin D溶液，两两组合配制成特定终浓度的任意2种化合物组合物；
2）在1ml DMSO中将上述 BX795、Ruxolitinib、Tofacitinib Citrate、和Actinomycin D溶液，三三组合配制成特定终浓度的任意3种化合物组合物；
3）在1ml DMSO中将上述 BX795、Ruxolitinib、Tofacitinib Citrate、和Actinomycin D溶液，组合配制成特定终浓度的4种化合物组合物，即为混合液A；
所述化合物BX795作用于细胞的终浓度范围可为0.01—10 μM；
所述化合物 Ruxolitinib作用于细胞的终浓度范围可为0.1—100μM；
所述化合物Tofacitinib Citrate作用于细胞的终浓度范围可为0.1—100 μM；
所述化合物Actinomycin D作用于细胞的终浓度范围可为0.1—100 nM；
3. 转染用质粒制备
用去除内毒素的质粒大提试剂盒（购自Qiagen公司）制备转染所需连有目的基因的表达质粒（本发明所用质粒为含有绿色荧光蛋白报告基因的表达载体pEGFP-N1，来源于Clontech公司），方法见厂家说明。用200 μl的ddH 2O缓冲液溶解质粒，紫外分光光度计测其浓度。
下面通过实施例对本发明进一步说明
实施例1 在L929细胞中验证混合液A对细胞转染效率的影响
试验方法如下：
本实验选用脂质体Lipofectamine 2000转染法转染L929细胞。按合适的浓度将L929细胞接种于6孔板，在含有5% CO 2和37℃培养箱中培养12小时，当细胞完全贴壁，长至60-90%即可开始转染；转染前1小时换液，用1 ml新鲜的含10% FBS的DMEM完全培养基换旧的培养基；DMEM完全培养基中加入混合液A，混合液与培养基的比例为1:1000；混合液A在培养基中终浓度为0.25μM BX795、5 μM Ruxolitinib、5μM Tofacitinib Citrate、5 nM actinomycin D。准备转染混合物：在1.5 ml的圆底无菌管中，将40μl的Opti-MEM溶液和2 μl的Lipofectamin 2000（Thermo Fisher公司）混合，放于漩涡震荡仪振匀，静置5分钟；加入2 μg的pEGFP-N1质粒，轻轻晃动混匀，静置20分钟；将DNA-Lipofecatamine混合液均匀加入L929细胞中，边加边轻轻晃动混匀；12小时后，吸去培养基与DNA沉淀，用1倍的PBS溶液清洗2次，然后换上新鲜培养基；培养基中需含有混合液A。48小时后使用荧光显微镜观察实验组和对照组（只用溶剂不含药物处理）细胞的荧光表达情况、利用流式细胞仪检测L929细胞GFP表达效率，并通过免疫印迹的方法检测GFP蛋白在细胞中的表达量。
试验结果如下：将未处理的L929细胞设为第一组，单一组分预处理L929细胞为第二组对照组，混合液A预处理的L929细胞作为实验组。将pEGFP-N1质粒转染对照组和实验组，结果如图1所示。图1结果表明在明场条件下，无论是对照组还是实验组，细胞的贴壁牢，虽然实验组的细胞增殖稍弱于对照组，但其细胞状态好，死细胞少；在暗场条件下观察细胞GFP的表达情况，预处理混合液A的实验组细胞荧光数目高于两个对照组，且荧光强度较强，说明在转染时预处理混合液A能大幅度提高L929细胞的转染效率和GFP表达效率，其结果远优于单一组分预处理的或未处理的细胞。之后通过免疫印迹实验检测GFP蛋白的表达情况(图2)，结果表明在内参一致的情况下(表明细胞数量大致相同) GFP蛋白在实验组的表达量远高于两组对照组，证明在转染时预处理混合液A可有效促进GFP质粒在L929细胞中的转染和表达。
实施例2在L929细胞中验证两两混合药物对转染效率的影响
试验方法如下：
本实验按比例配置BX795（终浓度0.25 μM）、Ruxolitinib（终浓度5 μM）、Tofacitinib Citrate（终浓度2.5 μM）、Actinomycin D（终浓度5 nM）的两两混合液，共6组，对照组为DMEM完全培养基中加入溶剂DMSO（1μl DMSO/ml DMEM培养基）；实验组为DMEM完全培养基中加入两两混合药物的L929细胞：BX795+Ruxolitinib，BX795+Tofacitinib Citrate，BX795+Actinomycin D，Ruxolitinib+ Tofacitinib Citrate，Ruxolitinib+Actinomycin D，Tofacitinib Citrate+Actinomycin D 。
本实验仍然选用脂质体Lipofectamine 2000转染法转染L929细胞。将L929细胞接种于6孔板，在含有5% CO 2和37℃培养箱中培养12小时，当细胞完全贴壁，长至60-90%即开始转染；转染前1小时换液，用1 ml新鲜的含10% FBS的DMEM完全培养基换旧的培养基；DMEM完全培养基中分别加入上述7组混合液，混合液与培养基的比例为1:1000；准备转染混合物：在1.5 ml的圆底无菌管中，将40 μl的Opti-MEM溶液和2 μl的Lipofectamine 2000（Thermo Fisher公司）混合，放于漩涡震荡仪振匀，静置5分钟；加入2 μg的pN1-GFP质粒，轻轻晃动混匀，静置20分钟；将DNA-Lipofectamine混合液均匀加入L929细胞中，边加边轻轻晃动混匀；12小时后，吸去培养基与DNA沉淀，用1倍的PBS溶液清洗2次，然后换上新鲜培养基；培养基中需含有各组混合液。48小时后使用荧光显微镜观察实验组和对照组细胞的荧光表达情况。
试验结果如下：如图3所示，用药物两两混合后预处理的L929的转染表达GFP的效率大于对照组，证明在转染时药物两两混合后可有效促进GFP质粒在L929细胞中的转染和表达。
实施例3在L929细胞中验证三种药物混合对转染效率的影响
试验方法如下：
本实验按比例将BX795（终浓度0.25 μM）、Ruxolitinib（终浓度5 μM）、Tofacitinib Citrate（终浓度2.5 μM）、 Actinomycin D（终浓度5 nM）的任意三种药物混合液，共分为4组，体系分别为：1对照组，为DMEM完全培养基中加入溶剂DMSO (1μl DMSO/ml DMEM）；2-4实验组为DMEM完全培养基中加入三种药物混合的L929细胞：BX795+Ruxolitinib+Tofacitinib Citrate，BX795+Ruxolitinib+Actinomycin D，BX795+ Tofacitinib Citrate +Actinomycin D，Ruxolitinib+ Tofacitinib Citrate+Actinomycin D。
本实验仍然选用脂质体Lipofectamine 2000转染法转染L929细胞。按合适的浓度将L929细胞接种于6孔板，在含有5% CO 2和37℃培养箱中培养12小时，当细胞完全贴壁，长至60-90%即可开始转染；转染前1小时换液，用1 ml新鲜的含10% FBS的DMEM完全培养基换旧的培养基；DMEM完全培养基中分别加入上述4组混合液，混合液与培养基的比例为1:1000；准备转染混合物：在1.5 ml的圆底无菌管中，将40 μl的Opti-MEM溶液和2 μl的Lipofectamine 2000（Thermo Fisher公司）混合，放于漩涡震荡仪振匀，静置5分钟；加入2 μg的pEGFP-N1，轻轻晃动混匀，静置20分钟；将DNA-Lipofectamine混合液均匀加入L929细胞中，边加边轻轻晃动混匀；12小时后，吸去培养基与DNA沉淀，用1倍的PBS溶液清洗2次，然后换上新鲜培养基；培养基中需含有各组混合液。48小时后使用荧光显微镜观察实验组和对照组细胞的荧光表达情况。
试验结果如下：将pEGFP-N1质粒转染对照组和实验组，结果如图4所示，0.25 μM BX795、 5 μM Ruxolitinib、2.5 μM Tofacitinib Citrate、10 nM Actinomycin D三种药物互相混合预处理L929细胞与两两混合的效果类似，与未处理的L929细胞相比较，极大提高GFP的表达，证明在转染时药物互相混合后采用合适的浓度可以极大发挥药物的联合作用，更有效促进GFP质粒在L929细胞中的转染和表达。
实施例4在鼠原代肺纤维细胞中验证混合液A对细胞转染效率的影响
试验方法如下：
前期数据表明药物混合后对L929细胞的转染效率有大幅度提高，但对于原代细胞的效果还未可知。以小鼠原代肺纤维细胞为例，检测药物混合后对原代贴壁细胞转染效率的影响。本实验分为两组，对照组为DMEM完全培养基中加入溶剂DMSO (1μl DMSO/ml DMEM）的小鼠肺纤维细胞，实验组为DMEM完全培养基中加入混合液A的小鼠肺纤维细胞。混合液A在培养基中终浓度为5 μM BX795、5 μM Ruxolitinib、5μM Tofacitinib Citrate、5 nM Actinomycin D。
对本实验选用脂质体Lipofectamine 2000转染法转染小鼠肺纤维细胞。按合适的浓度将小鼠肺纤维细胞细胞接种于6孔板，在含有5% CO 2和37℃培养箱中培养12小时，当细胞完全贴壁，长至60-90%即开始转染；转染前1小时换液，用1 ml新鲜的含10%FBS的DMEM完全培养基换旧的培养基；DMEM完全培养基中加入上述混合液A，混合液与培养基的比例为1:1000；混合液A的配比为5 μM BX795、5 μM Ruxolitinib、2.5 μM Tofacitinib Citrate、10 nM Actinomycin D。准备转染混合物：在1.5 ml的圆底无菌管中，将40 μl的Opti-MEM溶液和2 μl的Lipofectamine 2000（Thermo Fisher公司）混合，放于漩涡震荡仪振匀，静置5分钟；加入2μg的pEGFP-N1，轻轻晃动混匀，静置20分钟；将DNA-Lipofectamine混合液均匀加入小鼠肺纤维细胞中，边加边轻轻晃动混匀；12小时后，吸去培养基与DNA沉淀，用1倍的PBS溶液清洗2次，然后换上新鲜培养基；培养基中需含有如上所述的混合液A。48小时后使用荧光显微镜观察实验组和对照组细胞的荧光表达情况，利用流式细胞仪检测小鼠肺纤维细胞细胞GFP表达效率。
试验结果如下：
将pEGFP-N1质粒转染对照组和实验组，结果如图5所示。结果表明pEGFP-N1质粒转染小鼠肺纤维细胞时，转染效率极低，一个视野下转染成功的细胞个数为个位数，而预处理混合液A的细胞，转染后表达GFP的细胞数量大大增多。该结果说明混合液A对原代贴壁细胞，如小鼠肺纤维细胞能提高其转染效率，促进外源基因在细胞内的表达。
实施例5在原代T细胞中验证混合液A对细胞转染效率的影响
试验方法如下：
本实验选用慢病毒法感染人原代T细胞。具体实验方法如下：
1）T细胞的分离及培养
外周血单个核细胞(PBMC)，是指外周血中具有单个核的细胞，包含淋巴细胞、单核细胞等。为了进行PBMC分离，抽取人外周血，1：1与PBS稀释，稀释后的混合液体加入密度为1.077的Ficoll，体积比为2：1，1000 rpm离心，18℃ 30分钟。吸取界面的浑浊层，用2倍体积PBS洗涤，2000 rpm 离心5分钟，并弃上清，重复-3次，所得的细胞即为PBMC。PBMC培养：用含20% FBS 2.5μg/ml CD3，0.5 μg/ml CD28 1000 U/ml IL-2,10 ng/ml IL-7的1640培养液重悬（每5 -10 ml血可加入1 ml培养基）置于6孔板内（或其他贴壁处理的平板上），37℃ 5% CO 2温育2小时后收集细胞悬液，此时已经去除大部分的巨噬细胞。将6孔板内悬液转入未贴壁处理培养瓶内，培养4天，视细胞密度添加含10% FBS、1000 U IL-2的1640培养基。期间镜检，细胞应呈现聚团现象。
2）T细胞慢病毒感染步骤
1. 慢病毒的制备：用去除内毒素的质粒大提试剂盒（购自Qiagen公司）制备转染所需连有目的基因的表达质粒（本发明所用质粒为含有绿色荧光蛋白报告基因的表达载体pEGFP-N1，病毒组装所需质粒为psp2AX,VSVG）；在10cm平板中培养293T细胞，所用培养基为含10% FBS 的DMEM。待细胞长至70%-80%可开始慢病毒包装。选用的质粒***为三质粒***。以pNL:psp2AX:VSVG=4:3:2的比例进行包装，质粒加入总量为20 μg，溶解于250μl Opti-MEM溶液。分管配置60 μg PEI，溶解于Opti-MEM溶液。将所配置的溶液混合并剧烈震荡15秒，静置15分钟后，均匀滴入培养皿中。14小时后换液，之后间隔24小时和48小时收集上清，即为病毒原液。所收集的病毒原液2000 rpm离心5分钟以去除较大的细胞碎片。上清液用0.45μM滤膜过滤，超速离心进一步浓缩，获得的病毒液存放在-80℃的冰箱中。
3）T细胞的感染：感染所需平板的制备：待感染的前一天，用Retronectin（TAKARA，100T-A）包被未贴壁处理的12孔板。将Retronectin用PBS稀释为20 μg/ml，每孔加入1 ml，4℃过夜。使用前，吸取孔内液体，并加入含有2% BSA的PBS，室温放置30分钟。吸取培养瓶内培养的PBMC，离心并计数。感染时，每孔加入2×10 6个细胞以及含500 U/ml IL-2、20% FBS 的1640培养基和病毒液，终体积在2 ml。培养5天左右，根据生长情况补充含500 U/ml IL-2、20% FBS 的1640培养基，4天后可在荧光显微镜下观察到明显的荧光。
4）混合液A的添加：在T细胞感染前，在培养基中加入上述混合液A，混合液与培养基的比例为1:1000；混合液A的终浓度为5 μM BX795、5 μM Ruxolitinib、5 μM Tofacitinib Citrate、5 nM Actinomycin D。此外，在慢病毒感染过程中，培养基中需保持这些药物的浓度。
试验结果如下：
如图6所示，人的T细胞用慢病毒感染后，可以呈现部分荧光，说明慢病毒成功感染人的T细胞，混合液A预处理的人的T细胞经过慢病毒感染后，其荧光较对照组有显著增强，说明混合液A对于T细胞的慢病毒感染及外源基因表达效率有显著的增强效应。
以上所述仅为本发明的较佳实施例，凡依本发明申请专利范围所做的均等变化与修饰，皆应属本发明的涵盖范围。

Claims (3)

  1. 用于提高细胞转染效率的试剂组合物，其特征在于：所述试剂组合物包含：BX795、鲁索利替尼、托法替布、放线菌素D。
  2. 根据权利要求1所述的试剂组合物，其特征在于：所述试剂组合物由BX795、鲁索利替尼、托法替布和放线菌素D中的2种或2种以上物质组成。
  3. 根据权利要求1或2所述的试剂组合物，其特征在于，组合物中各物质浓度如下：BX795 为0.01-10 μM，鲁索利替尼为0.11-100μM，托法替布为 0.1-100μM，放线菌素D 0.1-100 nM。
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