Method for improving cell liposome transfection efficiency by increasing hydrostatic pressure of circulating cells and application

Abstract

The invention relates to the technical field of cell transfection experiment methods, and aims at effectively solving the cell liposome transfection tolerance phenomenon by increasing circulating cell hydrostatic pressure treatment in a cell liposome transfection experiment to improve the liposome transfection efficiency. In a cell liposome transfection experiment, the liposome transfection efficiency is improved by adding cell hydrostatic pressure treatment of a circulating mode; the circulation mode is that the hydrostatic pressure of the cells is maintained to be periodically changed in a set pressure interval value under a set frequency. The cell lipidosome transfection tolerance phenomenon is improved, and especially the transfection efficiency of lipidosome transfection tolerance cells such as primary culture cells and suspension culture cells is improved.

Description

Technical Field

[0001] The invention relates to the technical field related to cell transfection, and in particular to a method and application for improving cell liposome transfection efficiency by increasing the hydrostatic pressure of circulating cells. Background Art

[0002] Cell transfection is an experimental technique for introducing exogenous DNA and RNA molecules into eukaryotic cells. It is a common method for studying and controlling the gene function of eukaryotic cells and is widely used in basic medicine, biology, regenerative medicine and other fields. Cell transfection can be roughly divided into three types: physical mediation, chemical mediation and biological mediation. Physical mediation includes electroporation, microinjection, gene gun, etc.; chemical mediation includes calcium phosphate precipitation, liposome transfection, cationic polymer method; biological mediation includes protoplast transfection and virus-mediated method. These three types of transfection pathways have their own characteristics and are suitable for different experimental needs. Compared with traditional physical and biological mediation transfection pathways, which have high experimental costs, cumbersome processes and long cycles, chemical mediation is currently the most widely used method for transient gene transfection in laboratories.

[0003] Liposome transfection is the process of combining positively charged cationic liposomes with negatively charged DNA through electrostatic interaction to form a liposome-DNA complex, which enters cells through cell membrane fusion and clathrin-mediated endocytosis (Mol Ther. 2005; 12: 468-474.). In recent years, liposome transfection technology has made great progress, such as the widely used Lipofectamine TM 3000 liposome transfection reagent (Thermo Fisher Biotechnology, USA) has a high transfection efficiency in commonly used tool cells such as HEK293T and Hela (the positive rate can reach more than 80%), and is one of the most commonly used liposome transfection reagents in laboratories. However, there are still two important problems to be solved in liposome transfection technology. First, the transfection efficiency of different types of cells varies greatly, especially some primary culture cells, suspension culture cells, vascular endothelial cells, and vascular smooth muscle cells have liposome transfection tolerance. The transfection efficiency of this type of transfection-tolerant cells is not ideal and cannot meet the experimental requirements, which limits the application scope of liposome transfection technology; second, the liposome reagent itself has a certain cytotoxicity, which leads to the liposome transfection reagent cannot be used in large doses. Although there have been reports of methods for increasing the transfection efficiency of liposomes by adding other chemical reagent components (CN 111500637A), the addition of other chemical components may have a potential effect on the normal growth of cells and affect the experimental results. Therefore, it is necessary to develop new and safer technical methods to improve the transfection efficiency of liposome transfection-tolerant cells. Summary of the invention

[0004] The present invention provides a method for improving the efficiency of cell liposome transfection:

[0005] In the cell liposome transfection experiment, the cell liposome transfection efficiency was improved by increasing the cell hydrostatic pressure treatment.

[0006] The cell liposome transfection comprises the following steps: S1. preparing cells to be transfected; S2. configuring a liposome transfection system; and S3. treating the cells with hydrostatic pressure.

[0007] Preferably, in the cell liposome transfection experiment, the cell liposome transfection efficiency is improved by increasing the cell hydrostatic pressure treatment in a cyclic mode; the cyclic mode is to maintain the cell hydrostatic pressure to change periodically between set pressure interval values ​​at a set frequency.

[0008] The cell hydrostatic pressure of the cyclic mode changes periodically at a frequency of 0.0033-0.0166 Hz within a pressure range of 0 mmHg to 50-200 mmHg.

[0009] Furthermore, the pressure interval of the circulating cell hydrostatic pressure is 0-50 mmHg or 0-100 mmHg or 0-200 mmHg.

[0010] Preferably, the frequency of the periodic change is 0.0083 Hz.

[0011] Furthermore, the cell hydrostatic pressure treatment time in the circulation mode is 4-24 hours.

[0012] Preferably, the cell hydrostatic pressure treatment time in the circulation mode is 8-24 hours.

[0013] The cells to be transfected with liposomes are primary cultured cells or suspended cultured cells or vascular endothelial cells or vascular smooth muscle cells.

[0014] The above-mentioned method for improving the efficiency of liposome transfection can effectively improve the efficiency of cell liposome transfection when applied in cell liposome transfection.

[0015] The cell liposome transfection comprises the following steps: S1. preparing cells to be transfected; S2. configuring the liposome transfection system; S3. treating the cells with hydrostatic pressure;

[0016] Preferably, the cell hydrostatic pressure treatment is a cell hydrostatic pressure treatment in a cycle mode; the cells to be transfected are primary cultured cells or suspension cultured cells or vascular endothelial cells or vascular smooth muscle cells.

[0017] Compared with the prior art, the beneficial effects of the present invention are:

[0018] 1. The present invention is based on the existing liposome transfection technology, and the cyclic cell hydrostatic pressure auxiliary treatment with different frequencies, different pressure intervals and different action times is applied, which improves the efficiency of cell liposome transfection to a certain extent, indicating that in the cell liposome transfection experiment, by increasing the cell hydrostatic pressure treatment of the cyclic mode, the liposome transfection efficiency can be effectively improved. Wherein under the treatment of the cyclic cell hydrostatic pressure (0.0083Hz, 0-100mmHg), the transfection efficiency of the transfection-tolerant cell liposome can be increased by about 3 times, which greatly improves the cell liposome transfection tolerance problem, greatly improves the transfection efficiency of the exogenous target gene, solves the problem of unsatisfactory transfection efficiency of liposome transfection-tolerant cells, expands the scope of application of cell liposome transfection, and provides a new technical solution for improving the efficiency of cell liposome transfection for the majority of laboratories.

[0019] 2. Considering the cytotoxicity of liposome reagents themselves, users can reduce the amount of liposomes used and apply circulating cell hydrostatic pressure to assist treatment, so that a lower dose of liposome reagents can be used to achieve the transfection effect that originally required a high dose of liposome reagents. This not only reduces the cost of liposome use, but also avoids the cytotoxicity caused by high doses of liposome reagents.

[0020] 3. This invention is based on the existing chemical transfection method, and adds a physical factor (increasing the hydrostatic pressure of the circulating mode cells), practices a combined transfection method of chemical and physical strategies, and achieves the purpose of increasing the efficiency of cell transfection. The present invention creatively expands the existing classification method of cell transfection pathways, and has great scientific significance and economic value. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is an example diagram of the pressure change (0.0083Hz, 0-100mmHg) of the cyclic mode cell hydrostatic loading system.

[0022] Figure 2 Western Blotting experiments were performed to analyze the effects of three different cyclic patterns with the same frequency (0.0083 Hz) on the expression of GFP protein in primary human aortic smooth muscle cells (HASMC) after liposome transfection of FUGW plasmid (data are shown as mean ± SD, n = 3, ** P<0.01 vs. control).

[0023] Figure 3 Western Blotting experiments were performed to analyze the effect of hydrostatic pressure changes (0-100 mmHg) on ​​GFP protein expression in HASMC cells after liposome transfection of FUGW plasmid at different frequencies (data are shown as mean ± SD, n = 3, * P<0.05,** P<0.01 vs. control).

[0024] Figure 4 Western Blotting experiments were performed to analyze the effects of different treatment times of cyclic hydrostatic pressure (0.0083 Hz, 0-100 mmHg) on ​​the expression of GFP protein in HASMC cells after liposome transfection of FUGW plasmid (data are shown as mean ± SD, n = 3, * P<0.05, ** P<0.01 vs. control).

[0025] Figure 5 Fluorescence photos were used to analyze the effect of hydrostatic pressure (0.0083 Hz, 0-100 mmHg) on ​​the expression intensity of GFP fluorescent protein in HASMC cells after liposome transfection of FUGW plasmid (data are shown as mean ± SD, n = 6, * P<0.05, ** P<0.01 vs. control).

[0026] Figure 6 Flow cytometry was used to analyze the effect of changes in cyclic mode cell hydrostatic pressure (0.0083Hz, 0-100mmHg) on ​​the proportion of GFP-positive cells after HASMC cells were lipofected with FUGW plasmid.

[0027] Figure 7 Real-time fluorescence quantitative PCR (RT-PCR) was used to analyze the effect of hydrostatic pressure (0.0083 Hz, 0-100 mmHg) on ​​the expression level of GFP gene mRNA in HASMC cells after liposome transfection of FUGW plasmid (data are shown as mean ± SD, n = 6, ** P<0.01 vs. control).

[0028] Figure 8 The CCK-8 experiment was used to analyze the effect of changes in cyclic cell hydrostatic pressure (0.0083Hz, 0-100mmHg) on ​​HASMC cell proliferation.

[0029] Fig. 9 This is the result of taking white light photos with a microscope.

[0030] Fig.10 This is a diagram of Western Blotting experiment results (primary human umbilical vein endothelial cells, HUVEC).

[0031] Fig.11 This is a diagram of Western Blotting experiment results (human T cell leukemia cell line, CCRF-CEM).

[0032] Fig.12 This is the result of FAM-siRNA fluorescence photography. DETAILED DESCRIPTION

[0033] Cells in organisms are in a fluid environment and are constantly stimulated by various mechanical pressures. Among them, cell hydrostatic pressure refers to the pressure formed between the cell membrane and the cell wall by water molecules in the cytoplasm. Studies have revealed that the hydrostatic pressure of the cell microenvironment regulates various biological behaviors of cells, including differentiation, migration, apoptosis and proliferation. Under physiological conditions, vascular endothelial cells and vascular smooth muscle cells are not only subjected to the tensile force caused by periodic fluctuations and the shear force generated by blood flow, but also to the hydrostatic pressure perpendicular to the blood vessel wall (Sci China Life Sci. 2021; 64: 1677-1690). Based on this, we have developed for the first time a technical method to improve the efficiency of cell liposome transfection by increasing the cell hydrostatic pressure in a cyclic mode.

[0034] At present, laboratories mostly use the air pressure method to control cell hydrostatic pressure changes. The cell hydrostatic pressure control system (RHP-3000) is a cell pressure loading system that adopts the principle of the air pressure method. The system directly uses the ambient gas in a conventional CO2 cell culture chamber, and maintains the stable air pressure in the pressure chamber through the operation of an air pump. It can simulate a relatively large pressure change range and provide two pressure change modes: continuous or cyclic. The present invention relates to an example experiment using a cell hydrostatic pressure control system (RHP-3000) to regulate cell hydrostatic pressure changes. In order to solve the problem of cell liposome transfection tolerance and further improve the application scope of cell liposome transfection. The present invention relates to a method for improving the efficiency of transfection of transfection-tolerant cells by assisted application of a cyclic mode cell hydrostatic pressure change treatment.

[0035] The technical solution of the present invention is described in detail with reference to the embodiments. The reagents and biological materials used below are all commercial products unless otherwise specified.

[0036] Main reagents:

[0037] Dulbecco's Modified Eagle Medium (DMEM) cell culture medium (L110KJ), RPM1640 cell culture medium (L210KJ), and Fetal bovine serum (FBS) (S615JY) were purchased from Shanghai Yuanpei Biological Co., Ltd. (Shanghai, China). Endothelial cell culture medium (#ZQ-1304) was purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd. (Shanghai, China).

[0038] Lipofectamine TMLipo 3000 (#L3000015, Lipo3000) was purchased from Thermo Fisher (California, USA).

[0039] The 9.955-kb lentiviral plasmid FUGW (#14883) with hUbC-driven EGFP was purchased from Addgene (From D. Baltimore). FUGW was amplified in E. coli, and sufficient amounts of FUGW plasmid were isolated using the TIANprep Mini Plasmid Kit (#DP103) (Beijing, China) and used as a reporter gene vector.

[0040] FAM-labeled negative control siRNA (FAM-siRNA) was purchased from Shanghai Jima Pharmaceutical Co., Ltd. (Shanghai, China) and used as an indicator.

[0041] Anti-β-actin (#sc-47778) antibody was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA).

[0042] Anti-GFP (#ET1607-31) antibody was purchased from Hua Biotechnology (Hangzhou, China).

[0043] Cell counting kit (CCK-8) (#40203ES76) was purchased from Yisheng Biotechnology Co., Ltd. (Shanghai, China).

[0044] Cell culture:

[0045] Primary human aortic smooth muscle cells (HASMC) (#PCS-100-012) and human T cell leukemia cell line (CCRF-CEM) (#CCL-119) were purchased from ATCC Cell Preservation Center (ATCC, Manassas, VA, USA). Primary human umbilical vein endothelial cells (HUVECs) (DFSC-EC-01) were purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd. (Shanghai, China). The above cells were cultured in a CO2 cell culture incubator at 37°C and a CO2 concentration of 5%. HASMC cells were cultured in DMEM medium containing 10% FBS and 1% penicillin / streptomycin, HUVEC cells were cultured in endothelial cell-specific medium, and CCRF-CEM cells were cultured in RPM1640 cell culture medium containing 10% FBS and 1% penicillin / streptomycin.

[0046] General Example:

[0047] The purpose of the present invention is to provide an experimental method for improving the efficiency of liposome transfection of transfection-tolerant cells by auxiliary application of cyclic mode cell hydrostatic pressure change treatment. The present invention analyzes the effect of regulating cyclic mode cell hydrostatic pressure change on liposome transfection, and shows that under a specific cell hydrostatic pressure change mode (0.0033-0.0166Hz, 0-100mmHg), the efficiency of liposome transfection of transfection-tolerant cells can be increased by about 3 times, greatly improving the cell liposome transfection tolerance problem and greatly improving the transfection efficiency of exogenous target genes.

[0048] The technical solution adopted by the present invention to solve the above technical problems is as follows:

[0049] The following steps are involved:

[0050] S1: Cell (adherent, suspension) subculture.

[0051] S2: Liposome cell transfection steps: (1) Prepare cells to be transfected; (2) Configure the liposome transfection system; (3) Cyclic cell hydrostatic pressure treatment. Taking the cell hydrostatic pressure control system (RHP-3000) as an example, this system directly uses the normal CO2 cell culture incubator environment gas and maintains the air pressure in the pressure chamber stable through the operation of the air pump. The cell hydrostatic pressure change mode can generally be divided into continuous mode and cyclic mode. For example, the cyclic mode refers to maintaining the cell hydrostatic pressure at a set frequency and periodically changing between the set pressure range values ​​(0.0033-0.0166Hz, 0-100mmHg) (reference Figure 1 ).

[0052] S3: Transfection efficiency evaluation index: Analysis of the expression intensity of the target gene (at the mRNA and / or protein level).

[0053] Embodiment 1:

[0054] The efficiency of normal liposome transfection of primary human aortic smooth muscle cells (HASMC) (adherent cells) is not high (the transfection positive rate is about 10-15%). In this example, we analyze the effect of changes in circulating cell hydrostatic pressure (0.0083Hz, 0-100mmHg) on ​​the efficiency of HASMC liposome transfection. After the HASMC cells are fully grown in a 10cm culture dish, wash the cells twice with PBS (4°C), add 1mL of 0.25% trypsin, digest at room temperature for 2 minutes, add 3mL of DMEM complete medium (10% serum), collect the cell digestion solution (4mL in total) in a 15mL sterile centrifuge tube, centrifuge at 1000rpm at room temperature for 3 minutes, discard the cell supernatant, add 3mL of DMEM complete medium, mix the cells, and count the cells. Take 2mL of cell suspension and adjust the cell density to 1.5×10 with DMEM complete medium. 4cells / mL, the diluted cell suspension was inoculated into 4 6-well plates (2 mL cell suspension / well), and grown overnight in a 37°C, 5% CO2 cell culture incubator. The next day, the cell medium was changed and 1.75 mL DMEM complete medium was added to each well. Lipofectamine transfection system configuration: (1) Refer to Lipofectamine TM 3000 reagent product manual, taking 6-well plate as an example, press 5μL Lipofectamine TM 3000+120μL serum-free DMEM / well with liposome reagent, after preparation, let it stand at room temperature for 5 minutes; (2) Eukaryotic cell GFP green fluorescent protein expression plasmid (FUGW plasmid, purchased from Addgene, USA) is diluted to 125μL / well with serum-free DMEM culture medium at 2.5μg / well. The liposome system (125μL) and the plasmid reagent system (125μL) are mixed in equal volumes, gently mixed and let stand at room temperature for 20 minutes. After static, the mixed transfection system reagent is added to the 6-well plate at 250μL / well, and the liquid is not changed during transfection. According to the experimental grouping, the transfected cells are assisted with cyclic mode cell hydrostatic pressure change treatment. The pressure change frequency and pressure change range ( Figure 1 ). 24 hours after transfection, cell samples were collected to evaluate the effect of changes in cell hydrostatic pressure on the liposome transfection efficiency of HASMC cells from three levels: cell, protein, and molecule.

[0055] Furthermore, we analyzed the effects of changes in cell hydrostatic pressure in cyclic patterns with different frequencies (0.0333 Hz, 0.0083 Hz, 0.0167 Hz), different pressure ranges (0-50 mmHg, 0-100 mmHg, 0-200 mmHg), and different action times (4 h, 8 h, 24 h) on the HASMC liposome transfection efficiency.

[0056] Results Figure 2-9 :

[0057] like Figure 2 Western Blotting experiments were performed to analyze the effects of three different cyclic patterns of hydrostatic pressure on GFP protein expression in primary human aortic smooth muscle cells (HASMC) after liposome transfection of FUGW plasmid. The results showed that the cyclic pattern of hydrostatic pressure (0.0083Hz, 0-100mmHg) had the most significant effect, significantly improving the protein expression efficiency of the GFP gene (P<0.01).

[0058] like Figure 3Western Blotting experiments were performed to analyze the effects of different frequencies and the same cyclic mode hydrostatic pressure range (0-100 mmHg) on ​​the expression of GFP protein in HASMC cells after liposome transfection of FUGW plasmid. The results showed that compared with the control group, the cyclic mode hydrostatic pressure (0.0083 Hz, 0-100 mmHg) treatment group significantly increased the protein expression efficiency of the GFP gene (P<0.01).

[0059] like Figure 4 Western blotting experiments were performed to analyze the effect of changes in cyclic hydrostatic pressure (0.0083 Hz, 0-100 mmHg) on ​​the expression of GFP protein in HASMC cells after liposome transfection of FUGW plasmid. The results showed that compared with the control group, changes in cyclic hydrostatic pressure (0.0083 Hz, 0-100 mmHg) enhanced the expression of GFP protein in HASMC cells in a time-gradient manner ( * P<0.05, ** P<0.01).

[0060] like Figure 5 Fluorescence intensity analysis of the effect of cyclic mode cell hydrostatic pressure (0.0083Hz, 0-100mmHg) changes on the fluorescence intensity of GFP protein after HASMC cells were transfected with FUGW plasmid by liposomes. Consistent with the results of Western blotting experiments, cyclic mode cell hydrostatic pressure (0.0083Hz, 0-100mmHg) changes in a time-dependent manner enhanced the intensity of green fluorescent protein in HASMC cells ( * P<0.05, ** P<0.01).

[0061] like Figure 6 Flow cytometry analysis of the effect of changes in cyclic mode cell hydrostatic pressure (0.0083Hz, 0-100mmHg) on ​​the liposome transfection efficiency of HASMC cells. The flow analysis results (representative figure) showed that the GFP cell positive rate in the control group was 13.6%, and the GFP cell positive rate in the cell hydrostatic pressure (0.0083Hz, 0-100mmHg) treatment group increased to 26.8%, indicating that changes in cell hydrostatic pressure (cyclic pressure, 0.0083Hz, 0-100mmHg) effectively improved the liposome transfection efficiency of HASMC cells.

[0062] like Figure 7Real-time fluorescence quantitative PCR (RT-PCR) was used to analyze the effect of changes in cyclical cell hydrostatic pressure (0.0083 Hz, 0-100 mmHg) on ​​the expression level of GFP gene mRNA in HASMC cells after liposome transfection of FUGW plasmid. RT-PCR results showed that compared with the control group, changes in cyclical cell hydrostatic pressure (0.0083 Hz, 0-100 mmHg) significantly upregulated the GFP mRNA level (P<0.01).

[0063] like Figure 8 Effect of changes in cyclic mode cell hydrostatic pressure (0.0083Hz, 0-100mmHg) on ​​HASMC cell proliferation. The results of CCK-8 cell proliferation experiment showed that compared with the control group, changes in cyclic mode cell hydrostatic pressure (0.0083Hz, 0-100mmHg) for 24 hours did not affect HASMC cell proliferation.

[0064] like Fig. 9 The results of white light photography under the microscope showed that treating HASMC cells with cyclic cell hydrostatic pressure (0.0083Hz, 0-100mmHg) for 24 hours had little effect on cell morphology.

[0065] Conclusion: The analysis results of GFP marker gene mRNA molecules, protein expression, fluorescence intensity and other aspects showed that compared with the control group, the cyclic mode cell hydrostatic pressure (0.0083Hz, 0-100mmHg) treatment significantly improved the liposome transfection efficiency of HASMC cells, and did not affect the normal growth and cell morphology of HASMC cells. The cyclic cell hydrostatic pressure changes with different frequencies (0.0333Hz, 0.0083Hz, 0.0167Hz), different pressure ranges (0-50mmHg, 0-100mmHg, 0-200mmHg), and different action times (4h, 8h, 24h) all had a certain improvement on the HASMC liposome transfection efficiency, indicating that in the cell liposome transfection experiment, by increasing the cyclic mode cell hydrostatic pressure treatment, the liposome transfection efficiency can be effectively improved.

[0066] Embodiment 2:

[0067] Referring to previous studies, the liposome transfection efficiency of primary human umbilical vein endothelial cells (HUVEC) (adherent cells) is generally very low (the transfection positive rate is less than 5%). In this example, we analyze the effect of changes in the hydrostatic pressure of cells in the cyclic mode (0.0083Hz, 0-100mmHg) on ​​the liposome transfection efficiency of HUVEC cells. After the HUVEC cells are full in the 10cm culture dish, wash the cells twice with PBS, add 1mL of 0.05% trypsin, digest at room temperature for 30 seconds, add 3mL of endothelial cell-specific culture medium (10% serum), collect the cell digestion solution (4mL) in a 15mL sterile centrifuge tube, centrifuge at 1000rpm at room temperature for 5 minutes, discard the cell supernatant, add 3mL of endothelial cell-specific culture medium, mix the cells, and count the cells. Take 2mL of cell suspension and adjust the cell density to 2×10 with endothelial cell-specific culture medium. 4 cells / mL, the diluted cell suspension was inoculated into 4 6-well plates (2 mL cell suspension / well), and grown overnight in a 37°C, 5% CO2 cell culture incubator. On the second day, the cell medium was changed and 1.75 mL of endothelial cell-specific culture medium was added to each well. Lipofectamine transfection system configuration: (1) Refer to Lipofectamine TM 3000 reagent product manual, taking 6-well plate as an example, 5μL Lipofectamine TM 3000+120μL serum-free DMEM / well with liposome reagent, let it stand at room temperature for 5 minutes after preparation; (2) Eukaryotic cell GFP green fluorescent protein expression plasmid (FUGW plasmid, purchased from Addgene, USA) is diluted to 125μL / well with serum-free DMEM medium at 2.5μg / well. Liposome reagent (125μL) and FUGW plasmid reagent (125μL) are mixed in equal volumes, gently mixed and let stand at room temperature for 20 minutes. After standing, the mixed transfection reagent is added to the 6-well plate at 250μL / well. According to the experimental requirements, one 6-well plate is placed in a normal incubator for culture as a normal pressure control group. The remaining 3 6-well plates are placed in the pressure chamber of the cell hydrostatic pressure control system (RHP-3000) for a specific time (4h, 8h, 24h), and the pressure mode is set to the cyclic mode pressure (0.0083Hz, 0-100mmHg) ( Figure 1 ), after a specific period of pressure treatment, the 6-well plate was transferred to a conventional cell culture incubator for continued culture without changing the medium. 24 hours after transfection, the cells were collected and protein samples were obtained. The results of Western Blotting experiments showed that compared with the control cells, the cyclic mode cell hydrostatic pressure (0.0083Hz, 0-100mmHg) treatment enhanced the GFP protein expression of HUVEC cells after liposome transfection of FUGW plasmid in a time-dependent manner (P<0.01) ( Fig.10 ).

[0068] Embodiment 3:

[0069] The transfection efficiency of suspension cultured cells by liposome transfection is significantly lower than that of adherent cells. Here, we take suspension cultured cells (CCRF-CEM) as an example to analyze the effect of changes in the hydrostatic pressure of cells in the cyclic mode (0.0083Hz, 0-100mmHg) on ​​the liposome transfection efficiency of CCRF-CEM suspension cultured cells. For subculture of suspension cultured cells, directly add the culture medium containing the cells to a 15mL sterile centrifuge tube, centrifuge at 1000rpm for 3 minutes at room temperature, discard the cell supernatant, add 3mL of RPMI-1640 complete medium (10% serum), mix the cells, and count the cells. Take 2mL of cell suspension and adjust the cell density to 1×10 with RPMI-1640 complete medium. 5 cells / mL, the diluted cell suspension was inoculated into two 6-well plates (1.75 mL / well) and incubated at 37°C in a 5% CO2 cell culture incubator. Lipofectamine transfection system configuration: (1) Refer to Lipofectamine TM 3000 reagent product manual, taking 6-well plate as an example, 5μL Lipofectamine TM 3000+120μL serum-free RPMI-1640 / well was prepared with liposome reagent, and then allowed to stand at room temperature for 5 minutes; (2) Eukaryotic cell GFP green fluorescent protein expression plasmid (FUGW plasmid, purchased from Addgene, USA) was diluted to 125μL / well with serum-free RPMI-1640 medium at 2.5μg / well. Liposome reagent (125μL) and FUGW plasmid reagent (125μL) were mixed in equal volumes, gently mixed and allowed to stand at room temperature for 20 minutes. After standing, the mixed transfection reagent was added to the 6-well plate at 250μL / well. According to the experimental requirements, one 6-well plate was placed in a normal incubator for culture as a normal pressure control group, and the other 6-well plate was placed in the pressure chamber of the cell hydrostatic pressure control system (RHP-3000) for culture for 24 hours. The pressure mode was set to the cyclic mode pressure (0.0083Hz, 0-100mmHg), and the medium was not changed during the period. After 24 hours of transfection, the cell protein sample was collected. Western Blotting results showed that compared with the control group, the GFP protein expression in the group treated with cyclic hydrostatic pressure (0.0055Hz, 0-100mmHg) was significantly upregulated (P<0.01) ( Fig.11 ).

[0070] Embodiment 4:

[0071] In this example, we analyze the effect of changes in cell hydrostatic pressure (0.0083Hz, 0-100mmHg) in a cyclic mode on the efficiency of FAM-labeled siRNA transfection in HASMC liposomes. FAM-labeled siRNA is generally used as a fluorescent labeling control for siRNA transfection to determine whether siRNA has entered the cells. After HASMC cells (purchased from ATCC in the United States) are fully grown in a 10cm culture dish, wash the cells twice with PBS (4°C), add 1mL of 0.25% trypsin, digest at room temperature for 3 minutes, add 3mL of DMEM complete medium (10% serum), collect the cell digestion solution (4mL in total) in a 15mL sterile centrifuge tube, centrifuge at 1000rpm at room temperature for 3 minutes, discard the cell supernatant, add 3mL of DMEM complete medium, mix the cells, and count the cells. Take 2mL of cell suspension and adjust the cell density to 1.5×10 with DMEM complete medium. 4 cells / mL, the diluted cell suspension was inoculated into two 6-well plates (2 mL cell suspension / well), and grown overnight in a 37°C, 5% CO2 cell culture incubator. On the second day, the cell medium was changed and 1.75 mL DMEM complete medium was added to each well. Lipofectamine transfection system configuration: (1) Refer to Lipofectamine TM 3000 reagent product manual, taking 6-well plate as an example, press 5μL Lipofectamine TM 3000+120μL serum-free DMEM / well prepared with liposome reagent, and placed at room temperature for 5 minutes after preparation; (2) FAM-labeled siRNA (purchased from Shanghai Jima Biotechnology) was diluted to 125μL / well with serum-free DMEM culture medium according to the working concentration of 100μM. The prepared liposome reagent (125μL) and FAM-labeled siRNA mixture (125μL) were mixed in equal volumes, gently mixed and placed at room temperature for 20 minutes. After static, the mixed transfection reagent was added to the 6-well plate at 250μL / well. According to the experimental requirements, a 6-well plate was placed in a normal incubator for culture as a normal pressure control group, with 3 replicates in each group. Another 6-well plate was placed in the pressure chamber of the cell hydrostatic pressure control system (RHP-3000) for culture for 24 hours, and the pressure mode was set to the cyclic mode pressure (0.0083Hz, 0-100mmHg) ( Figure 1 ), and the medium was not changed during the period. 24 hours after transfection, the cells were washed twice with PBS and then observed and photographed under a fluorescence microscope. The results showed that ( Fig.12 ), the fluorescence photography results showed that compared with the control group, the cyclic mode cell hydrostatic pressure (0.0083Hz, 0-100mmHg) treatment significantly enhanced the FAM-labeled siRNA transfected by liposomes in HASMC cells (P<0.01).

[0072] Summary: The present invention is based on the existing liposome transfection technology, and further applies a cyclic mode cell hydrostatic pressure (0.0055-0.0166Hz, 0-100mmHg) auxiliary treatment, which can greatly improve the liposome transfection efficiency of liposome transfection-resistant cells, solve the problem of unsatisfactory transfection efficiency of liposome transfection-resistant cells, expand the application scope of liposome transfection, and provide a new technical solution for improving liposome transfection efficiency for the majority of laboratories. Secondly, considering the cytotoxicity of the liposome reagent itself, the user can reduce the amount of liposome reagent while applying a cyclic cell hydrostatic pressure auxiliary treatment, so that a lower dose of liposome reagent can be used to achieve the transfection effect that originally required a high dose of liposome reagent, which to a certain extent reduces the cytotoxicity of the liposome reagent.

[0073] Traditional cell transfection is roughly divided into three methods: chemical, physical, and biological. The present invention creatively expands this classification. This invention is based on the traditional chemical transfection method, and an auxiliary physical factor (increasing the hydrostatic pressure of circulating cells) is applied. Therefore, the present invention is actually a combined transfection method that combines chemical and physical strategies, thereby achieving the purpose of improving the efficiency of liposome cell transfection.

[0074] The above are only some preferred embodiments of the present invention, and the present invention is not limited to the contents of the embodiments. For those skilled in the art, various changes and modifications can be made within the scope of the technical solution of the present invention, and any changes and modifications made are within the protection scope of the present invention.

Claims

1. A method for improving cell liposome transfection efficiency, characterized in that: In the cell liposome transfection experiment, the cell liposome transfection efficiency is improved by increasing the cell hydrostatic pressure treatment; the cell liposome transfection includes the following steps: S1. preparing cells to be transfected; S2. configuring the liposome transfection system; S3. treating the cells with hydrostatic pressure.

2. The method according to claim 1, characterized in that: In the cell liposome transfection experiment, the cell liposome transfection efficiency is improved by increasing the cell hydrostatic pressure treatment in a cyclic mode; the cyclic mode is to maintain the cell hydrostatic pressure to change periodically between the set pressure interval values ​​at a set frequency.

3. The method according to claim 2, characterized in that: The cell hydrostatic pressure of the cyclic mode changes periodically at a frequency of 0.0033-0.0166 Hz within a pressure range of 0 mmHg to 50-200 mmHg.

4. The method according to claim 3, characterized in that: The pressure interval of the circulating cell hydrostatic pressure is 0-50 mmHg or 0-100 mmHg or 0-200 mmHg.

5. The method according to claim 3, characterized in that: The frequency of the periodic change is 0.0083 Hz.

6. The method according to claim 2, characterized in that: The cell hydrostatic pressure treatment time in the circulation mode is 4-24 hours.

7. The method according to claim 6, characterized in that: The cell hydrostatic pressure treatment time in the circulation mode is 8-24 hours.

8. The method according to claim 1, characterized in that: The cells to be transfected are primary cultured cells or suspension cultured cells or vascular endothelial cells or vascular smooth muscle cells.

9. The method for improving liposome transfection efficiency according to any one of claims 1 to 8, used in cell liposome transfection; the cell liposome transfection comprises the following steps: S1. Prepare cells to be transfected; S2. Liposome transfection system configuration; S3. Cell hydrostatic pressure treatment.

10. The use according to claim 9, characterized in that: The cell hydrostatic pressure treatment is a cell hydrostatic pressure treatment in a cycle mode; the cells to be transfected are primary cultured cells or suspended cultured cells or vascular endothelial cells or vascular smooth muscle cells.

Images