Small nucleic acid drug in-vitro screening method

Abstract

The invention provides a small nucleic acid drug in-vitro screening method, and belongs to the technical field of biological detection. Aiming at the problem that small nucleic acid drugs are difficult to screen quickly in the prior art, primary hepatocytes of cynomolgus monkeys are used as a screening basic model, to-be-detected small nucleic acid drugs are transfected, ACTB, ASGR1 and ASGR2 are used as target genes, cell lysates are collected at different time points to extract mRNA, and the mRNA is subjected to qPCR amplification reaction, so that the small nucleic acid drugs are screened quickly. And evaluating the regulation effect of the drug on the target gene according to the amplification product amount so as to screen the drug. The method can quickly and accurately screen small nucleic acid drugs, and provides powerful support for drug research and development.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to an in vitro screening method for small nucleic acid drugs. Background Art

[0002] Small nucleic acid drugs are an emerging type of drug, mainly including small interfering RNA (siRNA), microRNA (miRNA), etc. They play a role in disease treatment by complementary binding with the mRNA of the target gene and regulating gene expression. Small nucleic acid drugs have the advantages of high specificity and clear targets, and have shown great potential in the treatment of many diseases such as cancer, genetic diseases, and cardiovascular diseases.

[0003] At present, drug screening models mainly include cell line models and animal models. Cell line models, such as commonly used tumor cell lines, have the advantages of being easy to culture and available in large quantities. However, after long-term subculture, the biological characteristics of cell lines are different from those of primary cells, and they cannot completely simulate the physiological environment in the body, which may lead to drug screening results that are inconsistent with the actual in vivo situation. Although animal models can reflect the effects of drugs at the overall level, they have problems such as high cost, long cycle, and large individual differences, which are not conducive to large-scale and rapid drug screening. Crab-eating macaques are highly similar to humans in physiology and genetics, and their primary hepatocytes retain many physiological characteristics of liver cells in the body, such as cell metabolic function, receptor expression, etc., and are an ideal in vitro drug screening model.

[0004] However, the existing technology of drug screening using primary hepatocytes of crab-eating macaques still has many shortcomings. For example, during the cell culture process, the isolation and extraction methods of primary hepatocytes are not mature enough, and cell survival rate and activity are difficult to guarantee, which affects the accuracy of the screening results. At the same time, during the transfection process, there is a lack of efficient and optimized transfection methods, resulting in unstable transfection efficiency and difficulty in accurately regulating the dosage and effect of drugs entering cells. In addition, in terms of detection, there is a lack of systematic and accurate detection indicators and methods, and it is impossible to comprehensively and quickly evaluate the regulatory effects of drugs on multiple key genes in cells, making it difficult to meet the needs of rapid screening of small nucleic acid drugs. Summary of the invention

[0005] The purpose of the present invention is to provide an in vitro screening method for small nucleic acid drugs, which can achieve rapid screening of small nucleic acid drugs.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides an in vitro screening method for small nucleic acid drugs, comprising the following steps:

[0008] The small nucleic acid drug to be detected is mixed and incubated with a transfection reagent to obtain a transfection complex;

[0009] After washing the cells, the transfection complex and cell culture medium are added to a cell culture plate in which cynomolgus monkey primary hepatocytes are cultured for culturing;

[0010] ACTB, ASGR1, and ASGR2 were used as target genes, and cell lysates were collected at different time points of cell culture to extract mRNA, and qPCR amplification reactions were performed to obtain the amount of amplified products;

[0011] The amount of amplified product obtained is used to evaluate the regulatory effect of small nucleic acid drugs on target genes, thereby achieving drug screening.

[0012] Preferably, the primers used in the qPCR amplification reaction include the NHP-ACTB2 primer set, and the NHP-ACTB2 primer set includes the following primers:

[0013] An upstream primer having a nucleotide sequence as shown in SEQ ID NO.1;

[0014] A downstream primer having a nucleotide sequence as shown in SEQ ID NO.2;

[0015] The primers used in the qPCR amplification reaction include the NHP-ACTB9 primer set, and the NHP-ACTB9 primer set includes the following primers:

[0016] An upstream primer having a nucleotide sequence as shown in SEQ ID NO.3;

[0017] The nucleotide sequence of the downstream primer is shown as SEQ ID NO.4.

[0018] Preferably, the primers used in the qPCR amplification reaction include a Human-ASGR1-1 primer set, and the Human-ASGR1-1 primer set includes the following primers:

[0019] An upstream primer having a nucleotide sequence as shown in SEQ ID NO.5;

[0020] A downstream primer having a nucleotide sequence as shown in SEQ ID NO.6;

[0021] The primers used in the qPCR amplification reaction include the Human-ASGR1-2 primer set, and the Human-ASGR1-2 primer set includes the following primers:

[0022] An upstream primer having a nucleotide sequence as shown in SEQ ID NO.7;

[0023] The nucleotide sequence of the downstream primer is shown as SEQ ID NO.8.

[0024] Preferably, the primers used in the qPCR amplification reaction include a Human-ASGR2-1 primer set, and the Human-ASGR2-1 primer set includes the following primers:

[0025] An upstream primer having a nucleotide sequence as shown in SEQ ID NO.9;

[0026] A downstream primer having a nucleotide sequence as shown in SEQ ID NO.10;

[0027] The primers used in the qPCR amplification reaction include the Human-ASGR2-2 primer set, and the Human-ASGR2-2 primer set includes the following primers:

[0028] An upstream primer having a nucleotide sequence as shown in SEQ ID NO.11;

[0029] The nucleotide sequence of the downstream primer is shown as SEQ ID NO.12.

[0030] Preferably, the transfection reagent is RNAiMAX Transfection Reagen.

[0031] Preferably, the small nucleic acid drug to be detected and / or the transfection reagent are dispersed in the cell culture medium before mixing;

[0032] The cell culture medium is Opti-MEM;

[0033] And / or, the cells are washed using DPBS.

[0034] Preferably, the cynomolgus monkey liver primary hepatocytes are obtained by a preparation method comprising the following steps:

[0035] S1, perfusing the isolated liver tissue with anticoagulant wash solution;

[0036] S2, perfusion with irrigating fluid;

[0037] S3, perfusion digestion using digestion solution;

[0038] S4, after peeling off the capsule of the isolated liver tissue, rinse it with resuspension culture medium and collect the rinse fluid;

[0039] S5. Filter the flushing solution, terminate the digestion, perform density gradient centrifugation, obtain primary hepatocytes of cynomolgus monkey liver, and inoculate them into a cell culture plate.

[0040] Preferably, the anticoagulant wash solution contains DPBS without calcium and magnesium ions, heparin sodium and double antibody;

[0041] The lavage fluid contains EDTA or EGTA, DPBS without calcium and magnesium ions, and double antibody;

[0042] The digestive fluid contains collagenase II;

[0043] The resuspension medium contains DMEM and FBS.

[0044] Preferably, during the perfusion process, a perfusion tube is pre-inserted, and the perfusion solution or digestion solution is slowly perfused through the perfusion tube by a syringe;

[0045] The perfusion digestion time is within 15 minutes.

[0046] Preferably, the cell culture plate is coated with rat tail collagen.

[0047] Beneficial effects of the present invention:

[0048] The present invention uses primary cynomolgus monkey hepatocytes to construct a screening model, combined with a specific transfection scheme and detection method, to quickly obtain the regulatory effect of the drug on the target gene, effectively shortening the screening cycle. The present invention uses ACTB, ASGR1 and ASGR2 as target genes, and uses carefully designed primers to perform qPCR amplification reactions, which can accurately detect changes in gene expression, provide accurate data support for drug screening, and improve the reliability of screening results.

[0049] The present invention specifies in detail the steps of preparation of primary hepatocytes of crab-eating macaque liver, formation of transfection complexes, cell culture and detection, including the use of reagents, cell treatment conditions, etc., so as to standardize and normalize the entire screening process and facilitate repeated operations and result comparisons in different laboratories.

[0050] The present invention is applicable to the screening of a variety of small nucleic acid drugs. Whether it is the drug development for liver diseases or other diseases that may be related to liver metabolism, it can provide an effective screening method and help promote the development of the field of small nucleic acid drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 This is the microscopic observation of cynomolgus monkey primary liver cell culture at 0h;

[0052] Figure 2 This is a microscopic observation of cynomolgus monkey liver primary cells cultured for 16 hours;

[0053] Figure 3 The graph shows the expression changes of ASGR1 and ASGR2 mRNA during the culture of primary hepatocytes of cynomolgus monkeys;

[0054] Figure 4 This is a bar graph showing the expression of β-Actin gene after siRNA transfection at different cell densities. DETAILED DESCRIPTION

[0055] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0056] Example 1

[0057] Extraction of primary liver cells from cynomolgus monkeys

[0058] Solution preparation

[0059] Anticoagulant wash solution (precooled before use): DPBS (without calcium and magnesium) + sodium heparin + 1% double antibody

[0060] Prepare 40 mL: 4 mL (heparin sodium solution 2*12500 units) + 36 mL (DPBS containing 1% PS).

[0061] Irrigation fluid (precooled before use): 2.5mM EDTA (or EGTA) + DPBS (without calcium and magnesium) + 1% double antibody

[0062] Prepare 50mL / 100mL. 50mL: 0.25mL (0.5M EDTA) + 50mL (DPBS containing 1% PS).

[0063] Since EDTA / EGTA will form chelates with calcium and magnesium ions, the DPBS used in the lavage fluid does not contain calcium and magnesium. EDTA is used to chelate calcium and magnesium ions on the cell surface, reducing the adhesion between cells and making it easier for digestive enzymes to digest cells.

[0064] Digestion solution (needs to be preheated to 39°C before use): collagenase II (1×, 1 mg / mL)

[0065] Prepare a total of 200 mL from both right lobes of the liver.

[0066] Note: Collagenase II powder was first prepared into a 100 mg / mL (100×) stock solution with HBSS / DPBS (containing calcium and magnesium), and then dispensed into 1 mL / tube and stored at -20° away from light. On the day of the experiment, HBSS / DPBS (containing calcium and magnesium, calcium ions can increase collagenase activity; containing 10% FBS) was used to dilute the solution to 1 mg / mL (1×), filter and sterilize it for later use.

[0067] Resuspension medium (pre-cooled before use): DMEM + 5% FBS

[0068] Adhesion medium (preheated at 37°C before use): DMEM + 20% FBS

[0069] 100%Percoll: 9mLPercoll+1mL 10×PBS

[0070] 30%Percoll: 3mL 100%Percoll+7mL 1×PBS

[0071] Coat cell culture plates one day in advance

[0072] Rat tail collagen was diluted with ddHO at a ratio of 1:40 2 Dilute with 500 μL of HCl and add to the culture dish / plate (500 μL per well of a 24-well plate and 1 mL per well of a 6-well plate). Incubate at 37° for 3 hours, discard the liquid, and wash with ddHO. 2 Wash 1-2 times with 5% CO.

[0073] Blow dry in a biosafety cabinet and leave uncovered until use the next day.

[0074] On the day of the experiment, turn on the ice maker and water bath in advance, pre-cool the perfusion fluid and culture medium on ice, pre-heat the digestion solution to 39°C, disinfect and sterilize the surgical instruments (2 sets of forceps, scissors, and hemostatic forceps), and pre-cool the centrifuge at 4°C.

[0075] Extraction steps

[0076] Take out the ex vivo liver tissue (right lobe, retain the main blood vessels, and the cuts on the blood vessels must be neat and clear in shape), rinse with anticoagulant wash solution on ice, perfuse the anticoagulant wash solution into the liver until the liver swells and turns yellow to white, and quickly transfer to the biosafety cabinet.

[0077] Cover the mouth of a 500mL beaker with sterile gauze in advance, place the liver on the gauze, and use lavage fluid to perfuse into the liver tissue along the blood vessels (pre-insert the perfusion tube, and slowly perfuse the lavage fluid through a 50mL syringe through the perfusion tube to prevent the capsule from being ruptured by excessive pressure). During the perfusion process, apply uniform pressure to the liver with the index finger and thumb, and perfuse repeatedly until the liver is uniformly swollen and whitened, and there is no blood in the outflowing fluid.

[0078] Replace the lavage fluid with digestion fluid and continue perfusion. During the perfusion process, apply uniform pressure to the liver with the index finger and thumb until the tissue inside the liver becomes soft and inelastic. Use the tail of a sterile syringe to gently and evenly squeeze the entire liver tissue to fully digest it.

[0079] During perfusion digestion, the flow rate of the digestion solution was controlled and the digestion time was controlled within 15 minutes.

[0080] Transfer the liver to a culture dish, peel off the liver capsule with forceps, rinse with resuspension culture medium to disperse the minced meat tissue, and collect the rinse fluid. Do not apply mechanical force to the liver.

[0081] The digestion solution was filtered through a 100 μm filter and collected into a 50 mL centrifuge tube. An appropriate amount of resuspension medium was added to the tube to terminate the digestion.

[0082] Divide the filtered cell suspension into 15mL centrifuge tubes (no more than 10mL per tube, otherwise it may affect the centrifugal efficiency), centrifuge at 4℃, 50*g for 5min (↑:5, ↓:1), discard the supernatant, add 5mL of resuspension medium to resuspend, slowly overlay the cell suspension on an equal volume of 30% Percoll top layer, centrifuge at 4℃, 1200*g for 15min (↑:5, ↓:1). Discard the supernatant, add 10mL of resuspension medium to resuspend, centrifuge at 4℃, 50*g for 5min (↑:5, ↓:1), discard the supernatant, add 3ml of adhesion medium to resuspend, and count.

[0083] Laying the board and adding medicine:

[0084] Based on the counting results, inoculate cells according to the table below.

[0085] Table 1 Cell inoculation status

[0086] Cell seeding density Inoculation volume Final volume 24-well plate <![CDATA[About 0.5*10 6 cells / mL]]> 0.5mL 0.5 6-well plate <![CDATA[Approximately 1.0*10 6 CFU / mL]]> 1mL 2mL

[0087] Observe the cell growth status under the microscope at 0h of culture. Figure 1 As shown; liver cells can attach to the wall in about 16 hours. Figure 2 As shown, primary cynomolgus monkey liver cells were obtained.

[0088] Example 2

[0089] siRNA transfection

[0090] The small nucleic acid drug (siRNA) to be tested was centrifuged at 12000×g for 2 min, and an appropriate amount of ddH 2 O, prepare a 100 μM stock solution, take 5 μL and add 45 μL ddHO 2 O, prepared into 10 μM working solution and stored at -20°C.

[0091] Transfection steps

[0092] 1. According to 1.0×10 5 The prepared cynomolgus monkey primary hepatocytes were inoculated into 24-well plates, and CO 2 Incubator (37 ± 2 °C, 5% ± 2% CO 2 ) overnight. According to the experiment, a total of n wells are set up (negative control, fluorescent control, siRNA). Transfection is performed when the cell confluence reaches 80%.

[0093] 2. Prepare tube A: Opti-MEM 30μL*n+RNAiMAX 1.8μL*n;

[0094] 3. Prepare B tube (different B tube for each siRNA): Opti-MEM 30μL*m + siRNA (10μM) 0.6μL*m

[0095] 4. Add an equal volume of tube A (30 μL*m) into tube B (30 μL*m) and incubate at room temperature for 5 min to obtain a complex solution.

[0096] 5. Take out the cell plate, discard the culture medium, wash once with DPBS, add 450 μL Opti-MEM to each well, and add 50 μL complex solution to each well.

[0097] 6. 24 hours after transfection, samples were collected for quantitative polymerase chain reaction (qPCR) to evaluate the gene knockdown effect;

[0098] Note: After transfection, observe the expression of the fluorescent control (Qingke's fluorescent control, green light).

[0099] In this example, cell lysates were collected at different time points of cell culture to extract mRNA, and qPCR amplification reactions were performed with designed PCR primers. The mRNA expression levels of related genes under different experimental conditions (such as different transfection doses, etc.) were quantitatively analyzed by detecting the amount of amplified products, thereby evaluating the regulatory effect of small nucleic acid drugs on target genes, determining whether transfection was successful, and studying gene function. In this example, the expression variation patterns of ACTB, ASGR1, and ASGR2 during the culture of primary hepatocytes of cynomolgus monkeys were analyzed to provide data support for small nucleic acid drug screening and determine whether the constructed screening platform is effective and reliable.

[0100] Table 2 Target gene information

[0101] Gene name Gene or transcript ID Species APOB-1 102121781 Macaca APOB-2 102121781 Macaca APOB-3 102121781 Macaca APOB-1 102121781 Macaca APOB-2 102121781 Macaca APOB-3 102121781 Macaca ACTB-1 102139853 other ACTB-2 102139853 other ACTB-3 102139853 other

[0102] Table 3 Primer information

[0103]

[0104]

[0105] Table 4 siRNA information used in this example

[0106]

[0107]

[0108] Cell lysates were collected at 0, 16, 24, 40, and 48 h of cell culture to extract mRNA, and the ASGR1 and ASGR2 mRNA levels were quantified relative to GAPDH. Figure 3As shown, ASGR1 and ASGR2 mRNA were down-regulated in freshly extracted cynomolgus monkey primary hepatocytes. The results show that the expression levels of ASGR1 and ASGR2 genes can be detected by the present invention, which can be used as an indicator for screening the effects of small nucleic acid drugs on cells. For example, a certain small nucleic acid drug may affect the expression of these two genes. By observing the difference in gene expression between the drug-treated group and the control group at different time points, the effectiveness and duration of action of the drug can be evaluated.

[0109] After siRNA transfection at different cell densities, PCR detection was performed on the expression of β-Actin gene. The bar graph of β-Actin gene expression level is shown in Figure 4 shown. Figure 4 In the figure, P: siRNA Positive QC; N: Negative QC; C1: 1*10^5cells / well; C2: 2*10^5cells / well; C3: 4*10^5cells / well, by showing the relative expression levels of β-Actin genes of the positive control (P) and the negative control (N) at different cell densities (C1, C2, C3) and different time points (Day 1, Day 2, Day 3), it reflects the influence of cell density and time on gene expression in the siRNA transfection experiment. The results show that the expression level of the β-Actin gene can be detected by the present invention, which can be used as an indicator for screening the effects of small nucleic acid drugs on cells. For example, a certain small nucleic acid drug may affect the expression of this gene. By observing the difference in gene expression between the drug-treated group and the control group at different time points, the effectiveness and duration of action of the drug can be evaluated.

[0110] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for in vitro screening of small nucleic acid drugs, characterized in that: The following steps are involved: The small nucleic acid drug to be detected is mixed and incubated with a transfection reagent to obtain a transfection complex; After washing the cells, the transfection complex and cell culture medium are added to a cell culture plate in which cynomolgus monkey primary hepatocytes are cultured for culturing; ACTB, ASGR1, and ASGR2 were used as target genes, and cell lysates were collected at different time points of cell culture to extract mRNA, and qPCR amplification reactions were performed to obtain the amount of amplified products; The amount of amplified product obtained is used to evaluate the regulatory effect of small nucleic acid drugs on target genes, thereby achieving drug screening.

2. The in vitro screening method for small nucleic acid drugs according to claim 1, characterized in that: The primers used in the qPCR amplification reaction include the NHP-ACTB2 primer set, and the NHP-ACTB2 primer set includes the following primers: An upstream primer having a nucleotide sequence as shown in SEQ ID NO.1; A downstream primer having a nucleotide sequence as shown in SEQ ID NO.2; The primers used in the qPCR amplification reaction include the NHP-ACTB9 primer set, and the NHP-ACTB9 primer set includes the following primers: An upstream primer having a nucleotide sequence as shown in SEQ ID NO.3; The nucleotide sequence of the downstream primer is shown as SEQ ID NO.

4.

3. The in vitro screening method for small nucleic acid drugs according to claim 1, characterized in that: The primers used in the qPCR amplification reaction include the Human-ASGR1-1 primer set, and the Human-ASGR1-1 primer set includes the following primers: An upstream primer having a nucleotide sequence as shown in SEQ ID NO.5; A downstream primer having a nucleotide sequence as shown in SEQ ID NO.6; The primers used in the qPCR amplification reaction include the Human-ASGR1-2 primer set, and the Human-ASGR1-2 primer set includes the following primers: An upstream primer having a nucleotide sequence as shown in SEQ ID NO.7; The nucleotide sequence of the downstream primer is shown as SEQ ID NO.

8.

4. The in vitro screening method for small nucleic acid drugs according to claim 1, characterized in that: The primers used in the qPCR amplification reaction include the Human-ASGR2-1 primer set, and the Human-ASGR2-1 primer set includes the following primers: An upstream primer having a nucleotide sequence as shown in SEQ ID NO.9; A downstream primer having a nucleotide sequence as shown in SEQ ID NO.10; The primers used in the qPCR amplification reaction include the Human-ASGR2-2 primer set, and the Human-ASGR2-2 primer set includes the following primers: An upstream primer having a nucleotide sequence as shown in SEQ ID NO.11; The nucleotide sequence of the downstream primer is shown as SEQ ID NO.

12.

5. The in vitro screening method for small nucleic acid drugs according to claim 1, characterized in that: The transfection reagent is RNAiMAX Transfection Reagen.

6. The in vitro screening method for small nucleic acid drugs according to claim 1, characterized in that: The small nucleic acid drug to be detected and / or the transfection reagent are dispersed in the cell culture medium before mixing; The cell culture medium is Opti-MEM; And / or, the cells are washed using DPBS.

7. The in vitro screening method for small nucleic acid drugs according to any one of claims 1 to 6, characterized in that: The primary cynomolgus monkey liver hepatocytes are obtained by a preparation method comprising the following steps: S1, perfusing the isolated liver tissue with anticoagulant wash solution; S2, perfusion with irrigating fluid; S3, perfusion digestion using digestion solution; S4, after peeling off the capsule of the isolated liver tissue, rinse it with resuspension culture medium and collect the rinse fluid; S5. Filter the flushing solution, terminate the digestion, perform density gradient centrifugation, obtain primary hepatocytes of cynomolgus monkey liver, and inoculate them into a cell culture plate.

8. The in vitro screening method for small nucleic acid drugs according to claim 7, characterized in that: The anticoagulant washing solution contains DPBS without calcium and magnesium ions, heparin sodium and double antibody; The lavage fluid contains EDTA or EGTA, DPBS without calcium and magnesium ions, and double antibody; The digestive fluid contains collagenase II; The resuspension medium contains DMEM and FBS.

9. The in vitro screening method for small nucleic acid drugs according to claim 7, characterized in that: During the perfusion process, a perfusion tube is pre-inserted, and the perfusion solution or digestion solution is slowly perfused through the perfusion tube by a syringe; The perfusion digestion time is within 15 minutes.

10. The in vitro screening method for small nucleic acid drugs according to claim 7, characterized in that: The cell culture plate is coated with rat tail collagen.

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