An in vitro culture method of Golgi apparatus and its application in constructing a drug toxicity screening system

Abstract

The present invention belongs to the fields of drug screening and biological medicine technology, and particularly relates to a method for in vitro culture of Golgi apparatus and its application in constructing a drug toxicity screening system. Specifically, by improving the ATP regeneration buffer system and exploring the appropriate ratio of cytoplasmic matrix to Golgi apparatus, the present invention uses Western blotting and immunofluorescence techniques to measure the expression of Golgi apparatus function-related markers under this system, and confirms that the activity maintenance state of the Golgi apparatus is good under this system. Based on this culture system, a toxicity and sensitivity evaluation platform system for a variety of hepatotoxic drugs including triptolide is further constructed. The present invention greatly reduces the difficulty of in vitro culture of Golgi apparatus, shortens the operation time and has strong repeatability. It reflects the damage status of drug hepatotoxicity to organelles at the functional level, and can be used for more extensive evaluation of drug toxicity and sensitivity at the microscopic level. Therefore, it has good practical application value.

Description

Technical Field

[0001] The present invention belongs to the technical fields of drug screening and biomedicine, and particularly relates to a method for culturing Golgi bodies in vitro and its application in constructing a drug toxicity screening system. Background Art

[0002] Disclosing the information of this background art section is only intended to enhance the understanding of the overall background of the present invention, and is not necessarily regarded as an admission or any form of implication that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] The Golgi apparatus is a polarized secretory organelle composed of multiple flattened vesicles, mainly responsible for transporting, modifying, and packaging the proteins and lipids synthesized by the endoplasmic reticulum into vesicles, and then transporting them to specific parts of the cell or secreting them outside the cell. This process is an important stage for quality control and sorting of proteins and is crucial for maintaining intracellular homeostasis. In addition to being a major transportation hub for intracellular macromolecules, the Golgi apparatus is also a factory for intracellular sugar synthesis and plays various important roles in cell life activities. When drugs damage liver cells, the vesicle structure of the Golgi apparatus in the cell will swell and expand or disintegrate into scattered small fragments. At the same time, the functions of the Golgi apparatus, such as protein and lipid secretion, transportation, and glycosylation modification, will be inhibited. Therefore, characterizing the structure and function of the Golgi apparatus can be used as an important indicator for drug toxicity screening and evaluation.

[0004] The existing in vivo and in vitro drug toxicity screening models still have limitations in application: the time cycle for drug screening and evaluation at the tissue and organ levels is long, the cost is high, and the operation is complex; the drug screening at the molecular and enzyme levels cannot directly evaluate the in vivo effect of drugs. Compared with the above models, the drug screening at the cell and subcellular levels uses whole cells as the object of drug action to observe the effects of drugs on whole cells and subcellular organelles, and is currently the most widely used drug screening method. Compared with the cell screening model, the subcellular organelle screening model can not only perform functional screening, but also analyze the drug action at a more microscopic level, with higher drug sensitivity, lower cost, and stronger operability. Therefore, based on the important position of the Golgi apparatus among the target subcellular organelles of drug action, establishing an in vitro organelle culture system represented by the Golgi apparatus is of great significance for establishing a drug toxicity evaluation and screening system.

[0005] Currently, the main method for in vitro culture of the Golgi apparatus is to incubate the extracted Golgi membrane with different factors or buffer solutions in stages and add various cytokines to the culture system to induce in vitro culture. However, the inventors found that: (1) The existing Golgi culture system is complex and has poor reproducibility, and only maintains the activity of the Golgi apparatus through a chemical buffer system of ATP and various inorganic salts; (2) The existing Golgi culture system is difficult to maintain its complete structure and activity, so the application effect is poor and it has not been widely used in in vitro toxic drug screening. Summary of the Invention

[0006] In view of the above deficiencies of the prior art, through long-term technical and practical exploration, the inventors provide a method for in vitro culture of the Golgi apparatus and its application in constructing a drug toxicity screening system. By improving the ATP regeneration buffer system and exploring the appropriate ratio of cytoplasmic matrix to Golgi apparatus, the inventors used Western blotting, immunofluorescence technology, and transmission electron microscopy to measure the expression of markers related to the morphology and function of the Golgi apparatus under this system, and confirmed that the activity of the Golgi apparatus is well maintained under this system. Based on this culture system, a toxicity and sensitivity evaluation platform system for various hepatotoxic drugs including triptolide, stilbene glucoside, chrysophanol, and icariside I was further constructed. Based on the above research results, the present invention was completed.

[0007] To achieve the above technical objectives, the present invention adopts the following technical solutions:

[0008] In the first aspect of the present invention, a method for in vitro culture of the Golgi apparatus is provided. The culture method includes separating and extracting the Golgi apparatus using autologous primary cells, adding a cytoplasmic matrix solution thereto to complete the assembly of the Golgi apparatus and the cytoplasmic matrix, and then adding an ATP support system solution thereto.

[0009] Among them, the cytoplasmic matrix solution is taken from the same tissue source as the Golgi apparatus.

[0010] The ATP support system solution contains creatine phosphate, ATP, cytochalasin B, and creatine kinase.

[0011] In the second aspect of the present invention, an application of the above culture method in constructing a drug toxicity screening system is provided.

[0012] Specifically, the application is to construct a hepatotoxic drug screening system; the hepatotoxic drugs include but are not limited to stilbene glucoside, chrysophanol, icariside I, and triptolide.

[0013] In a third aspect of the present invention, a hepatotoxic drug screening system is provided. The system is constructed and obtained through the above-mentioned in vitro culture method of the Golgi apparatus. When the hepatotoxic drug screening system is in operation, the drug to be tested is premixed with the above-mentioned buffer solution, and then the buffer solution containing the drug to be tested is added thereto. The screening time can be controlled to 6 hours.

[0014] Compared with the prior art solutions, the above one or more technical solutions have the following beneficial effects:

[0015] 1. The above technical solution optimizes the currently complex in vitro ATP energy supply system of the Golgi apparatus organelle, and innovatively adopts the method of compounding active factors with a specific proportion of cytoplasmic matrix to maintain more comprehensive basic functions of the Golgi apparatus without the support of cell structure.

[0016] 2. The above technical solution extracts and separates the Golgi apparatus and extracellular matrix from the same mouse liver. This autologous primary tissue separation and assembly of the Golgi apparatus technology is more stable and closer to the actual in vivo physiological environment.

[0017] 3. Based on the in vitro culture system of the Golgi apparatus, the above technical solution develops a high-throughput new drug toxicity screening platform, shortens the screening time, realizes the possibility of large-scale in vitro screening, and at the same time reduces the screening cost. Therefore, it has good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The specification drawings forming a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation to the present invention.

[0019] Figure 1 It is an electron micrograph of the Golgi apparatus obtained in the embodiment of the present invention, and its scale is 500 nm.

[0020] Figure 2 It is the functional realization situation of the Golgi apparatus and cytoplasmic matrix in the ratio of 1:25 in the embodiment of the present invention. GM130 is the Golgi apparatus shaping protein on the cis surface of the Golgi apparatus; Golgin-45 (Gol45) is the coiled coil protein on the trans surface of the Golgi apparatus; Syntaxin 6 is a Golgi apparatus structure-related protein, which is related to Golgi apparatus translocation and can be used as a Golgi apparatus internal reference; COPB is a vesicle marker and can be used as a reference for vesicle transport conditions.

[0021] Figure 3This shows the maintenance of the structure and function of the Golgi apparatus under different culture systems in the embodiments of the present invention. GM130 is a Golgi matrix protein on the cis surface of the Golgi apparatus; Golgin-45 (Gol45) is a coiled-coil protein on the trans surface of the Golgi apparatus; Syntaxin 6 is a protein related to the structure of the Golgi apparatus, associated with Golgi translocation, and can be used as a Golgi internal reference; COPB is a vesicle marker and can be used as a reference for vesicle trafficking.

[0022] Figure 4 This shows the comparison of the evaluation effects of the Golgi apparatus on potentially hepatotoxic drugs in the embodiments of the present invention. GM130 is a Golgi matrix protein on the cis surface of the Golgi apparatus; Golgin-45 (Gol45) is a coiled-coil protein on the trans surface of the Golgi apparatus; Syntaxin 6 is a protein related to the structure of the Golgi apparatus, associated with Golgi translocation, and can be used as a Golgi internal reference; COPB is a vesicle marker and can be used as a reference for vesicle trafficking.

[0023] Figure 5 This shows the maintenance of the Golgi apparatus function after HepG2 cells are given different reported hepatocyte-damaging drugs in the embodiments of the present invention. GM130 is a Golgi matrix protein on the cis surface of the Golgi apparatus; Golgin-45 (Gol45) is a coiled-coil protein on the trans surface of the Golgi apparatus; Syntaxin 6 (Syn6) is a protein related to the structure of the Golgi apparatus, associated with Golgi translocation, and can be used as a Golgi internal reference; β-actin is a cytoskeleton internal reference; COPB is a vesicle marker and can be used as a reference for vesicle trafficking.

[0024] Figure 6 This shows the fluorescence staining of Golgi-related markers of the Golgi apparatus under the action of drug TP in the embodiments of the present invention. Among them, Syntaxin 6 is a protein related to the structure of the Golgi apparatus, associated with Golgi translocation, and can be used as a Golgi internal reference; COPB is a vesicle marker and can be used as a reference for vesicle trafficking function. Detailed implementation manners

[0025] It should be noted that the following detailed description is exemplary and is intended to provide further illustration of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present application belongs.

[0026] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should also be understood that when the terms "comprise" and / or "include" are used in this specification, they specify the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0027] As mentioned above, the in vitro culture method of the Golgi apparatus mainly involves stagewise incubation of the extracted Golgi membrane with different factors or buffer solutions and the addition of various cytokines to the culture system to induce in vitro recombination. However, this method has low stability, and adding too many substances easily damages the structure and function of the Golgi apparatus, and the Golgi activity can only be maintained briefly.

[0028] In view of this, in a typical specific embodiment of the present invention, a method for culturing the Golgi apparatus in vitro is provided. The culturing method includes isolating and extracting the Golgi apparatus using autologous primary cells, adding a cytoplasmic matrix solution thereto to complete the assembly of the Golgi apparatus and the cytoplasmic matrix, and then adding an ATP support system solution thereto;

[0029] In another specific embodiment of the present invention, the cytoplasmic matrix solution is taken from the same tissue source as the Golgi apparatus. The total protein mass ratio in the Golgi apparatus and the cytoplasmic matrix solution is 1:20 - 30; through experimental verification, when the total protein mass ratio of the Golgi apparatus to the cytoplasmic matrix solution is 1:25, the performance of the assembly system is optimal.

[0030] The ATP support system solution contains creatine phosphate, ATP, cytochalasin B, and creatine kinase.

[0031] In another specific embodiment of the present invention, the isolating and extracting the Golgi apparatus using autologous primary cells specifically includes selecting a non-human animal liver tissue to obtain the Golgi apparatus; the specific method is as follows: adding the pretreated fresh non-human animal liver tissue to a buffer solution, breaking it and then washing it, adding reagent A, homogenizing and performing the first centrifugation to remove the precipitate, obtaining the supernatant and then performing the second centrifugation, then removing the precipitate, obtaining the supernatant and continuing to perform the third centrifugation to remove the precipitate, adding a WT solution to the supernatant and continuing to perform the fourth centrifugation, discarding the supernatant and retaining the precipitate, adding reagent B to the precipitate and mixing well; performing the fifth centrifugation, and the precipitate is the Golgi apparatus.

[0032] Among them, the non-human animal is selected from non-human mammals, such as rats, mice, monkeys, orangutans, etc. In a specific embodiment of the present application, the non-human animal is selected from mice.

[0033] The reagent A, reagent B, and WT solution are all selected from commercially available Golgi extraction kits. In a specific embodiment of the present invention, the Golgi extraction kit is selected from the Golgi extraction kit with model number HR0247 produced by Boleibao.

[0034] The buffer solution can be PBS buffer solution, and the pretreatment can be washing the liver tissue with PBS buffer solution.

[0035] During the homogenization process, attention should be paid to gentle operation to ensure the integrity of the Golgi structure, and homogenize for 15 - 20 strokes.

[0036] The specific conditions for the first centrifugation are centrifuging at 500 - 1500 g (preferably 1000 g) for 1 - 10 minutes (preferably 5 minutes);

[0037] The specific conditions for the second centrifugation are centrifuging at 2000 - 4000 g (preferably 3000 g) for 5 - 20 minutes (preferably 10 minutes);

[0038] The specific conditions for the third centrifugation are centrifuging at 4000 - 6000 g (preferably 5000 g) for 5 - 20 minutes (preferably 10 minutes);

[0039] The specific conditions for the fourth centrifugation are centrifuging at 10000 - 30000 g (preferably 20000 g) for 10 - 30 minutes (preferably 20 minutes);

[0040] The specific conditions for the fifth centrifugation are centrifuging at 10000 - 30000 g (preferably 20000 g) for 10 - 60 minutes (preferably 30 minutes).

[0041] By centrifuging at multiple different speeds, high - purity Golgi can be obtained to the greatest extent.

[0042] It should be noted that to ensure the activity of the Golgi, the reagents and instruments used in the above - mentioned Golgi preparation process need to be pre - cooled at low temperature (such as 4°C) for at least 1 hour. At the same time, during the extraction and preparation process, it should be ensured as much as possible to carry out under low - temperature (such as 4°C) conditions.

[0043] The preparation method of the cytoplasmic matrix solution is as follows: After crushing the liver tissue of non - human animals after pretreatment, add sodium citrate buffer solution for homogenization treatment, then centrifuge the mixture, take the supernatant and then perform ultra - high - speed centrifugation, and take the supernatant to obtain it.

[0044] Among them, the pretreatment method of the liver tissue includes: perfusing normal saline into the liver until the blood in the liver blood vessels is completely replaced, and when the liver turns yellow - white and there is no obvious blood clot accumulation, the perfusion is completed.

[0045] During the homogenization process, sufficient homogenization is performed for at least 30 strokes.

[0046] The specific conditions for centrifugation are centrifugation at 5000 - 20000 g (preferably 10000 g) for 10 - 30 minutes (preferably 20 minutes).

[0047] The specific conditions for the ultra - high - speed centrifugation are centrifugation at 90000 - 200000 g (preferably 105000 g) for 30 - 120 minutes (preferably 60 minutes).

[0048] The specific composition of the ATP - supporting system solution is as follows: 50 - 200 mM phosphocreatine, 0.5 - 5 mM ATP, 0.1 - 0.5 mg / ml cytochalasin B, and 0.1 - 0.5 mg / ml creatine kinase.

[0049] In another specific embodiment of the present invention, the culture method further includes adding a buffer solution or a buffer solution containing the drug to be tested. The buffer solution includes: 10 - 100 mM Tris - HCl (pH = 7.4), 0.1 - 0.5 M sucrose, 10 - 100 mM potassium chloride, 10 - 50 mM β - glycerophosphate, 10 - 30 mM EGTA, 5 - 20 mM magnesium chloride, 1 - 5 mM ATP, 0.5 - 2 mM GTP, and 0.5 - 2 mM glutathione.

[0050] In another specific embodiment of the present invention, an application of the above - mentioned culture method in constructing a drug toxicity screening system is provided.

[0051] Specifically, the application is to construct a hepatotoxic drug screening system; the hepatotoxic drugs include but are not limited to stilbene glucoside, chrysophanol, icariside II, and triptolide.

[0052] In another specific embodiment of the present invention, a hepatotoxic drug screening system is provided. The system is constructed by the above - mentioned in vitro culture method of the Golgi apparatus. When the hepatotoxic drug screening system is in operation, the drug to be tested is pre - mixed with the above - mentioned buffer solution, and then the buffer solution containing the drug to be tested is added thereto. The screening time can be controlled to 6 hours.

[0053] In summary, the present invention uses autologous primary cells to isolate and extract the Golgi apparatus and adopts a new in vitro culture system of the Golgi apparatus that optimizes the ATP functional system and the cell matrix biological support system. It greatly reduces the difficulty of in vitro culture of the Golgi apparatus, shortens the operation time and has strong repeatability. This system aims to establish a stable and active Golgi apparatus culture system in vitro, reflect the damage of drug hepatotoxicity to organelles at the functional level, and further be used for drug toxicity and sensitivity evaluation at a broader microscopic level.

[0054] The present invention will be further described below in conjunction with specific examples. The following examples are only for explaining the present invention and do not limit its content. If the specific experimental conditions are not specified in the examples, they are usually in accordance with conventional conditions or the conditions recommended by the sales company; without special limitation in the present invention, they can all be obtained through commercial channels.

[0055] Example

[0056] 1 Extraction of cytoplasmic matrix

[0057] 1.1 Experimental materials: physiological saline, 0.1M sodium citrate solution, Dounce homogenizer, ordinary 2ml EP centrifuge tube, scissors for animal experiments, forceps for animal experiments, fresh liver tissue of mice, small animal anesthesia instrument, peristaltic pump, rubber hose, ultracentrifuge, ultracentrifuge tube.

[0058] 1.2 Pretreatment before experiment

[0059] (1) All solutions and surgical instruments need to be pre-cooled for at least 1 hour.

[0060] (2) Pretreatment of mice: Select mice over 7 weeks old, pre-fill the small animal anesthesia box with isoflurane for 2 minutes and then put the mice in. Select an anesthesia ventilation volume of 0.4 for more than 3 minutes. After ensuring that the mice are completely anesthetized, select the small animal face mask anesthesia mode for continuous anesthesia.

[0061] 1.3 Experimental method

[0062] (1) Cut open the extraperitoneal skin of the mouse's abdomen along the midline of the abdomen and cut open the inner peritoneum. Use an indwelling needle to pour the pre-cooled physiological saline into the inferior vena cava. After the liver slightly stands up, cut open the portal vein of the liver and continuously perfuse at least 20 mL of physiological saline until the blood in the liver blood vessels has been completely replaced and the liver turns yellowish-white without obvious blood clot accumulation, indicating that the perfusion is completed.

[0063] (2) Roughly cut the whole liver (between 1.0 - 1.7 g according to the mouse difference) and place it in the Dounce homogenizer for homogenization. At the same time, add 3 mL of 0.1M sodium citrate buffer solution and homogenize for at least 30 strokes. Until the liver has no obvious lumps and becomes a yellowish-brown homogeneous liquid.

[0064] (3) After homogenization, transfer all the solutions to a centrifuge tube. Centrifuge at 10,000 g for 20 minutes at 4°C and take the supernatant.

[0065] (4) Transfer the centrifuged supernatant and evenly load it into the ultracentrifuge tube. The rotation speed of the ultracentrifuge is 105,000 g and the centrifugation time is 1 hour. It can be seen that the upper layer of the centrifuge tube is a transparent and homogeneous solution, and this part of the solution is sucked out.

[0066] (5) Protein quantification: Measure the total protein content in the cytoplasmic matrix.

[0067] 2 Golgi apparatus extraction

[0068] 2.1 Experimental materials: Golgi apparatus extraction kit (BioLabo, HR0247), PBS solution, Dounce homogenizer, scissors for animal experiments, forceps for animal experiments, fresh liver tissue of mice

[0069] 2.2 Pretreatment before experiment:

[0070] (1) Pre-cooling: To maintain the activity of the Golgi apparatus, all reagents and surgical instruments during the experiment need to be pre-cooled at 4°C for at least 1 hour.

[0071] 2.3 Formal extraction of the Golgi apparatus

[0072] (1) Take 100 mg of fresh animal liver tissue sample and wash the tissue surface thoroughly with cold PBS.

[0073] (2) Add 500 μl of cold PBS to a centrifuge tube, and then add the liver. Cut the liver into pieces as small as possible with scissors and wash it twice with cold PBS.

[0074] (3) Add 500 μl of pre-cooled Reagent A in the above Golgi apparatus extraction kit and place it on ice for 10 minutes.

[0075] (4) Homogenize thoroughly on ice with a homogenizer for 15 - 20 strokes. During the homogenization process, pay attention to gentle movements to ensure the integrity of the Golgi apparatus structure and do not exceed 15 strokes. Then centrifuge at 4°C and 1000 g for 5 minutes.

[0076] (5) Discard the precipitate and aspirate the supernatant into another pre-cooled clean centrifuge tube. Centrifuge at 4°C and 3000 g for 10 minutes. (6) Discard the precipitate and aspirate the supernatant into another pre-cooled clean centrifuge tube. Centrifuge at 4°C and 5000 g for 10 minutes.

[0077] (7) Discard the precipitate and aspirate the supernatant into another pre-cooled clean centrifuge tube. Add 10 μl of Reagent WT in the above Golgi apparatus extraction kit to the supernatant and centrifuge at 4°C and 20000 g for 20 minutes.

[0078] (8) Discard the supernatant and keep the precipitate. Add 500 μl of cold Reagent B in the above Golgi apparatus extraction kit to the precipitate and mix well. After mixing, take 10 μl of the mixed solution for protein quantification

[0079] (9) Centrifuge at 4°C and 20000 g for 30 minutes. The precipitate is the Golgi apparatus, and measure the total protein content in the supernatant.

[0080] 3 Golgi apparatus assembly

[0081] 3.1 Experimental materials: Golgi apparatus, Golgi apparatus cytoplasmic matrix, ATP support system, buffer solution, enzyme-free and sterile water, 96-well plate, constant temperature incubator. Tris–HCl, sucrose, potassium chloride, β-glycerophosphate, EGTA, magnesium chloride, ATP, GTP, glutathione, creatine phosphate, ATP, cytochalasin B, creatine kinase.

[0082] 3.2 Preparation of key solutions

[0083] (1) Preparation of buffer solution: Tris-HCl (50 mM, pH = 7.4), sucrose (0.2 M), potassium chloride (50 mM), β-glycerophosphate (20 mM), EGTA (15 mM), magnesium chloride (10 mM), ATP (2 mM), GTP

[0084] (1 mM), glutathione (1 mM).

[0085] (2) Preparation of ATP support system solution: creatine phosphate (100 mM), ATP (1 mM), cytochalasin B (0.2 mg / ml), creatine kinase (0.2 mg / ml).

[0086] 3.3 Assembly process

[0087] (1) Assembly of Golgi apparatus and cytoplasmic matrix: This assembly process is carried out in a 96-well plate. The culture system is a 100 μl solution system per well, and the assembly ratio is (i.e., 1:25) Golgi apparatus - cytoplasmic matrix. After calculating based on the total protein amount of the cytoplasmic matrix and the total protein amount of the Golgi apparatus obtained in the previous steps 1 and 2, according to the total protein amount of the Golgi apparatus precipitate, add the corresponding cytoplasmic matrix solution, gently resuspend and mix well, and then add it to the wells of the 96-well plate.

[0088] (2) Addition of ATP system: According to the required number of wells, mix the mother liquors of each factor of the ATP system required for this experiment according to the dilution ratio. Note that the concentration of the mother liquor preparation should not be too low to prevent the addition of the ATP system-related solution from exceeding the total solution volume (100 μl). Then, add it evenly to the 96-well plate according to the actual number of wells used.

[0089] (3) If no drug needs to be administered, buffer solution can be directly added to make up the liquid volume to 100 μl / well. Incubate in a 37 °C constant temperature incubator.

[0090] 4 Construction of drug screening platform

[0091] (1) According to existing research, four hepatotoxic drugs, namely stilbene glucoside, chrysophanol, icariside I, and triptolide, with a final administration concentration of 10 μM are selected.

[0092] (2) If drug loading is required, during assembly, the Golgi apparatus - cytoplasmic matrix - ATP system can be assembled first and then placed in an incubator at 37°C for stabilization. The drug can be pre-mixed with the buffer solution in equal proportion, and then added to the 96-well plate. During the addition process, it should be ensured that the drug is evenly dispersed in the entire culture solution.

[0093] (3) The screening time of this drug platform is 6 hours, and further research and verification can be carried out 6 hours after drug administration.

[0094] Test results:

[0095] We first extracted the Golgi apparatus and fixed it with 2.5% glutaraldehyde solution, placed it at 4°C overnight, then sent the sample for projection electron microscopy photography. As Figure 1 shown, clear Golgi vesicles can be seen at a magnification of 12,000, proving that the organelle extraction process of this Golgi drug evaluation system is good.

[0096] We explored the optimal ratio of co-culture of cytoplasmic matrix and Golgi apparatus. As Figure 2 shown, on the premise that the total protein mass ratio of Golgi apparatus to cytoplasmic matrix is (1:25), this in vitro Golgi apparatus can express a variety of Golgi markers, proving that the construction of this Golgi drug evaluation system is good. After administration of the hepatotoxic drug triptolide, a significant decrease in the level of the trans-Golgi marker Gol45 can be seen.

[0097] Subsequently, we compared different culture systems, namely the in vitro complete culture system of the Golgi apparatus with separate culture of the ATP support system, separate culture of the cytoplasmic matrix, and co-culture of the ATP support system and the cytoplasmic matrix system (hereinafter referred to as the complete culture system). The cytoplasmic matrix was assembled according to Figure 2 the test ratio (1:25), and the ATP system was based on the final concentration of the corresponding factor in the technical solution, and the total solution volume was made up with the buffer. The results showed that compared with the simple ATP support system, the complete culture system could express higher levels of Golgi markers and was more stable.

[0098] In the in vitro complete culture system of the Golgi apparatus, we selected traditional Chinese medicine monomers that have been reported to potentially have hepatotoxic adverse reactions for drug administration. The administration concentration was 10 μM and the action time was 6 hours. It can be seen that icariside II and triptolide can effectively reduce the expression of the Golgi marker GM130 at this administration concentration, suggesting that liver Golgi toxicity damage may occur at this time.

[0099] HepG2 cells, as a hepatocellular carcinoma cell line, are currently commonly used in vitro evaluation cell systems for hepatotoxicity. However, compared with Figure 4The results show that after administration of various hepatotoxic traditional Chinese medicine monomers at the same dose and for the same time, only triptolide reduces the Golgi markers, and the in vitro evaluation system of the Golgi constructed in this example is more sensitive.

[0100] When administering Tripterygium wilfordii, a traditional Chinese medicine with reported hepatotoxic adverse reactions, compared with the blank control results, it can be seen that after administration of triptolide, the expression of the vesicle transport-related protein COPB decreases significantly, and the vesicle transport function of the Golgi is damaged. It can be seen that this system can ensure the realization of the normal function of the Golgi and effectively simulate the impairment of the Golgi function.

[0101] The above are only the preferred embodiments of the present application and are not used to limit the present application. For those skilled in the art, various changes and modifications can be made to the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included within the protection scope of the present application.

Claims

1. A method for in vitro culture of Golgi apparatus, characterized in that, The culture method includes extracting Golgi apparatus by centrifugal separation at five different rotation speeds using autologous primary cells, adding a cytoplasmic matrix solution thereto to complete the assembly of the Golgi apparatus and the cytoplasmic matrix, and then adding an ATP support system solution thereto; The cytoplasmic matrix solution is taken from the same tissue source as the Golgi apparatus; The total protein mass ratio in the Golgi apparatus and the cytoplasmic matrix solution is 1:20 - 30; The ATP support system solution contains creatine phosphate, ATP, cytochalasin B, and creatine kinase; The preparation method of the cytoplasmic matrix solution is as follows: After crushing the liver tissue of a non-human animal after pretreatment, add sodium citrate buffer solution for homogenization treatment, then centrifuge the mixture, take the supernatant and perform ultra-high speed centrifugation, and take the supernatant to obtain it; The pretreatment method of the liver tissue includes: perfusing physiological saline into the liver until the blood in the liver blood vessels is completely replaced, and when the liver turns yellowish-white and there is no obvious blood clot accumulation, the perfusion is completed; The specific conditions for centrifugation treatment are centrifugation at 5000 - 20000g for 10 - 30 minutes; The specific conditions for the ultra-high speed centrifugation treatment are centrifugation at 90000 - 200000g for 30 - 120 minutes; The specific composition of the ATP support system solution is as follows: 100 mM creatine phosphate, 1 mM ATP, 0.2 mg / ml cytochalasin B, and 0.2 mg / ml creatine kinase; The culture method further includes adding a buffer solution or a buffer solution containing a drug to be tested thereto. The buffer solution includes: 10 - 100 mM Tris-HCl, 0.1 - 0.5 M sucrose, 10 - 100 mM potassium chloride, 10 - 50 mM β-glycerophosphate, 10 - 30 mM EGTA, 5 - 20 mM magnesium chloride, 1 - 5 mM ATP, 0.5 - 2 mM GTP, 0.5 - 2 mM glutathione.

2. The method for in vitro culture of Golgi apparatus according to claim 1, characterized in that, The total protein mass ratio in the Golgi apparatus and the cytoplasmic matrix solution is 1:25; The separation and extraction of the Golgi apparatus using autologous primary cells specifically includes selecting the liver tissue of a non-human animal to obtain the Golgi apparatus; the specific method is as follows: Add the fresh liver tissue of a non-human animal after pretreatment to a buffer solution, crush it and wash it, add reagent A, homogenize and perform the first centrifugation, remove the precipitate, obtain the supernatant and perform the second centrifugation, then remove the precipitate, obtain the supernatant and continue to perform the third centrifugation, remove the precipitate, add WT solution to the supernatant and continue to perform the fourth centrifugation, discard the supernatant, retain the precipitate, add reagent B to the precipitate, and mix well; Perform the fifth centrifugation, and the precipitate is the Golgi apparatus; Reagent A, reagent B, and WT solution are all selected from commercially available Golgi apparatus extraction kits.

3. The method for in vitro culture of Golgi apparatus according to claim 2, wherein, The non-human animal is selected from non-human mammals, including rats, mice, monkeys, and chimpanzees.

4. The method for in vitro culture of Golgi apparatus according to claim 2, wherein The buffer solution is PBS buffer solution, and the pretreatment is to wash the liver tissue with PBS buffer solution.

5. The in vitro culture method of the Golgi apparatus according to claim 2, wherein, The specific conditions for the first centrifugation are centrifugation at 500 - 1500g for 1 - 10 minutes; The specific conditions for the second centrifugation are centrifugation at 2000 - 4000 g for 5 - 20 minutes; The specific conditions for the third centrifugation are centrifugation at 4000 - 6000 g for 5 - 20 minutes; The specific conditions for the fourth centrifugation are centrifugation at 10000 - 30000 g for 10 - 30 minutes; The specific conditions for the fifth centrifugation are centrifugation at 10000 - 30000 g for 10 - 60 minutes.

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