Method for separating and purifying haploid nucleus and polyploid nucleus of polyploid hepatocyte

Abstract

The invention discloses a method for separating and purifying haploid nucleuses and polyploid nucleuses of polyploid hepatocytes, and belongs to the field of molecular biology, biological medicine and medical health care, and the method comprises the following steps: S1, performing perfusion by using an improved two-step collagenase digestion method to obtain primary hepatocytes; s2, dyeing the primary hepatocytes and carrying out first flow sorting; s3, respectively grinding the diploid hepatocytes, the tetraploid hepatocytes and the octaploid hepatocytes after the first flow sorting to obtain hepatocyte nucleuses; s4, the hepatocyte nucleuses obtained after grinding are subjected to second-time flow type sorting; therefore, the application of the invention can break through the limitation that the polyploidy hepatocyte nucleus cannot be distinguished in the traditional hepatocyte separation, so that the separation of the hepatocyte nucleus is more accurate, the bottleneck of polyploidy karyotype separation of the hepatocyte nucleus is broken through, and the development of the research field of the hepatocyte nucleus is quickly promoted.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of molecular biology and biomedicine, medical care, and relates to the separation of hepatocyte nuclei, and in particular to a method for separating and purifying haploid nuclei and diploid nuclei of polyploid hepatocytes. BACKGROUND

[0002] Hepatocytes are different from other diploid somatic cells in that they have multiple ploidy forms. Hepatocytes have both diploid and polyploid forms, such as diploid hepatocytes, tetraploid hepatocytes, and octoploid hepatocytes. The nuclei of hepatocytes are also different from those of diploid somatic cells, and hepatocyte nuclei have multiple forms, such as mononuclear hepatocytes, binuclear hepatocytes, and multinuclear hepatocytes. The nuclei of polyploid hepatocytes are also divided into diploid nuclei, tetraploid nuclei, and octoploid nuclei. Therefore, polyploid hepatocytes are divided into mononuclear polyploid hepatocytes, binuclear polyploid hepatocytes, and multinuclear polyploid hepatocytes. The nuclei of polyploid hepatocytes are divided into diploid nuclei, tetraploid nuclei, and polyploid nuclei. The nuclei of tetraploid hepatocytes contain both 2 diploid hepatocyte nuclei and 1 tetraploid hepatocyte nucleus. The nuclei of octoploid hepatocytes contain 1 octoploid nucleus, 2 tetraploid nuclei, 1 tetraploid nucleus plus 2 diploid nuclei, and 4 diploid nuclei. Different ploidy hepatocytes have different physiological functions, and the functions of diploid nuclei and tetraploid nuclei of polyploid hepatocytes are also different. To study the functions of the multiple ploidy nuclei of polyploid hepatocytes, it is necessary to isolate the multiple ploidy forms of the nuclei of polyploid hepatocytes. However, it is very difficult to separate and distinguish the multiple ploidy nuclei of polyploid hepatocytes, which is a bottleneck problem that hinders the study of polyploid hepatocytes and polyploid hepatocyte nuclei. Directly isolating nuclei by grinding cells cannot distinguish the ploidy forms of the nuclei, and directly sorting polyploid hepatocytes according to their ploidy and then grinding them cannot separate diploid nuclei and polyploid nuclei in polyploid hepatocytes.

[0003] In order to distinguish different ploidy nuclei of different ploidy hepatocytes, we constructed a method of twice flow sorting combined with cell nucleus grinding to distinguish and separate monoploid nuclei and polyploid nuclei of polyploid hepatocytes. The first flow sorting of polyploid hepatocytes obtained hepatocytes of different ploidy, and then the hepatocytes of different ploidy were ground to obtain hepatocyte nuclei, and the hepatocyte nuclei of different ploidy were subjected to the second flow sorting to distinguish the ploidy of hepatocyte nuclei. For example, 4-ploidy hepatocytes were obtained by the first flow sorting of primary hepatocytes, and then the 4-ploidy hepatocytes were ground to obtain 4-ploidy hepatocyte nuclei, and then the second flow sorting was performed to obtain 2-ploidy nuclei and 4-ploidy nuclei in 4-ploidy hepatocytes. The 2-ploidy nuclei of 2-ploidy hepatocytes obtained by the above method were marked as (2n), the 2-ploidy nuclei of 4-ploidy hepatocytes were marked as (2n×2), the 4-ploidy nuclei were marked as (4n), the 2-ploidy nuclei of 8-ploidy hepatocytes were marked as (2n×4), the 4-ploidy nuclei were marked as (4n×2), and the 8-ploidy nuclei were marked as (8n). The method of twice flow sorting combined with cell nucleus grinding to distinguish and separate monoploid nuclei and polyploid nuclei of polyploid hepatocytes not only distinguishes hepatocytes of different ploidy, but also distinguishes different ploidy nuclei contained in hepatocytes of different ploidy. This method of separating hepatocyte nuclei distinguishes hepatocytes of different ploidy and distinguishes different ploidy nuclei contained in hepatocytes of different ploidy, and provides a method for separating and distinguishing hepatocyte nuclei, which can break through the limitation of traditional hepatocyte separation that cannot distinguish polyploid hepatocyte nuclei, make the separation of hepatocyte nuclei more accurate, break through the bottleneck of hepatocyte nucleus ploidy separation, and rapidly promote the progress of hepatocyte nucleus research field. SUMMARY

[0004] The present application aims to provide a method for separating and purifying monoploid nuclei and polyploid nuclei of polyploid hepatocytes to solve the problems in the background art.

[0005] To achieve the above-mentioned purpose, the present application provides the following technical solutions:

[0006] A method for separating and purifying monoploid nuclei and polyploid nuclei of polyploid hepatocytes, the steps of the method comprising:

[0007] S1, obtaining primary hepatocytes by perfusion using a modified two-step collagenase digestion method;

[0008] S2, staining the primary hepatocytes and performing the first flow sorting to obtain 2-ploidy hepatocytes, 4-ploidy hepatocytes and 8-ploidy hepatocytes;

[0009] S3, grinding the 2-ploidy hepatocytes, 4-ploidy hepatocytes and 8-ploidy hepatocytes after the first flow sorting to obtain hepatocyte nuclei;

[0010] S4, the second flow sorting is performed on the obtained liver cell nucleus after grinding, to obtain 2-ploid nucleus in 2-ploid liver cells, 2-ploid nucleus and 4-ploid nucleus in 4-ploid liver cells, 2-ploid nucleus, 4-ploid nucleus and 8-ploid nucleus in 8-ploid liver cells.

[0011] Preferably, the step S1 specifically implements the steps comprising:

[0012] S11, perfusion separation of the liver is performed by using a modified two-step collagenase digestion method;

[0013] S12, a perfusion pump is used to adjust the rotating speed to 5 mL / min, PBS is filled into the pipeline, and then the indwelling needle is pierced into the superior vena cava (attention should be paid to not piercing the superior vena cava), then the needle is withdrawn into the hose, and is smoothly pushed forward and fixed, so that the needle does not slip out of the superior vena cava during the whole perfusion process to cause perfusion failure. PBS is peristaltically pumped into the liver by the perfusion pump, and it is observed that the liver starts to swell and become larger, and the color becomes lighter, which indicates that the superior vena cava is successfully entered, then the hepatic portal vein on the other side of the liver is cut to allow PBS to pass through the liver completely;

[0014] S13, three kinds of perfusion fluids are preheated in a 37℃ water bath, and the buffer I, II and III are sequentially replaced, and the whole operation process is not more than 30 min. It is necessary to pay close attention to the infusion tube at all times, if it is not replaced in time, a large amount of air will enter the infusion tube, and the liver capsule will be ruptured in advance, and the perfusion will fail. After all the liver perfusion fluids are finished, the indwelling needle is withdrawn in advance to prevent air from entering, and the liver is observed, and it is found that the liver tissue under the liver capsule becomes loose, which indicates that the liver capsule is separated from the liver parenchyma;

[0015] S14, the liver is cut off, the gallbladder is removed, and is placed in a 10 cm culture dish, 10 mL of pre-cooled liver perfusion buffer III (which can continue to digest) is added, two small forceps are used to slowly and multi-pointly pierce the liver capsule, and then the liver is clamped and slowly and gently shaken repeatedly, so that most of the liver parenchymal cells are washed out, and it can be seen that the liquid becomes turbid, and finally the liver only has a fibrous bundle of basic structure, and the above process is operated on ice to reduce the number of dead liver cells;

[0016] S15, a 70 um cell filter screen is placed on a 50 mL centrifuge tube, and the cell suspension is filtered by using a Pasteur dropper, and then further filtered by using a 40 um cell filter screen, so that large impurities are removed. The obtained liver cell suspension is subjected to low-speed centrifugation (4°, 50 g, 5 min), and the supernatant is discarded, and the lower layer of the precipitate is the liver parenchymal cells. The cell precipitate is resuspended by using low-sugar DMEM, and then subjected to low-speed centrifugation (4°, 50 g, 5 min), and the process is repeated for several times;

[0017] S16, cell counting, 4% trypan blue mixed with cell suspension 1:1, placed on a hemocytometer plate under a microscope, manual counting to determine the number of hepatocytes, calculation method: total cell number in 4 fields of view / 4x10 4 / 2, counting live cells and dead cells in turn, then calculating the survival rate of cells = live cell number / live cell number+dead cell number, measuring 3 times before and after to take the average value;

[0018] S17, if the cell activity is less than 70%, Percoll purification separation solution (Percoll / 10x EBSS=9:1) is used to remove dead cells and separate hepatocytes, and a series of centrifugation steps (2x50gx4min) are used to collect;

[0019] When the cell activity reaches more than 90% after separation and purification, it can be used for the next step of sorting;

[0020] The purified hepatocytes are diluted with PBS to a density of 10 6 cells / mL, stored in ice for a maximum of 1 hour, until flow sorting;

[0021] When sorting, a flow loading tube with a filter cover is used for further filtration, and the loading concentration of the hepatocyte sample is adjusted to 1 million / mL to prevent clogging of the flow sorting pipeline;

[0022] S18, the obtained primary hepatocytes are seeded in a 6-well plate coated with Matrigel glue (Corning, item number 356234) at an appropriate concentration, and after 24h of culture in maintenance medium, the live hepatocytes are extended, and under a 20x microscope, hepatocytes of various karyotypes can be seen.

[0023] Preferably, the step S2 is specifically implemented as follows:

[0024] S21, the primary hepatocytes obtained in step one are added with 10μL of Hoechst 33342 dye solution at a concentration of 5μg / mL in 1mL of hepatocyte resuspension, and stained at 37℃ in the dark for 30 minutes; 2n, 4n, and 8n hepatocytes show increasing fluorescence intensity in turn, which is suitable for karyotype differentiation;

[0025] S22, wash the cells with PBS for 2 times;

[0026] S23, dilute the stained primary hepatocytes to a concentration of 1 million / mL, then filter them with a 40um filter, and add 5uL of PI (propidium iodide) per mL before flow sorting;

[0027] S24, the fluorescence intensity of dead and live liver cells is different, which can cause experimental error. In order to avoid the experimental error caused by dead liver cells, PI is used to distinguish the dead and live liver cells during flow sorting of liver cells;

[0028] S25, the forward scattering light (FSC) voltage value of the flow cytometer is set to 245, the side scattering light (SSC) voltage value is set to 210, the area value (A) of FSC is taken as the abscissa, the area value (A) of SSC is taken as the ordinate, and the cell gate is circled. Then, the area value (A) of FSC is taken as the abscissa, the height value (H) of FSC is taken as the ordinate, and the single cell gate is circled to remove the adherent cells;

[0029] S26, the voltage value of the 488-576 channel (PI channel) of the flow cytometer is set to 350, and the 488-576 fluorescence is taken as the abscissa and the FSC-A is taken as the ordinate to plot. The PI positive cells are dead cells, and the PI negative cells are live cells. The gate of live cells is drawn;

[0030] S27, the voltage value of the 405-448 channel (PB450 channel) of the flow cytometer is set to 450, and the 405-448 fluorescence is taken as the abscissa and the SSC-A is taken as the ordinate to plot. The diploid liver cells, tetraploid liver cells and octoploid liver cells are circled;

[0031] S28, the sorting logic is set. The sorting logic is to circle the liver cells through the FSC and SSC gates to remove impurity signal interference. Then, according to the fluorescence intensity of the PI channel, the PI negative cells are circled as live cells. Finally, according to the fluorescence intensity of the PB450 channel, the gates of diploid liver cells, tetraploid liver cells and octoploid liver cells are set. The diploid liver cells, tetraploid liver cells and octoploid liver cells are sorted according to the sorting logic;

[0032] S29, during sorting, the sample loading amount is 1 mL, and the cells are manually resuspended every 10 min with a Pasteur pipette to prevent sedimentation. The nozzle of the sorter is 100 um, and the sample concentration for sorting is preferably 1 million / mL, and the loading speed is preferably 2000 events per second;

[0033] S210, after the first flow sorting, only diploid liver cells with single nucleus, tetraploid liver cells containing single nucleus and double nucleus, and octoploid liver cells containing single nucleus, double nucleus and 3 nuclei are obtained. The cells are inoculated in Matrigel gel coated 24-well plates at appropriate concentrations.

[0034] Preferably, the step S3 specifically comprises the following steps:

[0035] S31, centrifuge the 2-ploid, 4-ploid and 8-ploid hepatocytes collected after the first flow sorting at 50g for 5min at 4℃, discard the supernatant, and resuspend the cells with 300μL HB buffer;

[0036] S32, for the diploid and tetraploid hepatocytes, use a 1mL tissue homogenizer for grinding, first use loose type pestle (hammer gap 63.5-139.7um) to grind slowly for 5 times, then replace the tight type pestle (hammer gap 12.7-63.5um) to grind for 10 times, gently break the hepatocyte membrane without damaging the nuclear membrane; for octaploid hepatocytes, use a 2mL tissue homogenizer for grinding, first use loose type pestle (hammer gap 76.2-127um) to grind slowly for 5 times, then replace the tight type pestle (hammer gap 12.7-63.5um) to grind for 10 times, gently break the hepatocyte membrane without damaging the nuclear membrane;

[0037] S33, filter the homogenate through a 40μm filter screen, use 700μL HB buffer to rinse the homogenizer and continue filtering, recover the nuclei;

[0038] Preferably, the step S4 specifically implements the steps comprising:

[0039] S41, re-stain the nuclear liquid obtained in step S3 with Hoechst 33342;

[0040] S42, stain the hepatocyte nuclei with Hoechst 33342 for 10min at room temperature in the dark, respectively;

[0041] S43, after staining, perform second flow sorting on the nuclei of the 2-ploid hepatocytes, the nuclei of the 4-ploid hepatocytes, and the nuclei of the 8-ploid hepatocytes, respectively;

[0042] S44, first perform flow sorting on the nuclei of the ground 2-ploid hepatocytes;

[0043] S45, then perform flow sorting on the nuclei of the ground 4-ploid hepatocytes;

[0044] S46, finally perform flow sorting on the nuclei of the ground 8-ploid hepatocytes;

[0045] S47, through the above gating scheme and steps, perform second sorting on the flow cytometer to obtain the 2-ploid nuclei of the 2-ploid hepatocytes, the 2-ploid nuclei and 4-ploid nuclei of the 4-ploid hepatocytes, and the 2-ploid nuclei, 4-ploid nuclei and 8-ploid nuclei of the 8-ploid hepatocyte nuclei;

[0046] S48, the flow collection tube is coated with 10% FBS in advance and placed at 4°C overnight to prevent the adsorption of cell nuclei to the wall. The collection volume is set to 300 μL, and finally transferred to a 1.5 mL centrifuge tube, centrifuged at 1000g at 4°C for 8 minutes, the supernatant was discarded, and the complete cell nucleus was precipitated;

[0047] S49, the composition distribution of nuclei in diploid hepatocytes, tetraploid hepatocytes and octoploid hepatocytes is counted.

[0048] Preferably, the step S44 specifically implements the steps comprising:

[0049] S441, the forward scatter light voltage value of the flow sorter is set to 275, the side scatter light voltage value is set to 270, the area value A of FSC is taken as the abscissa, the area value A of SSC is taken as the ordinate, and the cell nucleus gate is circled;

[0050] S442, the area value A of FSC is taken as the abscissa, and the height value H of FSC is taken as the ordinate, the single cell nucleus gate is circled, and the adherent cell nucleus is removed;

[0051] S443, the voltage value of the 405-448 channel (PB450 channel) of the flow sorter is set to 450, and the 405-448 fluorescence is taken as the abscissa and FSC-A as the ordinate to plot, the diploid nucleus of diploid hepatocytes is circled, and the sorting logic is set, and the diploid nucleus of diploid hepatocytes is sorted out;

[0052] Preferably, the step S45 specifically implements the steps comprising:

[0053] S451, the forward scatter light voltage value of the flow sorter is set to 275, the side scatter light voltage value is set to 270, the area value A of FSC is taken as the abscissa, the area value A of SSC is taken as the ordinate, and the cell nucleus gate is circled;

[0054] S452, the area value A of FSC is taken as the abscissa, and the height value H of FSC is taken as the ordinate, the single cell nucleus gate is circled, and the adherent cell nucleus is removed;

[0055] S453, the voltage value of the 405-448 channel (PB450 channel) of the flow sorter is set to 450, and the 405-448 fluorescence is taken as the abscissa and FSC-A as the ordinate to plot, the diploid nucleus of diploid hepatocytes is circled, and the sorting logic is set, and the diploid nucleus of diploid hepatocytes is sorted out;

[0056] Preferably, the step S46 specifically implements the steps comprising:

[0057] S461, set the forward scatter light voltage value of the flow sorter to 275, the side scatter light voltage value to 270, take the area value A of FSC as the horizontal coordinate, take the area value A of SSC as the vertical coordinate, and circle the nucleus gate;

[0058] S462, again take the area value A of FSC as the horizontal coordinate, take the height value H of FSC as the vertical coordinate, circle the single cell and gate, and remove the adherent cell nucleus;

[0059] S463, set the voltage value of the 405-448 channel (PB450 channel) of the flow sorter to 450, plot the 405-448 fluorescence as the horizontal coordinate and FSC-A as the vertical coordinate, circle the 8-ploid liver cell nucleus 2-ploid nucleus, 4-ploid nucleus and 8-ploid nucleus, and perform sorting logic setting, and respectively sort the 2-ploid nucleus, 4-ploid nucleus and 8-ploid nucleus of the 8-ploid liver cell.

[0060] Compared with the prior art, the present application has the following beneficial effects:

[0061] 1. The liver cell nucleus type is distinguished by twice flow sorting. The first flow sorting of the primary liver cell can distinguish different ploid liver cells. The second flow sorting after the liver cell nucleus grinding can distinguish different ploid nucleus types of different ploid liver cells. The twice flow sorting can significantly distinguish the 2-ploid nucleus (2n) in the 2-ploid liver cell, the 2-ploid nucleus (2n*2) and the 4-ploid nucleus (4n) in the 4-ploid liver cell, and the 2-ploid nucleus (2n*4), the 4-ploid nucleus (4n*2) and the 8-ploid nucleus (8n) in the 8-ploid liver cell.

[0062] 2. When perfusion separating the liver by using the improved two-step collagenase digestion method, the operation time is accurately controlled, and the survival rate of the primary liver cell is improved. The rotation speed of the perfusion pump is adjusted to 5 mL / min, and the volumes of the liver perfusion buffer I, II and III are adjusted: I is changed from 50 mL to 30 mL, II is about 50 mL, and III is changed from 100 mL to 60 mL and about 10 mL is reserved in advance and pre-cooled on the ice (for continuing the digestion after the liver is cut off), so that the whole operation process is ensured to be less than 30 min. If the time is too short, the liver has not been digested by collagenase sufficiently, and the liver capsule has not been separated from the liver parenchyma, so that the subsequent mechanical crushing of the liver is needed, which can obviously reduce the cell survival rate and greatly increase the cell fragments. If the time is too long, the liver is over-digested, and the survival rate of the liver cell is also obviously reduced. The three kinds of perfusion fluids are preheated in a 37℃ water bath, so as to ensure the activity of collagenase, and the infusion tube must be closely observed at any time, and the buffer I, II and III are replaced in time. If the replacement is not in time, a large amount of air will enter the infusion tube, and the liver capsule will be broken in advance when the air enters the liver, so that the perfusion fails.

[0063] 3、Optimization of the processing method after cutting the liver at the end of perfusion separation of the liver can significantly reduce cell debris, improve the activity and yield of primary hepatocytes, and ensure the activity and number of hepatocytes for subsequent sorting. After cutting the liver at the end of perfusion separation of the liver, use two small forceps to slowly puncture the liver capsule in multiple points, then clamp the liver and slowly and gently shake it repeatedly, so that most of the liver parenchymal cells are washed out. The liquid gradually becomes turbid, until the liver is left with only the basic structure of fibrous bundles. The whole process is operated on ice to avoid mechanical crushing of the liver with small scissors, which would increase the damage and death of hepatocytes, significantly reducing cell survival rate. At the same time, the degree of liver digestion can also be judged according to the ease of washing out of liver parenchymal cells. If the liver parenchymal cells are difficult to wash out, continue to digest in liver perfusion buffer III for a period of time; if the liver parenchymal cells are easily washed out, it means that the liver has been fully digested, and the cells need to be quickly processed to get rid of the digestion environment.

[0064] 4、When flow sorting polyploid hepatocytes, use Hoechst33342 dye to stain live hepatocytes. It can penetrate the cell membrane and bind to DNA double strands without damaging the cells, with low cytotoxicity. After Hoechst33342 dye penetrates the cell membrane and binds to DNA, it emits blue fluorescence, and the fluorescence intensity is proportional to the DNA content. The fluorescence intensity of 4-ploid hepatocytes is twice that of 2-ploid hepatocytes, and the fluorescence intensity of 8-ploid hepatocytes is twice that of 4-ploid hepatocytes. Utilize the linear difference of fluorescence intensity to sort 2-ploid hepatocytes, 4-ploid hepatocytes and 8-ploid hepatocytes.

[0065] 5. During flow cytometry sorting of polyploid hepatocytes, propidium iodide (PI) is used to distinguish live and dead hepatocytes. Primary hepatocytes obtained by perfusion are very fragile and easily die due to damage. When primary hepatocytes are obtained by perfusion using a two-step collagenase digestion method, the viability of the hepatocytes is generally 70-90%. Of these, approximately 10% are dead. Due to changes in cell membrane permeability, the fluorescent dye can more easily penetrate into the cell nucleus, resulting in a difference in blue fluorescence intensity between dead and live cells after Hoechst 33342 staining. To avoid experimental errors caused by dead hepatocytes, we use PI to distinguish live and dead hepatocytes during flow cytometry sorting of primary hepatocytes, sorting only live hepatocytes, not dead ones. PI is a nucleic acid dye that intercalates into double-stranded DNA and emits red fluorescence. However, PI cannot penetrate intact cell membranes. The cell membranes of live cells are intact, so PI cannot pass through. However, the cell membrane integrity of dead cells is damaged, and PI intercalates into double-stranded DNA, emitting red fluorescence. Therefore, in order to avoid the influence of dead hepatocytes on the sorting of polyploid hepatocytes, PI staining was added at the same time as Hoechst33342 staining. Hepatocytes that did not emit red fluorescence were living cells. Under the living cell gating condition, hepatocytes of different ploidy were further distinguished according to the intensity of blue fluorescence.

[0066] 6. When sorting hepatocytes and hepatocyte nuclei, take advantage of the difference in size between hepatocytes and hepatocyte nuclei to set appropriate parameters to ensure accurate sorting of hepatocytes and hepatocyte nuclei. When setting sorting conditions for flow cytometry, the forward scattered light (FSC) and side scattered light (SSC) voltages reflect cell size and internal granularity, respectively. The larger the cell, the higher the FSC value; the more complex the cell's internal structure, the higher the SSC value. Hepatocytes are 20-30 μm in size, much larger than other cells (3-10 μm). Because hepatocytes have multiple karyotypes and a more complex internal structure, they have higher SSC values. Therefore, when setting the FSC and SSC voltages for hepatocyte sorting, the voltages should be lower (the Moflo Astrisos flow cytometer has an FSC voltage of 245 and an SSC voltage of 210; commonly used FSC voltages for other cells are 270-280 and SSC voltages of 270-300). If the commonly used FSC and SSC voltage parameters are set, hepatocytes will exceed the detection range, resulting in cell loss and sorting failure. When setting the sorting parameters for hepatocyte nuclei, because the size of hepatocyte nuclei is much smaller than that of hepatocytes, the FSC and SSC voltage parameters for hepatocyte nuclei are set higher than those for hepatocytes (the FSC voltage value for hepatocyte nuclei sorting Moflo Astrisos is 275, and the SSC voltage value is 270). Otherwise, the hepatocyte nucleus image will be located in the lower left corner of the image, making it impossible to distinguish it from cell debris, reducing sorting accuracy.

[0067] 7. When sorting hepatocytes and hepatocyte nuclei, use the FSC area value (A) and height value (H) to distinguish adherent hepatocytes and hepatocyte nuclei. When preparing hepatocytes and hepatocyte nuclei, we need to try to keep the hepatocytes and hepatocyte nuclei in a single suspension state. However, in actual operation, it is impossible to achieve 100% of hepatocytes and hepatocyte nuclei in a single suspension state, and adherent hepatocytes or hepatocyte nuclei will affect the accuracy of hepatocyte and hepatocyte nuclei sorting. Therefore, during sorting, use the FSC area value (A) and height value (H) to draw a graph to distinguish adherent cells from single cells. With the FSC area value (A) as the horizontal axis and the height value (H) as the vertical axis, single cells will be located on the diagonal line of the graph, while adherent cells will be located off the diagonal line. This allows us to distinguish between adherent and non-adherent cells and ensure the accuracy of the sorting results.

[0068] 8. Precisely control the Hoechst 33342 staining time to ensure accurate, reproducible, and universally applicable results for the isolation of hepatocytes and nuclei. Because the cell membrane of live hepatocytes is selectively permeable, Hoechst 33342 can penetrate the cell membrane, enter the nucleus, and bind to DNA. However, during our experiments, we observed that the blue fluorescence intensity of live hepatocytes stained with Hoechst 33342 increased with increasing staining time. Therefore, to improve the accuracy of hepatocyte and nucleus isolation, we need to strictly control the Hoechst 33342 staining time. After trial and error, we found that 30 minutes at room temperature is an appropriate staining time. If flow cytometry is performed after 10 minutes of staining, the fluorescence will be very weak and unable to distinguish polyploid hepatocytes. However, prolonged staining will significantly reduce hepatocyte viability. Therefore, after staining for 30 minutes at room temperature, the cells should be washed twice with PBS to remove any Hoechst 33342 that has not entered the cells. This operation can not only ensure that enough fluorescent dye enters the cells to distinguish polyploid hepatocytes, but also ensure that when the flow cytometry is applied, the dye that has not entered the cells will not continue to enter the cells, resulting in excessive fluorescence intensity and affecting the experimental results.

[0069] 9、Adjust the appropriate liver cell sorting sample loading mode to ensure the activity, accuracy and efficiency of the liver cells. The liver cells are large in size and settle quickly, etc. (3-8 min after resuspension, the liver cells settle to the bottom of the tube, while other cells such as lymphocytes settle to the bottom of the tube in 1-2 h), which leads to the flow cytometer being easily blocked during sorting, making it extremely difficult to sort the live liver cells. Meanwhile, the liver cells are very fragile and prone to death. The method of resuspending the liver cells by using the sample injection needle oscillation function of the flow cytometer will cause the liver cells to die due to the excessive force of the sample injection needle. Therefore, in order to ensure the cell activity of the liver cell sorting, the nozzle of 100 um is used for the flow cytometric sorting of the liver cells in the present application. At the same time, the sample loading concentration of the liver cells is adjusted to 1 million / mL. A too high concentration will block the flow cytometer, and a too low concentration will prolong the sorting time and cause the liver cells to die. Finally, the total volume of the cells in the sample injection tube does not exceed 1 mL during each sorting sample loading, to avoid excessive liver cell settlement. At the same time, instead of using the sample injection needle oscillation function of the instrument, the method of manually and gently resuspending the liver cells every 10 min by using a dropper is used for the flow cytometric sorting of the live liver cells. After many experiments, such a sample loading mode can ensure the activity, accuracy and efficiency of the liver cell sorting.

[0070] 10、In the process of extracting liver cell nuclei, the present application adopts a two-stage mild mechanical homogenization combined with a specific low-osmotic buffer treatment of the cell nuclei grinding technology. According to the different sizes of the different ploidy liver cells, different specifications of tissue homogenizers are selected to effectively damage the cell membrane without damaging the nuclear membrane, ensuring the integrity of the cell nuclear membrane. The liver cell membrane is ruptured without damaging the nuclear membrane of the liver cell, so that the liver cell nuclei with complete structure can be obtained for further flow sorting and functional research.

[0071] 11、When sorting and recovering the liver cell nuclei, the flow collection tube is pre-coated with serum, and the volume of the collection buffer and the centrifugation conditions are controlled, which effectively improves the recovery rate and sorting stability of the cell nuclei. At the same time, the flow sorting technology is combined to realize the high-purity separation of the polyploid liver cell nuclei, which is suitable for subsequent functional experiments such as protein expression detection and chromatin state analysis. BRIEF DESCRIPTION OF DRAWINGS

[0072] Figure 1 The schematic diagram of the cell morphology of the primary liver cells obtained by perfusion before sorting (after 24 h of culture in the maintenance medium, various liver cells with different nuclear types can be seen under a 20x microscope, such as mononuclear, binuclear and trinuclear cells; two different fields of view are selected, and the blue circles represent mononuclear cells, the yellow circles represent binuclear cells, and the red circles represent trinuclear cells);

[0073] Figure 2Flow cytometry histogram of 2-ploid, 4-ploid and 8-ploid hepatocytes (P7 green represents 2-ploid hepatocytes, P8 yellow represents 4-ploid hepatocytes, P9 blue represents 8-ploid hepatocytes);

[0074] Figure 3 Flow cytometry histogram and scatter plot of 2-ploid, 4-ploid and 8-ploid hepatocytes (A: flow cytometry scatter plot of 2-ploid, 4-ploid and 8-ploid hepatocytes; B: flow cytometry histogram of 2-ploid, 4-ploid and 8-ploid hepatocytes; C: flow cytometry scatter plot of 2-ploid, 4-ploid and 8-ploid hepatocytes, red represents live hepatocytes, green represents dead hepatocytes. D: flow cytometry histogram of 2-ploid, 4-ploid and 8-ploid hepatocytes, red represents live hepatocytes, green represents dead hepatocytes. P3, P7 represent 2-ploid hepatocytes, P4, P8 represent 4-ploid hepatocytes, P5, P9 represent 8-ploid hepatocytes);

[0075] Figure 4 Flow cytometry scheme for sorting 2-ploid, 4-ploid and 8-ploid hepatocytes (A: flow cytometry scatter plot of FSC and SSC, R1 gate is for cells; B: flow cytometry scatter plot of FSC(A) and FSC(H), R2 is for single cells; C: flow cytometry scatter plot of PI channel and SSC, R5 is for live hepatocytes, D: flow cytometry scatter plot of 2-ploid, 4-ploid and 8-ploid hepatocytes, abscissa is fluorescence channel of Hoechst33342, 2n represents 2-ploid hepatocytes, 4n represents 4-ploid hepatocytes, 8n represents 8-ploid hepatocytes);

[0076] Figure 5 Schematic diagram of the morphology of live hepatocytes after 24h culture after obtaining 2-ploid hepatocytes, 4-ploid hepatocytes and 8-ploid hepatocytes by first flow sorting (A: 2-ploid hepatocytes obtained by first sorting, B: 4-ploid hepatocytes obtained by first sorting, C: 8-ploid hepatocytes obtained by first sorting. Two different fields are selected respectively, and the blue circle is a single nucleus, the yellow circle is a double nucleus, and the red circle is a triple nucleus);

[0077] Figure 6 Flow cytometry scheme for sorting nuclei of 2-ploid hepatocytes (A: flow cytometry scatter plot of FSC and SSC, R1 gate is for nuclei of 2-ploid hepatocytes; B: flow cytometry scatter plot of FSC(A) and FSC(H), R2 is for single nuclei; C: flow cytometry scatter plot of nuclei of 2-ploid hepatocytes, abscissa is fluorescence channel of Hoechst33342, Nu-2n represents 2-ploid nuclei of 2-ploid hepatocytes);

[0078] Figure 7Figure 1 shows a schematic diagram of a flow cytometry scheme for sorting nuclei of 4-ploid hepatocytes (A: flow cytometry scatter plot composed of FSC and SSC, R1 gate for nuclei contained in 4-ploid hepatocytes; B: flow cytometry scatter plot composed of FSC(A) and FSC(H), R2 for single nuclei; C: flow cytometry scatter plot of nuclei of 4-ploid hepatocytes, abscissa for fluorescence channel of Hoechst 33342, Nu-2n for 2-ploid nuclei of 4-ploid hepatocytes, Nu-4n for 4-ploid nuclei of 4-ploid hepatocytes);

[0079] Figure 8 Figure 2 shows a schematic diagram of a flow cytometry scheme for sorting nuclei of 8-ploid hepatocytes (A: flow cytometry scatter plot composed of FSC and SSC, R1 gate for nuclei contained in 8-ploid hepatocytes; B: flow cytometry scatter plot composed of FSC(A) and FSC(H), R2 for single nuclei; C: flow cytometry scatter plot of nuclei of 8-ploid hepatocytes, abscissa for fluorescence channel of Hoechst 33342, Nu-2n for 2-ploid nuclei of 8-ploid hepatocytes, Nu-4n for 4-ploid nuclei of 8-ploid hepatocytes, Nu-8n for 8-ploid nuclei of 8-ploid hepatocytes);

[0080] Figure 9 Figure 3 shows a diagram showing the proportion of nuclei of polyploid hepatocytes (A: pie chart showing the proportion of nuclei of 2-ploid hepatocytes, Nu-2n for 2-ploid nuclei of 2-ploid hepatocytes; B: pie chart showing the proportion of nuclei of 4-ploid hepatocytes, Nu-2n for 2-ploid nuclei of 4-ploid hepatocytes, Nu-4n for 4-ploid nuclei of 4-ploid hepatocytes; C: pie chart showing the proportion of nuclei of 8-ploid hepatocytes, Nu-2n for 2-ploid nuclei contained in 8-ploid hepatocytes, Nu-4n for 4-ploid nuclei of 8-ploid hepatocytes, Nu-8n for 8-ploid nuclei of 8-ploid hepatocytes; D: statistics of the proportion of nuclei of 2-ploid hepatocytes, Nu-2n for 2-ploid nuclei of 2-ploid hepatocytes; E: statistics of the proportion of nuclei of 4-ploid hepatocytes, Nu-2n for 2-ploid nuclei of 4-ploid hepatocytes, Nu-4n for 4-ploid nuclei of 4-ploid hepatocytes; F: statistics of the proportion of nuclei of 8-ploid hepatocytes, Nu-2n for 2-ploid nuclei contained in 8-ploid hepatocytes, Nu-4n for 4-ploid nuclei of 8-ploid hepatocytes, Nu-8n for 8-ploid nuclei of 8-ploid hepatocytes);

[0081] Figure 10 Figure 4 shows a flow chart of a method for separating and purifying haploid nuclei and polyploid nuclei of polyploid hepatocytes. DETAILED DESCRIPTION

[0082] The present application will be further described below in conjunction with the accompanying drawings and embodiments:

[0083] As Figure 1As shown, a method for separating and purifying haploid nuclei and diploid nuclei of primary hepatocytes, the steps include:

[0084] S1, using a modified two-step collagenase digestion method to perfuse to obtain primary hepatocytes;

[0085] S2, staining the primary hepatocytes and performing the first flow sorting to obtain diploid hepatocytes, tetraploid hepatocytes and octoploid hepatocytes;

[0086] S3, grinding the diploid hepatocytes, tetraploid hepatocytes and octoploid hepatocytes after the first flow sorting to obtain hepatocyte nuclei;

[0087] S4, performing the second flow sorting on the hepatocyte nuclei obtained after grinding to obtain diploid nuclei in diploid hepatocytes, diploid nuclei and tetraploid nuclei in tetraploid hepatocytes, and diploid nuclei, tetraploid nuclei and octoploid nuclei in octoploid hepatocytes.

[0088] The specific implementation process is as follows:

[0089] Step one, using a modified two-step collagenase digestion method to perfuse to obtain primary hepatocytes

[0090] (1) Using a modified two-step collagenase digestion method to perfuse to separate the liver; taking 6-8 weeks of C57BL / 6J mice, first injecting the mice with three bromine ethanol anesthesia (according to the weight of the mouse, generally 0.4-0.5mL per mouse), opening the abdominal cavity, connecting the indwelling needle and the infusion tube of the perfusion pump in advance, turning on the perfusion pump, adjusting the speed to 5mL / min, filling the PBS pipeline, then inserting the indwelling needle into the superior vena cava (pay attention to not to pierce the superior vena cava), then withdrawing the needle into the hose, and then pushing forward steadily and fixing it, to ensure that the needle does not slip out of the superior vena cava during the whole perfusion process, resulting in perfusion failure.

[0091] (2) Turn on the perfusion pump, and PBS will peristalsis in the liver with the perfusion pump, and when the liver starts to swell and become larger and the color becomes lighter, it means that the superior vena cava has been successfully entered, then cut the hepatic portal vein on the other side of the liver, so that the PBS can pass through the liver completely. Three kinds of perfusion fluid are preheated in a 37℃ water bath, about 30mL of liver perfusion buffer I is used, until the liver blood is washed clean, and the liver turns from red to light pink, then replace about 50mL of liver perfusion buffer II (containing 0.5M EGTA without Ca 2+ / Mg 2+ 1% HEPES), then replace the liver perfusion buffer III about 60 mL (reserve 10 mL on ice for subsequent digestion), namely add 0.5-0.6 mg / mL of collagenase type IV (purchased from Sigma, item number V900893) in EBSS + 1% HEPES. The whole operation process does not exceed 30 min, and it is necessary to pay close attention to the infusion tube at all times and replace the buffer I, II, III in time. If it is not replaced in time, a large amount of air will enter the infusion tube, which will cause the liver capsule to rupture prematurely and the perfusion to fail.

[0092] (3) After all the liver perfusion fluid is finished, the indwelling needle is pulled out in advance to prevent air from entering. The liver is observed, and when the liver tissue under the liver capsule becomes loose, it means that the liver capsule and liver parenchyma have separated. Then the liver is cut off, the gallbladder is removed, and placed in a 10 cm culture dish, 10 mL of pre-cooled liver perfusion buffer III (which can continue to digest) is added, and the liver capsule is slowly punctured at multiple points with two small forceps, and then the liver is slowly and gently shaken repeatedly to allow most of the liver parenchymal cells to be washed out. The liquid becomes turbid, and finally the liver is only left with a fibrous bundle of basic structure. The above process is operated on ice to reduce the number of dead liver cells.

[0093] (4) Place a 70-um cell filter on a 50-mL centrifuge tube, and use a Pasteur pipette to suck the cell suspension for filtration, and then further filter using a 40-um cell filter to ensure that large impurities are removed. The obtained liver cell suspension is centrifuged at low speed (4°, 50g, 5min), and the supernatant is discarded. The lower layer of the precipitate is the liver parenchymal cells. The cell precipitate is resuspended in low-sugar DMEM and then centrifuged at low speed (4°, 50g, 5min). Repeat this process several times.

[0094] (5) Perform cell counting. Mix 4% trypan blue with the cell suspension 1:1 and place it on a hemocytometer plate under a microscope. Manually count the number of liver cells and calculate the total number of cells in 4 fields under the microscope / 4 x 10 4 / 2, and then calculate the survival rate of the cells = number of living cells / number of living cells + number of dead cells. Measure three times and take the average value.

[0095] (6) If the cell activity is less than 70%, use Percoll purification solution (Percoll / 10 x EBSS = 9:1) to remove dead cells and separate the liver cells. After a series of centrifugation steps (2 x 50g x 4min), collect. When the cell activity reaches more than 90% after purification and separation, it can be used for the next step of sorting. Dilute the purified liver cells with PBS to contain 10 6The cells were stored in ice for up to 1 hour until flow cytometry sorting. During sorting, the hepatocyte sample was filtered again using a flow cytometry loading tube with a filter cap. The loading concentration of the hepatocyte sample was adjusted to 1 million / mL to prevent clogging of the flow cytometry tube.

[0096] (7) The primary hepatocytes obtained by perfusion were seeded at appropriate concentrations in a 6-well plate coated with Matrigel (Corning, Cat. No. 356234). After 24 h of culture, the living hepatocytes were expanded and various karyotypes of hepatocytes were observed under a 20x microscope, such as mononuclear, binuclear, and trinuclear (e.g. Figure 1 shown).

[0097] Step 2: Primary hepatocytes were stained and sorted by flow cytometry to obtain diploid hepatocytes, quadruploid hepatocytes, and octoploid hepatocytes.

[0098] (1) Add 10 μL of Hoechst33342 dye (purchased from Shanghai Biyuntian Biotechnology Co., Ltd., with a final concentration of 5 μg / mL) to the primary hepatocytes obtained in step 1, and stain in the dark at 37°C for 30 minutes. Hoechst33342 is a membrane-permeable dye that can enter living cells and bind to double-stranded DNA. After binding, it emits blue fluorescence, and its fluorescence intensity is proportional to the DNA content. Diploid, quadruple, and octaploid hepatocytes show increasing fluorescence intensity in turn, which is suitable for ploidy differentiation. Figure 2 shown.

[0099] (2) Wash the cells twice with PBS.

[0100] (3) The stained primary hepatocytes were diluted to a concentration of 1 million / mL, and then filtered with a 40um filter. 5uLPI (propidium iodide) was added to each mL of the filtered hepatocytes before flow cytometry. PI was used to distinguish between live and dead hepatocytes. The primary hepatocytes obtained by perfusion are very fragile and easily die due to damage. When primary hepatocytes were obtained by perfusion using a two-step collagenase digestion method, the activity of the hepatocytes was generally 70-90%, of which about 20% were dead hepatocytes. Due to changes in the permeability of the cell membrane, the fluorescent dye can more easily penetrate into the cell nucleus, resulting in a difference in blue fluorescence intensity between dead cells and live cells after Hoechst33342 staining. The distribution of 2-ploid, 4-ploid, and 8-ploid hepatocytes after Hoechst33342 staining of live hepatocytes is shown in the figure. Figure 3 As shown in A and 3B, the fluorescence intensity of dead hepatocytes is stronger than that of living cells. Figure 3 C, 3D (red indicates live cells, green indicates dead cells).

[0101] (4) The fluorescence intensity of dead and live liver cells is different, which can cause experimental error. To avoid the experimental error caused by dead liver cells, we use PI to distinguish between live and dead liver cells when flow sorting liver cells. PI is a nucleic acid dye that can embed double-stranded DNA and emit red fluorescence, but PI cannot penetrate the intact cell membrane. The cell membrane of live cells is intact, so PI cannot pass through; the integrity of the cell membrane of dead cells is damaged, and PI embeds double-stranded DNA to emit red fluorescence. Therefore, to avoid the influence of dead liver cells on the sorting of polyploid liver cells, PI staining is added at the same time as Hoechst 33342 staining. Liver cells that do not emit red fluorescence are live cells, and further according to the intensity of blue fluorescence, different liver cell ploidy is distinguished under the conditions of live cell circle gate.

[0102] (5) Set the forward scatter light (FSC) voltage value of the flow cytometer to 245, and the side scatter light (SSC) voltage value to 210. Take the area value (A) of FSC as the horizontal coordinate, and the area value (A) of SSC as the vertical coordinate, and circle the cell gate (such as Figure 4 A). Figure 4 B) as the vertical coordinate, and remove the adherent cells.

[0103] (6) Set the voltage value of the 488-576 channel (PI channel) of the flow cytometer to 350. Take the 488-576 fluorescence as the horizontal coordinate and FSC-A as the vertical coordinate to plot, PI positive is dead cells, and PI negative is live cells. Draw the gate of live cells (such as Figure 4 C).

[0104] (7) Set the voltage value of the 405-448 channel (PB450 channel) of the flow cytometer to 450. Take the 405-448 fluorescence as the horizontal coordinate and SSC-A as the vertical coordinate to plot, and circle the 2-ploid liver cells, 4-ploid liver cells and 8-ploid liver cells as shown in Figure 4 D.

[0105] (8) Set the sorting logic, and the sorting logic is shown by the arrow as follows: Figure 4

[0106] ​(9) When sorting, the sample loading amount is 1 mL, and the cells are manually resuspended every 10 min with a Pasteur pipette to prevent sedimentation. The nozzle of the sorter is 100 um, and the sample concentration for sorting is preferably 1 million / mL. The loading speed is preferably 2000 events per second. If the speed is too fast, it will be easy to block. Due to the characteristics of large volume (20-30 um) and fast sedimentation (3-8 min after resuspension, the bottom of the tube, while other cells such as lymphocytes will settle to the bottom of the tube in 1-2 h), it is very difficult to sort live liver cells, which are prone to block the flow cytometer. In order to ensure the cell viability of liver cells during sorting, we use a 100 um nozzle to sort liver cells. At the same time, the sample loading concentration of liver cells is adjusted to 1 million / mL. If the concentration is too high, it will block the sorter. If the concentration is too low, it will prolong the sorting time and cause liver cell death. Finally, the total volume of cells in the sample tube during each sorting loading is not more than 1 mL to avoid excessive liver cell sedimentation. Instead of using the instrument's built-in sample needle shaking function to resuspend the cells, we use a dropper to manually resuspend the liver cells every 10 min. After many experiments, this loading method not only ensures the activity, accuracy and efficiency of liver cell sorting, but also ensures the activity, accuracy and efficiency of liver cell sorting.

[0107] (10) After the first flow cytometry sorting, 2-ploid liver cells (only single nucleus), 4-ploid liver cells (single nucleus and double nucleus, no triple nucleus) and 8-ploid liver cells (single nucleus, double nucleus and triple nucleus) are obtained. The cells are inoculated in Matrigel-coated 24-well plates at an appropriate concentration, and the morphology of the cells after 24 h of culture is shown in Figure 5 .

[0108] Step three, grinding the 2-ploid liver cells, 4-ploid liver cells and 8-ploid liver cells obtained after the first flow cytometry sorting to obtain liver cell nuclei

[0109] (1) Collect the 2-ploid, 4-ploid and 8-ploid liver cells obtained after the first flow cytometry sorting, centrifuge at 50g at 4°C for 2 min, discard the supernatant, resuspend the cells with 300 μL HB buffer (formula as follows), and the composition of the HB buffer is shown in Table 1:

[0110] Table 1: HB buffer information

[0111]

[0112]

[0113] (2) 1 mL and 2 mL Dounce Homogenizer (Wheaton) were used for grinding, 1 mL Dounce Homogenizer was used for grinding for diploid and tetraploid hepatocytes, and the loose pestle (hammer gap 63.5-139.7 um) was used for slow grinding for 5 times, and then the tight pestle (hammer gap 12.7-63.5 um) was used for grinding for 10 times, which gently ruptured the hepatocyte membrane without damaging the nuclear membrane; for octaploid hepatocytes, 2 mL Dounce Homogenizer was used for grinding, and the loose pestle (hammer gap 76.2-127 um) was used for slow grinding for 5 times, and then the tight pestle (hammer gap 12.7-63.5 um) was used for grinding for 10 times, which gently ruptured the hepatocyte membrane without damaging the nuclear membrane;

[0114] (3) The homogenate was filtered through a 40 μm filter screen, and the homogenizer was rinsed with 700 μL of HB buffer and continued to be filtered, and the nuclei were recovered.

[0115] Step four, the hepatocyte nuclei obtained after grinding were subjected to a second flow sorting

[0116] (1) The nuclear liquid obtained in step three was re-stained with Hoechst 33342.

[0117] (2) The hepatocyte nuclei were stained with Hoechst 33342 for 10 min at room temperature in the dark.

[0118] (3) The stained hepatocyte nuclei were subjected to a second flow sorting, and the nuclei of 2-fold hepatocytes, 4-fold hepatocytes and 8-fold hepatocytes after grinding were subjected to a second flow sorting.

[0119] (4) The nuclei of 2-fold hepatocytes after grinding were subjected to flow sorting, and the steps were as follows:

[0120] ① The forward scatter light (FSC) voltage value of the flow sorter was set to 275, the side scatter light (SSC) voltage value was set to 270, the area value (A) of FSC was taken as the abscissa, and the area value (A) of SSC was taken as the ordinate, and the cell nucleus gate (such as Figure 6 A) was circled;

[0121] ② The area value (A) of FSC was taken as the abscissa, and the height value (H) of FSC was taken as the ordinate, and the single cell nucleus gate (such as Figure 6 B) was circled, and the adherent cell nuclei were removed;

[0122] ③ The voltage value of the 405-448 channel (PB450 channel) of the flow sorter was set to 450, and the 405-448 fluorescence was taken as the abscissa, and FSC-A was taken as the ordinate, and the graph was plotted, as shown in Figure 6Circle the gate of diploid hepatocyte nuclei as shown in C, and Figure 4 The sorting logic is set to sort out the diploid nuclei contained in the diploid hepatocytes.

[0123] (5) The nuclei of the ground 4-ploid hepatocytes are then flow-sorted as follows:

[0124] ① Set the forward scattered light (FSC) voltage value of the flow sorter to 275, the side scattered light (SSC) voltage value to 270, use the FSC surface value (A) as the horizontal axis and the SSC area value (A) as the vertical axis, and circle the gate of the cell nucleus (such as Figure 7 A);

[0125] ② Then use the FSC value (A) as the horizontal axis and the FSC height value (H) as the vertical axis to circle the gate of single cell nuclei (such as Figure 7 B), removal of adherent nuclei;

[0126] ③ Set the voltage value of the 405-448 channel (PB450 channel) of the flow cytometry instrument to 450, and plot the fluorescence of 405-448 as the horizontal axis and FSC-A as the vertical axis, as shown in the figure below: Figure 7 Circle the gate of the tetraploid hepatocyte nucleus as shown in C, and Figure 5 The sorting logic is set to sort out the diploid nuclei and tetraploid nuclei of the tetraploid hepatocytes respectively.

[0127] (6) Finally, the nuclei of the ground octaploid hepatocytes were flow cytometry sorted as follows:

[0128] ① Set the forward scattered light (FSC) voltage value of the flow sorter to 275, the side scattered light (SSC) voltage value to 270, use the FSC surface value (A) as the horizontal axis and the SSC area value (A) as the vertical axis, and circle the gate of the cell nucleus (such as Figure 8 A);

[0129] ② Then use the FSC value (A) as the horizontal axis and the FSC height value (H) as the vertical axis to circle the gate of single cell nuclei (such as Figure 8 B), removal of adherent nuclei;

[0130] ③ Set the voltage value of the 405-448 channel (PB450 channel) of the flow cytometry instrument to 450, and plot the fluorescence of 405-448 as the horizontal axis and FSC-A as the vertical axis, as shown in the figure below: Figure 8 Circle the gate of the octaploid hepatocyte nucleus as shown in C, and Figure 6 The sorting logic was set to sort out the diploid nuclei, quadruploid nuclei and octoploid nuclei of the octoploid hepatocytes respectively.

[0131] (7) The second sorting was performed on Beckman moflo astrisos flow cytometer by the above gating strategy and steps, and the 2n nuclei of 2n hepatocytes, 2n x 2 nuclei of 4n hepatocytes, 4n nuclei of 4n hepatocytes, 2n x 4 nuclei of 8n hepatocytes, 4n x 2 nuclei of 8n hepatocytes and 8n nuclei of 8n hepatocytes were obtained.

[0132] (8) The flow collection tube was coated with 10% FBS in advance and placed at 4°C overnight to prevent the nuclei from adsorbing to the wall. The collection volume was set to 300 μL, and finally transferred to a 1.5 mL centrifuge tube, centrifuged at 1000 g at 4°C for 8 min, the supernatant was discarded, and the intact nuclei pellet was obtained.

[0133] (9) The composition distribution of nuclei in 2n hepatocytes, 4n hepatocytes and 8n hepatocytes was counted, and the results were as follows Figure 9 A, 9D, showed that the proportion of 2n nuclei in 2n hepatocytes was about 87.08%, and the remaining about 12%. About 92% were other nuclei (aneuploid nuclei and other conditions). As Figure 9 B, 9E, showed that the proportion of 2n nuclei in 4n hepatocytes was about 57.28%, the proportion of 4n nuclei in 4n hepatocytes was about 32.12%, and the remaining about 10.85% were other nuclei (aneuploid nuclei and other conditions). As Figure 9 C, 9F, showed that the proportion of 2n nuclei in 8n hepatocytes was about 57.81%, the proportion of 4n nuclei in 8n hepatocytes was about 27.03%, the proportion of 8n nuclei in 8n hepatocytes was about 2.58%, and the remaining about 12.58% were other nuclei (aneuploid nuclei and other conditions).

[0134] The above is only an embodiment of the present application, and the specific technical solutions and / or common knowledge of the scheme are not described in detail. It should be noted that for those skilled in the art, without departing from the technical solutions of the present application, a number of modifications and improvements can be made, which should also be considered as the protection scope of the present application, which will not affect the effect and practicality of the patent. The protection scope of the present application should be subject to the content of its claims, and the specific embodiments in the specification can be used to explain the content of the claims.

Claims

1. A method for separating and purifying haploid nuclei and polyploid nuclei of polyploid hepatocytes, characterized by: The steps of the method include: S1. Primary hepatocytes were obtained by perfusion using a modified two-step collagenase digestion method. S2. Primary hepatocytes were stained and subjected to the first flow cytometry sorting to obtain diploid hepatocytes, quadruploid hepatocytes, and octoploid hepatocytes; S3, after the first flow cytometry sorting, the diploid hepatocytes, quadruploid hepatocytes, and octoploid hepatocytes were ground to obtain hepatocyte nuclei; S4. The hepatocyte nuclei obtained after grinding were subjected to a second flow cytometry sorting to obtain diploid nuclei in diploid hepatocytes, diploid nuclei and quadruple nuclei in quadruple hepatocytes, and diploid nuclei, quadruple nuclei and octaploid nuclei in octaploid hepatocytes.

2. The method for separating and purifying haploid nuclei and polyploid nuclei of polyploid hepatocytes according to claim 1, wherein: The specific implementation steps of step S1 include: S11, perfusion isolation of the liver using a modified two-step collagenase digestion method; S12. Use the perfusion pump and adjust the speed to 5 mL / min to fill the tube with PBS. Then insert the indwelling needle into the superior vena cava, making sure not to puncture the superior vena cava. Then, retract the needle into the hose and steadily advance it forward and secure it to ensure that the needle does not slip out of the superior vena cava and cause perfusion failure during the entire perfusion process. The PBS is pumped into the liver with the perfusion pump. When the liver begins to swell and become lighter in color, it means that it has successfully entered the superior vena cava. Then, cut the portal vein on the other side of the liver to allow the PBS to pass through the liver intact. S13. Preheat the three perfusion solutions in a 37°C water bath and replace buffers I, II, and III in sequence. The entire operation should take no more than 30 minutes. Always pay close attention to the infusion tubing. If not replaced promptly, a large amount of air will enter the tubing, which can cause premature rupture of the liver capsule and perfusion failure. After all liver perfusion solutions have been removed, remove the indwelling needle in advance to prevent air from entering. Observe the liver and observe if the liver tissue under the liver capsule becomes loose, indicating that the liver capsule and liver parenchyma have separated. S14. Excise the liver, remove the gallbladder, and place it in a 10 cm culture dish. Add 10 mL of pre-chilled liver perfusion buffer III. Continue digestion. Use two small forceps to slowly puncture the liver capsule at multiple points. Grasp the liver and gently shake it repeatedly to flush out most of the hepatic parenchymal cells. The fluid will become turbid, until only the basic structure of the liver, consisting of fibrous bundles, remains. Perform the above steps on ice to reduce the number of dead hepatocytes. S15. Place a 70um cell strainer on a 50mL centrifuge tube, use a Pasteur pipette to draw up the cell suspension for filtration, and then further filter it with a 40um cell strainer to ensure that any large impurities are removed. The obtained hepatocyte suspension is centrifuged at low speed (4°, 50g, 5min), the supernatant is discarded, and the lower sediment is the hepatocytes. Resuspend the cell pellet in low-glucose DMEM and centrifuge it at low speed (4°, 50g, 5min), and repeat this process several times. S16. Perform cell counting. Mix the cell suspension with 4% trypan blue in a 1:1 ratio and place the mixture on a hemocytometer. Observe the mixture under a microscope and manually count the number of hepatocytes. The calculation method is the total number of cells in 4 fields of view under the microscope / 4×10 4 / 2, count the number of live cells and dead cells in turn, and then calculate the cell survival rate = number of live cells / number of live cells + number of dead cells, measure three times before and after and take the average value; S17. If the cell viability is lower than 70%, the hepatocytes are separated and decapped using Percoll purification buffer (Percoll / 10×EBSS=9:1) and collected by a series of centrifugation steps (2×50g×4min). When the cell activity reaches more than 90% after separation and purification, it can be used for the next step of sorting; The purified hepatocytes were diluted with PBS to a concentration of 10 6 The cells were stored in ice for a maximum of 1 hour until flow cytometry sorting. During sorting, filter the sample once more using a flow cytometry loading tube with a filter cap and adjust the loading concentration of the hepatocyte sample to 1 million / mL to prevent clogging of the flow cytometry tube. S18. The primary hepatocytes obtained by perfusion were seeded at an appropriate concentration in a Matrigel-coated 6-well plate. After culturing with the culture medium for 24 hours, the living hepatocytes were expanded. Hepatocytes of various karyotypes were observed under a 20x microscope.

3. The method for separating and purifying haploid nuclei and polyploid nuclei of polyploid hepatocytes according to claim 1, wherein: The specific implementation steps of step S2 include: S21. Add 10 μL of Hoechst 33342 dye (5 μg / mL) to 1 mL of the primary hepatocyte resuspension obtained in step 1, and stain in the dark at 37°C for 30 minutes. Diploid, quadruple, and octaploid hepatocytes will show increasing fluorescence intensity, which is suitable for ploidy differentiation. Wash cells twice with S22 and PBS; S23. Dilute the stained primary hepatocytes to a concentration of 1 million / mL, then filter with a 40 μm filter. Add 5 μL of LPI per mL of filtered hepatocytes before loading on flow cytometry. S24. Different fluorescence intensities of dead and living hepatocytes can cause experimental errors. To avoid experimental errors caused by dead hepatocytes, PI was used to distinguish between dead and living hepatocytes during flow cytometry sorting. S25. Set the forward scattering light voltage value of the flow sorter to 245 and the side scattering light voltage value to 210. Use the FSC value (A) as the horizontal axis and the SSC area value (A) as the vertical axis to circle the cell gate. Then, use the FSC value (A) as the horizontal axis and the FSC height value (H) as the vertical axis to circle the single cell gate and remove adherent cells. S26. Set the voltage value of the 488-576 channel of the flow cytometry instrument to 350, plot the fluorescence of 488-576 as the horizontal axis and FSC-A as the vertical axis, define PI-positive cells as dead cells and PI-negative cells as live cells, and draw a gate for live cells; S27. Set the voltage of the 405-448 channel of the flow cytometry instrument to 450, plot the fluorescence intensity at 405-448 as the horizontal axis and SSC-A as the vertical axis, and circle the diploid hepatocytes, quadruploid hepatocytes, and octoploid hepatocytes. S28. Set the sorting logic. The sorting logic is to circle out hepatocytes through FSC and SSC gates to remove interference from impurity signals; then, based on the fluorescence intensity of the PI channel, circle out PI-negative cells as live cells; finally, based on the fluorescence intensity of the PB450 channel, set gates for diploid hepatocytes, quadruple hepatocytes, and octaploid hepatocytes, and sort out diploid hepatocytes, quadruple hepatocytes, and octaploid hepatocytes, respectively, according to the sorting logic; S29. During sorting, the sample loading volume is 1 mL, and cells are manually resuspended with a Pasteur pipette every 10 minutes to prevent sedimentation. The nozzle of the sorter is 100 μm, the sample concentration is preferably 1 million / mL, and the loading speed is preferably 2000 events per second. S210. After the first flow cytometry sorting, diploid hepatocytes with only mononuclei, quadruple hepatocytes with mononuclei and binuclei, and octaploid hepatocytes with mononuclei, binuclei, and triploids were obtained, which were inoculated into Matrigel-coated 24-well plates at appropriate concentrations.

4. The method for separating and purifying haploid nuclei and polyploid nuclei of polyploid hepatocytes according to claim 1, wherein: The specific implementation steps of step S3 include: S31. Collect diploid, quadruploid, and octoploid hepatocytes after the first flow cytometry sorting and centrifuge at 50 g for 5 min at 4°C. Discard the supernatant and add 300 μL HB buffer to resuspend the cells. S32. For diploid and tetraploid hepatocytes, use a 1 mL tissue homogenizer for grinding. First, use a loose pestle with a pestle gap of 63.5-139.7 μm and grind slowly for 5 times. Then, change to a tight pestle with a pestle gap of 12.7-63.5 μm and grind for 10 times. Gently rupture the hepatocyte membrane without damaging the nuclear membrane. For octoploid hepatocytes, use a 2 mL tissue homogenizer for grinding. First, use a loose pestle with a pestle gap of 76.2-127 μm and grind slowly for 5 times. Then, change to a tight pestle with a pestle gap of 12.7-63.5 μm and grind for 10 times. Gently rupture the hepatocyte membrane without damaging the nuclear membrane. S33. The homogenate was filtered through a 40 μm filter, and the homogenizer was rinsed with 700 μL HB buffer and filtered again to recover the cell nuclei.

5. The method for separating and purifying haploid nuclei and polyploid nuclei of polyploid hepatocytes according to claim 1, characterized in that: The specific implementation steps of step S4 include: S41, the nuclear fluid obtained in step S3 was stained again with Hoechst33342 at room temperature in the dark for 10 min; S42, after staining, the nuclei of diploid hepatocytes, the nuclei of quadruple hepatocytes, and the nuclei of octaploid hepatocytes were subjected to a second flow cytometry sorting; S43, firstly, the nuclei of the ground diploid hepatocytes were flow sorted; S44, then the nuclei of the ground 4-ploid hepatocytes were flow sorted; S45. Finally, the nuclei of the ground octaploid hepatocytes were flow sorted; S46. Perform a second sorting on a flow cytometer using the above gating scheme and steps to obtain diploid nuclei of diploid hepatocytes of clear origin, diploid nuclei and quadruple nuclei of quadruple hepatocytes, and diploid nuclei, quadruple nuclei, and octaploid nuclei of octaploid hepatocytes; S47. Flow cytometry collection tubes were pre-coated with 10% FBS and incubated at 4°C overnight to prevent nuclei from adhering to the tube walls. The collection volume was set to 300 μL, and the cells were finally transferred to a 1.5 mL centrifuge tube. Centrifuged at 1000 g for 8 minutes at 4°C, the supernatant discarded, and the intact nuclei pellet obtained. S48. Statistically analyze the distribution of nuclear composition in diploid, quadruple, and octaploid hepatocytes.

6. The method for separating and purifying haploid nuclei and polyploid nuclei of polyploid hepatocytes according to claim 5, characterized in that: The specific implementation steps of step S44 include: S441. Set the forward scattered light voltage value of the flow sorter to 275 and the side scattered light voltage value to 270, use the FSC area value A as the horizontal axis and the SSC area value A as the vertical axis, and circle the gate of the cell nucleus; S442, use the FSC face value A as the horizontal axis and the FSC height value H as the vertical axis to circle the gate of single cell nuclei and remove the adherent cell nuclei; S443. Set the voltage value of the 405-448 channel of the flow sorter to 450, plot with 405-448 fluorescence as the horizontal axis and FSC-A as the vertical axis, circle the diploid nuclei of the diploid hepatocytes, and set the sorting logic to sort out the diploid nuclei of the diploid hepatocytes.

7. The method for separating and purifying haploid nuclei and polyploid nuclei of polyploid hepatocytes according to claim 5, characterized in that: The specific implementation steps of step S45 include: S451. Set the forward scattered light voltage value of the flow sorter to 275 and the side scattered light voltage value to 270, use the FSC area value A as the horizontal axis and the SSC area value A as the vertical axis, and circle the cell nucleus gate; S452, use the FSC face value A as the horizontal axis and the FSC height value H as the vertical axis to circle the gate of single cell nuclei and remove the adherent cell nuclei; S453. Set the voltage value of the 405-448 channel of the flow sorter to 450, plot with 405-448 fluorescence as the horizontal axis and FSC-A as the vertical axis, circle the diploid nuclei and quadruple nuclei of the quadruple hepatocytes respectively, and set the sorting logic to sort out the diploid nuclei and quadruple nuclei of the quadruple hepatocytes respectively.

8. The method for separating and purifying haploid nuclei and polyploid nuclei of polyploid hepatocytes according to claim 5, characterized in that: The specific implementation steps of step S46 include: S461. Set the forward scattered light voltage value of the flow sorter to 275 and the side scattered light voltage value to 270, use the FSC area value A as the horizontal axis and the SSC area value A as the vertical axis, and circle the gate of the cell nucleus; S462, use the FSC face value A as the horizontal axis and the FSC height value H as the vertical axis to circle the gate of single cell nuclei and remove the adherent cell nuclei; S463. Set the voltage value of the 405-448 channel of the flow sorter to 450, plot with 405-448 fluorescence as the horizontal axis and FSC-A as the vertical axis, circle the 2-ploid nuclei, 4-ploid nuclei, and 8-ploid nuclei of the 8-ploid hepatocytes, and set the sorting logic to sort out the 2-ploid nuclei, 4-ploid nuclei, and 8-ploid nuclei of the 8-ploid hepatocytes, respectively.

Images