CN111549019A - Method for preparing high-quality single cell suspension by remarkably improving plaque digestion - Google Patents
Method for preparing high-quality single cell suspension by remarkably improving plaque digestion Download PDFInfo
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Abstract
The invention discloses an arterial plaque digestion method for remarkably improving the quality of single cell suspension, which comprises collagen hydrolase I, collagen hydrolase II, collagen hydrolase IV, collagen hydrolase XI, calcium chloride, deoxyribonuclease I, phosphate buffer salt solution, bovine serum albumin solution and RPMI 1640 cell culture solution. The invention also discloses an application prospect of the polypeptide in digesting plaque tissues. The invention is invented mainly aiming at the digestion treatment of plaque tissues, and the plaque tissues can be digested into single cell suspension with higher activity ratio through the combination of several enzymes, and the biological reaction of the plaque tissues is researched on the basis of single cells, thereby avoiding the condition that a plurality of calcified cell fragments, tissue impurities and the like influence a single cell sequencing machine or the subsequent data analysis result.
Description
Technical Field
The invention relates to the field of methods for plaque digestion, in particular to a method for preparing high-quality single cell suspension by using plaque tissue digestion and application thereof.
Background
The Chinese cardiovascular disease report 2018 shows that the morbidity and mortality of Chinese cardiovascular and cerebrovascular diseases are still in a continuously rising stage, and the death of the cardiovascular and cerebrovascular diseases is the first place in the death of resident diseases. At present, cardiovascular and cerebrovascular diseases become a great public health problem, and the control of atherosclerosis is closely related to the prevention and treatment of the cardiovascular and cerebrovascular diseases. Atherosclerosis is the main pathological basis of cardiovascular and cerebrovascular diseases, risk factors such as hypertension, smoking and hyperhomocysteinemia can cause the organism to generate atherosclerosis, even the primary state of the atherosclerosis, namely lipid streaks, can be observed in the fetal aorta at the earliest time due to the hypercholesterolemia of a mother body, and the pathogenesis of the atherosclerosis is multifactorial, so that the realization of plaque reversion and the delay of the pathogenesis are extremely complex. With the continuous update of scientific research technology, particularly in the field of life science, a new research method is developed in an iterative way, and now in the research process, arterial plaque tissues need to be digested into single cell suspensions, so that cell classification, cell grouping, cell subset, cell phenotype conversion, cell migration and the like in the plaque tissues are analyzed through a single cell sequencing method, and temporal-spatial information in the development process of atherosclerosis is revealed. The analysis and research on the level of single cell population can more accurately illustrate the pathogenesis of atherosclerosis.
In the plaque processing process, if mechanical cutting and enzyme digestion are not well processed, a plurality of cell fragments and a large number of dead cells can be generated, so that the operation time is too long when the cell suspension is filtered, and the phenomenon of pipeline blockage can be generated when the single cell transcriptome sequencing machine is subsequently carried out, thereby causing the damage of a precision instrument. Although a plurality of tissue digestion kits exist in the market at present, due to the particularity of plaques, digestion is not thorough, a large amount of cell fragments and dead cells exist, a good digestion effect cannot be achieved, and the quality of the prepared single cell suspension cannot be guaranteed. At present, the problems of thorough digestion of plaque tissues and elimination of calcified fragments and cell fragments are not well solved.
Disclosure of Invention
The invention aims to provide a method for preparing high-quality single cell suspension by remarkably improving plaque digestion so as to overcome the defects of the prior art.
The invention adopts the following technical scheme:
the present invention provides an enzyme combination comprising: collagen hydrolase I, collagen hydrolase II, collagen hydrolase IV and collagen hydrolase XI;
preferably, the concentration ratio of the collagen hydrolase I, the collagen hydrolase II, the collagen hydrolase IV and the collagen hydrolase XI is 1: 1: 1: 1;
more preferably, the enzyme combination comprises dnase i.
The invention provides a digestive fluid comprising the enzyme combination as described above.
Further, the digest comprises RPMI 1640 cell culture fluid.
Furthermore, the concentration of collagenase in the enzyme combination of the digestive juice is 1mg/mL, and the concentration of DNA I enzyme is 200U/mL.
Still further, the digestive juice comprises calcium chloride.
In a particular embodiment of the invention, the final concentration of calcium chloride in the digestive juice is 10 mM.
The present invention provides a method of digesting carotid plaque comprising the use of a combination of enzymes as hereinbefore described or the use of a digestive fluid as hereinbefore described.
Further, the method comprises the steps of:
(1) collecting tissues: selecting a case sample, and placing the case sample in MACS tissue preservation solution at 4 ℃ after the plaque is stripped and separated in vitro in a plaque operation;
(2) separating a sample: placing the plaque sample in PBS to clean blood clots, removing calcified blocks and separating;
(3) tissue digestion: transferring the sample into digestive juice, cutting into pieces, and digesting;
(4) termination of digestion: after digesting for 1h, repeatedly pumping and uniformly mixing the digestive system by using a micro pipette, and standing for 1min at room temperature. Centrifuging at 200g, 4 deg.C for 5 min;
(5) sieving and centrifuging: centrifuging the liquid of the digestion system, removing the supernatant, re-suspending with 15ml of PBS, and sieving with a 40-micron cell sieve;
(6) and (3) optimizing: red blood cell cracking, removing dead cell kit by adopting Meitian and whirlpool, removing debris kit by adopting Meitian and whirlpool and the like, and filtering impurities;
(7) and (3) activity detection: detection was performed using a Countstar Instrument ID, WIN-7RFTLHV6T 5L.
The invention provides the use of a combination of enzymes as hereinbefore described in the preparation of a digestive fluid as hereinbefore described.
The invention provides the use of a combination of enzymes as hereinbefore described or a digestive fluid as hereinbefore described in the preparation of a reagent for the digestion of carotid plaques.
Further, the carotid plaque is human carotid plaque tissue.
The invention has the beneficial effects that:
the method is invented mainly aiming at completely digesting the plaque tissue, the plaque tissue can be completely digested into single cell suspension by combining a plurality of enzymes and a mechanical method, the pathophysiological mechanism of the plaque can be researched at the single cell level, and red blood cells, cell fragments, impurities and the like in the single cell suspension can be removed without influencing the activity and the concentration of the single cell by various optimization schemes, so that the conditions that calcified cell fragments, impurities, cell conglomerations and the like influence the subsequent on-cell sequencing and sequencing database analysis of the single cell and the subsequent flow cytometry and cell culture verification are not easy are avoided.
Drawings
FIG. 1 is the detection picture information of plaque tissue of the treated control group 1 by a cell activity detection instrument Countstar;
FIG. 2 is the detection picture information of plaque tissue of the treated control group 2 by the cell activity detection instrument Countstar;
FIG. 3 is the detection picture information of plaque tissues of the treatment experimental group by a cell activity detection instrument Countstar;
FIG. 4 is a statistical plot of cell activity after plaque tissue digestion.
Detailed Description
The invention is further explained below with reference to experimental groups and figures. The following examples are provided only for illustrating the present invention and do not limit the scope of the present invention.
EXAMPLES plaque tissue digestion
First, experimental material
The following test groups, control group 1 and control group 2 all involved enzymes, Collagenase (collagen I, sigma, C0130-100MG), Collagenase II (collagen II, sigma, C6885-100MG), Collagenase III (collagen III, Solarbio, C8490-100MG), Collagenase IV (collagen IV, sigma, C5138-100MG) and Collagenase XI (collagen XI, sigma, C7657-100MG), at concentrations of 1 MG/ml. Trypsin (0.25% Trypsin-EDTA (1X), Gibco-25200072-500ml), Hyaluronidase (Hyaluronidase, sigma, H3506-50 MG).
Calcium chloride (CaCl)2，sigma，C1016-500g)。
Deoxyribonuclease I (DNase I, neb, M0303S).
Phosphate buffered saline (PBS, Hycone, SH30256.01B).
Bovine serum albumin solution (BSA, gentle, 130-.
1640 medium (RPMI 1640, life, 11875093).
Collagen hydrolase I, II, IV, XI: the cells were lysed in 1640 cell culture medium to a final concentration of 1 mg/ml. In control 1, collagen hydrolase iv: the cells were lysed in 1640 cell culture medium to a final concentration of 2 mg/ml. In control 2, trypsin, collagen hydrolase i, ii: the cells were lysed in 1640 cell culture medium to a final concentration of 2 mg/ml.
PBS 1% BSA
DNase I enzyme: the cells were lysed in 1640 cell culture medium to a final concentration of 200U/ml.
Step two, step
Preparing before experiment, putting sterilized experimental apparatus and consumables into a super clean bench, irradiating by ultraviolet rays for 1 hour, and preparing enzyme required by the experiment in the super clean bench to prepare a plaque digestion mixed enzyme system, which comprises: collagen hydrolase I, II, IV, XI (each 1mg/ml), calcium chloride (10mM), and deoxyribonuclease I (200U/ml). And dispensed 0.5ml of the cocktail into 2ml EP tubes.
1. Material taking: a carotid plaque Tissue specimen is obtained when a patient undergoes carotid plaque denudation operation in vitro, and is preserved and transported at 4 ℃ in MACS Tissue preservation Solution (the cat # 130-.
2. Washing: pouring the tissue from the preservation solution into one of the six-hole plate, firstly cleaning the blood in the preservation solution and removing the serious calcified part, then washing the remaining 5 holes filled with 1640 culture medium one by one, and removing the preservation solution, the blood stain, the calcified piece and the like.
3. Measurement: plaque tissue was separated into approximately 1cm x 1cm samples in six well plates for final washing.
4. Shearing the tissues: aseptically operating in a super clean bench, burning tweezers and ophthalmic scissors on an alcohol lamp, wiping the alcohol lamp clean, transferring a sample into a subpackaged EP (EP) tube by using the tweezers, mechanically cutting plaque tissues into 1mm multiplied by 1mm by using the ophthalmic scissors, adding 1ml of mixed enzyme system, and preparing into a 1.5ml reaction system. Taking no more than 10 minutes, the digestion is started ready.
5. Preparation of digestion: the prepared reaction system (1.5 ml in total) was rewarmed for 2 minutes in a 2ml EP tube in a 37 ℃ water bath with shaking, and then transferred to a 37 ℃ incubator and digested for 1 hour with shaking on an HS vertical mixer.
6. Termination of digestion: after digesting for 1h, repeatedly blowing and uniformly mixing the digestive system by using a micro liquid transfer gun, and standing for 1min at room temperature; transfer the digestion solution to a 15ml centrifuge tube, add PBS (containing 1% BSA) to 15ml, and centrifuge at 200g, 4 ℃ for 5 min.
7. Sieving by using a filter screen: after centrifugation of the digestion liquid, the supernatant was discarded and resuspended in 15ml of PBS. And repeatedly and softly blowing and beating the solution by using the miniature liquid-transfering gun head, then sieving the solution by using a 40-micron cell sieve, and filtering the solution around the filter screen during liquid transfer.
8. And (3) red blood cell lysis: mixing the digested single cell suspension, removing supernatant, adding erythrocyte lysate 4ml, standing for 10min, centrifuging at 4 deg.C for 5min at 200g, and resuspending in 1ml 1640.
9. Meitian whirlwind deceased cell kit: the above system was subjected to dead cell removal, resuspended in 500ml 1640+ BSA and centrifuged at 200g at 4 ℃ for 5 min.
10. Beautiful day and whirlpool the shard kit: and 9, centrifuging at the temperature of 300g and 4 ℃ for 5min, discarding supernatant after centrifugation, resuspending PBS, operating the preparation system according to the instruction, centrifuging at the temperature of 3000g and 4 ℃ for 10min, and centrifuging at the temperature of 1000g and 4 ℃ for 10 min.
11. Density gradient centrifugation kit: following step 10, the preparative system was subjected to gradient centrifugation according to the instructions of the reagent procedure of Meitian and whirly.
12. Cell activity detection and counting: detection was performed using a Countstar Instrument ID, WIN-7RFTLHV6T 5L.
Control group 1: the mixed enzyme system contains collagen hydrolase IV: dissolving with 1640 cell culture solution to final concentration of 2mg/ml,
DNase I enzyme: the cells were lysed in 1640 cell culture medium to a final concentration of 200U/ml.
In example step 4, the mixed enzyme system was changed to collagen hydrolase IV 2mg/ml and DNase I200U/ml, and the same experimental groups were used.
Control group 2: the mixed enzyme system comprises two steps of treatment, namely treating the mixed enzyme system by trypsin at 37 ℃ for 30min, and then treating the mixed enzyme system by collagen hydrolase I, collagen hydrolase III: the cells were lysed in 1640 cell culture medium to a final concentration of 2 mg/ml.
Specifically, in the step 4 of the example, trypsin is used for digesting the mixture for 30min at 37 ℃, the pancreatin is removed by centrifugation, and then collagenase I, collagenase III and hyaluronidase are used for digesting the mixture for 30min at 37 ℃ in a system of 2ml, and the rest of the experiment groups are the same.
Third, experimental results
The information of the plaque tissues of the control group 1, the control group 2 and the experimental group (example) detected by the cell viability detector Countstar is shown in fig. 1 to 3, respectively.
The analysis results are shown in table 1 and fig. 1.
TABLE 1 Countstar test results of plaque single cell suspensions
Table 1 shows that the cell activity of the experimental group (example) was significantly increased compared to the control group.
Tests show that the plaque tissue subjected to mechanical cutting treatment can be thoroughly digested into single cell suspension, and the number and activity of living cells of the single cell suspension are remarkably improved.
Drawings
Drawing notes: FIG. 1 control group 1; FIG. 2 control 2; FIG. 3 experimental group; FIG. 4 comparison of control group and experimental group
A: BR bright field; b: FL1 green fluorescence; c: FL2 red fluorescence; d: fusing; e: a diameter profile; f: FL1 fluorescence intensity profile; g: FL2 fluorescence intensity profile.
Claims (10)
1. An enzyme combination, comprising: collagen hydrolase I, collagen hydrolase II, collagen hydrolase IV and collagen hydrolase XI;
preferably, the concentration ratio of the collagen hydrolase I, the collagen hydrolase II, the collagen hydrolase IV and the collagen hydrolase XI is 1: 1: 1: 1;
more preferably, the enzyme combination comprises dnase i.
2. A digestive fluid comprising the enzyme combination of claim 1.
3. The digestive juice according to claim 2, wherein the digestive juice comprises RPMI 1640 cell culture fluid.
4. The digestive juice according to claim 3, wherein the concentration of the collagenolytic enzymes I, II, IV, XI in the enzyme combination of the digestive juice is 1mg/mL, and the concentration of the DNase I is 200U/mL.
5. The digestive juice according to claim 4, wherein the digestive juice comprises calcium chloride.
6. The digestive fluid according to claim 4, wherein the final concentration of calcium chloride in the digestive fluid is 10 mM.
7. A method of digesting carotid plaque, comprising the use of the enzyme combination of claim 1 or the use of the digestive fluid of any of claims 2-6.
8. The method according to claim 7, characterized in that it comprises the steps of:
(1) collecting tissues: selecting a case sample, and placing the case sample in MACS tissue preservation solution at 4 ℃ after the plaque is stripped and separated in vitro in a plaque operation;
(2) separating a sample: placing the plaque sample in PBS to clean blood clots, and removing calcified blocks;
(3) tissue digestion: transferring the sample into digestive juice, shearing, and digesting;
(4) termination of digestion: after digestion for 1h, repeatedly pumping and uniformly mixing a digestion system by using a micro pipette, standing for 1min at room temperature, and centrifuging for 5min at 200g at 4 ℃;
(5) centrifugal sieving: after centrifugation, the supernatant was discarded, the pellet was resuspended in 15ml PBS and filtered through a 40 μm cell sieve;
(6) and (3) optimizing: after the filtrate is subjected to erythrocyte lysis, filtering out dead cells and impurities by using a Meitian and whirlpool dead cell removal kit and a Meitian and whirlpool debris removal kit;
(7) and (3) activity detection: activity measurements were performed using a Countstar instrument and counted.
9. Use of the enzyme combination according to claim 1 for the preparation of a digestive fluid according to any one of claims 2 to 6.
10. Use of the enzyme combination of claim 1 or the digestive juice of any one of claims 2 to 6 in the preparation of a reagent for digesting carotid plaque.
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Cited By (2)
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| CN111514167A (en) * | 2020-05-09 | 2020-08-11 | 北京航空航天大学 | Application of donkey-hide gelatin in product for relieving oxidative stress injury of cells |
| WO2022262102A1 (en) * | 2021-06-16 | 2022-12-22 | 浙江大学 | Method for dissociation of bovine rumen epithelial tissue for single-cell sequencing |
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