阅读说明：本技术 一种显著提升斑块消化制备高质量单细胞悬液的方法 (Method for preparing high-quality single cell suspension by remarkably improving plaque digestion ) 是由 朱尚明 吉训明 张永彪 石小峰 成军 裴葆青 谷涌泉 刘会生 于 2020-01-13 设计创作，主要内容包括：本发明公开了一种显著提升单细胞悬液质量的动脉斑块消化的方法,包括胶原蛋白水解酶Ⅰ、胶原蛋白水解酶Ⅱ、胶原蛋白水解酶Ⅳ、胶原蛋白水解酶Ⅺ、氯化钙、脱氧核糖核酸酶I、磷酸缓冲盐溶液、牛血清白蛋白溶液、RPMI 1640细胞培养液。本发明同时公开了其在消化斑块组织中的应用前景。本发明主要针对斑块组织进行消化处理而发明,通过几种酶的组合,可将斑块组织消化成为活性比例较高单细胞悬液,在单细胞基础上研究斑块组织的生物学反应,从而避免出现存在许多钙化细胞碎片、组织杂质等影响单细胞测序上机或后续数据分析结果的情况。(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.)

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.

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.