阅读说明：本技术 一种白细胞的分离纯化方法 (Separation and purification method of leucocyte ) 是由 张衡 阮亮亮 徐晨 徐斌 刘妍婷 于 2018-08-06 设计创作，主要内容包括：本发明提供一种白细胞的分离纯化方法,所述分离纯化方法包括步骤：(1)收集血液样本；(2)离心分离出白细胞粗提物；(3)在白细胞粗提物中加入红细胞裂解液裂解红细胞,离心去上清；(4)重复步骤(3)至少一次,分离获得白细胞。本发明方法操作方便,时间短,成本低,可以大规模处理样本。(The invention provides a method for separating and purifying leucocytes, which comprises the following steps: (1) collecting a blood sample; (2) centrifuging to separate out leukocyte crude extract; (3) adding erythrocyte lysate into the crude extract of the white blood cells to lyse the erythrocytes, and centrifuging to remove supernatant; (4) repeating the step (3) at least once, and separating to obtain the white blood cells. The method has the advantages of convenient operation, short time and low cost, and can process samples in a large scale.)

1. A method for separating and purifying white blood cells is characterized in that red blood cell lysate is used for separation and purification in the method.

2. A method for separating and purifying leukocytes, comprising the steps of:

(1) collecting a blood sample;

(2) centrifuging to separate out leukocyte crude extract;

(3) adding erythrocyte lysate into the crude leukocyte extract, fully lysing erythrocytes, and centrifuging to remove supernatant;

(4) repeating the step (3) at least once, and separating to obtain a finished product of the white blood cells.

3. The method for separating and purifying leukocytes according to claim 2, wherein in the step (3), the erythrocyte lysate is added and then mixed well, and the mixture is left for 5 to 10 minutes, and the container is inverted every 3 minutes for a plurality of times.

4. The method for separating and purifying leukocytes according to claim 2, wherein in the step (3), the erythrocyte lysate is added thereto, the mixture is mixed well, and the mixture is left to stand for 10 minutes while the vessel is inverted 5 times every 3 minutes.

5. The method for separating and purifying leukocytes according to any one of claims 1 to 4, wherein in the step (1), an EDTA anticoagulation tube is used to collect the blood sample.

6. The method for separating and purifying leukocytes according to any one of claims 1 to 4, further comprising the steps of:

(5) adding PBS into the leukocyte finished product, gently mixing the leukocyte, sucking supernatant and freezing for storage.

7. The method for separating and purifying leukocytes according to claim 5, further comprising the steps of: (5) adding PBS into the leukocyte finished product, gently mixing the leukocyte, sucking supernatant and freezing for storage.

8. The method for separating and purifying leukocytes according to claim 5, wherein in the step (2), the centrifugation parameters are 800g for 20 minutes.

9. The method for separating and purifying leukocytes according to claim 1 to 4 or 7, wherein in the step (2), the centrifugation parameters are 800g for 20 minutes; the volume ratio of the white blood cell lysate to the red blood cell lysate in the steps (3) and (4) is 1: 2-4.

10. The method for separating and purifying leukocytes according to claim 8, wherein in the step (3) (4), the centrifugation parameters are 400g for 5 minutes.

Technical Field

The invention relates to the field of biological medicine, in particular to a cell separation method.

Background

Leukocytes are derived from hematopoietic stem cells in bone marrow, enter blood and lymph circulation after development in bone marrow, are also present in tissues outside blood vessels and lymph vessels, function to protect the body from pathogens (bacteria and viruses), cancer cells, and foreign bodies, and are guardians of the human body. There are eight types of leukocytes in humans, including macrophages, dendritic cells, B lymphocytes, T lymphocytes, natural killer cells, neutrophils, eosinophils, and basophils.

Macrophages are the first largest leukocytes and develop from monocyte differentiation. Macrophages phagocytose and digest cellular residues and pathogens, participate in professional antigen presentation for adaptive immunity, and promote germ cell development, sex hormone, bone tissue resorption, and blood vessel formation. Dendritic cells are also a type of monocyte that helps the body recognize pathogens by taking up, processing, and presenting antigens to lymphocytes in lymph nodes and lymphoid organs, initiating specific immune responses. B cells are one of the components of lymphocytes. After being stimulated by an antigen, B cells synthesize and secrete antibodies against the pathogen. The binding of the antibody to the antigen helps the body to recognize the pathogen and make it the target cell for clearance by other immune cells. B cells, known as memory cells, can maintain memory of pathogen biomolecular markers, leaving the same antigen free of infection when re-entered the body. T cells are also one of the lymphocytes, including cytotoxic T cells, helper T cells, suppressor T cells, natural killer T cells, and memory T cells. Cytotoxic T cells directly kill target cells, helper T cells help B cells to produce antibodies, and suppressor T cells suppress the immune response of B cells and other T cells to antigens. Natural killer cells are lymphocytes distributed in the blood circulation, which recognize infected cells and senescent cells and destroy diseased cells by releasing chemical particles to phagocytize. Neutrophils are the most abundant granulocytes in the blood circulation and can rapidly appear at the site of infection and injury when the body encounters pathogen invasion. Eosinophils are phagocytic leukocytes which increase significantly upon allergic reactions and parasitic infections. Eosinophils have large eosinophil granules that release chemicals to eliminate pathogens. Basophils are granulocytes (with granulocytes) containing substances such as histamine and heparin in granules, and the heparin can dilute blood, inhibit the formation of blood clots, and expand capillary vessels and increase blood flow.

In previous experiments, peripheral blood leukocytes were generally isolated using lymphocyte isolates. Leukocytes in blood differ from other cells in their volume, morphology and density. The density of erythrocytes and granulocytes is about 1.090g/ml, while the density of lymphocytes and monocytes is 1.075-1.090 g/ml and the density of platelets is 1.030-1.035 g/ml. The lymphocyte separating medium is a solution with density of 1.075-1.090 g/ml and similar to isosmosis, and is used for density gradient centrifugation to ensure that cells with certain density are distributed according to corresponding density gradient, thereby separating various blood cells.

The operation steps of separating the white blood cells in the whole blood by using the lymphocyte separating medium are as follows: the lymphocyte separation solution is returned to room temperature, 4ml of the lymphocyte separation solution is placed in a 15ml centrifuge tube, 4ml of whole blood is slowly added into the centrifuge tube along the tube by a pipette, the upper layer of the blood can be seen, and the separation solution is arranged at the lower layer. Putting the mixture into a centrifuge, centrifuging the mixture for 30 minutes at normal temperature at 400 g; taking out the centrifugal tube from the centrifuge, and dividing the tube into 4 layers: the upper layer is plasma, the second layer is a leucocyte layer, the third turbid layer is a lymphocyte separation liquid layer, and the lower layer is a erythrocyte layer; sucking the second layer of turbid layer of white blood cells by a liquid transfer device, placing the second layer of turbid layer of white blood cells in a 15ml centrifuge tube, adding 8ml of phosphate buffer solution to wash the cells, slightly bouncing and uniformly mixing the white blood cells, centrifuging at normal temperature, and performing centrifugation for 250g for 10 minutes; removing supernatant, adding 1ml of phosphate buffer solution to wash cells, gently mixing white blood cells uniformly, transferring all the solution into a 2ml centrifuge tube, centrifuging for 5 minutes at 250 g; the supernatant was aspirated and the leukocytes were stored in a freezer at-80 ℃.

The operation process of separating the white blood cells by using the lymphocyte separating medium is complex and tedious, the operation time is long, and the sample cannot be processed on a large scale. Therefore, there is an urgent need in the art to develop a method for separating leukocytes, which is simple in operation, short in time, and low in cost.

Disclosure of Invention

The invention provides a method for separating the white blood cells, which has the advantages of simple operation, short time and low cost.

The invention provides a method for separating and purifying leukocytes, which is characterized in that a lysate of erythrocytes is used for separation and purification in the method for separating and purifying leukocytes.

In a second aspect, the present invention provides a method for separating and purifying leukocytes, comprising the steps of:

(1) collecting a blood sample;

(2) centrifuging to separate out leukocyte crude extract;

(3) adding erythrocyte lysate into the crude leukocyte extract, fully lysing erythrocytes, and centrifuging to remove supernatant;

(4) repeating the step (3) at least once, and separating to obtain a finished product of the white blood cells.

In the step (3), the erythrocyte lysate is added and fully mixed, and the mixture is kept stand for 5 to 10 minutes, and the container is inverted for a plurality of times every 3 minutes.

In the step (3), the erythrocyte lysate is added and fully mixed, the mixture is kept stand for 10 minutes, and the container is turned upside down for 5 times every 3 minutes.

And (2) collecting the blood sample by adopting an EDTA anticoagulation tube in the step (1).

The separation and purification method further comprises the following steps:

(5) adding PBS into the leukocyte finished product, gently mixing the leukocyte, sucking supernatant and freezing for storage.

In the step (2), the centrifugation parameters were 800g for 20 minutes.

The volume ratio of the white blood cells to the red blood cell lysate in the steps (3) and (4) is 1: 3-4. The leucocyte is crude leucocyte extract.

In the above steps (3) and (4), the centrifugation parameters were 400g for 5 minutes.

The method has the advantages of convenient operation, short time and low cost, and can process samples in a large scale. The commercially available erythrocyte lysate can be used in the separation and purification method, and the obtained leucocytes meet the quality requirement.

Drawings

FIG. 1 is a technical scheme of a leukocyte separation and purification method;

FIG. 2 shows the result of separating white blood cells from a lysate of red blood cells.

FIG. 3 shows the results of measurement of the ratio of rRNA of leukocytes after 18 blood samples were separated from the erythrocyte lysate.

FIG. 4 shows the result of detecting the leukocyte rRNA integrity index after 18 blood samples are separated from erythrocyte lysate.

Detailed Description

Hereinafter, the present invention is described in more detail and specifically with reference to examples, but the following examples are not intended to limit the present invention.