CN108753685B

CN108753685B - Separation, screening, culture and function identification method of human aortic vessel wall stem cells expressing c-Kit

Info

Publication number: CN108753685B
Application number: CN201810635146.1A
Authority: CN
Inventors: 于宝琪; 曲爱娟
Original assignee: Capital Medical University
Current assignee: Capital Medical University
Priority date: 2018-06-20
Filing date: 2018-06-20
Publication date: 2022-06-28
Anticipated expiration: 2038-06-20
Also published as: CN108753685A

Abstract

The invention discloses a method for separating, screening, culturing and functionally identifying human aortic vessel wall stem cells expressing c-Kit. The invention provides a Kit for obtaining human aortic vessel wall stem cells, which comprises a substance for combining c-Kit positive cells. In the Kit, the substance which binds to the c-Kit positive cells comprises a c-Kit antibody; and/or the c-Kit antibody is a c-Kit immunomagnetic bead antibody. The invention separates the vascular adventitia from the human aorta tissue, digests the vascular adventitia with collagenase, obtains cell suspension through cell screening, and obtains the c-Kit positive vascular wall stem cell through the screening of the c-Kit immunomagnetic bead antibody.

Description

Separation, screening, culture and function identification method of human aortic vessel wall stem cells expressing c-Kit

Technical Field

The invention belongs to the technical field of stem cell and tissue engineering, and particularly relates to a method for separating, screening, culturing and functionally identifying human aortic vessel wall stem cells expressing c-Kit.

Background

Adult stem cells are undifferentiated cells existing in differentiated tissues in adult animals or human bodies, have the capacity of continuous proliferation and self-renewal, and can be differentiated into cells of specific types of the tissues, so that damaged mature cells in the tissues can be timely supplemented, and the dynamic balance of growth, aging, damage and repair of tissues and organs can be maintained. In recent years, a large number of studies have shown that a population of adult stem cells reside in the adventitia of the arterial and venous blood vessels of mice and humans, and that under normal physiological conditions they maintain vascular homeostasis and in time replenish and renew the senescent apoptotic vessel wall cells. In pathological conditions, such as in endothelial injury and vascular wall lesion, these stem cells can be activated by various factors such as active oxygen, chemokines, cytokines, growth factors, metalloproteinases, etc., and can timely supplement injured endothelial cells or smooth muscle cells by means of proliferation, migration, differentiation, etc., so as to participate in the process of repair after vascular injury or vascular remodeling. At present, in many studies, vascular wall stem cells have been isolated from various blood vessels such as mouse aorta, transplanted blood vessel and the like, and successfully cultured in vitro, and applied to the research of pathophysiological mechanisms of various mouse vascular disease models. However, with the continuous and deep research on vascular wall stem cells in recent years, if the vascular wall stem cells can be obtained by separating from human vascular tissues, the research on the pathophysiological mechanisms of various vascular diseases of a human body can be directly and deeply carried out, and a normal and various diseases vascular wall stem cell bank is clinically established in the future, so that sufficient resources are provided for the application of the vascular wall stem cells in tissue engineering and clinical stem cell therapy.

At present, for the research on stem cells in human vascular wall, some documents only demonstrate the existence of cells expressing stem cell marker molecules on blood vessel sections of human aortic tissues in normal and different disease groups by immunohistochemical staining methods, and do not perform cell isolation and in vitro culture. In other part of documents, although it has been reported that cells isolated from normal human aortic blood vessels express mesenchymal stem cell marker molecules, the stem cell marker molecules are not screened in the isolation process, so that the purity of the finally obtained stem cells is not high.

The c-Kit belongs to one of tyrosine kinase receptor protein family members, is used as a stem cell factor receptor, and can be combined with a stem cell factor so as to start a corresponding signal path and regulate multiple biological functions of proliferation, differentiation and the like of stem cells. The c-Kit is an important marker molecule for identifying the specific type of the bone marrow hematopoietic stem cells, and the detection of the c-Kit can be used for the classified diagnosis and the type-dividing diagnosis of clinical acute leukemia.

Disclosure of Invention

An object of the invention is to provide a kit for obtaining human aortic vessel wall stem cells.

The Kit provided by the invention comprises a substance which binds to c-Kit positive cells.

In the Kit, the substance capable of binding to the c-Kit positive cells comprises a c-Kit antibody;

and/or the c-Kit antibody is a c-Kit immunomagnetic bead antibody.

In the kit, the kit further comprises erythrocyte lysate for lysing erythrocytes.

The kit also comprises a separation buffer and a separation column.

The isolation buffer was a sterile PBS solution containing 0.5% BSA and 2mM EDTA.

The Kit also comprises a culture medium for culturing the c-Kit positive cells,

the culture medium consists of the following components: DMEM-F12+ GlutaMAX-1 basal medium, 20% by volume of FBS, 100U/ml streptomycin, 10ng/ml human LIF, 0.1mM beta-mercaptoethanol and 20ng/ml human bFGF.

The kit also comprises a module or a comparison card for recording the separation method;

the separation method comprises the following steps:

1) separating the vascular adventitia from the isolated human aortic tissue, and digesting the vascular adventitia into cells;

2) and (3) combining the cells with the substance which is combined with the c-Kit positive cells in the Kit with the c-Kit on the c-Kit positive cells to separate the c-Kit positive cells, namely obtaining the human aortic vascular wall stem cells.

Between steps 1) and 2), the following steps are also included: filtering the digested cells through a cell sieve, collecting filtrate, and centrifuging to obtain cell precipitates in the filtrate; finally, removing the red blood cells in the cell sediment.

The application of c-Kit in obtaining human aortic vessel wall stem cell markers is also the protection scope of the invention.

The application of the kit in preparing the human aortic vessel wall stem cell products is also within the protection scope of the invention;

or, the application of the above-mentioned kit in obtaining human aortic vessel wall stem cells is also within the scope of protection of the present invention.

Or, the application of the substance combining the c-Kit positive cells in the preparation of the product for obtaining the human aortic vessel wall stem cells is also within the protection scope of the invention;

or, the application of the substance combining with the c-Kit positive cells in obtaining the human aortic vessel wall stem cells is also the protection scope of the invention;

or, the application of the kit in preparing the product for obtaining the human aortic vessel wall stem cells is also within the protection scope of the invention;

or, the application of the kit in obtaining human aortic vessel wall stem cells is also the protection scope of the invention;

Alternatively, the use of the above-described medium for culturing c-Kit positive cells is also within the scope of the present invention.

The second purpose of the invention is to provide a method for obtaining human aortic vessel wall stem cells.

The method provided by the invention comprises the following steps: and c-Kit is used as an aortic vessel wall stem cell marker to separate the aortic vessel wall stem cells.

In the above method, the separation step is as follows:

1) separating vascular adventitia from isolated human aortic tissue, and digesting the vascular adventitia into cells;

Digesting the adventitia of the blood vessel into cells by adopting collagenase;

the substance for combining the c-Kit positive cells is an antibody of c-Kit and the like, and specifically is a c-Kit immunomagnetic bead antibody;

the method also comprises the following steps between the step 1) and the step 2): filtering the cells by a cell sieve, collecting filtrate, and centrifuging to obtain cell precipitates in the filtrate; finally, removing the red blood cells in the cell sediment.

The method also comprises the following steps: the c-Kit positive cells were cultured in the above-mentioned medium for culturing c-Kit positive cells.

The 3 rd object of the present invention is to provide a medium for culturing c-Kit positive cells.

The culture medium provided by the invention is the culture medium in the kit.

The invention separates the vascular adventitia from the human aorta tissue, uses collagenase to digest, obtains cell suspension through cell screening and filtration, obtains the c-Kit positive vascular wall stem cell through the screening of the c-Kit immunomagnetic bead antibody, the stem cell has good in vitro proliferation and migration capacity, has little apoptosis, not only has the differentiation potential of the mesenchymal stem cell (can be differentiated into fat cell, osteoblast and chondrocyte), but also has the tissue specificity differentiation capacity that the vascular wall stem cell can be differentiated into the vascular smooth muscle cell; in a word, the method of the invention overcomes the problems of low purity and slow growth of the separated stem cells caused by lack of specific stem cell marker screening in the prior separation method, and provides a novel separation culture method which can obviously improve the purity and the cell activity of the stem cells on the wall of the human aortic blood vessel.

Drawings

FIG. 1 is a diagram showing the results of flow cytometry to identify the expression level of stem cell marker molecules and c-Kit positive cells marked by immunofluorescence staining;

Wherein A-I is a cell separated from the adventitial tissue of the blood vessel wall of the human aorta, and the expression level of a stem cell marker molecule is identified by flow cytometry before c-Kit immunomagnetic bead screening; j is the c-Kit positive cell marked by immunofluorescent staining after the screening of the c-Kit immunomagnetic bead antibody.

FIG. 2 shows the expression level of proliferation cell-associated nuclear antigen ki67 in c-Kit-positive vascular wall stem cells of normal donors and patients after aortic replacement after 24 hours of culture.

FIG. 3 is a graph showing the identification of apoptosis levels in c-Kit positive vascular wall stem cells from normal donors and patients with aortic replacement after 72 hours of culture; (A) AnnexinV/PI assay, (B) TUNEL staining.

FIG. 4 is a graph showing the level of migration of c-Kit positive vascular wall stem cells measured in normal donors and patients with aortic replacement for 6-8 hours; a is a Transwell migration test, and B is a scratch test.

FIG. 5 shows the level of differentiation of c-Kit positive vascular wall stem cells into adipocytes, osteoblasts, chondrocytes and smooth muscle cells in normal donors and patients with aortic replacement.

FIG. 6 is a graph showing the level of differentiation of c-Kit positive vascular wall stem cells into vascular smooth muscle cells in normal donors and patients with aortic replacement.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 isolation, screening and culture of human aortic vessel wall Stem cells

Separation and screening of human aortic vessel wall stem cells

1. Isolation of human aortic vessel wall stem cells

1) Isolating adventitia

Collecting aorta tissue of a patient with normal organ donation donor or aorta replacement (patient, donor or donor family informed), with area of about 2 × 5cm²Immediately put into 20ml of ice-cold sterile tissue preservation solution (containing 1mg of EDTA-Na)₂And 3% FBS in sterile PBS). Taking out vascular tissue from the tissue preservation solution with sterile forceps in a clean bench, and adding EDTA-Na containing 1mg₂The sterile PBS solution rinses the vascular tissue for a plurality of times, and removes the residual bloodstain until the rinsing solution is clear and has no visible bloodcolor. Gently separating the adventitia from the media with two pairs of sterile forceps, retaining adventitia tissue to obtain the adventitia, rinsing again for three times, sucking up liquid as much as possible, and cutting the adventitia into small pieces of 2-3mm with sterile scissors.

2) Collagenase digestion

The tissue fragments were placed in about 4ml of 37 ℃ preheated collagenase (Liberase) at 0.25mg/ml^TMTL Research Grade,5401020001, ROCHE) in serum-free DMEM/F12 medium (0.1mg collagenase: 1 cm)²Aortic tissue) was shaken on a shaker for 3-4 hours in a 37 ℃ incubator.

When the digestion suspension is turbid into chyle and has no obvious tissue blocks, the digestion can be stopped. Repeatedly pumping the digestive juice by a pipette to disperse the tissue cell lumps as much as possible. The tissue suspension was then filtered sequentially through sterile cell sieves with a pore size of 100 μm and 70 μm and each filter was rinsed twice three times with the complete medium. The filtrate was collected, centrifuged at 1000rpm for 5 minutes, the supernatant was discarded, and the cell pellet was collected.

2. Screening of C-Kit positive human aortic vessel wall stem cells

And (2) screening the c-Kit labeled molecules of the human aortic vessel wall stem cells obtained in the step (1), wherein the method comprises the following steps:

1) lysing erythrocytes

In order to avoid the situation of separation column blockage caused by non-specific binding of a large amount of residual red blood cells in tissue cell suspension to magnetic beads, before screening C-Kit positive cells by using a separation column, 5ml of red blood cell lysate (Biyuntian, C3702) (the amount of the added red blood cell lysate is 5 times of the amount of the cells) is added into a cell precipitate obtained in the previous step, the cell precipitate is incubated at room temperature for 10 minutes, 20ml of PBS is added and mixed uniformly, the centrifugation is carried out at 1000rpm for 5 minutes, the supernatant is discarded until no obvious red precipitate exists, and the cell precipitate with the red blood cells removed is obtained.

2) c-Kit antibody (CD117MicroBead Kit, human, Miltenyi Biotec, 130-

Erythrocyte-free cell pellet (about 1X 10) obtained in 1) above⁷Individual cells) was added to a pre-cooled 300. mu.l of isolation buffer (consisting of 0.5% BSA, 2mM EDTA and sterile PBS by volume) and counted on a cell counting plate with a cell count of less than 10⁸For each cell (as the total number of cells before selection), 100. mu.l of FcR blocking solution (provided in CD117MicroBead Kit) and 100. mu. l c-Kit immunomagnetic bead-conjugated antibody (CD117MicroBead Kit) were added at the same time, mixed well, and incubated at 4 ℃ for 15 minutes in a refrigerator to obtain a cell suspension after incubation.

3) Magnetic bead adsorption

Taking out the incubated cell suspension obtained in the above 2), adding 2ml of separation buffer solution, centrifuging for 10 minutes at 300g, completely sucking up the supernatant, and discarding.

While cell centrifugation was performed, the separation column was fixed to a MACS separator, 500. mu.l of separation buffer was added, and the column was rinsed to avoid mixing air bubbles and clogging the column.

After centrifugation, 500. mu.l of separation buffer was added to the cell pellet, the pellet was mixed with a pipette, and the cell suspension was slowly applied to a separation column (MS Columns, Miltenyi Biotec, 130-. At this time, the positive cells labeled with the c-Kit immunomagnetic bead antibody are magnetically adsorbed in the column, while the negative cells not labeled with the c-Kit are discharged out of the column with the liquid.

After the cell suspension is completely dripped, 500 mul of separation buffer solution is added again to wash the negative cells remained in the separation column, the operation is repeated twice, and the obtained positive cells are adsorbed in the separation column.

4) c-Kit Positive cell isolation

Gently removing the separation column after adsorbing the cells obtained in the step 3) from the MACS separator, placing the separation column on a new 15ml sterile centrifuge tube, adding 1ml separation buffer solution into the separation column, rapidly pumping the cell suspension in the separation column into a centrifuge tube below the separation column by using a matched piston, and collecting the c-Kit positive cell suspension, namely the c-Kit positive human vessel wall stem cells (derived from normal organ donation donors or aorta tissues of patients who undergo aortic replacement).

The collected c-Kit positive cells were counted by a cell counting plate, and the percentage of c-Kit positive cells was calculated by comparing the total number of cells before screening (the number of cells before binding of c-Kit immunomagnetic beads coupled with antibodies).

The percentage of human vascular wall stem cells (from normal organ donation donors) was about 10%;

the percentage of human vascular wall stem cells (derived from the aortic tissue of a patient undergoing aortic replacement) is about 5% -12%.

Culture of human aortic vessel wall stem cells

Culturing the human aortic vessel wall stem cells obtained by the method as follows:

Adding 3ml of complete culture medium into the human vascular wall stem cells obtained in the previous step, uniformly mixing the cells, adding a culture dish which is paved with 0.04% B type bovine skin collagen and has the diameter of 35mm, placing the culture dish at 37 ℃ and 5% CO₂An incubator. After 24 hours, cells were observed, more than 90% of which had adhered, and which were spindle or spindle shaped. The culture medium was replaced with new one to remove nonadherent cells. The medium was changed once for 3 days, and after 3-7 days, when the cells grew to 90%, they were digested with 0.05% trypsin EDTA, and then passaged at a ratio of 1: 3.

The complete culture medium consists of the following components: DMEM-F12+ GlutaMAX-1 basal medium (Gibco)^TM10565018), 20% (volume percent) FBS, 100U/ml streptomycin, 10ng/ml human LIF, 0.1mM beta-mercaptoethanol and 20ng/ml human bFGF.

Functional verification of human aortic vessel wall stem cells

1. Separation purity identification of human aortic vessel wall stem cells

Identifying a plurality of molecular markers of cells digested with collagenase obtained from the above one of 1 by flow cytometry: respectively incubating the cells on ice for 1 hour in a dark place by using different molecular antibodies and corresponding IgG thereof, centrifuging at 1500rpm for 5 minutes, washing by using PBS once, re-suspending by using PBS, and immediately detecting the expression level of each antibody by using a flow cytometer; comprises stem cell marker molecules CD29, CD73, CD105, CD44, CD90 and c-Kit, endothelial cell marker molecule CD31, leukocyte marker molecule CD45 and macrophage marker molecule CD11 b.

As shown in FIGS. 1A-I, it can be seen that the cell pellet obtained in the first step 1 of the present invention expresses a large amount of stem cell marker molecules, about 10% of which are c-Kit positive cells and hardly express endothelial, leukocyte and macrophage marker molecules, indicating that the stem cells obtained by the present separation method have high purity and are rarely mixed into other tissues and blood cells.

And (3) detecting the c-Kit positive cell suspension cells obtained in the step (2) by an immunofluorescence staining method, fixing the cell slide by 4% paraformaldehyde for 10 minutes, sealing donkey serum at room temperature for 60 minutes, adding rabbit anti-c-Kit primary antibody, incubating overnight at 4 ℃, adding donkey anti-rabbit secondary antibody, incubating at room temperature for 1 hour, washing with PBS for three times, dyeing with DAPI for 5 minutes, sealing the slide, observing the result by a fluorescence microscope, and showing that more than 90% of cells express c-Kit protein, wherein the purity of the c-Kit dry cells obtained by the extraction and screening method is very high.

2. Human aortic vessel wall stem cell proliferation capacity identification

In order to identify the proliferation capacity of the c-Kit positive human aortic vessel wall stem cells, the second two human vascular wall stem cells are subjected to fourth generation cell digestion and then seeded on a 4-hole glass plate, cell nucleus related antigen ki67 is subjected to immunofluorescence staining, after the cells are cultured for 24 hours, 4% formalin is fixed for 5 minutes, donkey serum is sealed for 1 hour at room temperature, ki67 antibody is incubated overnight at 4 ℃, PBS is washed for three times, corresponding secondary antibody is incubated for 1 hour at room temperature, PBS is washed for three times, DAPI is stained for 5 minutes, and the result is sealed and observed by a fluorescence microscope.

As a result, as shown in FIG. 2, human vascular wall stem cells (c-Kit positive) isolated from normal donor and aortic replacement patients had significant cell proliferation capacity.

3. Identification of human aortic vessel wall stem cell apoptosis level

In order to identify the apoptosis level of the C-Kit positive human aortic vessel wall stem cells, after the cells are cultured in vitro for 72 hours according to the method II, the cells are stained by an Annexin V/PI apoptosis Kit and TUNEL, and the number of apoptotic cells is detected by flow cytometry and a fluorescence microscope respectively (the Annexin V/PI Kit, Biyun day, C1063, the TUNEL Kit, Biyun day, C1088) (the methods are operated according to the instruction of the corresponding Kit).

The results are shown in FIG. 3, and the human vascular wall stem cells (c-Kit positive) isolated and extracted from normal donor and aortic replacement patients are at the level of apoptosis and necrosis with very few cells under the culture conditions provided by the present invention, and only less than 4% of the cells are apoptotic or dead, which indicates that the activity of the c-Kit stem cells obtained by the extraction and screening method of the present invention is also very high.

4. Identification of migration capacity of stem cells in human aortic vessel wall

In order to identify the migration capacity of the c-Kit positive human aortic vessel wall stem cells, cells migrating to the lower bottom surface of the Transwell chamber and in the area within the scratch are counted after 6-8 hours through a Transwell migration experiment and a scratch experiment respectively.

As a result, as shown in FIG. 4, human vascular wall stem cells (c-Kit positive) isolated from normal donor and aortic replacement patients had strong cell migration ability.

5. Positive identification of differentiation capability of human aortic vessel wall stem cells

To identify the differentiation capacity of the c-Kit positive Human aortic vessel wall Stem cells, the conventional adipogenic, osteogenic and chondrogenic Cell culture medium was used for stimulated differentiation (Human sensory Stem Cell Functional Identification Kit, R & D SC006, was used in the differentiation experiments) (the method steps were performed as described in the specification).

The immunofluorescent staining results are shown in fig. 5, and the expression level of the adipocyte marker molecules is increased 14 days after differentiation. After 21 days, the expression levels of the chondroblast and osteoblast marker molecules were increased.

In addition, according to the experimental results shown in FIG. 6, after 7 days of differentiation stimulation in specific smooth muscle cell differentiation culture medium (DMEM-F12+ GlutaMAX-1 medium with 1% FBS and 10ng/ml TGF-. beta.1), through qPCR analysis, the level of the smooth muscle cell marker molecule, Calponin mRNA, was significantly increased compared to that before differentiation, and further through immunofluorescence staining, the Calponin protein expression level was also significantly increased.

The above-described embodiments, objects, technical solutions and advantages of the present invention are further described in detail, and it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principle and spirit of the present invention, the scope of which is defined by the appended claims and their equivalents.

Claims

The application of the c-Kit in obtaining the human aortic vessel wall stem cell marker;

or, the c-Kit antibody is applied to the preparation of the human aortic vessel wall stem cell product;

or, the application of the c-Kit antibody in obtaining human aortic vessel wall stem cells.
2. The kit is applied to the preparation of products for obtaining human aortic vessel wall stem cells;

or, the kit is applied to obtaining human aortic vessel wall stem cells;

the Kit comprises a c-Kit antibody;

the c-Kit antibody is specifically a c-Kit immunomagnetic bead antibody;

the Kit also comprises a culture medium for culturing the c-Kit positive cells, a red blood cell lysate for lysing red blood cells and a module or a comparison card for recording the separation method,

the culture medium consists of the following components: DMEM-F12+ GlutaMAX-1 basal medium, 20% by volume of FBS, 100U/ml streptomycin, 10ng/ml human LIF, 0.1mM beta-mercaptoethanol and 20ng/ml human bFGF;

the separation method comprises the following steps:

1) separating the vascular adventitia from the isolated human aortic tissue, and digesting the vascular adventitia into cells;

2) and (3) combining the cells with the substance combining with the c-Kit positive cells with the c-Kit on the c-Kit positive cells to separate the c-Kit positive cells, namely obtaining the human aortic vascular wall stem cells.
3. A method for obtaining stem cells of an aortic blood vessel wall of a human, comprising the following steps: c-Kit is used as an aortic vessel wall stem cell marker to separate aortic vessel wall stem cells; the separation steps are as follows:

1) separating the vascular adventitia from the isolated human aortic tissue, and digesting the vascular adventitia into cells;

2) and (3) combining the cells with c-Kit on the c-Kit positive cells by using a c-Kit antibody, and separating the c-Kit positive cells to obtain the human aortic vascular wall stem cells.
4. The method of claim 3, wherein:

the method further comprises the steps of: culturing the c-Kit positive cells in a culture medium for culturing the c-Kit positive cells;

the culture medium consists of the following components: DMEM-F12+ GlutaMAX-1 basal medium, 20% by volume of FBS, 100U/ml streptomycin, 10ng/ml human LIF, 0.1mM beta-mercaptoethanol and 20ng/ml human bFGF.