CN109055300B - Separation culture method of endothelial progenitor cells derived from human endometrial tissue - Google Patents
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Abstract
The present invention relates to human endometrial tissue-derived endothelial progenitor cells isolated from human endometrial tissue. Digesting human endometrial tissue by an enzymatic hydrolysis method to obtain a single cell suspension, separating by adopting a density gradient centrifugation method to obtain a cell population containing endothelial progenitor cells, inoculating the cell population into an endothelial progenitor cell culture medium, and carrying out culture and subculture purification to finally obtain the new endothelial progenitor cells with super-strong angiogenesis capacity. Experiments prove that the endothelial progenitor cells have the capacity of self-proliferation and clone-like growth, have surface markers of the endothelial progenitor cells, have the capacity of taking Dil-ac-LDL and combining with FITC-UEA-1 and forming tubular structures in vitro, and can be applied to the preparation of medicines for preventing or treating ischemic diseases.
Description
Technical Field
The present invention relates to a new kind of endothelial ancestral cell, and is especially one kind of endothelial ancestral cell with strong vascularizing capacity extracted from human endometrial tissue. The invention also relates to a method for separating and culturing the endometrial endothelial progenitor cells.
Background
With the change of living standard and living habit of people in recent years, the number of patients suffering from various ischemic diseases including cardiovascular and cerebrovascular diseases and the medical social problems caused by the ischemic diseases are continuously increased, the incidence rate is high, the fatality rate and the disability rate are high, and the ischemic diseases become main diseases harmful to human health.
Ischemic diseases are diseases in which poor tissue perfusion is caused by stenosis or occlusion of a blood vessel, which results in insufficient blood supply to ischemic local tissue cells, ischemic injury, necrosis, and further damage to local or systemic tissues and organs. Aiming at ischemic diseases, the existing treatment means mainly comprises drug treatment, interventional therapy and surgical operation treatment, and mainly aims to recover tissue blood supply of ischemic local parts, obtain better treatment effect in most patients and greatly improve the prognosis of patients with ischemic diseases. However, these treatments still have their limitations, such as time limitation of drug treatment, recurrence of intervention, risk of surgical treatment, etc., so that the fatality and disability rate of the diseases remain high. Therefore, there is an urgent need to actively discuss a new, safer and more effective treatment strategy for ischemic diseases, especially for people with poor therapeutic effect on the conventional therapy, so as to supplement and improve the existing treatment means.
The tissue injury caused by various ischemic diseases is basically poor local tissue perfusion, and the blood supply of ischemic local tissue cells is insufficient. Promoting the ischemia local angiogenesis of patients is a strategy for targeting the root of the attack. In fact, the process of angiogenesis exists in normal human body, and it acts as a self-compensatory mechanism for ischemia and hypoxia injury. In the early stage of ischemic diseases, such as early stage of myocardial ischemia, the density of local capillaries is increased complementally, new blood vessels are formed around obstructed blood vessels, and new collateral circulation is established, which is a self-repair and protection process of the body against ischemic injury. This self-repair is not sufficient to counter injury, resulting in ischemia or even necrosis of local tissue cells, but only during the progression of the ischemic disease. Therefore, the enhancement of angiogenesis, the promotion of injury and the inclination of the balance of angiogenesis repair to the repair direction, are the key to the treatment of ischemic diseases.
Therapeutic angiogenesis is a new concept for the treatment of ischemic diseases. Among the various approaches to promote angiogenesis in patients, intensive studies on stem cells and stem cell transplantation have provided promise.
The stem cell/progenitor cell is a cell with self-replication, renewal and multidirectional differentiation potential, has the potential of repairing damaged tissue cells, has become an ideal seed cell in the fields of disease treatment and tissue engineering in recent years, and is a possible new effective treatment method for various diseases, particularly various intractable diseases.
According to different development stages, stem cells are divided into embryonic stem cells and adult stem cells, wherein the adult stem cells have wide application prospect due to the characteristics of wide sources, relatively low tumorigenicity and the like.
The progenitor cells have more definite directional differentiation capability than stem cells, wherein the endothelial progenitor cells are precursor cells of vascular endothelial cells, can self-proliferate and have the capability of directional differentiation to the vascular endothelial cells, and have very important functions of participating in damaged blood vessel repair under the stimulation of various physiological and pathological factors. Research shows that endothelial progenitor cells play an important role in the repair process of various vascular injuries including cardiovascular diseases, cerebrovascular diseases, peripheral vascular diseases and the like, and become a new treatment idea of various ischemic diseases. However, the existing problems of limited source of endothelial progenitor cells, limited angiogenesis capacity and the like still exist.
Disclosure of Invention
The invention aims to provide a novel endothelial progenitor cell with super angiogenesis capacity derived from human endometrial tissue.
It is another object of the present invention to provide a method for culturing and isolating said human endometrial tissue-derived endothelial progenitor cells.
The invention also aims to provide the application of the novel endothelial progenitor cells.
The inventors noticed an interesting clinical phenomenon: the incidence of cardiovascular events in premenopausal women is significantly lower than in men of the same age group, but this advantage of low incidence in women is lost post-menopause. This phenomenon has brought new hopes for our treatment of ischemic diseases.
The existing research results show that although the hormone level of women before and after menopause is greatly changed, the situation of increasing the incidence rate of cardiovascular events of postmenopausal women cannot be obviously inhibited by only supplementing hormone. On the other hand, endometrium has super-strong regeneration capability, and its functional layer can be periodically proliferated, secreted and desquamated, i.e. the physiological period of premenopausal female is periodically changed. The above phenomena all suggest that the human uterus has stem cells with strong regeneration capability.
In previous mouse studies, the inventors have observed that, after myocardial infarction, cells derived from uterus home to ischemic injury of heart, participate in angiogenesis and repair of ischemic injury, and further improve cardiac function of ischemic heart. Therefore, whether cells participating in angiogenesis exist in the human uterus or not can be used as a beneficial supplement of bone marrow-derived angiogenic cells, and how to separate and culture the cells with strong angiogenic capacity in the human uterus and use the cells for treating ischemic diseases to realize therapeutic angiogenesis is a task with great clinical application prospect and challenge.
First, the present invention proposes a novel human endometrial tissue-derived endothelial progenitor cell having superior angiogenesis ability, which can be isolated from human endometrial tissue.
Secondly, the invention provides a separation culture method of the new human endometrial tissue-derived endothelial progenitor cells with super-strong angiogenesis capacity, which comprises the steps of digesting human endometrial tissue by an enzymatic hydrolysis method to obtain a single cell suspension, separating by adopting a density gradient centrifugation method to obtain a cell population containing the endothelial progenitor cells, inoculating the cell population into an endothelial progenitor cell culture medium, culturing, subculturing and purifying to finally obtain the new endothelial progenitor cells with super-strong angiogenesis capacity.
Specifically, the method for isolating and culturing endothelial progenitor cells derived from human endometrial tissue according to the present invention is performed as follows.
1) Taking PBS buffer solution containing 250-350 mug/ml collagenase III and 30-50 mug/ml DNase I as digestive juice, mixing the digestive juice and human endometrial tissue according to the volume ratio of 2-4: 1, performing shake digestion in water bath at 37 ℃ for 40-60 min, extracting supernatant of a digestion product, adding 10vol% FBS-DMEM/F12 according to the volume ratio of 1: 1 for neutralization, and obtaining single cell suspension after digestion is stopped.
2) And filtering the single cell suspension by using a 200-mesh cell filter screen, and centrifuging at 1000-1200 r/min to obtain cell masses.
3) Three gradient density buffer solutions containing Percoll and Ads with the densities of 1.048-1.052 g/ml, 1.058-1.062 g/ml and 1.070-1.084 g/ml are respectively prepared and are respectively marked as solution A, solution B and solution C, wherein the solution B is marked by 0.01g/l phenol red.
4) And (3) transferring the liquid B with the same volume in the centrifuge tube added with the liquid A, gently inserting the centrifuge tube into the bottom of the centrifuge tube, and slowly dripping the liquid B below the liquid A to form a two-layer liquid separation interface.
5) Re-suspending the cell mass obtained in the step 2) by using a liquid C with the same volume as the liquid A to obtain a cell suspension, inserting the cell suspension into the bottom of a centrifuge tube, and slowly adding the cell suspension below the liquid B to form a gradient density buffer solution with a colorless-red-colorless three-layer liquid clear separation interface.
6) Centrifuging at 1300-1700 r/min, dispersing cells in different density areas of the buffer solution with gradient density, sucking cell suspensions in areas below the solution B and above the solution C, putting the cell suspensions into a centrifuge tube which is added with 15-30 ml of 10vol% FBS-DMEM/F12 in advance, and gently and uniformly mixing.
7) Centrifuging at 1000-1300 r/min to obtain cell masses, adding 10-20 ml of 10vol% FBS-DMEM/F12, gently mixing uniformly, centrifuging at 1000-1300 r/min and collecting the cell masses.
8) Taking 45-55 mg/L human fibronectin as a coating solution to cover the bottom surface of the culture dish, standing in a cell culture box at 37 ℃ for 0.5-1.5 h, and coating the culture dish.
9) The cell mass collected in the step 7) is re-suspended in EGM-2 medium, inoculated in a coated culture dish, and 5% CO at 37 DEG C 2 Culturing in a cell incubator, subculturing and purifying for not less than 1 time, and separating to obtain the endothelial progenitor cells derived from the human endometrial tissue.
In the process of the isolated culture of the endothelial progenitor cells derived from human endometrial tissue, the other operations are carried out at room temperature except for the specified temperature.
In the above isolation culture method of the present invention, the human endometrial tissue used is a tissue that has been washed with PBS and sufficiently minced.
Wherein the human endometrial tissue is digested with the digestive juice no less than 2 times, and the resulting cell suspensions are combined and digested.
The human endometrial endothelial progenitor cells obtained by separation, culture and purification have strong angiogenesis capacity and can be applied to the preparation of medicaments for preventing or treating ischemic diseases.
In vitro cell experiments prove that the endothelial progenitor cells derived from the human endometrium obtained by the in vitro isolated culture method can grow in a clone shape and have the basic characteristics of stem cells/progenitor cells.
Further, surface markers CD133, CD34, CD309 and CD45 characteristic to endothelial progenitor cells were selected, and the flow cytometry detection was performed on the human endometrial endothelial progenitor cells isolated and cultured in vitro, and the results of identification showed that CD133 was present + 、CD34 + 、CD309 + And CD45 - And meets the surface marker characteristics of endothelial progenitor cells.
The cells obtained by the invention are further proved to be endothelial progenitor cells through Dil-ac-LDL uptake of the endothelial progenitor cells and FITC-UEA-1 binding experiments.
The formation experiment of the stroma hose-shaped structure shows that the human endometrial endothelial progenitor cells obtained by the invention have the capacity of forming a tubular structure in vitro, namely participating in angiogenesis. The invention proves the angiogenesis capacity of the human endometrial endothelial progenitor cells for the first time, and brings hope to patients with ischemic diseases.
Drawings
FIG. 1 is a human endometrial endothelial progenitor cell clonogram.
FIG. 2 is a bar graph of flow identification of enriched human endometrial progenitor cells.
FIG. 3 is a graph showing the results of Dil-ac-LDL uptake and FITC-UEA-1 binding by human endometrial progenitor cells.
FIG. 4 is a graph showing the results of an in vitro tube structure formation experiment for human endometrial endothelial progenitor cells.
Detailed Description
The following examples are only preferred embodiments of the present invention and are not intended to limit the present invention in any way. Various modifications and alterations to this invention will become apparent to those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Example 1: and (3) separating and culturing the human endometrial endothelial progenitor cells.
1) Human endometrial tissue was harvested, placed in sterile 1 x PBS and moved to the clean bench as soon as possible.
2) In an aseptic culture vessel, human endometrial tissue was repeatedly washed with PBS 3 times to remove impurities such as surface blood.
3) Sufficiently crushing the cleaned human endometrial tissue to less than 1mm 3 。
4) Adding 3 times volume of PBS digestion solution containing 300 mug/ml collagenase III and 40 mug/ml DNase I to the minced human endometrial tissue, performing shake digestion for 45min in a water bath at 37 ℃ at a shaking speed of 110rpm, extracting a digestion product supernatant, and adding 10% FBS-DMEM/F12 in a volume ratio of 1: 1 for neutralization to terminate the digestion.
5) Adding the digestive liquid into the undigested tissue fragments again, and repeating the digestion process of the step 4).
6) And (3) filtering the single cell suspension obtained after the two-step digestion and neutralization by using a 200-mesh cell filter screen, and centrifuging for 5min at the speed of 1000r/min to obtain cell masses.
7) Preparing 3 gradient density buffers (A solution \ B solution \ C solution) containing Percoll and Ads, wherein the densities of the buffers are 1.050g/ml, 1.060g/ml and 1.082g/ml respectively, and the B solution is marked as red by 0.01g/l phenol red.
8) 10ml of solution A was added to the bottom of a new 50ml centrifuge tube.
9) And (3) gently inserting the pipette absorbed with 10ml of the liquid B into the bottom of the centrifuge tube, and gently and slowly dripping to form an interface for separating two layers of liquid.
10) And re-suspending the obtained cell mass by using 10ml of the solution C to obtain a cell suspension, sucking the cell suspension, inserting the cell suspension into the bottom of a centrifugal tube, and slowly adding the cell suspension below the solution B to form a gradient density buffer solution with a colorless-red-colorless three-layer liquid clear separation interface.
11) And (3) ensuring the acceleration and deceleration to be 0 at the rotating speed of 1500r/min, and centrifuging for 35min at room temperature.
12) After centrifugation, the cell suspensions in the areas below the solution B and above the solution C were aspirated into 50ml centrifuge tubes previously filled with 20ml of 10% FBS-DMEM-F12, and gently mixed.
13) And continuously centrifuging at the rotating speed of 1200r/min for 10min at room temperature.
14) Discarding the supernatant, adding 10ml 10% FBS-DMEM/F12, mixing gently, centrifuging at 1200r/min for 5min at room temperature to obtain cell mass.
15) 1ml of coating solution (50 mg/L human fibronectin) was added to a 60mm cell culture dish, and the dish was shaken to allow the coating solution to cover the entire bottom surface of the dish.
16) Standing in a cell culture box at 37 ℃ for 1h, and coating a culture dish.
17) Sucking up the coating solution, suspending the cell pellet obtained in step 14) with EGM-2 medium, inoculating in coated 60mm culture dish, and culturing at 37 deg.C with 5% CO 2 Culturing in a cell culture box.
18) And when the cells grow to 80-90% and are fused, digesting the cells by PBS digestive juice containing 0.25% of pancreatin and 0.02% of EDTA, carrying out passage, and continuously culturing the cells in an EGM-2 culture medium to obtain the purified endothelial progenitor cells derived from the endometrial tissue of the human.
The separated, cultured and purified human endometrial progenitor cells are photographed by an inverted phase contrast microscope under a normal growth state, and the culture form of living cells is shown in figure 1. It can be seen that the human endometrial endothelial progenitor cells obtained have a typical clonal-like growth cell morphology, with the characteristics of stem/progenitor cells.
Example 2: flow identification of human endometrial endothelial progenitor cells.
1) The human endometrial endothelial progenitor cell surface markers are selected from the group consisting of CD133, CD34, CD309 and CD 45.
2) Endothelial progenitor cells cultured to day 7, 14, and 21 were selected, and stained with the above-mentioned surface marker-corresponding antibody after conventional digestion.
3) Flow cytometry detects human endometrial endothelial progenitor cell surface markers.
The results of the flow assays at day 14 of culture are given in FIG. 2, and the bar graph shows the percent positivity for various surface markers.
FIG. 2 shows that human endometrial endothelial progenitor cells obtained by isolated culture exhibit CD133 + ，CD34 + ，CD309 + And CD45 - The surface marker characteristics of (a), which conform to the surface marker characteristics of endothelial progenitor cells.
Example 3: functional identification of the uptake of Dil-ac-LDL and binding of FITC-UEA-1 by human endometrial endothelial progenitor cells.
1) And (3) paving the human endometrial progenitor cells in a 6-well plate, putting a glass slide in the 6-well plate in advance, adding EGM-2 culture solution, observing the growth condition of the cells, and when the cells are fused to 50-70%.
2) 1ml of EGM-2 containing 10. mu.g/ml Dil-ac-LDL was added to the culture well and cultured in a cell culture chamber at 37 ℃ for 18 hours in the dark.
3) After 18h, the EGM-2 containing Dil-ac-LDL was aspirated, washed 3 times with PBS, and fixed with 4% paraformaldehyde at room temperature for 30 min.
4) PBS was washed 3 times.
5) Adding 1ml of FITC-UEA-1 working solution with the concentration of 10 mug/ml, and incubating for 2h at 37 ℃ in a dark place.
6) After 2h, the FITC-UEA-1 working solution was aspirated and washed 3 times with PBS.
7) 1ml of 3. mu.g/ml DAPI was added and incubated at 37 ℃ for 10min in the dark.
8) PBS was washed 3 times.
9) The slide is taken out and mounted with an anti-fluorescence quenching mounting agent.
10) A confocal laser microscope photograph is taken.
Fig. 3 shows the microscope photograph results under different wavelength laser excitation. Wherein A is a fluorescence pattern under 549nm wavelength laser excitation, and shows that human endometrial endothelial progenitor cells phagocytose Dil-ac-LDL into cytoplasm and show red fluorescence around cell nucleus. The fluorescence of the same visual field under excitation of laser with 494nm wavelength is shown in B, which shows that human endometrial progenitor endothelial cells can be combined with FITC-UEA-1 and show green fluorescence. In the fluorescence capture image C of the same field under 358nm wavelength laser excitation, nuclei are shown in blue. D is A, B and C are superposed fluorescence pictures. According to the results of the pictures in FIG. 3, it is suggested that the endothelial progenitor cells of human endometrium obtained by separation culture can take Dil-ac-LDL and combine FITC-UEA-1, and have the function characteristics of typical endothelial progenitor cells.
Example 4: and (3) identifying the in vitro angiogenesis capacity of the human endometrial endothelial progenitor cells.
1) And (3) sucking 100 mu l of Matrigel, and paving the Matrigel in a precooled 96-well cell culture plate.
2) After plating, the 96-well culture plate was placed at 37 ℃ in 5% CO 2 Incubating the cells in the incubator for 45min to gelatinize the cells.
3) Human endometrial endothelial progenitor cells are routinely digested to obtain a cell suspension.
4) Cell count at 3X 10 4 The density of each cell/well is inoculated on a 96-well cell culture plate, and the plate is placed at 37 ℃ and 5% CO 2 Culturing in the cell culture box.
5) After 3h, the tubule formation state of the human endometrial endothelial progenitor cells on Matrigel was observed under an inverted phase contrast microscope and photographed.
The apparent ring structure, i.e. the tubular structure surrounded by human endometrial endothelial progenitor cells, is shown in fig. 4, which shows that the human endometrial endothelial progenitor cells cultured on matrigel can form a characteristic tubular structure, which is also a typical functional characteristic of endothelial progenitor cells and endothelial cells of terminal cells.
In summary, the clonal cell growth pattern and the in vitro rapid expansion behavior observed in FIG. 1 suggest that the cells isolated and cultured by the method of the present invention have the characteristics of stem cells/progenitor cells, while the surface marker identification result in FIG. 2 indicates that the cells have typical surface markers of endothelial progenitor cells, and both results suggest that the endothelial progenitor cells are isolated and cultured. The functional identification result (figure 3) of taking Dil-ac-LDL and combining FITC-UEA-1 and the tubular structure form experiment (figure 4) show that the isolated and cultured human endometrial endothelial progenitor cells are functional endothelial progenitor cells with activity and angiogenesis capacity. The acquisition of the human endometrial endothelial progenitor cells brings hope for the treatment of patients with ischemic diseases.
Claims (2)
1. A separation and culture method of endothelial progenitor cells from human endometrial tissue is characterized in that single cell suspension is obtained by digesting human endometrial tissue through an enzymatic hydrolysis method, a cell population containing the endothelial progenitor cells is obtained by adopting a density gradient centrifugation method, the cell population is inoculated in an endothelial progenitor cell culture medium and is cultured and subcultured to obtain the endothelial progenitor cells, wherein collagenase III and DNase I are used in the enzymatic hydrolysis method, and the method is characterized in that
The human endometrial tissue used was cleaned with PBS and fully minced tissue;
the method comprises the following steps:
1) mixing the digestive liquid and human endometrial tissue according to a volume ratio of 2-4: 1 by taking PBS buffer solution containing 250-350 mug/ml collagenase III and 30-50 mug/ml DNase I as digestive liquid, performing shock digestion in water bath at 37 ℃ for 40-60 min, extracting supernatant of a digestion product, adding 10vol% FBS-DMEM/F12 according to a volume ratio of 1: 1 for neutralization, and obtaining a single cell suspension after digestion is stopped;
2) filtering the single cell suspension by using a 200-mesh cell filter screen, and centrifuging at 1000-1200 r/min to obtain cell masses;
3) respectively preparing three gradient density buffer solutions containing Percoll and Ads with the densities of 1.048-1.052 g/ml, 1.058-1.062 g/ml and 1.070-1.084 g/ml, wherein the three gradient density buffer solutions are respectively marked as solution A, solution B and solution C, and the solution B is marked by 0.01g/l phenol red;
4) Moving the liquid B with the same volume into a centrifugal tube added with the liquid A, gently inserting the centrifugal tube into the bottom of the centrifugal tube, and slowly dripping the liquid B below the liquid A to form a two-layer liquid separation interface;
5) re-suspending the cell mass obtained in the step 2) by using a liquid C with the same volume as the liquid A to obtain a cell suspension, inserting the cell suspension into the bottom of a centrifugal tube, and slowly adding the cell suspension below the liquid B to form a gradient density buffer solution with a colorless-red-colorless three-layer liquid clear separation interface;
6) centrifuging at 1300-1700 r/min, dispersing cells in different density areas of a gradient density buffer solution, sucking cell suspensions in areas below the solution B and above the solution C, putting the cell suspensions into a centrifuge tube which is added with 15-30 ml of 10vol% FBS-DMEM/F12 in advance, and gently and uniformly mixing;
7) centrifuging at 1000-1300 r/min to obtain cell masses, adding 10-20 ml of 10vol% FBS-DMEM/F12, gently mixing uniformly, centrifuging at 1000-1300 r/min and collecting the cell masses;
8) taking 45-55 mg/L human fibronectin as a coating solution to cover the bottom surface of the culture dish, standing in a cell culture box at 37 ℃ for 0.5-1.5 h, and coating the culture dish;
9) the cell mass collected in the step 7) is re-suspended in EGM-2 medium, inoculated in a coated culture dish, and 5% CO at 37 DEG C 2 Culturing in a cell culture box, subculturing and purifying for not less than 1 time, and separating to obtain the human endometrial tissue-derived endothelial progenitor cells.
2. The method of claim 1, wherein the method further comprises the step of
Digesting the human endometrial tissue with the digestive juice for at least 2 times, and combining cell suspensions obtained by digestion.
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