WO2020135345A1 - Use of human endometrial mesenchymal stem cells for improving damaged myocardial cells - Google Patents

Abstract

Use of human endometrial mesenchymal stem cells for improving damaged myocardial cells. A stem cell formulation is prepared by resuspending human endometrial mesenchymal stem cells in PBS, and then is injected into a mesenchymal injury site of model rats. Observation of cardiac function changes by using cardiac color Doppler ultrasound, observation of myocardial cell activity and energy metabolism by using PET-CT and observation of the degree of fibrosis by using Masson staining show that compared with human bone marrow mesenchymal stem cells, human endometrial mesenchymal stem cells have better effects of improving cardiomyocytes activity, energy metabolism, cardiac fibrosis, etc., and finally achieve the purpose of repairing injured myocardium.

Description

Application of human endometrial mesenchymal stem cells in improving injured cardiomyocytes Technical field

The invention relates to mesenchymal stem cells, in particular to mesenchymal stem cells derived from human endometrium. The human endometrial mesenchymal stem cells of the present invention can improve the activity and function of damaged myocardial cells, and thus can better repair heart diseases caused by dysfunction of myocardial cells.

Background technique

With the accelerated development of the economy, human lifestyles and dietary structures are constantly changing, and coronary atherosclerotic heart disease continues to grow. It can cause local myocardial cell apoptosis, myocardial cell fibrosis, energy metabolism disorders, etc., and eventually heart failure. Heart failure seriously affects people's quality of life and is an important cause of human death.

Stem cells are a kind of cells with the potential of self-replication, renewal and multi-directional differentiation, and have the potential to repair damaged tissue cells. In recent years, they have become an ideal seed cell in the field of disease treatment and tissue engineering. Possible new and effective treatment for curative diseases.

Regenerative medicine continues to develop, and stem cell transplantation to repair damaged cardiomyocytes has become the most promising treatment for cardiovascular diseases. Among them, the remodeling of myocardial function is the key, including the improvement of cardiomyocyte activity, the improvement of cardiomyocyte energy metabolism, and the improvement of myocardial fibrosis.

The ideal seed cells that can effectively improve the activity and function of damaged myocardium through regenerative medicine should have strong proliferative ability, good differentiation, low immunogenicity, easy access and strong secretion ability. The most important thing is that they should have strong The ability to form blood vessels.

The 201810930181.6 patent application relates to a method for separating and culturing mesenchymal stem cells derived from human endometrial tissue, which isolates and cultures a human endometrial mesenchymal stem cell with strong repair and regeneration ability from human endometrial tissue, and It is proved that the mesenchymal stem cells have stronger proliferative ability, that is, have stronger vitality and the ability to quickly obtain more seed cells.

technical problem

The object of the present invention is to provide the application of human endometrial mesenchymal stem cells in improving damaged myocardial cells.

Technical solution

The inventors have shown through mouse studies that after ligating the left coronary artery of the mouse, the uterine cells of the mouse homing to the myocardial injury area participate in the repair of myocardial cells and improve cardiac function, and uterine-derived cells can significantly increase blood vessel formation. Therefore, there are stem cells with strong repair and regeneration ability in the human uterus.

The inventor has isolated and cultured human endometrial mesenchymal stem cells from human endometrial tissue. The present invention further verifies that such human endometrial mesenchymal stem cells have a stronger ability to promote blood vessel formation, can better improve the activity of injured myocardial cells, and improve the energy metabolism and fibrosis degree of injured myocardium.

Based on this, the present invention uses the cultured human endometrial mesenchymal stem cells as medicines for improving the damage to cardiomyocytes.

Specifically, the present invention resuspends the cultured human endometrial mesenchymal stem cells in PBS and mixes them to prepare a human endometrial mesenchymal stem cell preparation for improving damaged myocardial cells.

More specifically, the present invention uses the cultured human endometrial mesenchymal stem cells of the P3 to P6 generation to prepare the human endometrial mesenchymal stem cell preparation.

In the human endometrial mesenchymal stem cell preparation prepared by the present invention, it is preferable to contain (3-7)×10 ⁷ human endometrial mesenchymal stem cells per 1 ml of the stem cell preparation.

Beneficial effect

The present invention has conducted verification studies on the above-mentioned human endometrial mesenchymal stem cell preparations to improve the activity and function of injured myocardial cells in vitro and in vivo.

A single cell suspension of human umbilical vein endothelial cells supplemented with human endometrial mesenchymal stem cell culture supernatant was inoculated on Matrigel Matrigel, and in vitro experiments proved that human endometrial mesenchymal stem cells can promote umbilical vein endothelial cells in Matrigel The tubules on the formation.

The left anterior descending coronary artery of nude rats was ligated for modeling, and the endometrial mesenchymal stem cell preparation was locally injected into the myocardial injury of the successfully modeled nude rats. Cardiac ultrasonography was used to observe changes in cardiac function, PET-CT to observe myocardial cell activity and Energy metabolism and Masson staining to observe the degree of fibrosis. In vivo experiments have shown that human endometrial mesenchymal stem cell preparation can better repair damaged myocardium by better improving the activity of injured myocardial cells, energy metabolism and fibrosis. The role.

BRIEF DESCRIPTION

FIG. 1 is a morphological diagram of human endometrial mesenchymal stem cells and bone marrow mesenchymal stem cells in promoting blood vessel formation in vitro.

Figure 2 is the result of echocardiographic evaluation of left ventricular function changes in nude rats before and after modeling in each group.

Figure 3 is a PET-CT assessment of the myocardial cell activity and energy metabolism of nude rats before and after modeling in each group.

Figure 4 is Masson's three-color staining showing the changes of fibrosis in the injured myocardial area of nude rats in each group after treatment.

Embodiments of the invention

The following embodiments are only preferred technical solutions of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Example 1: Preparation of human endometrial mesenchymal stem cell preparation.

Human endometrial mesenchymal stem cells were cultured in 10% FBS-DMEM/F12 medium containing 1% penicillin, and the culture bottle was placed in a 37°C 5% CO ₂ incubator every 3 to 4 Change the medium every day. When the cell fusion reaches 80-90%, it is digested with 0.05% trypsin solution (containing 0.02% EDTA) and passaged at a ratio of 1:3 to obtain the first generation of human endometrial mesenchymal stem cells, denoted as P1. Repeat the last operation to prepare P3～P6 generation endometrial mesenchymal stem cells.

Select P3～P6 generation cells with good growth status. When the cell fusion reaches 80～90%, rinse with PBS buffer three times. After washing FBS, add 0.05% trypsin solution (containing 0.02% EDTA) for digestion for 3～4min. Observe the cells rounded under a microscope, add 10% FBS-DMEM/F12 to stop the digestion, inhale into a 15ml centrifuge tube and centrifuge, discard the supernatant to retain the cell mass.

After washing the residual serum with the PBS solution, the resulting cell mass was resuspended with PBS solution at a concentration of (3-7)×10 ⁷ /ml to prepare (3-7)×10 ⁶ /100µl human endometrial interstitial filling. Qualitative stem cell preparation.

All the above operations for preparing human endometrial mesenchymal stem cell preparations are performed in a sterile operation table, and the principles of aseptic operation are performed in the production, transportation, and use.

Example 2: Human endometrial mesenchymal stem cells Matrigel Matrigel promotes angiogenesis in vitro.

Human endometrial mesenchymal stem cells were used as the experimental group, and human bone marrow mesenchymal stem cells were used as the control group.

Human endometrial mesenchymal stem cells and human bone marrow mesenchymal stem cells were taken and seeded on 12-well plates, respectively, with 10% FBS-DMEM/F12 or 10% FBS-IMDM normal culture medium, when the cells grow to 70 ~ 80% confluence, replace with 2% FBS-DMEM/F12. At the same time add 2% to the blank board FBS-DMEM/F12 as a blank group.

After the above groups continued to cultivate for 4 days, the cell culture supernatants of each group were collected to conduct an in vitro tubular structure formation experiment.

Before the experiment, the Matrigel Matrigel was placed in a refrigerator at 4°C overnight and melted to a liquid state. The loaded pipette tips and 96-well plates were kept in the refrigerator at -20°C overnight, keeping the temperature low.

Aspirate 100µl Matrigel with a pre-chilled pipette tip and lay it on a pre-chilled 96-well cell culture plate. All the above operations are performed on ice.

After plating, the 96-well cell culture plate was placed in an incubator at 37°C and 5% CO ₂ for more than 45 minutes and allowed to solidify.

While waiting for the gel, 0.05% trypsin was used to digest human umbilical vein endothelial cells, and the suspensions of human umbilical vein endothelial cells were prepared with the cell culture supernatant of each group collected above.

For cell counting, prepare the cell suspension to 3×10 ⁵ /ml, and inoculate 3×10 ⁴ cells per well on the 96-well cell culture plate paved with Matrigel matrigel, and place at 37℃, 5% CO ₂ Incubate in the incubator.

After culturing for 3h, the tube formation status of endothelial cells on Matrigel was observed and photographed under an inverted microscope. The results are shown in Figure 1.

In Figure 1, A is the tubular structure of human endometrial mesenchymal stem cell supernatant to promote the formation of umbilical vein endothelial cells; B is the human bone marrow mesenchymal stem cell supernatant to promote the formation of umbilical vein endothelial cells; C is the blank culture medium to promote the umbilical cord A tubular structure formed by vein endothelial cells. The greater the number of tubular structures in the figure, the stronger the paracrine function of promoting the formation of tubular structures of umbilical vein endothelial cells, and the stronger the ability of forming blood vessels.

Matrigel Matrigel's in vitro angiogenesis test proves that human endometrial mesenchymal stem cells have stronger ability to promote angiogenesis of human umbilical vein endothelial cells than human bone marrow mesenchymal stem cells of the same age and same generation.

Example 3: Experiment of repairing damaged myocardium by human endometrial mesenchymal stem cells.

The left anterior descending coronary artery of the nude rat was ligated to model. One week later, the injured myocardial tissue was injected with human endometrial mesenchymal stem cells to treat myocardial infarction.

Take 200～250g nude rats, prepare the skin by electric push before operation, and inhale 3% isoflurane mixed with oxygen for general anesthesia.

After the anesthesia was sufficient, the nude rats were intubated with an 18G intravenous indwelling needle, connected to a small animal ventilator V8S to assist mechanical ventilation, tidal volume 5-7ml, frequency 75 times/min. Fix the extremities on the operating table covered with a 37°C constant temperature pad in the supine position. The skin of the surgical field is disinfected with 75% alcohol, cut the skin, subcutaneous, and muscular layer and open the chest layer by layer. Cut the third and fourth ribs along the front line of the left armpit. The saline gauze blocks the lung tissue, cuts the pericardium along the heart, and separates the pericardium to expose the heart. Find the pink anterior descending coronary artery between the cone of the pulmonary artery and the root of the left atrial appendage, and ligature the left anterior descending coronary artery with a 7/0 round needle nylon thread at a depth of 1 to 2 mm at 3 to 5 mm of the lower edge of the left atrial appendage. Width 3mm. The damaged myocardium is pale and the movement is weakened, which indicates that the modeling is successful.

After successful modeling, the chest cavity was closed layer by layer with 3/0 nylon sutures. Before pulling out the endotracheal tube, subcutaneous injection of long-acting penicillin 150000U/kg. Observe the reaction of the nude rats closely, and give oxygen inhalation when the nude rats show strong spontaneous breathing and try to get out of the ventilator by themselves. When the breathing and heart rate were observed to be relatively stable, the endotracheal tube was removed.

The modeled nude rats were placed on a 37°C thermostat for 30 minutes, re-warmed in a separate cage, and then placed in a large cage when they were completely free to move.

After 1 week of echocardiography, 18 nude rats that meet the enrollment criteria were selected and randomly divided into an experimental group, a control group, and a blank group, and proceeded to the next experiment.

Repeating the thoracotomy step of the above model, all nude rats were selected 3 injection points, 2 injured myocardial border area and 1 injured myocardial central area. Insulin syringes were used to locally inject 60 µl (1.8 to 4.2×10 ⁶ cells) of stem cell preparations into the myocardium at the above three locations. The experimental group was injected with human endometrial mesenchymal stem cell preparation, the control group was injected with human bone marrow mesenchymal stem cell preparation, and the blank group was injected with an equal volume of PBS solution. After the injection is completed, repeat the steps of modeling and closing the chest cavity.

Example 4: Echocardiography was used to detect the cardiac function changes of the nude rats modeled in each group.

Nude rats were modeled before and after 1 week, and 1 or 2 weeks after transplantation of stem cell preparations. Echocardiography was used to evaluate the cardiac function of nude rats, and the nude rats were successfully screened.

Induce anesthesia in nude rats with 2.0% isoflurane mixed oxygen, remove the median hair, take the supine position on a far infrared thermostatic pad at 37°C, and continue to inhale the isoflurane mixed oxygen in the nasal mask.

Echocardiographic probes take long-axis and short-axis B-ultrasound images and short-axis M-ultrasound images. Ultrasound records analyze left ventricular end-systolic diameter (LVIDs) and left ventricular end-diastolic diameter (LVIDd). The left ventricular ejection is automatically calculated by the ultrasound machine Score (EF), left ventricular short axis shortening rate (FS).

A typical model of a nude rat showed a thinning of the anterior wall of the left ventricle after 1 week. The short-axis view of the left ventricle showed that the myocardial structure of the anterior wall and anterior wall of the left ventricle disappeared, the echo increased, and the movement amplitude decreased or disappeared.

The criteria for successful modeling into cell transplantation experiments are: objective indicators FS <30%, EF <60%; subjective indicators judge that there is at least one level of anomalous contraction of the anterior wall. Those who meet the two indicators at the same time are selected to enter the next experiment.

In Fig. 2, A is LVIDd, indicating the left ventricular end-diastolic diameter, B is LVIDs, indicating the left ventricular end-systolic diameter, C is EF, indicating the ejection fraction, and D is FS, indicating the left ventricular short axis shortening rate.

If the experimental results of nude rats immediately after modeling show that FS <30% and EF <60%, it indicates that the modeling was successful.

With the extension of time, the values of LVIDd and LVIDs in the experimental group and the control group gradually decreased compared with those after modeling, while EF and FS increased gradually after modeling, while the blank group showed no significant improvement. At the same time, the experimental group increased significantly compared to the control group, and this difference became more pronounced with time.

The above experimental conclusions prove that cell transplantation can significantly improve the damaged heart function, and human uterine mesenchymal stem cells are better than human bone marrow mesenchymal stem cells.

Example 5: PET-CT evaluation of cardiomyocyte activity and energy metabolism of nude mice modeled in each group.

PET-CT was used to observe the effect of cell transplantation on glucose metabolism in myocardial infarction area before and after 1 week of model building in nude rats and 2 weeks after transplantation of stem cell preparations, so as to reflect the activity of cardiomyocytes and energy metabolism of cardiomyocytes.

Nude rats in each group were anesthetized with 2.0% isoflurane mixed with oxygen. ¹⁸ F-FDG 6MBq/body was injected into the tail vein. After 40 minutes, they were placed in a constant temperature Minerve small animal compartment at 37°C to maintain the animal’s temperature and placed in Mico-PET/ On the CT scan bed, the neck to the upper abdomen were selected for PET-CT scan acquisition imaging, during which 2.0% isoflurane mixed oxygen was used to maintain anesthesia.

The 3D Order Subsets Expectation Maximization (OSEM) algorithm based on the Monte Carlo system model was used for image reconstruction. The acquisition time is set to 20 minutes, the isotope selection is F-18, and the remaining parameters are selected by the system default. After the acquisition is completed, enter the radiopharmaceutical dose and measurement time in the pop-up dialog box, and the system will automatically complete the reconstruction. Data reconstruction first selects the filter back projection method, and then uses the two-dimensional ordered subset maximum expected value method to reconstruct the image again. Use PMOD software for image analysis.

The experimental results in Figure 3 show that the myocardial metabolic activity in the infarcted area of the blank group (PBS) is significantly reduced, and the FDG uptake is significantly reduced; and after cell transplantation, the myocardial metabolic activity in the infarcted area can be improved and the myocardial uptake of FDG in the infarcted area can be improved, and The improvement effect of the experimental group is more obvious. It is suggested that transplantation of human endometrial mesenchymal stem cells can improve the cell activity and energy metabolism of injured myocardium, thereby promoting the repair of injured myocardium.

Example 6: Masson trichrome staining was used to detect the myocardial cell fibrosis of nude rats modeled in each group.

At the end of the fourth week after cell transplantation, at the end of the aforementioned experiment, all nude rats were sacrificed by intravenous injection of cardiac arrest solution under anesthesia induced by isoflurane mixed oxygen.

The heart was taken out, and the damaged myocardium in the left anterior descending branch supply area of the left ventricle was taken out and placed in paraformaldehyde for immersion and fixation. After 24 hours, it was taken out, dehydrated, transparent, wax-immersed, embedded, and sliced.

After dewaxing the tissue section with xylene and dehydrating with gradient alcohol, it was fixed in Bouin solution and washed with water. Weiger's Working solution (A+B=1:1) dyed black, washed clean; Biebrich dyed red, washed, P/P Acid treatment; Methly Blue dyed blue, washed water. Finally, they were washed with 1% glacial acetic acid, washed with water, and dehydrated with an alcohol gradient. Xylene was transparent and sealed with neutral gum. Place in a fume hood to air dry overnight, take a photo scan, and assess the degree of fibrosis in the injured myocardial area of each group.

Figure 4 shows the scanned image of Masson's three-color staining. The blue area in the figure is the fibrous tissue, which represents fibrosis. The results showed that the blue area of the experimental group after transplantation of human uterine mesenchymal stem cells (EMSCs) decreased, while the blue area of the blank group transplanted with PBS was large, suggesting that the degree of cardiac fibrosis of human endometrial mesenchymal stem cell transplantation was obvious Lighten.

Claims (4)

1. The application of human endometrial mesenchymal stem cell preparation as a drug for improving the damage of myocardial cells.
2. The application according to claim 1, wherein human endometrial mesenchymal stem cells are resuspended in PBS and mixed to prepare a human endometrial mesenchymal stem cell preparation for improving damaged myocardial cells.
3. The use according to claim 1 or 2, characterized in that the human endometrial mesenchymal stem cells are cultured human endometrial mesenchymal stem cells of generations P3 to P6.
4. The use according to claim 2, characterized in that each 1 ml of stem cell preparation contains (3-7)×10 ⁷ human endometrial mesenchymal stem cells.