阅读说明：本技术 一种间充质干细胞和外泌体联合制剂在制备心肌梗死药物中的应用 (Application of mesenchymal stem cell and exosome combined preparation in preparation of myocardial infarction medicament ) 是由 蒲锋星 于 2020-11-30 设计创作，主要内容包括：本发明提供了一种间充质干细胞和外泌体联合制剂在制备心肌梗死药物中的应用,间充质干细胞和外泌体联合制剂包括间充质干细胞和外泌体。间充质干细胞在培养过程中会分泌大量的外泌体,间充质干细胞分泌的外泌体能够调节局部组织微环境,促进血管新生,改善局部供血,恢复心室壁厚度、改善心梗大鼠的左心室射血分数,从而达到改善大鼠心脏功能的效果。并且间充质干细胞本身具有一定的治疗心肌梗死效果,外泌体悬液能够为间充质干细胞提供更好的生存环境,能够最大限度地维持细胞的活性和功能。(The invention provides an application of a mesenchymal stem cell and exosome combined preparation in preparation of a myocardial infarction medicament. The mesenchymal stem cells can secrete a large amount of exosomes in the culture process, and the exosomes secreted by the mesenchymal stem cells can adjust the local tissue microenvironment, promote angiogenesis, improve local blood supply, recover the ventricular wall thickness and improve the left ventricular ejection fraction of the myocardial infarction rat, so that the effect of improving the heart function of the rat is achieved. And the mesenchymal stem cells have a certain effect of treating myocardial infarction, and the exosome suspension can provide a better living environment for the mesenchymal stem cells and can maintain the activity and the function of the cells to the maximum extent.)

1. An application of a mesenchymal stem cell and exosome combined preparation in preparing a myocardial infarction medicament is characterized in that the mesenchymal stem cell and exosome combined preparation comprises mesenchymal stem cells and exosomes.

2. The use of claim 1, wherein the exosomes are homologous exosomes of mesenchymal stem cells.

3. The use of claim 1, wherein the mesenchymal stem cell is selected from a bone marrow mesenchymal stem cell, an umbilical cord mesenchymal stem cell, an adipose mesenchymal stem cell, or a dental pulp mesenchymal stem cell.

4. A preparation method of a combined preparation of mesenchymal stem cells and exosomes is characterized by comprising the following steps:

(1) inducing, culturing and differentiating the mesenchymal stem cells, and recovering the stem cells;

(2) obtaining homologous exosomes from the mesenchymal stem cells in the step (1) and preparing an exosome suspension;

(3) and (3) resuspending the stem cells obtained in the step (1) in the exosome suspension obtained in the step (2) to prepare a combined preparation.

5. The method according to claim 4, wherein the mesenchymal stem cells of 3-5 generations are selected in the step (1) and are induced and cultured in the alpha-MEM culture medium containing 10% fetal bovine serum, and when the cell fusion rate reaches 85-95%, the cells are digested and recovered.

6. The method according to claim 4, wherein the mesenchymal stem cells of 3 to 5 generations are selected in the step (2) and are induced and cultured in an alpha-MEM culture medium containing 10% fetal bovine serum, when the cell fusion rate reaches 70%, the alpha-MEM culture medium containing no serum is replaced, the culture supernatant is recovered after incubation for 72 hours in an incubator at 37 ℃, and the exosome is obtained after filtration and separation and purification by an ultracentrifugation method.

7. The method of claim 6, wherein the ultracentrifugation comprises the steps of: centrifuging at 4 deg.C for 5min at 800 Xg, and recovering supernatant; centrifuging at 2000 Xg for 10min, and recovering supernatant; 16000 Xg for 30 min, and recovering the supernatant; the supernatant was centrifuged at 150000 Xg for 90 minutes, and then discarded, and the supernatant was centrifuged at 150000 Xg for 90 minutes, and then discarded.

8. The method according to claim 4, wherein the stem cells are contained in the combined preparation in the step (3) in an amount of 0.5X 10⁶～2×10⁶One/100. mu.L.

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to an application of a mesenchymal stem cell and exosome combined preparation in preparation of a myocardial infarction medicine.

Background

Myocardial infarction is the necrosis of the heart muscle caused by acute and persistent hypoxia of the coronary arteries. Clinically, severe and persistent poststernal pain, rest and incomplete relief of nitrate medicines are caused, and the increased activity of serum myocardial enzyme and progressive electrocardiogram change are accompanied with arrhythmia, shock or heart failure. Acute myocardial infarction is a serious disease which endangers human health, is called as the first killer in developed countries, and is one of the main death reasons worldwide. The increase in the incidence of AMI places a heavy burden on individuals, families and society, controls the incidence of AMI, increases the level of AMI rescue, and is an important issue in the cardiovascular field.

Mesenchymal stem cells are multipotent stem cells with self-renewal and multi-item differentiation potential as mesenchymal stem cells of a tissue source, and are ideal seed cells in regenerative medicine due to convenient material acquisition and no immunological rejection. Research shows that the most important way for the stem cells to travel the biological functions is to regulate the functions of pathological tissues, promote the regeneration of blood vessels, accelerate the regeneration and repair of tissues and the like by secreting a large amount of bioactive factors. The secretion pathway of a large number of bioactive factors is mainly through exosomes, a kind of secretory vesicles coated by biological membranes and with diameters varying from 40 to 100 nm. More and more researches show that exosomes secreted by stem cells can reduce apoptosis, relieve inflammatory reaction, reduce myocardial infarction area in an acute stage, promote angiogenesis in a later stage, inhibit fibrosis and relieve myocardial reconstruction so as to improve the cardiac function after myocardial infarction.

The current cell transplantation therapy can promote local angiogenesis and myocardial regeneration of infarction so as to improve the cardiac function after myocardial infarction, and the safety and the effectiveness of the cell transplantation therapy on the treatment of cardiovascular diseases are proved to provide great hope for the repair of damaged heart, but the problems of low survival rate and colonization rate after transplantation, difficult differentiation of cardiovascular cell lineage and the like become key factors for restricting the curative effect of the cell transplantation therapy.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides an application of a mesenchymal stem cell and exosome combined preparation in preparation of a myocardial infarction medicament.

In order to realize the purpose of the invention, the invention is realized by adopting the following technical scheme:

an application of a combined preparation of a mesenchymal stem cell and an exosome in preparing a myocardial infarction medicament.

In the aspect of treating myocardial infarction, mesenchymal stem cells can migrate to the damaged part to secrete a large number of bioactive factors, so that the function of pathological tissues is regulated, the regeneration of blood vessels is promoted, and the regeneration and repair of tissues are accelerated. The exosome can conduct molecular signal conduction and biological signal information transmission among myocardial cells, cardiac fibroblasts, endothelial cells and cardiac stem cells, and is widely involved in physiological and pathological processes of a cardiovascular system, such as cell survival, myocardial infarction, ventricular remodeling and cardiac regeneration. Therefore, the combined use of the two can improve the survival of the mesenchymal stem cells and make the mesenchymal stem cells better play an effective role, thereby better treating the myocardial infarction.

Preferably, the exosomes are homologous exosomes of mesenchymal stem cells. In the process of culturing the mesenchymal cells, the method of starvation culture of the mesenchymal stem cells promotes the secretion of the bioactive substances of the mesenchymal stem cells to obtain homologous exosomes, and the recovery mode of the bioactive substances secreted by the homologous stem cells in the form of the exosomes is simpler.

Preferably, the mesenchymal stem cell is selected from a bone marrow mesenchymal stem cell, an umbilical cord mesenchymal stem cell, an adipose mesenchymal stem cell or a dental pulp mesenchymal stem cell.

The invention provides a preparation method of a combined preparation of mesenchymal stem cells and exosomes, which comprises the following steps:

(1) inducing, culturing and differentiating the mesenchymal stem cells, and recovering the stem cells;

(2) obtaining homologous exosomes from the mesenchymal stem cells in the step (1) and preparing an exosome suspension;

(3) and (3) resuspending the stem cells obtained in the step (1) in the exosome suspension obtained in the step (2) to prepare a combined preparation.

Preferably, the mesenchymal stem cells of 3-5 generations are selected in the step (1), and are induced and cultured in an alpha-MEM culture medium containing 10% fetal bovine serum, and when the cell fusion rate reaches 85% -95%, the cells are digested and recovered.

Preferably, the mesenchymal stem cells of 3-5 generations are selected to be induced and cultured in an alpha-MEM culture medium containing 10% fetal bovine serum, when the cell fusion rate reaches 70%, the alpha-MEM culture medium without serum is replaced, the culture supernatant is recovered after incubation for 72h in an incubator at 37 ℃, the culture supernatant is filtered and separated and purified by an ultracentrifugation method to obtain exosomes, and the exosomes are stored for later use at the temperature of 2-8 ℃. The culture supernatant was filtered through a 0.22 μm pore size filter.

And (2) washing the cells prepared in the step (1) twice by using normal saline, and suspending the cells in an exosome suspension which is re-warmed to room temperature to prepare a combined preparation. According to the invention, the method of starvation culture of the mesenchymal stem cells is adopted, the extract of the mesenchymal exosome can be obtained in a short time, the mesenchymal stem cell exosome with high concentration is obtained by concentration through an ultracentrifugation method, and the mesenchymal stem cells are resuspended in the concentrated mesenchymal stem cell exosome, so that the treatment effect is greatly improved.

Preferably, the ultracentrifugation method comprises the steps of: centrifuging at 4 deg.C for 5min at 800 Xg, and recovering supernatant; centrifuging at 2000 Xg for 10min, and recovering supernatant; 16000 Xg for 30 min, and recovering the supernatant; the supernatant was centrifuged at 150000 Xg for 90 minutes, and then discarded, and the supernatant was centrifuged at 150000 Xg for 90 minutes, and then discarded.

Preferably, the content of the stem cells in the exosome conjugate in the step (3) is 0.5 × 10⁶～2×10⁶One/100. mu.L.

Compared with the prior art, the invention has the beneficial effects that:

the invention provides an application of a mesenchymal stem cell and exosome combined preparation in preparation of a myocardial infarction medicament. The mesenchymal stem cells can secrete a plurality of exosomes in the culture process, the exosomes secreted by the mesenchymal stem cells can adjust the local tissue microenvironment, promote angiogenesis, improve local blood supply, restore the ventricular wall thickness and improve the left ventricular ejection fraction of the myocardial infarction rat, so that the effect of improving the heart function of the rat is achieved. By combining the mesenchymal stem cells with exosomes, the prepared drug can obviously improve the cardiac function of the myocardial infarction rat, recover the ventricular wall thickness and rhythmic motion, improve the left ventricular ejection fraction of the myocardial infarction rat and obviously reduce the infarct area of the myocardial infarction rat.

Drawings

FIG. 1A is a diagram showing the result of normal rat detection by an ultrasonic apparatus in an embodiment of the present invention;

FIG. 1B is a diagram showing the result of the ultrasonic apparatus detecting the myocardial infarction model rat in the embodiment of the present invention;

FIG. 2A is the cardiac ultrasound test results of rats in saline group according to the embodiment of the present invention;

FIG. 2B shows the results of the ultrasonic testing of the heart of rats in the combined preparation group according to the example of the present invention;

FIG. 3A is a statistical plot of left ventricular ejection fraction before and after treatment in an infarcted rat according to an example of the invention;

FIG. 3B is a statistical graph of the change in the short axis shortening rate of myocardial infarction rats before and after treatment in accordance with an embodiment of the present invention;

FIG. 4A shows Masson staining results of a saline group in an example of the present invention;

fig. 4B is the Masson staining results of the combined preparation groups in the examples of the present invention.

Detailed Description

The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.

Example 1

First, separation of umbilical cord mesenchymal stem cells

And (3) discarding redundant umbilical cord preservation solution in the obtained fresh umbilical cord, pouring 75% alcohol for disinfection for three minutes, taking out the umbilical cord, putting the umbilical cord into physiological saline containing double antibiotics for repeated cleaning, and removing residual alcohol on the surface. Cutting off the ligation part and discarding, dividing the remaining umbilical cord into small segments, cleaning repeatedly the residual blood, removing umbilical vein and umbilical artery, peeling off Wharton's gum, and cutting into pieces of 1mm³Tissue mass of size. Adding appropriate amount of conventional culture medium according to the amount of tissue block, mixing, spreading in 15cm culture dish, standing at 37 deg.C and 5% CO₂And culturing for 24h in a humidity saturation incubator, and supplementing 8mL of culture solution for each culture dish. Fluid infusion on day 5, fluid change on day 8, and cell replacementAnd after the fusion degree reaches more than 40-60%, discarding the supernatant, adding normal saline to wash twice, digesting for about 1.5min by using 0.25% pancreatin, adding stop solution to blow and collect cells, centrifuging, discarding the supernatant, adding a fresh culture medium to a 10cm culture dish to perform subculture, performing subculture amplification when the cell fusion reaches 80%, and taking the 3 rd-5 th-generation mesenchymal stem cells to perform subsequent experiments.

Extraction of umbilical cord mesenchymal stem cell exosome

Selecting 3-5 generation umbilical cord mesenchymal stem cells with good growth state, culturing with alpha-MEM culture medium containing 10% fetal bovine serum by volume fraction, when cell fusion rate reaches about 70%, replacing alpha-MEM culture medium without serum, incubating in incubator at 37 deg.C for 72h, recovering all culture supernatant, filtering with 0.22 μm filter membrane, centrifuging the filtered culture supernatant in centrifuge at 4 deg.C under gradient, centrifuging at 800 × g for 5min, and recovering supernatant; centrifuging at 2000 Xg for 10min, and recovering supernatant; 16000 Xg for 30 min, and recovering the supernatant; the supernatant was centrifuged at 150000 Xg for 90 minutes, and then discarded, and the supernatant was centrifuged at 150000 Xg for 90 minutes, and then discarded. Resuspending the precipitate with appropriate amount of normal saline, and storing at 2-8 deg.C as exosome suspension for preparing myocardial infarction therapeutic agent.

Culture of umbilical cord mesenchymal stem cells

Selecting 3-5 generation umbilical cord mesenchymal stem cells with good growth state according to 6000-8000/cm²The cells were inoculated in a cell culture flask and cultured in an α -MEM medium containing 10% by volume of fetal bovine serum. After inoculating and culturing for 2-4 days, the cell fusion rate reaches 80-90%, the cell growth state is good, all cells are recovered, the cells are washed for 2-3 times by using normal saline, and finally obtained cells are subjected to 2 × 10⁶The cell density of the cells is resuspended in 200 mu L of exosome suspension which is rewarmed to 18-24 ℃ to prepare a combined preparation.

Fourth, establishment and identification of myocardial infarction model of SD rat

Selecting an SD rat with the weight of 180-: 1. removing hair from the left anterior chest, disinfecting a drape, cutting the skin in the gap between the 3 rd and 4 th ribs beside the sternum, separating subcutaneous tissues and muscles bluntly, then cutting the 3 rd rib, separating and exposing the heart by using a suture, ligating the left anterior descending branch of the coronary artery at the 2mm position of the lower edge of the left auricle, observing that the front wall of the left ventricle and the apex of the heart are whitish after the ligation is finished correctly, then suturing the chest cavity from inside to outside in sequence, discharging the gas in the cavity to recover the negative pressure state in the chest cavity, and finally carrying out postoperative care on the model-making rat. After 1-2 weeks of SD rat modeling operation, an ultrasonic instrument is used for detecting the result of cardiac ultrasound to identify whether the myocardial infarction model is successfully modeled.

The model identification results are shown in fig. 1A-1B, fig. 1A is the identification result of a normal rat detected by an ultrasonic instrument, fig. 1B is the identification result of a myocardial infarction model rat detected by an ultrasonic instrument, and it can be seen that compared with the identification result of a normal rat, the motion function of the anterior wall of the left ventricle of a model-making rat is weakened and the wall of the ventricle is thinned, so that the success of the model can be determined.

And fifthly, treatment and ultrasonic detection of the SD rat myocardial infarction model.

And carrying out intramyocardial treatment of the combination of the mesenchymal stem cells and the exosomes on the rat with the success of myocardial infarction modeling determined by ultrasonic detection. Carrying out intraperitoneal injection anesthesia by using 1% sodium pentobarbital (40 mg/kg), then, fixing the patient on a test bench in a supine position, connecting an animal artificial respirator, wherein the respiratory frequency is about 86 times/minute, the tidal volume is 15ml, and the respiratory ratio is set as 1: 1. removing hair from left chest, sterilizing, spreading towel, cutting skin in the gap between 3 rd and 4 th ribs beside sternum, separating subcutaneous tissue and muscle, cutting 4 th rib, separating and exposing heart with suture, and performing intramyocardial injection to the combined preparation at 2-3 points at the edge of myocardial infarction region to treat myocardial infarction rats with injection dose of 150 μ L. And finally, suturing the thoracic cavity from inside to outside in sequence, exhausting gas in the cavity, carrying out postoperative care on the treated rat, and detecting the treatment effect of the myocardial infarction rat by ultrasonic at different time points after treatment.

The ultrasonic testing results are shown in fig. 2A-3B, wherein fig. 2A is the cardiac ultrasonic testing result of the rat in the physiological saline group, and fig. 2B is the cardiac ultrasonic testing result of the rat in the combined preparation group; the results show that compared with a normal saline control group, the mesenchymal stem cells combined with the exosome preparation can obviously improve the ventricular wall thickness and rhythmic motion after the myocardial infarction rats are treated; FIG. 3A is a statistical chart of left ventricular ejection fraction before and after treatment of physiological saline group/combined preparation group myocardial infarction rats; FIG. 3B is a statistical chart of the change of the short axis shortening rate of a physiological saline group/combined preparation group myocardial infarction rat before and after treatment; the results show that the left ventricular ejection fraction can be obviously improved after the mesenchymal stem cell combined with the exosome preparation is used for treating the myocardial infarction rats compared with the normal saline group.

Masson trichrome staining of heart tissue of six, SD rat

After the experimental period is finished, the rat is euthanized, the heart is taken and fixed in 4% paraformaldehyde solution for about 12 hours, the heart tissue is taken out, the heart area with infarction is cut out, after the heart area is fixed for 12 hours, the fixing solution is discarded, PBS is added for repeated cleaning until no paraformaldehyde fixing solution is left, PBS is discarded, 30% of cane sugar is added for dehydration until the heart tissue sinks to the bottom of the tube. Then, the heart tissue is embedded and sliced by using a frozen embedding medium. Finally, dyeing the slices with a mixture of Weigart iron hematoxylin A, B liquid in equal proportion for 5-10min, and washing with running water; then 1% hydrochloric acid alcohol differentiation liquid is used for differentiation for several seconds to tens of seconds, and the mixture is washed clean by running water; dyeing with ponceau acid fuchsin dye solution for 5-10min, and washing with running water; treating with phosphomolybdic acid solution for 5min, pouring off phosphomolybdic acid solution on the slide, counterstaining with aniline blue dye solution for about 90s, pouring off dye solution on the slide, treating with 1% glacial acetic acid for about 1min, washing the section until no blue color is removed, dehydrating according to the program, sealing with neutral resin, and collecting the result with a body type microscope. The results are shown in fig. 4A and 4B, wherein fig. 4A is the Masson staining result of the normal saline group; FIG. 4B shows Masson staining results for the combined preparation group; the results show that the combined preparation group can significantly reduce the infarct area of the infarcted area of the rat after treating the myocardial infarction rat compared with the normal saline control group, thereby indicating that the combined preparation of the mesenchymal stem cells and the exosomes prepared by the invention can be effectively applied to the field of myocardial infarction diseases.

The above description is only an embodiment of the present application, but the scope of the present application is not limited thereto, and any changes or substitutions within the technical scope of the present disclosure should be covered by the scope of the present application. Therefore, the protection scope of the present application shall be subject to the protection scope of the claims.