CN108210928B - Composition containing mesenchymal stem cells and vitamins and application thereof in tissue repair - Google Patents
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Abstract
The invention relates to a composition containing mesenchymal stem cells and vitamins and application thereof in tissue repair, belonging to the field of medicine. In the composition, the concentration of the mesenchymal stem cells is (1-7) x 105Per ml; the concentration of the vitamin is 0.1-10 mg/ml. In addition, the composition can also comprise 10-100ng/ml of natriuretic peptide and 0.1-0.5mg/ml of angiotensin II receptor antagonist (AngII receptor antagonist).
Description
Technical Field
The invention relates to a composition containing mesenchymal stem cells and vitamins and application thereof in tissue repair, belonging to the field of medicine.
Background
Mesenchymal Stem Cells (MSCs), which are non-hematopoietic stem cells having various differentiation potential, have attracted considerable interest to experts in the field of stem cell therapy and are considered as potential tools for cell regeneration therapy because they are easily isolated from bone marrow, adipose tissue, synovium, periostium, teeth, placenta, umbilical cord, etc., and have high differentiation potential, nutritional activity, immunoregulatory properties, and a huge donor pool. Nevertheless, the source and decontamination procedure of MSCs remains crucial for their therapeutic potential, and standardization of the optimal MSCs isolation procedure undoubtedly contributes to their optimal clinical use. MSCs, in their particular context, not only differentiate into multiple cell lineages, but also have immunosuppressive potency that makes them successful in allogeneic transplantation. In addition, the MSCs have the characteristics of actively tending to inflammatory or tumor tissues, promoting angiogenesis and the like in vivo, and have the advantages of easy separation, culture and amplification, easy transfection and stable expression by exogenous genes and the like in vitro.
Although stem cells always show good prospects in treatment of many diseases, current researches show that the detection rate ratio of transplanted MSCs in target tissues is low, and the repair effect of the MSCs on the target organ tissues is seriously influenced. This may be associated with colonization of MSCs in the target tissue, but a low proportion of MSCs that migrate through the blood vessels to the target tissue and colonize (i.e., home to) is a more important factor. Thus, improving the homing of MSCs to the target organs is critical for the treatment of disease by MSCs. Generally, MSCs are clinically used by infusion routes including vein transplantation, artery transplantation, and local transplantation. The homing rate of vein transplantation is low, but the method is high in safety. The homing rate of arterial grafts is significantly higher than that of venous grafts, but arterial grafts increase the probability of arteriolar occlusion. Local transplantation, although capable of injecting MSCs directly into the target organ, is associated with a high risk in clinical use, and local MSCs often die due to ischemia and malnutrition before they are therapeutically effective.
Liver cirrhosis is the terminal manifestation of various chronic liver diseases, and is characterized by liver fibroplasia and pseudolobule, at the moment, a large amount of normal liver cells are damaged and destroyed, the number of liver parenchymal cells is reduced, and when the damage of the liver exceeds the compensation function of the liver, the liver is seriously deficient in various functions of synthesis, storage, detoxification, immunity and the like, and then a series of corresponding clinical symptoms and manifestations are generated, so that the health of people is seriously threatened. Research and exploration in the stem cell field enable MSCs with the advantages of wide sources, weak immunogenicity, strong proliferation and differentiation capacity and the like to show attractive clinical application values in the aspect of treating end-stage liver diseases, and are expected to bring breakthrough to the treatment of related end-stage liver diseases.
However, in addition to the above-mentioned problem of low homing rate after transplantation, induced differentiation of stem cells after homing presents a problem in the application of MSCs to the treatment of cirrhosis. It has been shown that MSCs differentiate into myofibroblasts after transplantation into an individual, leading to collagen deposition, thus exacerbating liver fibrosis and leading to an increased disease status of the individual. Therefore, how to provide the homing rate of the target organ and induce its differentiation into the target tissue cell is an urgent technical problem to be solved in the art.
Disclosure of Invention
In a first aspect of the invention, a composition is provided that includes mesenchymal stem cells and a vitamin.
In one embodiment, the concentration of the mesenchymal stem cells in the composition is (1-7) x 105Per ml; the concentration of the vitamin is 0.1-10 mg/ml.
In another embodiment, the vitamin is selected from: one or more of vitamin A, vitamin B3, vitamin B5, vitamin B6, vitamin D, vitamin E, vitamin M (folic acid) and coenzyme Q10.
In further embodiments, the vitamins consist of vitamin B3 and vitamin M; the weight ratio of the two is 1: 0.1-0.5.
In yet another embodiment, the composition further comprises 10-100ng/ml of a natriuretic peptide and 0.1-0.5mg/ml of an angiotensin II receptor antagonist (AngII receptor antagonist).
In particular embodiments, the natriuretic peptide is Atrial Natriuretic Peptide (ANP), Brain Natriuretic Peptide (BNP), or C-type natriuretic peptide; preferably, C-type natriuretic peptide.
In yet another embodiment, the angiotensin II receptor antagonist is selected from eprosartan (eprosartan) and telmisartan (telmisartan).
In another embodiment, the mesenchymal stem cell may be a bone marrow, adipose tissue, synovium, periostium, tooth, placenta, or umbilical cord mesenchymal stem cell.
In a more specific embodiment, the composition comprises (1-7). times.105Mesenchymal stem cells per ml, vitamin B3 at 1-3mg/ml, vitamin M at 0.1-1.5mg/ml, natriuretic peptide at 30-50ng/ml and angiotensin II receptor antagonist at 0.2-0.4 mg/ml.
The second aspect of the invention provides a pharmaceutical preparation containing the composition, which consists of the composition and pharmaceutically acceptable auxiliary materials. Further, the pharmaceutical preparation is an injection.
In a third aspect, the invention provides the use of the above composition in the preparation of a medicament for tissue repair. The tissue repair is tissue repair after liver fibrosis.
In the use of the above-mentioned composition, the above-mentioned pharmaceutical composition can be prepared into an appropriate pharmaceutical preparation for convenient administration according to the condition of the animal and the site of administration, and for the present invention, the administration time and the administration frequency of the composition are determined according to the specific diagnosis result of the condition, which is within the technical scope of those skilled in the art. For example, it will be apparent to one of ordinary skill in the art that the effective dose of all drugs to humans can be converted to the effective dose of the drug to mice when the therapeutic regimen for mice is applied to humans.
In a fourth aspect, the present invention provides a process for the preparation of the above composition, which comprises
1) Preparing mesenchymal stem cells;
2) diluting the obtained mesenchymal stem cells to a treatment concentration by using a buffer solution, adding vitamin B3, vitamin M, natriuretic peptide and angiotensin II receptor antagonist into the cell suspension according to corresponding concentrations, and placing the cell suspension in an incubator for incubation for 1h to obtain the compound.
The invention discovers that vitamin B3 can improve the homing rate of stem cells to liver tissues and inhibit the stem cells from differentiating to fiber cells; the natriuretic peptide and angiotensin II receptor antagonist have the function of dilating blood vessels, which is beneficial to the homing of mesenchymal stem cells to target organ tissues through blood vessels and can prevent the vascular embolism phenomenon caused by high-concentration stem cells in the infusion process; furthermore, the invention also discovers that the natriuretic peptide and the angiotensin II receptor antagonist can also induce mesenchymal stem cells to differentiate into liver cells; the vitamin M (folic acid) has the inherent antioxidation function to improve hepatic fibrosis injury, and further has the synergistic effect on the three substances.
Detailed Description
The invention may be further understood by reference to the following examples, which illustrate some methods of making or using. However, it is to be understood that these examples do not limit the present invention. Variations of the invention, now known or further developed, are considered to fall within the scope of the invention as described herein and claimed below.
In the present invention, the concentration of mesenchymal stem cells, vitamin B3, vitamin M, natriuretic peptide and angiotensin II receptor antagonist in the composition of the present invention are only the concentrations of the stem cell preparation immediately after preparation, and the concentration of mesenchymal stem cells after preparation for a certain period of time cannot be understood as the concentration after preparation.
In addition, the mesenchymal stem cells used in the present invention have a wide source, and are not limited to mesenchymal stem cells of a specific source, which can be prepared by a method known in the art or obtained commercially.
For the composition of the present invention, other compositions than mesenchymal stem cells may be used alone as a stem cell inducer, or 1-3mg/ml vitamin B3, 0.1-1.5mg/ml vitamin M, 30-50ng/ml natriuretic peptide and 0.2-0.4mg/ml angiotensin II receptor antagonist
Example 1 preparation of adipose tissue mesenchymal stem cells (ADMSCs)
(1) Taking human adipose tissue, repeatedly centrifuging and washing with D-Hank's balanced salt solution with pH of 7.2-7.4, centrifuging to remove excessive blood;
(2) mincing fat tissue to 1-2mm3Adding digestive juice with the same volume as the adipose tissue into small pieces, digesting in a shaker at 37 deg.C and 190rpm for 30 min; digestion was stopped by adding an equal volume of BME medium containing 15% FBS; the digestive juice is D-Hank's balanced salt solution of pancreatin-EDTA with the concentration of 0.1 percent and collagenase type I with the concentration of 0.3 percent respectively;
(3) standing for layering, repeatedly beating bottom layer cells with D-Hank's balanced salt solution with pH of 7.2-7.4, and cleaning; filtering the washed liquid by a 100-mesh screen, removing undigested tissues, centrifuging and removing supernate, and mixing the adipose stem cell suspension and the erythrocyte lysate in a ratio of 1: 1, mixing and incubating for 2 minutes, centrifuging for 5min at 4 ℃ under the centrifugal force of 450g, and suspending adipose-derived stem cells by BME (basal adipose-derived activated carbon) culture medium;
(4) the obtained stem cells are processed according to 2-3 × 104/cm2Inoculating to culture flask, adding culture solution (BME culture medium containing L-glutamine 1mmol/L, basic fibroblast growth factor 20ng/ml, epidermal growth factor 5ng/ml, and leukemia inhibitory factor 5 ng/ml), standing at 37 deg.C and CO2Culturing in an incubator with the concentration of 5% and the humidity of 95%, absorbing and discarding the original culture solution after 1 day, replacing the fresh culture solution, discarding the non-adherent cells, replacing the culture solution every 24 hours later, adding 0.25% of pancreatin-EDTA into a culture bottle for digestion when the cells grow to reach 80% fusion, and carrying out passage according to the ratio of 1:3 to obtain the human adipose tissue mesenchymal stem cell stem cells after passage culture.
The obtained adipose tissue mesenchymal stem cells are subjected to antigen detection, and the adipose tissue mesenchymal stem cells with CD34, CD31 and CD45 account for less than 1 percent of the total stem cells; the proportion of adipose tissue mesenchymal stem cells with CD29, CD73, CD90, CD105 and CD49d in total stem cells is higher than 95%.
EXAMPLE 2 preparation of the composition
ADMSCs obtained in example 1 were diluted to 3X 10 in PBS buffer5Adding 2mg/ml vitamin B3, 0.8mg/ml vitamin M, 40ng/ml C-type natriuretic peptide and 0.3mg/ml eprosartan into the cell suspension according to corresponding concentrations, and placing the cell suspension in an incubator for incubation for 1h to obtain the medicine.
Example 3 preparation of the composition
ADMSCs obtained in example 1 were diluted to 6X 10 in PBS buffer5Adding 3mg/ml of vitamin B3, 1.5mg/ml of vitamin M, 50ng/ml of natriuretic peptide and 0.2mg/ml of eprosartan into the mixture, and placing the mixture in an incubator for incubation for 1 hour to obtain the oral liquid.
Example 4 in vitro transformation study of ADMSCs
The ADMSCs obtained in example 1 were resuspended in RPMI1640 medium containing 10% FBS and adjusted to 3X 105And each sample is placed in a culture dish at a concentration of 5 ml/dish, different components are added into different culture dishes, and the transformation efficiency of the ADMSCs into the liver-like cells or the hepatic stellate cells is observed.
The specific addition components of each group are as follows (final concentration):
group 1: without adding any component
Group 2: 4mg/ml vitamin B3 and 1.6mg/ml vitamin M
Group 3: 80ng/ml C-type natriuretic peptide and 0.6mg/ml eprosartan
Group 4: 2mg/ml vitamin B3, 0.8mg/ml vitamin M, 40ng/ml C-type natriuretic peptide and 0.3mg/ml eprosartan
Group 5: 5 μ g/ml M-CSF, 2 μ g/ml HGF and 10 μ g/ml FGF-4
Culturing each group of culture dishes in an incubator for 14 days, sucking the culture medium in the culture dishes, adding 4% paraformaldehyde, fixing for 1 hour at room temperature, then determining the positive rate of each group of cells expressing ALB, AFP and GFAP by adopting an immunofluorescence method, repeating for 3 times in parallel, and taking an average value.
The specific results are as follows:
| ALB(％) | AFP(％) | GFAP(％) | |
| group 1 | 8.3±4.6 | 2.1±0.4 | 10.8±2.1 |
| Group 2 | 45.4±5.9** | 25.2±3.8** | 5.6±1.4** |
| Group 3 | 21.7±5.1** | 6.8±2.6* | 9.3±2.6 |
| Group 4 | 76.5±6.3**## | 49.4±4.9**# | 1.7±0.5**## |
| Group 5 | 49.8±4.7** | 36.7±3.2** | 19.6±2.9** |
And represents p <0.05 or 0.01 compared to group 1 by Oneway-ANOVA test; # represents P <0.01 compared to the other groups
It is well known in the art that ALB and AFP are protein markers for the transformation of mesenchymal stem cells into hepatocyte-like cells, and GFAP is a marker for the transformation into hepatic stellate cells; in vivo stellate cells form myofibroblasts by transdifferentiation, which in turn leads to liver fibrosis. Therefore, the stem cells are induced to be transformed into liver-like cells, and the transformation of the stem cells into stellate cells is inhibited. The results show that the added components of the composition can better induce the transformation of the stem cells to the liver-like cells and inhibit the transformation of the stem cells to the stellate cells; although conventional inducers in the art can induce transformation of liver-like cells, a certain proportion of the cells are transformed into stellate cells, which may be an important reason why conventional stem cell compositions cannot perform therapeutic functions in vivo.
Example 5 Effect of the composition on CCl4 model of liver fibrosis in rats
Adult SD male rats weighing about 200g were selected and randomly divided into a control group, a cirrhosis group and an administration group. Injecting normal saline subcutaneously into control group, injecting 60% carbon tetrachloride-soybean oil subcutaneously into hepatocirrhosis group and administration group one, three, five weeks one, three, five, with dosage of 0.3ml/100g, and starting administration after 8 weeks; the tail vein of the control group and the liver cirrhosis group is infused with 2ml of normal saline, the tail vein of the administration group is injected with 2ml of composition solution, and the specific scheme of the administration group is as follows:
group 1: 3X 105PBS buffer of ADMSCs/ml
Group 2: 3X 105ADMSCs per ml, vitamin B3 4mg/ml and vitamin M1.6 mg/ml in PBS buffer
Group 3: 3X 105ADMSCs per ml, natriuretic peptide type C80 ng/ml and eprosartan 0.6mg/ml in PBS buffer
Group 4: 3X 105ADMSCs per ml, vitamin B3 2mg/ml, vitamin M0.8 mg/ml, natriuretic peptide type C40 ng/ml and eprosartan 0.3mg/ml in PBS buffer
Once weekly, 4 weeks after continuous dosing, rats were sacrificed, blood centrifuged, and serum Albumin (ALB), alkaline phosphatase (ALP), and alanine Aminotransferase (ALT) were measured. In addition, the liver tissue of each group of rats was cryosectioned, and the number of DAPI-labeled cells in the liver tissue was observed by randomly picking 5 fields under a fluorescence microscope.
The specific results are as follows:
1. composition for influencing ALB, ALP and ALT levels in serum
| ALB(g/L) | ALP(U/L) | ALT(U/L) | |
| Control group | 38.9±3.4 | 107±12 | 28±7 |
| Group of liver cirrhosis | 20.1±2.8## | 259±26## | 157±16## |
| Group 1 | 26.7±4.3** | 193±17** | 118±21** |
| Group 2 | 32.4±3.1** | 176±22** | 96±15** |
| Group 3 | 33.8±2.9** | 162±19** | 74±16** |
| Group 4 | 39.5±3.7** | 131±14** | 42±11** |
2. Effect of compositions on Stem cell homing Effect
This summary merely illustrates some embodiments which are claimed, wherein one or more of the features recited in the claims can be combined with any one or more of the embodiments, and such combined embodiments are also within the scope of the present disclosure as if they were specifically recited in the disclosure.
Claims (4)
1. A composition comprising mesenchymal stem cells and a vitamin; the concentration of the mesenchymal stem cells is (1-7) x 105Per ml; the concentration of the vitamin is 0.1-10 mg/ml;
the vitamins consist of vitamin B3 and vitamin M; the weight ratio of the two is 1: 0.1-0.5;
the composition also comprises 10-100ng/ml of C-type natriuretic peptide and 0.1-0.5mg/ml of eprosartan.
2. The composition of claim 1, wherein the composition comprises (1-7) x 105Mesenchymal stem cells per ml, vitamin B3 of 1-3mg/ml, vitamin M of 0.1-1.5mg/ml, natriuretic peptide type C of 30-50ng/ml and eprosartan of 0.2-0.4 mg/ml.
3. A pharmaceutical formulation comprising the composition of any one of claims 1-2, consisting of the composition and a pharmaceutically acceptable excipient.
4. Use of a composition according to any one of claims 1-2 for the manufacture of a medicament for tissue repair; the tissue repair is tissue repair after liver fibrosis.
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| CN106038596A (en) * | 2016-05-26 | 2016-10-26 | 深圳爱生再生医学科技有限公司 | Stem cell preparation capable of resisting hepatic fibrosis and preparation method of stem cell preparation |
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| 血管钠肽抑制肝纤维化的实验研究;赵鸽等;《中国普通外科杂志》;20110831;第830-834页 * |
| 重视肝纤维化的诊治;郭津生;《临床肝胆病杂志》;20180131;第16-18页 * |
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