Pharmaceutical application of human umbilical cord mesenchymal stem cells

Abstract

The invention provides pharmaceutical application of human umbilical cord mesenchymal stem cells, and belongs to the technical field of biological medicine. The research of the technical scheme finds that the Hcy content in blood can be effectively reduced by intravenous injection of the human umbilical cord mesenchymal stem cells. Benefited from the above beneficial hairstyle, the invention determines the application of the human umbilical cord mesenchymal stem cells to preparation of the hyperhomocysteinemia treatment medicine. According to the present invention, the drug is administered according to the single injection dose of 0.4-0.6 * 10 < 8 >, the Hcy content can be controlled at the normal level only by 3-4 times within 3 months to half a year, and the persistence is good; according to the application, the medical application of the human umbilical cord mesenchymal stem cells is expanded, a new optional scheme is provided for medicine treatment of hyperhomocysteinemia, folic acid, vitamin B6, vitamin B12 and the like do not need to be orally taken for a long time in the scheme, and a new treatment approach is provided for part of medicine taboo patients. The traditional Chinese medicine is remarkable in curative effect and has outstanding technical advantages.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to pharmaceutical use of human umbilical cord mesenchymal stem cells. Background Art

[0002] Mesenchymal stem cells are a type of pluripotent stem cells that have all the common characteristics of stem cells, namely, self-renewal and multidirectional differentiation capabilities. They are also the most widely used in clinical applications. When used in combination with hematopoietic stem cells, they can improve the success rate of transplantation and accelerate hematopoietic reconstruction. When patients receive high-dose chemotherapy, the infusion of mesenchymal stem cells together with hematopoietic stem cells can significantly accelerate the recovery time of blood cells in patients, and is safe and has no adverse reactions. Mesenchymal stem cells exist not only in the bone marrow, but also in skeletal muscle, umbilical cord blood, umbilical cord tissue, placental tissue, adipose tissue and other tissues. Because it can differentiate into a wide range of tissue types, it has a high clinical application value.

[0003] Mesenchymal stem cells are increasingly attracting attention due to their multidirectional differentiation potential, ability to support hematopoiesis and promote hematopoietic stem cell implantation, immune regulation, and easy isolation and culture. With the increasing maturity of mesenchymal stem cells and related technologies, clinical research has been carried out in many countries. As seed cells, they are mainly used clinically to treat a variety of refractory diseases of tissue cell and organ damage that the body cannot naturally repair; as immunoregulatory cells, they are used to treat immune rejection and autoimmune diseases. Specifically, umbilical cord mesenchymal stem cells are currently mainly used to repair or replace damaged tissues and organs, such as bones and muscles; they are also used to regulate immunity and treat immune-related diseases such as lupus erythematosus and scleroderma; in addition, studies have shown that umbilical cord mesenchymal stem cells can promote cerebral blood circulation and metabolism, delay aging, and improve memory; some studies have concluded that umbilical cord mesenchymal stem cells have a certain therapeutic effect on cerebrovascular diseases and Alzheimer's disease.

[0004] Hyperhomocysteinemia is a disease characterized by elevated homocysteine ​​in the blood, usually referring to homocysteine ​​content >15μmol / L. Homocysteine ​​is a non-protein amino acid that can be converted into methionine or cysteine ​​in a cycle with the help of B vitamins. Vitamin B deficiency can lead to hyperhomocysteinemia. Homocystinemia has been shown to be an independent risk factor for cardiovascular disease, cerebrovascular disease, cognitive impairment, and osteoporosis-related fractures. The treatment of hyperhomocysteinemia is mainly to reduce the level of homocysteine ​​in plasma, which is divided into general treatment and drug therapy. Among them, general treatment can increase the level of vitamin B in the body by improving eating habits, such as eating more foods rich in folic acid (such as green vegetables, oranges, and beans). However, the efficacy of most patients is not obvious, and drug therapy is still the first choice of treatment after diagnosis. At present, clinical treatment mainly involves oral folic acid or a combination of oral folic acid, vitamin B6, and vitamin B12. However, some patients still do not see definite efficacy, and those who do see definite efficacy also need to take medication for a long time to reduce HCY to normal levels, which often takes 2 to 3 years. In addition, there are certain limiting factors in the treatment of folic acid, vitamin B6, and vitamin B12. For example, large doses of folic acid may cause asthma, rash, etc., and increase the risk of prostate cancer; vitamin B12 is contraindicated for patients with Leber's disease. Summary of the invention

[0005] The first technical problem to be solved by the present invention is: how to expand the optional drug treatment options for hyperhomocysteinemia.

[0006] The second technical problem to be solved by the present invention is that the treatment course of the combined use of folic acid, vitamin B6 and vitamin B12 is relatively long and the efficacy needs to be improved.

[0007] The third technical problem to be solved by the present invention is: how to expand the medical use of human umbilical cord mesenchymal stem cells.

[0008] In order to achieve the above technical objectives, the present invention adopts the following technical solutions: Use of human umbilical cord mesenchymal stem cells for preparing a drug for treating hyperhomocysteinemia.

[0009] Preferably, the drug is a drug for reducing homocysteine ​​content in blood.

[0010] Preferably, the dosage form of the drug is an injection.

[0011] Preferably, the single injection dose of the drug is 0.4-0.6×10 8 indivual.

[0012] Preferably, the drug is injected 3 to 4 times, and the time interval between two adjacent injections is 1 to 1.5 months.

[0013] Preferably, the use includes: 1) Wash the umbilical cord with saline, cut it into segments, remove the arteries and veins, tear off the Wharton's jelly, cut it into pieces and add it to the culture bottle containing complete culture medium, and culture it statically for 6 to 8 days; 2) Use a pipette with a cut end to draw up half of the culture medium, discard it, and add half of the new culture medium; 3) Add trypsin to the culture flask, tap the flask wall after the cells become round, add stop solution, aspirate the cell suspension into a centrifuge tube, add physiological saline to the culture flask, blow and wash it into the centrifuge tube; centrifuge, discard the supernatant, add physiological saline to wash again by centrifugation, resuspend the precipitate with complete medium, filter through a cell sieve, rinse the sieve with complete medium, and count; spread the flask according to the number of cells so that the cell concentration is 10 4 ~2×10 4 / m1, cultured in an incubator; 4) Add trypsin digestion to the culture flask, add stop solution, collect all the liquid in a centrifuge tube, then add physiological saline to the culture flask, pipette and collect into the centrifuge tube; centrifuge and discard the supernatant, suspend the cell pellet with physiological saline to the target concentration, mix and take samples for counting and flow cytometry, and take the supernatant for endotoxin detection.

[0014] Preferably, in step 1), at least 8 mL of Wharton's gel is torn off, cut into pieces and divided equally into 4 bottles of 175 cm2 containers with 25 mL of complete culture medium. 2 In culture flask.

[0015] Preferably, in step 3), the centrifugation speed is 1200 rpm for 6 min.

[0016] Preferably, in step 4), the digestion temperature is 37° C., and the amount of the stop solution added is 10 mL.

[0017] As a preferred method, the method further comprises the following step 5): centrifuging and discarding the supernatant, suspending the precipitate with FBS, adding the cryopreservation mother solution, mixing and dispensing into cryopreservation tubes, and controlling the number of cryopreserved cells at 2×10 6 ~5×10 6 / mI range, cooled to -80℃ environment overnight and then transferred to liquid nitrogen tank for freezing.

[0018] The present invention provides a pharmaceutical use of human umbilical cord mesenchymal stem cells. The technical solution has found that intravenous injection of human umbilical cord mesenchymal stem cells can effectively reduce the Hcy content in the blood. Thanks to the above beneficial results, the present invention determines the use of human umbilical cord mesenchymal stem cells to prepare a drug for treating hyperhomocysteinemia; the drug is 0.4~0.6×10 8 The single injection dose of 100mg / mL is administered, and within 3 to 6 months, only 3 to 4 administrations are needed to control the Hcy content at a normal level, and the persistence is good. The present invention not only expands the medical use of human umbilical cord mesenchymal stem cells, but also provides a new optional solution for the drug treatment of hyperhomocysteinemia, which does not require long-term oral administration of folic acid, vitamin B6, vitamin B12, etc., and provides a new treatment approach for some patients with drug contraindications. The present invention has significant efficacy and outstanding technical advantages. DETAILED DESCRIPTION

[0019] The technical solutions in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application. In addition, ordinary technicians in this field know that with the development of technology and the emergence of new scenarios, the technical solutions provided in the embodiments of the present application are also applicable to similar technical problems.

[0020] In the description of the present application, it should be understood that, unless otherwise defined, all technical and scientific terms used in the present application have the same meaning as those commonly understood by those skilled in the art of the present application. In addition, any terminology used is only for the purpose of describing specific embodiments and is not intended to limit the present application.

[0021] In addition, in order to better illustrate the present application, numerous specific details are provided in the following specific embodiments. It should be understood by those skilled in the art that the present application can also be implemented without certain specific details. In some examples, methods, means, components and circuits well known to those skilled in the art are not described in detail in order to highlight the subject matter of the present application.

[0022] Preparation of human umbilical cord mesenchymal stem cells: 1. Wash the umbilical cord thoroughly with saline and cut it into small pieces. Remove the arteries and veins and tear off at least 8 ml of Wharton's Jelly. Cut it into pieces and divide it equally into 4 bottles of 175 cm2 tubes with 25 ml of complete culture medium. 2 Place in a culture bottle and culture for 7 days. On the 8th day, change the medium and perform subculture according to the growth situation.

[0023] 2. Decide whether to replace the medium in full or in half according to the cell growth and the color of the medium. Use a pipette with a cut end to discard half or all of the old medium, replace the pipette, and slowly add an equal amount of new medium to the non-cell culture surface of the culture bottle.

[0024] 3. Add 3 ml of 0.25% trypsin to each bottle. After the cells become round, tap the bottle wall. Add 10 ml of stop solution (2% FBS + a-MEM) to each bottle. Aspirate the cell suspension into two 50 ml centrifuge tubes. Add 10 ml of normal saline to each culture bottle, and wash and flush into a 50 ml centrifuge tube. Centrifuge at 1200 rpm for 6 minutes and discard the supernatant. Combine the precipitates into 1 tube, add 40 ml of normal saline and centrifuge and wash again. Resuspend the precipitate with 10 ml of complete culture medium, filter through a cell sieve, rinse the sieve with 5 ml of complete culture medium, and count. Spread the bottle according to the number of cells so that the cell concentration is 1~2×10 4 / m1, 37℃, 5% CO 2 Culture in an incubator.

[0025] 4. Add 3ml of 0.25% trypsin to each bottle, digest at 37℃ for 1min, add 10ml / bottle of stop solution, collect all the liquid into a 50ml centrifuge tube, then add 10ml of normal saline to each bottle, gently pipette and collect into a 50ml centrifuge tube. Centrifuge at 1200 rpm for 6min, discard the supernatant, suspend the cell pellet with 16ml of normal saline, mix well and take 1ml for counting and flow cytometry. Add normal saline to 40ml, take 500μl of supernatant for endotoxin detection, centrifuge at 1200rpm for 6min. Discard the supernatant, suspend the centrifugal pellet with FBS, then slowly add the cryopreservation mother solution, mix well and dispense into cryopreservation tubes, 1ml per tube. The number of frozen cells should be controlled between 2~5×10 6 / mI range. Place in a -80℃ medical refrigerator overnight using a program cooling box and then transfer to a liquid nitrogen tank.

[0026] 80 patients with moderate hyperhomocysteinemia were randomly divided into 4 groups after Hcy content was detected, with 20 patients in each group. The first group used the human umbilical cord mesenchymal stem cells prepared above at a concentration of 0.4-0.6×10 8 The second group used commercially available human umbilical cord mesenchymal stem cells (produced by Qingdao Aoke Biotechnology Co., Ltd.) at a dose of 0.4-0.6×10 8 The single injection dose was injected 4 times, and the interval between two adjacent injections was 1.5 months. The third group took folic acid, vitamin B6, and vitamin B12 orally, of which folic acid was 0.8 mg per day, vitamin B6 was 20 mg per day, and vitamin B12 was 0.1 mg per day until the end of the experiment. The fourth group was a blank control. Hcy content was regularly tested during the experiment.

[0027] Enzyme-linked immunosorbent assay was used to detect the Hcy content: the kit used a competitive method to detect the Hcy content in the sample; the sample was added to the enzyme-labeled well pre-coated with the antibody, and then the biotin-labeled recognition antigen was added, and incubated at 37°C for 30 minutes. The two competed with the solid-phase antibody to form an immune complex, and the unbound biotin antigen was removed by washing with PBST, and then avidin-HRP was added. The mixture was incubated at 37°C for 30 minutes, and the avidin-HRP was combined with the biotin antigen. After washing, the combined HRP catalyzed TMB (tetramethylbenzidine) into blue, which was then converted into yellow under the action of acid. There was an absorption peak at a wavelength of 450nm, and the absorbance value was negatively correlated with the concentration of the antigen in the sample.

[0028] Operation steps: Please equilibrate the kit at room temperature for half an hour before use.

[0029] Blank well: No sample is added, only color developing reagent A, B and stop solution are added for zero adjustment.

[0030] Standard wells: add 50 μl of diluted standard to each well; zero wells: add 50 μl of standard / sample diluent, and then add 50 μl of biotin antigen working solution.

[0031] Sample well: add 50 μl of sample, then add 50 μl of biotin antigen working solution.

[0032] Shake gently, cover with sealing film, and incubate in a 37°C incubator for 30 minutes.

[0033] Dilute the 25-fold concentrated washing solution with distilled water 25 times for later use.

[0034] First wash: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing solution, let it stand for 30 seconds and then discard, repeat this 5 times and pat dry.

[0035] Add 50 μl of avidin-HRP to the standard wells and sample wells, shake gently, cover with sealing film, and incubate in a 37°C incubator for 30 min.

[0036] Second wash: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing solution, let it stand for 30 seconds and then discard, repeat this 5 times and pat dry.

[0037] Color development: First add 50μl of color developer A to each well, then add 50μl of color developer B, gently shake to mix, and develop the color at 37℃ in the dark for 10 minutes.

[0038] Stop: Add 50 μl of stop solution to each well to terminate the reaction (the blue color immediately turns yellow).

[0039] Determination: Use the blank well as zero setting, and measure the absorbance (OD value) of each well in sequence at a wavelength of 450nm. The determination should be carried out within 10 minutes after adding the stop solution.

[0040] Calculation: The regression equation of the standard curve was calculated based on the concentration and OD value, and ELISAcalc was used for calculation. The logistic curve (four parameters) was selected as the fitting model.

[0041] The test results are shown in Table 1 below.

[0042] Table 1 Changes of mean plasma Hcy content (μmol / L) in patients in each experimental group over time

[0043] The above experimental results show that the injection of human umbilical cord mesenchymal stem cells can significantly reduce the Hcy content in the patient's plasma. Moreover, no adverse reactions occurred during the entire treatment, and the safety was good. In addition, 2 patients in Experimental Group 3 were always insensitive to oral medications and did not show definite therapeutic effects, while all subjects in Experimental Group 1 and Experimental Group 2 showed obvious therapeutic effects. It can be seen that the effectiveness of the present invention is higher than that of oral B vitamins. In addition, at 4.5 months of the experiment (that is, when Experimental Group 1 and Experimental Group 2 injected the present invention 3 times), the patient's Hcy content had reached a normal level. After 4 injections until 30 months of observation, the Hcy content did not rebound. It can be seen that the injection of umbilical cord mesenchymal stem cells 4 times can keep the patient's Hcy content at a normal level, and it can be considered that the degree of cure has been achieved. In contrast, after 4.5 months of oral B vitamins, the patient's Hcy content was still as high as 45.2μmol / L, and after 30 months of continuous oral medication, the Hcy content still did not reach a normal level. It can be seen that the efficacy of the present invention is better than oral B vitamins in all aspects, and the course of treatment is significantly shorter. In summary, after reading this detailed disclosure, it will be apparent to those skilled in the art that the aforementioned detailed disclosure may be presented only by way of example and may not be restrictive. Although not explicitly stated herein, it will be appreciated by those skilled in the art that this application is intended to encompass various reasonable changes, improvements and modifications to the embodiments. These changes, improvements and modifications are intended to be proposed by this application and are within the spirit and scope of the exemplary embodiments of this application.

[0044] It should be understood that in the foregoing description of the embodiments of the present application, in order to help understand a feature and to simplify the present application, the present application combines various features into a single embodiment or its description. However, this does not mean that the combination of these features is necessary, and it is entirely possible for those skilled in the art to extract some of the features and understand them as separate embodiments when reading the present application.

[0045] It should be understood that the embodiments disclosed herein are illustrations of the principles of the present application. Other modified embodiments are also within the scope of the present application. The embodiments disclosed herein are merely examples and not limitations, and the embodiments of the present application are not limited to the embodiments precisely described above.

Claims

1. Use of human umbilical cord mesenchymal stem cells for preparing drugs for treating hyperhomocysteinemia.

2. The use according to claim 1, characterized in that The drug is a drug that lowers the level of homocysteine ​​in the blood.

3. The use according to claim 1, characterized in that The dosage form of the drug is injection.

4. The use according to claim 3, characterized in that The single injection dose of the drug is 0.4~0.6×10 8 indivual.

5. The use according to claim 3, characterized in that: The drug is injected 3 to 4 times, and the time interval between two adjacent injections is 1 to 1.5 months.

6. The use according to claim 1, characterized in that: include: 1) Wash the umbilical cord with saline, cut it into segments, remove the arteries and veins, tear off the Wharton's jelly, cut it into pieces and add it to the culture bottle containing complete culture medium, and culture it statically for 6 to 8 days; 2) Use a pipette with a cut end to draw up half of the culture medium, discard it, and add half of the new culture medium; 3) Add pancreatin to the culture flask, tap the flask wall after the cells become round, add stop solution, aspirate the cell suspension into a centrifuge tube, add physiological saline to the culture flask, blow and wash it into the centrifuge tube; centrifuge, discard the supernatant, add physiological saline to centrifuge and wash again, resuspend the precipitate with complete medium, filter through a cell sieve, rinse the sieve with complete medium, and count; spread the flask according to the number of cells so that the cell concentration is 10 4 ~2×10 4 / m1, cultured in an incubator; 4) Add trypsin digestion to the culture flask, add stop solution, collect all the liquid in a centrifuge tube, then add physiological saline to the culture flask, pipette and collect into the centrifuge tube; centrifuge and discard the supernatant, suspend the cell pellet with physiological saline to the target concentration, mix and take samples for counting and flow cytometry, and take the supernatant for endotoxin detection.

7. The use according to claim 6, characterized in that In step 1), tear off at least 8 mL of Wharton's gel, cut it into pieces and divide it equally into 4 bottles of 175 cm2 tubes with 25 mL of complete culture medium. 2 In culture flask.

8. The use according to claim 6, characterized in that In step 3), the centrifugation speed is 1200 rpm and the duration is 6 min.

9. The use according to claim 6, characterized in that: In step 4), the digestion temperature is 37° C., and the amount of the stop solution added is 10 mL.

10. The use according to claim 6, characterized in that The following step 5 is also included): centrifuge and discard the supernatant, suspend the precipitate with FBS, add the cryopreservation mother solution, mix well and dispense into cryopreservation tubes, and control the number of cryopreserved cells at 2×10 6 ~5×10 6 / mI range, cooled to -80℃ environment overnight and then transferred to liquid nitrogen tank for freezing.

Images