CN114848674A - Application of stem cell particles in preparation of medicine for treating Parkinson's disease - Google Patents
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
- A61P25/16—Anti-Parkinson drugs
Abstract
The invention discloses an application of stem cell particles in preparing a medicament for treating Parkinson. The stem cell microparticle is a vesicular body with a phospholipid bilayer membrane structure, which is shed or secreted from a stem cell membrane, has a diameter ranging from 100nm to 1000nm, and contains components such as protein, lipid, mRNA, miRNA and the like. The invention discovers that the stem cell particles can effectively relieve the symptoms of the Parkinson's disease. Therefore, the stem cell microparticles have a prospect of being developed into a drug for treating Parkinson.
Description
Technical Field
The invention belongs to the field of biological medicines, and particularly relates to an application of stem cell particles in preparation of a medicine for treating Parkinson.
Background
Parkinson's disease is a chronic neurodegenerative disease affecting the central nervous system, mainly the motor nervous system. Its symptoms usually appear slowly over time, with the most obvious symptoms in the early stages being tremor, stiffness of the limbs, hypokinesia and gait abnormalities, as well as possible cognitive and behavioral problems; dementia is quite common in severely ill patients, with major depressive disorders and anxiety occurring in more than one third of cases. Other symptoms that may accompany include problems with perception, sleep, mood. Parkinson's treatment is a worldwide problem. At present, the treatment medicines, including dopamine supplement (MEDOPA, Benning, etc.) and anticholinergic medicines (dipheny, etc.), can only control symptoms, keep basic life quality and cannot delay the progress of diseases.
Mesenchymal stem cell-derived microparticles (MVs, abbreviated as stem cell microparticles) are vesicular bodies with a phospholipid bilayer membrane structure, which are shed or secreted from stem cell membranes, have diameters ranging from 100nm to 1000nm, and contain components such as proteins, lipids, mRNA, miRNA, and the like. And the expression of markers such as CD63 and CD81 can also be detected. MVs can interact with neighboring cells by plasma membrane fusion with target cells, or internalization through endocytosis, or interaction with cell surface receptors to affect intracellular signaling pathways, exchange bioactive molecules with neighboring cells, and participate in processes such as intercellular communication, cell migration, angiogenesis, and immunoregulation. Compared with stem cells, MVs have the advantages of small volume, stable property, easiness in storage and transportation, no cell activity, low immunogenicity, no risk of forming tumors and the like, and are high in safety and non-ethical dispute.
There is currently no report of stem cell microparticles on parkinson's treatment.
Disclosure of Invention
The invention aims to provide application of stem cell particles in preparing a medicament for treating Parkinson.
The above purpose of the invention is realized by the following technical scheme:
an application of stem cell microparticles in preparing a medicament for treating Parkinson's disease.
Further, the stem cell microparticle is a mesenchymal stem cell microparticle.
Still further, the stem cell microparticle is an umbilical cord mesenchymal stem cell microparticle.
Has the advantages that:
the invention discovers that the stem cell particles can effectively relieve the symptoms of the Parkinson's disease. Therefore, the stem cell microparticles have a prospect of being developed into a drug for treating Parkinson.
Drawings
Fig. 1 is a result of observing mesenchymal stem cells under a 100-fold microscope;
FIG. 2 is an electron microscope observation of stem cell microparticles;
FIG. 3 shows the results of stem cell microparticles CD63, TSG101, and CD9 protein detection;
FIG. 4 shows the NTA measurement results;
FIG. 5 shows the results of a rod fatigue test;
FIG. 6 shows the results of protein detection in striatum and substantia nigra of mice.
Detailed Description
The following examples are given to illustrate the essence of the present invention, but not to limit the scope of the present invention.
First, experimental material
The stem cell source is as follows: the 1 st generation stem cells (P0) are Wharton's jelly separated from umbilical cord tissue of newborn supplied by department of obstetrics in women's obstetrics and children's hospital of Huaxi, Sichuan university, and are obtained by tissue culture method. The maternal age was controlled at 20-32 years of age and both the maternal and infant were strictly screened for infectious and severe genetic diseases. The cell quality standard is as follows: CD105, CD90 and CD73 are more than or equal to 95 percent; CD45, CD19, CD34, CD11b and HLA-DR are less than or equal to 2 percent, and have the capacity of inducing tri-differentiation in vitro.
Stem cell culture medium: prepared according to the patent CN 112143695A.
Microscope and transmission electron microscope: microscope a leiacdmi 1 inverted microscope was used; hitachi (HT7800) was used as the transmission electron microscope.
Centrifuge and ultrahighspeed centrifuge: the centrifuge uses a Beckmann Coulter Allegrax-12 series centrifuge; the ultra-high speed centrifuge uses a Beckmann Coulter OptimaXPN series ultra-high speed centrifuge.
Nanoparticle Tracking Analysis (NTA): ZetaViewTwinPMX-220 from Particlemmetrix, Germany was used.
BCA protein quantification kit: purchased from Shanghai Biyun, cat # P0009.
SurePAGE-gel: purchased from aures biotechnology.
Detection of microparticle marker antibodies, CD63(AF5117), CD9(AF5193), TSG101(DF8427) were purchased from Affinity. Dopamine transporters (ab184451), tyrosine hydroxylases (ab137869), α -synuclein (ab138501), phosphorylated α -synuclein (ab51253), internal control GAPDH (ab8245) were purchased from abcam.
ChemiDoc TM A touch imaging system: the use of the Bio-Rad ChemiDoc TM MP。
MPTP (1-methyl-4-phenyl-1, 2,3, 6-tetrahydropyridine): MPTP was purchased from Tokyo and assigned the number M2690. 500mg of MPTP was dissolved in 50ml of PBS solution to constitute a solution of 10mg/ml for constructing a Parkinson's animal model.
Mice: c57BL/6 male 8 week mice were used for experiments, purchased from Beijing Huafukang Biotech GmbH.
Rotating rod fatigue tester: an XR1514 tester from Shanghai Soft information technology, Inc. was used.
Second, Experimental methods
1. Stem cell culture
Mixing 1ml of the 1 st generation mesenchymal stem cells with the culture medium, transferring the mixture into a 175mm cell culture dish by using a pipette, and placing the dish at 37 ℃ with 5% CO 2 And (5) incubating in a cell incubator. The culture medium was changed every three days and placed under a Leica inverted microscope to observe the morphology and the number of cells. After the cells had grown to approximately 70% -80% of the culture dish, 3ml of 0.25% trypsin was added and 5% CO was added at 37 deg.C 2 Digesting in incubator for 5 min. 5ml of PBS were then added and shaken well and the cell suspension was centrifuged for 3min at 20 ℃ in a 1000g centrifuge. After centrifugation, the supernatant was discarded, the pelleted cells were resuspended in medium and replated for subculture until passage 5 (P5).
2. Stem cell microparticle extraction and identification
When the 5 th generation mesenchymal stem cells are proliferated to more than 80% and fused, discarding cell supernatant, adding 5ml PBS, and washing for 3 times; the residual PBS in the flask was aspirated off, and the flask was incubated with serum-free medium at 37 ℃ in CO 2 Starvation culture is carried out in an incubator for 48 hours, and the stem cells are induced to release MVS. After starvation culture of the stem cells for 48 hours, the stem cells are placed in a sterile operating platform, the surfaces of the cells are blown and beaten for a plurality of times by an electric pipette gun, cell supernatants are collected into a 50ml centrifuge tube, the centrifuge tube is centrifuged at 400g at 4 ℃ for 10min, and then the centrifuge tube is centrifuged at 2000g at 4 ℃ for 20 min. The supernatant was carefully transferred to a new 50ml centrifuge tube and the pellet discarded. Transferring the collected supernatant to ultra-high speed centrifugeIn a centrifuge tube special for a heart machine; placing the centrifugal tube in an ultra-high speed centrifuge, strictly balancing by using an electronic scale, centrifuging at the temperature of 4 ℃ for 2 hours at 50000g, discarding the supernatant, washing the precipitate in the centrifugal tube by using PBS and transferring to another centrifugal tube, centrifuging again at the temperature of 4 ℃ for 2 hours at 50000g, discarding the supernatant, and resuspending the precipitate by using 1ml of PBS to obtain the extracted MVs, and storing at the temperature of-80 ℃ for later use. Taking a proper amount of MVs, observing under an electron microscope, measuring the expression of CD63, TSG101 and CD9 proteins by western blot, and measuring the inner diameter of the particle by Nanoparticle Tracking Analysis (NTA).
3. Parkinson animal model preparation, grouping and administration treatment
The construction of a parkinson animal model was performed using MPTP. The MPTP parkinsonian animal model is currently the accepted animal model for the most widely used parkinson's disease. The references include (DOI:10.1002/npr2.12060, DOI: 10.18632/ling.102221, DOI: 10.1016/j.neulet.2020.135032.).
First 8 week male C57BL/6 mice were randomized into 4 groups of 8 mice each, each group:
1. saline + saline group;
2. saline + microparticle group;
3. MPTP + brine group;
4. MPTP + microparticle group.
The preparation method of the Parkinson animal model comprises the steps of injecting MPTP (10mg/kg) into mice of a molding group in an intraperitoneal injection mode at 9 am, 11 am and 1 pm respectively on the day of molding, and simultaneously injecting saline with the same amount into the abdominal cavity of a control group. After one week of molding, the microparticles were used for treatment. The extracted mesenchymal stem cell microparticles were dissolved in saline to a final concentration of 2.5 μ g/ml, and the microparticles were injected into each mouse in the microparticle-treated group by tail vein injection, and the same amount of saline was injected into the control group.
4. Mouse behavioural test
One week after microparticle treatment, behavioral testing was performed. The test was carried out using an XR1514 model rat and mouse rotary rod fatigue tester (purchased from Shanghai Xin Soft information technology Co., Ltd.) at a rotation speed of 30r/min for 5 min. The time of the mice's movement on the rotarod was recorded and 3 tests were averaged. Mice were subjected to bar-rotation training 3 times prior to testing.
5. Detection of protein in striatum and substantia nigra of mouse brain
Grinding striatum of brain and substantia nigra in liquid nitrogen, adding cell lysate after grinding and crushing, and mixing well. The BCA protein assay kit was used to quantify the 50ug protein containing solution, which was then heated at 95 ℃ for 5 minutes with the addition of beta-mercaptoethanol and stored at-20 ℃ for use. The protein solution was subjected to gel electrophoresis using a SurePAGE-gel. After electrophoresis, proteins were transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were blocked in 2% BSA for 1 hour at room temperature and incubated with the corresponding antibodies (dopamine transporter, tyrosine hydroxylase, alpha-synuclein, phosphorylated alpha-synuclein), respectively, overnight at 4 ℃. The following day, the blot was washed 3 times in TBST and incubated with secondary antibody (GAPDH) for 1 hour at room temperature. After the last 3 washes, ChemiDoc was used TM Touch imaging systems capture images and analyze the images.
6. Statistical analysis
Data analysis was performed using statistical analysis software SPSS (Verison 20). The data were statistically analyzed using two-way analysis of variance (two-way ANOVA). Asterisks indicate statistical differences, P <0.05, P <0.01, P < 0.001.
Third, experimental results
1. Stem cell observations
The observation result of the mesenchymal stem cells under a 100-fold microscope is shown in figure 1, and the mesenchymal stem cells are fusiform and accord with the characteristics of the mesenchymal stem cells.
2. Stem cell microparticle identification results
The observation result of the stem cell particles by an electron microscope is shown in figure 2, the stem cell particles have good shapes and accord with the characteristics of the stem cell particles. The Western blot assay result is shown in FIG. 3, and the positive expression of CD63, TSG101 and CD9 proteins accords with the characteristics of stem cell microparticles.
The NTA measurement results are shown in FIG. 4, where the inner diameter of the particles is between about 100 and 1000, which corresponds to the length of the inner diameter of the particles.
3. Results of mouse behavioural test
The results of the rolling bar fatigue test are shown in fig. 5, the residence time of the MPTP-modeled mice on the rolling bar is obviously reduced, and the reduction can be alleviated by the stem cell microparticle treatment, which indicates that the stem cell microparticles can alleviate the behavior abnormality of the parkinson model mice.
4. Detection result of protein in striatum and substantia nigra of mouse brain
The results of protein detection in striatum and substantia nigra are shown in fig. 6, the expressions of dopamine transporters and tyrosine hydroxylases in striatum and substantia nigra of the Parkinson model mouse are reduced, the phosphorylation proportion of alpha-synuclein is increased, and the stem cell particles can improve the protein expression disorder of the Parkinson model mouse and restore the function of nerve cells.
The above experimental results show that the stem cell microparticles can effectively relieve the symptoms of the Parkinson's disease. Therefore, the stem cell microparticles have a prospect of being developed into a drug for treating Parkinson.
The above-described embodiments are intended to be illustrative of the nature of the invention, but those skilled in the art will recognize that the scope of the invention is not limited to the specific embodiments.
Claims (3)
1. An application of stem cell microparticles in preparing medicine for treating Parkinson's disease is disclosed.
2. The use of claim 1, wherein the stem cell microparticle is a mesenchymal stem cell microparticle.
3. The use of claim 2, wherein the stem cell microparticle is an umbilical cord mesenchymal stem cell microparticle.
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