CN113186157A - Application of mesenchymal stem cells in preparation of medicine for treating overactive bladder syndrome - Google Patents

Abstract

The invention discloses an application of mesenchymal stem cells in preparing a medicament for treating overactive bladder syndrome. The invention provides a method for separating and culturing bone marrow mesenchymal stem cells from bone marrow, which improves the proliferation capacity of the bone marrow mesenchymal stem cells of old organisms; in another aspect, the invention provides a cellular medicament that may be used to treat symptoms of OAB, including reducing frequency, ameliorating thickening of bladder smooth muscle layer caused by OAB, and improving urodynamics.

Description

Application of mesenchymal stem cells in preparation of medicine for treating overactive bladder syndrome

Technical Field

The invention relates to the technical field of treatment of overactive bladder syndrome, in particular to a method for preparing mesenchymal stem cells for treating overactive bladder syndrome and application of the mesenchymal stem cells.

Background

According to the international urinary control society, overactive bladder syndrome is a syndrome characterized by symptoms of urgency (OAB), often accompanied by symptoms of frequency and nocturia, with or without urge incontinence. The attack of OAB in China shows obvious age-dependent characteristics, the incidence rate is continuously increased, the OAB can seriously affect the life quality of patients, also can cause great psychological burden of the patients, directly affects families and work of the patients, and forms a vicious circle.

At present, the pathogenesis of OAB mainly comprises the abnormality of bladder nerve fiber transmitter and its receptor, the change of channels of calcium, sodium and potassium ions and the like of bladder detrusor smooth muscle cells, the abnormal communication among bladder detrusor cells, the increase of excitability of paced cells and the like. The existing OAB treatment mode mainly aims at relieving symptoms, wherein M receptor blocking agents are used mostly, the mechanism is mainly used for relaxing bladder smooth muscles so as to relieve OAB symptoms, certain side effects are caused on other organs, desensitization is possible to occur after long-time use, and the effective rate is low. In addition, the invention publication CN106265667B (chinese patent application No. CN201510254361.3, published 2017, 1 month, 14 days) of the applicant's invention team also describes that chloroquine has the effect of relaxing bladder smooth muscle, making it a potential drug for OAB treatment.

Mesenchymal Stem Cells (MSCs) belong to pluripotent stem cells, and have the advantages of multipotentiality, easy acquisition, easy amplification, stable genome after multiple passages, immunoregulatory ability, and the like, because they can self-renew in vivo and remain undifferentiated, and become ideal seed cells in stem cell transplantation therapy.

At present, the most used mesenchymal stem cells are derived from bone marrow, and the existing method for establishing a stem cell bank by separating the stem cells from the bone marrow still has many defects, such as great influence on cell viability due to age, easy cell aging in the culture process and the like, and needs to be improved. For example, the applicant's invention group's publication CN112111448A (chinese patent application No. CN202010847844.5, published 2020, 8/21) describes an improved method of culturing bone marrow mesenchymal stem cells (BMSCs) system, which has shown some excellent properties.

There is still a need in the art for improvements in methods for isolating mesenchymal stem cells, particularly for improving the effect of the age of mesenchymal stem cells on the culture results, in order to increase the efficiency of the method for isolating mesenchymal stem cells from bone marrow; in addition, there is a need in the art for new regimens for treating OAB, providing new drugs for OAB patients.

Disclosure of Invention

Objects of the invention include one or more of the following: on one hand, the method solves the defects and limitations of the existing preparation method of the mesenchymal stem cells, and provides a practical and efficient method for separating and culturing the mesenchymal stem cells from the bone marrow; on the other hand, the mesenchymal stem cells are used for treating overactive bladder syndrome, and the method and the application can effectively relieve OAB symptoms.

In order to achieve the above object, the present invention provides a method for preparing a mesenchymal stem cell drug, comprising the steps of:

(1) killing a mouse by a neck breaking method, soaking the mouse in 75% alcohol for disinfection for 5-10 minutes, placing the mouse in a culture dish, cutting skin from the abdomen along a coronal axis by using scissors to form a small opening, cutting a circle from the small opening along the waist, cutting the skin along two thigh groins of the mouse respectively, withdrawing the skin of the leg of the mouse to the ankle by using forceps, clamping the leg bone by using the forceps, separating the tendon between the femur and the patella by using the forceps, cutting the leg bone close to the patella by using the scissors, separating the femur from the muscle wrapped on the femur, cutting the sacrum by using the scissors, separating the whole femur from the mouse, and rapidly placing the femur in the culture dish filled with phosphate buffer solution; removing the muscle connected to the femur by using forceps, placing the muscle in a new culture dish containing phosphate buffer solution, and taking the culture dish containing the femur into a biological safety cabinet;

(2) collecting the thighbone in the culture dish obtained in the step (1), placing a sterile sieve at the opening of the tube, sucking 1mL of culture medium by using an injector for standby use, clamping one thighbone by using sterile forceps, shearing two ends of the thighbone by using scissors, replacing the injector containing the culture medium by the right hand, inserting the needle head of the injector into one end of the opening of the thighbone, pushing the injector to blow bone marrow contents onto the sterile sieve, naturally flowing the liquid into the 50mL tube, blowing 1-2mL of the liquid, and then blowing the other opening end of the thighbone until the thighbone is white;

(3) collecting the diluted bone marrow in the step (2), slowly adding the diluted bone marrow to the liquid surface of the Ficoll according to the proportion of adding 1 part of lymphocyte separation liquid (Ficoll separation liquid) to 2 parts of diluted bone marrow, centrifuging for 30 minutes at 2000r/min under the condition of 20 ℃ by using a horizontal centrifuge, washing the mesenchymal stem cells taken out from the interface for 2-3 times by using normal saline, and adding a culture medium for culturing.

Preferably, the culture medium is composed of a basal medium for providing necessary nutrition for the growth of the mesenchymal stem cells and selected from one of DMEM, EMEM, IMDM, GMEM, RPMI-1640 or alpha-MEM, and serum or serum substitute from fetal bovine serum and platelet lysate.

Preferably, the marrow mesenchymal stem cells are separated by using a mouse with the age of less than 10 months instead of a young mouse with the age of 10-14 days, preferably, the marrow mesenchymal stem cells are separated from the old mouse with the age of 8-10 months, and the marrow mesenchymal stem cells with the expansion capability close to that of the young mouse are obtained by adding additives; preferably, the bone marrow mesenchymal stem cells with approximate expansion capacity are obtained by the bone marrow separation culture of 8-10 month old mice and 10-14 day old mice.

Preferably, the additive comprises the following components in dosage: 0-500 muM L-carnitine, 0-500 muM lipoic acid, 0-500 muM pyrroloquinoline quinone, 0-20mM coenzyme Q10, 0-20mM melatonin and 0-20mM nicotinamide mononucleotide, 50-200 muM non-essential amino acids, 0-100 muM BMSC exosomes.

A method for preparing a medicament for treating overactive bladder syndrome comprises the following steps:

s1, selecting mesenchymal stem cells separated from organisms;

the prepared mesenchymal stem cells are from bone marrow, umbilical cord, placenta or fat;

the organism is derived from a whole-aged individual, including young individuals or old individuals;

and S2, culturing the mesenchymal stem cells, and adding additives into the culture medium, wherein after the 1 st-2 th generation cells are qualified by flow cytometry, the 3 rd-5 th generation cells are directly applied to the medicine for treating overactive bladder syndrome.

Preferably, the mesenchymal stem cells obtained by subculture are transferred to a centrifuge tube added with a medicinal solvent for centrifugation at 2000rpm for 5min, supernatant is discarded, and saline solution is added to resuspend the cells, so that the pharmaceutical preparation is prepared.

A medicine for treating overactive bladder syndrome comprises mesenchymal stem cells and medicinal solvent;

the medicinal solvent comprises one or more of diluent, excipient, filler, binder, disintegrant, surfactant and lubricant.

Preferably, the medicament is selected from injection, oral preparation or external preparation, preferably injection; more preferably, the drug is selected from intravenous, intramuscular, subcutaneous or intrathecal injection.

Preferably, the dosage for intravenous injection is 0.5-5 μ L/kg.

Preferably, the concentration of mesenchymal stem cells in the pharmaceutical preparation is 1-10 x10⁶one/mL, preferably 1.5-8 x10⁶one/mL, more preferably 2-5 x10⁶one/mL.

Preferably, the pharmaceutical vehicle comprises sodium chloride at a concentration of 7-10mg/mL, preferably at a concentration of 7.5-9.5mg/mL, more preferably at a concentration of 8-9 mg/mL;

and/or calcium pantothenate in a concentration of 0.25 to 5mg/mL, preferably in a concentration of 0.5 to 4mg/mL, more preferably in a concentration of 1 to 3 mg/mL;

and/or ornithine in a concentration of 5-40mg/mL, preferably in a concentration of 10-30mg/mL, more preferably in a concentration of 15-25 mg/mL.

An application of a mesenchymal stem cell medicament in preparing a medicament for treating overactive bladder syndrome.

Accordingly, the present invention provides a practical and efficient method for isolating and culturing mesenchymal stem cells from bone marrow. At present, a mouse bone marrow mesenchymal stem cell is usually separated by a mouse with 10-14 days, the proliferation speed of the bone marrow mesenchymal stem cell separated by a mouse with a large age is slow, the phenomenon of aging and the like easily occurs in the cell culture process, and the failure condition easily occurs. After the mesenchymal stem cells are obtained, sterile detection, mycoplasma detection and endotoxin detection are required, and subculture is performed after qualification. Flow cytometry is carried out for 1-2 generations, and the 3-5 generations are applied in medicine.

In a second aspect the invention provides the use of a mesenchymal stem cell for the treatment of overactive bladder syndrome (OAB). The mesenchymal stem cells are bone marrow mesenchymal stem cells with surface markers approved by the International society for cell therapy, and have no mycoplasma, endotoxin and fungus pollution.

The technical solution of the present invention is further described in detail by the accompanying drawings and embodiments.

Drawings

FIG. 1 is a photograph of primary isolated BMSCs cultured on day 7; a: group 1, B: group 2, C: group 3, D: group 4;

FIG. 2 is a second generation cell expansion curve;

FIG. 3 is a schematic representation of bladder wet weights of different groups in a mouse model simulating OAB;

FIG. 4 is a schematic representation of the detection of plaques in different groups of mice in the OAB model;

FIG. 5 is the results of HE staining of mice;

FIG. 6 is a schematic representation of urodynamic testing of different groups in a mouse model simulating OAB.

Detailed Description

In order to facilitate understanding of the invention, the invention will be described more fully and in detail with reference to the accompanying drawings and preferred embodiments, but the scope of the invention is not limited to the specific embodiments below.

Unless otherwise defined, all terms of art used hereinafter have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention.

Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be prepared by existing methods.

Example one

Bone marrow mesenchymal stem cell culture method

Sample treatment: healthy C57BL/6N mice of about 10 days and 10 months of age were selected and divided into 4 groups, and bone marrow mesenchymal stem cells were isolated, respectively. The first group used 10 day mice to isolate BMSCs (group 1); the second group used 5 mice of 10 months of age to isolate BMSCs (group 2); the third group used 3 10-day mice and 5 10-month-old mice to isolate BMSCs (group 3); fourth group BMSCs were isolated using 5 mice 10 months old and additional protective agents were added (group 4).

A culture system: 10-15% serum and 0-1% double antibody alpha MEM culture medium.

Additional protectant ingredients: 0-500 muM L-carnitine, 0-500 muM lipoic acid, 0-500 muM pyrroloquinoline quinone, 0-20mM coenzyme Q10, 0-20mM melatonin and 0-20mM nicotinamide mononucleotide, 50-200 muM non-essential amino acids, 0-100 muM BMSC exosomes.

Preparation work: two sets of surgical instruments are prepared, each set including two forceps and a pair of scissors. The surgical instruments were wrapped in tinfoil paper, placed in an aluminum box with 2 tubes of 15mL and 1 tube of 50mL, and autoclaved at 121 ℃ for 30 minutes in newspaper. Placing one set of instruments on a super clean bench, placing the rest articles in the aluminum box into a biological safety cabinet, and cooling to room temperature for later use; preparing phosphate buffer solution added with 1 thousandth double antibody, and adding 10mL of the buffer solution into a 10cm culture dish; preparing an alpha MEM culture medium added with 10-15% of serum and 1% of double antibodies, and placing the culture medium in a biological safety cabinet for later use; a sterile sieve and a 1mL sterile syringe are prepared and placed in a biological safety cabinet for later use.

And (3) a separation process: mice were sacrificed by cervical dislocation and were sterilized by soaking in 75% alcohol for 10 minutes. The mouse is placed in a culture dish, a small opening is cut through the skin from the abdomen along the coronal axis by scissors, a circle is cut through the small opening along the waist, the skin is cut through the two thigh groins of the mouse, and the skin of the leg of the mouse is retracted to the ankle by tweezers. The left hand pincette clamps the calf bone, the right hand pincette separates the tendon between the femur and the patella, and the scissors cut the calf bone close to the patella. The femur was separated from the muscle wrapped on it, cut at the sacrum with scissors, the whole femur was separated from the mouse, and the femur was quickly placed in a petri dish containing phosphate buffer.

The same procedure separates the other femur and places it in the petri dish. Muscles connected with the thighbone are removed as much as possible by using forceps, and the thighbone cannot be broken by taking care that the thighbone cannot be broken when the thighbone is placed in a new culture dish containing phosphate buffer solution. The culture dish containing the thighbone is taken into the biological safety cabinet, and a circle of sealing film can be wound outside the culture dish. A sterile sieve was placed at a 50mL nozzle and 1mL of media was aspirated with a syringe until needed. The left hand is used for clamping a femur by using tweezers in another set of instruments, and the right hand is used for cutting off the two ends of the femur by using scissors. And (3) changing the injector filled with the culture medium by the right hand, inserting the needle of the injector into one end of the opening of the femur, pushing the injector to blow the bone marrow content onto the sterile sieve, naturally flowing the liquid into a 50mL tube, and blowing 2mL, and then changing to blow the other opening end of the femur until the femur turns white.

Adding 1 part of lymphocyte separation liquid (Ficoll separation liquid) and 2 parts of diluted bone marrow, slowly adding the diluted bone marrow onto the Ficoll liquid surface, centrifuging at 2000r/min for 30 minutes (rising 9 and falling 7) by a horizontal centrifuge at 20 ℃, washing bone marrow mesenchymal stem cells taken out from an interface for 3 times by using normal saline, and adding culture solution for counting.

Primary culture of bone marrow mesenchymal stem cells: according to 0.3X 10⁶The mesenchymal stem cells with the density of/mL are inoculated in the culture medium and cultured for 3 days, and then the quantity of the mesenchymal stem cells is detected.

The photograph taken on day 7 of primary culture is shown in FIG. 1, which shows that group 1 has the best growth state, cells are fusiform, and proliferation is fast; while group 2 had a certain number of dead cells, the cells proliferated the slowest. The BMSC separation experiment is influenced by the age increase of the mice, and the slow cell proliferation, the easy aging and the like are caused. The proliferation rate of group 3 was significantly increased compared to group 2 due to the addition of a portion of the BMSCs derived from the bone marrow of the mice for 10 days, which suggests that the BMSCs of the young mice secrete certain signals to the BMSCs of the old mice, thereby promoting the proliferation of the BMSCs of the old mice. Mitochondrial activity becomes weaker with age, and intracellular involvement of increased mitochondrial mutation rate, increased mitochondrial swelling rate, and decreased mitochondrial function. Since mitochondria within mesenchymal stem cells can migrate between cells, it is presumed that mesenchymal stem cells of 10-day mice have a promoting effect on older mice. Group 4 added additives group proliferation capacity was not much faster than group addition, additional additives increased mitochondrial function and increased primary BMSC viability.

Example two

Culture of mesenchymal stem cells

Mesenchymal stem cells are cultured in a culture dish, alpha MEM is added into the culture dish as a culture medium, 10-15% fetal bovine serum and 0-1% double antibody, and the group 4 is additionally inoculated with additives: 0-500 muM L-carnitine, 0-500 muM lipoic acid, 0-500 muM pyrroloquinoline quinone, 0-20mM coenzyme Q10, 0-20mM melatonin and 0-20mM nicotinamide mononucleotide, 50-200 muM non-essential amino acids, 0-100 muM BMSC exosomes. And (5) carrying out passage when the cell density is more than or equal to 80 percent until the cell density reaches P5 generation for later use.

The second generation amplification curve after passage is shown in FIG. 2. MTT experiment shows that OD value is obviously increased on 2-3 days, peak is reached on 5-7 days, and source of 110 days mice in the group is obviously faster than that of older mice. Group 3 the proliferative capacity was between that of group 1 and group 2 due to the inclusion of a portion of the mouse-derived BMSCs for 10 days in older mice. Group 1 showed that the amplification of mouse-derived BMSCs was optimal for 10 days; group 3 second, group 2 had an amplification curve approaching 10 days mouse BMSC due to the addition of the additive. The method is shown to reduce the influence of age on the isolation of the mesenchymal stem cells.

In addition, each group of second generation streaming results are shown in table 1. The results show that the BMSCs of each group of separated groups meet the standard.

TABLE 1

Test of

Improved BMSCs had therapeutic effect on OAB, and this experiment was performed with BMSCs of group 4 and using passage 4 cells.

Construction of mouse bladder outlet obstruction model (PBOO): female mice, 12 weeks old, C57BL, were selected and grouped. Among them, 10 of the pseudo-surgery group, the surgery group and the surgery + treatment group were used. Mice in the operation group and the operation + treatment group were anesthetized with 5% chloral hydrate of 200 μ L, fixed in the supine position after anesthesia, and subjected to abdominal depilation, and the periphery of the urethral orifice was sterilized with 75% ethanol. The bladder was inserted transurethrally with a 26 gauge venous indwelling needle. The skin at the upper end of the vaginal opening is cut open, the proximal urethra is separated and the urethra near the bladder neck is ligated with a size 7 non-absorbable suture. The ligation standard is that the outer tube of the venous indwelling needle is tight, and the probe can be easily pulled out. The vein indwelling probe and the outer tube are pulled out successively, the incision is closed, and the skin is sutured. The sham group performed the above procedure at the same site, but without ligation of the urethra. The model can obviously increase the weight of the bladder and the thickness of the smooth muscle layer, and is convenient to observe.

Administration mode and dosing: the mice are injected 2 times in the second week after the operation, and before the mice enter a cage, the operation + treatment group adopts the intravenous injection of the bone marrow mesenchymal stem cells, wherein the bone marrow mesenchymal stem cells are injected by 1x10⁶Each was dissolved in 500. mu.L of physiological saline as an injection solution and injected into the tail vein at 0.75mg/kg body weight. The sham and surgical groups were both intravenously injected with equal volumes of PBS.

Detection of bladder weight

After 5 weeks of surgery, the wet weight of the bladder in the sham surgery group, the surgery group and the surgery + treatment was counted, and the data are respectively 0.0226g + -0.000957, 0.0386g + -0.002197 and 0.031g + -0.001726, as shown in fig. 3. The results show that BMSC have inhibitory effect on the compensatory increase in bladder weight by PBOO.

Experiment for detecting urine spot

The padding is removed from the cage bottom, the cage bottom is wiped dry, then the cage bottom is put into 12cm by 25cm filter paper, and the cage bottom is stuck with adhesive tape. The filter paper was removed after two hours of acclimation in the mouse and the same filter paper was replated for recording, allowing the mouse to eat freely without water, and after two hours the filter paper was removed and photographed using a gel imaging system. The parameters are as follows: uv reflected light, aperture 85, focal length 50, focus 100, gain 35.03dB, contrast 0, gamma 1.1, exposure time 1.1 s.

The experimental results of the mouse urinary spots are shown in FIG. 4, and the experimental data of the pseudo-operation group, the operation group and the operation + treatment group are 13.78 + -2.67, 26.75 + -3.67 and 11.70 + -1.30.

The urinary spot experiment shows that the urinary spots of the operation group show obvious overactive bladder symptoms, namely, the micturition is mainly performed by the urinary spots smaller than 0.5mm, the micturition of the pseudo-operation group is mainly performed by a single large amount of micturition, and the operation and treatment group has improvement compared with the operation group.

Detection III, histological detection

Preparation of Paraffin section of bladder tissue

Obtaining materials of bladder: after injecting 5% chloral hydrate (100. mu.L/10 g body weight) into the abdominal cavity of the mouse, the abdominal cavity was opened rapidly after the mouse was anesthetized, and the bladder was taken out and placed in PBS solution. Removing adipose tissue by micro-shearing, and cutting a notch at the neck of bladder to discharge urine from bladder. The bladder was then fixed by shaking in 4% paraformaldehyde solution (PBS as solvent) for 48 hours at 4 ℃. The fixed bladder tissue was dehydrated by placing it in a 75% ethanol solution. And (3) soaking the dehydrated tissues in a gradient xylene solution. Embedding the tissue in a paraffin embedding machine (Leica), heating at 60 deg.C with an iron groove of 6mm depth, placing the coronal surface of the bladder in the groove, dropping paraffin, and cooling. Slicing with a paraffin slicer (Leica), spreading in 45 deg.C water with a thickness of 5 μm, placing on an anti-peeling glass slide, and oven drying at 60 deg.C for 5 min.

HE staining:

(1) HE staining reagent:

and (3) hematoxylin: 2g of hematoxylin, 40mL of absolute ethyl alcohol, 100g of aluminum potassium sulfate, 600mL of distilled water, 0.4g of sodium iodate and 20mL of glacial acetic acid. Adding distilled water, adding aluminum potassium sulfate, slightly heating, and adding hematoxylin, anhydrous ethanol, sodium iodate, and glacial acetic acid to dissolve completely.

Eosin solution: eosin (alcohol soluble) 2.5g, 95% ethanol 1L, glacial acetic acid. Eosin was dissolved in 95% ethanol, stirred and adjusted to pH 4.5 with glacial acetic acid.

Ethanol hydrochloride differentiation solution: concentrated hydrochloric acid 1mL, 75% ethanol 200 mL.

(2) And (3) dyeing: and (3) conventional dewaxing: baking in oven at 60 deg.C for 15min, heating with xylene for 3 times, respectively for 10min, and respectively passing through 100% ethanol, 95% ethanol, 90% ethanol, 85% ethanol, and 75% ethanol for 5 min. Dyeing: soaking in hematoxylin for 5min, washing with tap water, differentiating with hydrochloric acid and ethanol for 30s, washing with tap water, soaking in eosin solution for 5min, and washing with tap water. And (3) dehydrating: quickly taking out 95% ethanol, separating 100% ethanol for 10s three times, separating xylene for 3 times for 1.5min, sealing with neutral gum, and covering with cover glass of 0.13-0.17mm thickness.

HE staining results are shown in fig. 5, with significant thickening of the bladder wall in the operative group compared to the sham operative group, and remission in the operative + BMSC treated group.

Detection of four, urine dynamic assay

Cystostomy and intubation: a PE10 tube (inner diameter 0.28mm, outer diameter 0.61mm) is cut into 35cm long, and the tail end is heated to be cuff-like and expanded, so that the PE10 tube can be conveniently fixed in the bladder after fistulization. Sterilizing with 75% ethanol. Before implantation, the implant is flushed with physiological saline. Surgical instruments were autoclaved before use, mice were anesthetized with chloral hydrate, fixed in the supine position, the abdomen was dehaired and sterilized with 75% ethanol, and the skin was opened on the abdomen. The fistulation was performed using an 18G needle at the contralateral tip of the bladder neck, and a 7-0 non-absorbable suture was sutured tightly around the fistulation along the 3-needle periadventitial suture (taking care not to penetrate the smooth muscle layer and the intimal layer to cause bladder leakage). The PE10 tube was implanted through the stoma, causing the cuff sample to bulge into the bladder. The purse-string sutures were tied and the catheter was pulled gently outward to check for fixation. 100-200 mu L of normal saline was slowly injected into the bladder along the PE10 tube, whether there was any leakage was checked carefully, the abdominal muscle was sutured with 3-0 after no error was confirmed, and the PE10 catheter was fixed at the upper part to prevent slipping. The mice were prone, depilated in the scapular area and a small incision was opened, the subcutaneous tissue was passed through the scapula to the abdomen with a hollow gavage needle, PE10 tubing was guided to the scapula through the gavage needle and secured with 3-0 sutures. The redundant catheter is wound into a ring with the length of about 2cm and is fixed by a 3-0 suture line, so that the PE10 pipe can be prevented from being bitten by an animal in the experimental process. The mice were placed on a hot plate (approximately 30 degrees) to wake up. Urodynamic tests were performed 3 days after recovery.

And (3) measuring the urine power: the mice were placed in metabolic cages and allowed to move freely, and the experiment was started half an hour after acclimation. The PE10 tube on the back of the mouse was connected to a three-way tube, and connected to a urodynamic instrument (ANDROMEDA) and a micro-injection pump through the three-way tube. The bladder perfusion rate was (1.2mL/h, physiological saline) and recorded for one hour as the conscious state urodynamics. The mice were then anesthetized with 0.3mL of 5% tribromoethanol and the urodynamics of the anesthesia continued to be recorded for half an hour. Urodynamic results were recorded using ANDROMEDA-AUDACT software and processed in excel. Results are expressed as x ± s, and treatment with t-test and analysis of variance was performed between groups.

The urodynamic results of the mice are shown in fig. 6, and the bladder pressures (mHg) of the pseudo-operation group, the operation group and the operation + treatment group are respectively: 3.819 + -1.004, 11.141 + -4.185 and 7.592 + -3.355. The results showed a significant drop in bladder pressure in the BMSC treated group relative to the model group, indicating that BMSC had a therapeutic effect on OAB.

Therefore, the application of the mesenchymal stem cell in preparing the medicine for treating overactive bladder syndrome can effectively relieve OAB symptoms, including reducing frequency of micturition, relieving bladder thickening caused by OAB and improving urodynamics.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting the same, and although the present invention is described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that: modifications and equivalents may be made to the invention without departing from the spirit and scope of the invention.

Claims (12)

1. A method for preparing a bone marrow mesenchymal stem cell medicament is characterized by comprising the following steps:

(1) killing a mouse by a neck breaking method, soaking the mouse in 75% alcohol for disinfection for 5-10 minutes, placing the mouse in a culture dish, cutting skin from the abdomen along a coronal axis by using scissors to form a small opening, cutting a circle from the small opening along the waist, cutting the skin along two thigh groins of the mouse respectively, withdrawing the skin of the leg of the mouse to the ankle by using forceps, clamping the leg bone by using the forceps, separating the tendon between the femur and the patella by using the forceps, cutting the leg bone close to the patella by using the scissors, separating the femur from the muscle wrapped on the femur, cutting the sacrum by using the scissors, separating the whole femur from the mouse, and rapidly placing the femur in the culture dish filled with phosphate buffer solution; removing the muscle connected to the femur by using forceps, placing the muscle in a new culture dish containing phosphate buffer solution, and taking the culture dish containing the femur into a biological safety cabinet;

(2) collecting the thighbone in the culture dish obtained in the step (1), placing a sterile sieve at the opening of the tube, sucking 1mL of culture medium by using an injector for standby use, clamping one thighbone by using sterile forceps, shearing two ends of the thighbone by using scissors, replacing the injector containing the culture medium by the right hand, inserting the needle head of the injector into one end of the opening of the thighbone, pushing the injector to blow bone marrow contents onto the sterile sieve, naturally flowing the liquid into the 50mL tube, blowing 1-2mL of the liquid, and then blowing the other opening end of the thighbone until the thighbone is white;

(3) collecting the diluted bone marrow in the step (2), slowly adding the diluted bone marrow to the liquid surface of the Ficoll according to the proportion of adding 1 part of lymphocyte separation liquid (Ficoll separation liquid) to 2 parts of diluted bone marrow, centrifuging for 30 minutes at 2000r/min under the condition of 20 ℃ by using a horizontal centrifuge, washing the mesenchymal stem cells taken out from the interface for 2-3 times by using normal saline, and adding a culture medium for culturing.

2. The method of claim 1, wherein the culture medium is selected from DMEM, EMEM, IMDM, GMEM, RPMI-1640 or α -MEM, and serum or serum replacement is derived from fetal bovine serum, platelet lysate, etc.

3. The method for preparing a mesenchymal stem cell medicament according to claim 1, wherein: separating the bone marrow mesenchymal stem cells by using a aged mouse within 10 months instead of a young mouse of 10-14 days, preferably, separating the bone marrow mesenchymal stem cells by using an aged mouse within 8-10 months, and adding additives to obtain the bone marrow mesenchymal stem cells with the capacity close to the amplification capacity of the young mouse; preferably, the bone marrow mesenchymal stem cells with approximate expansion capacity are obtained by the bone marrow separation culture of 8-10 month old mice and 10-14 day old mice.

4. The method for preparing a mesenchymal stem cell medicament according to claim 3, wherein the additive comprises the following components in dosage: 0-500 μ M L-carnitine, 0-500 μ M lipoic acid, 0-500 μ M pyrroloquinoline quinone, 0-20mM coenzyme Q10, 0-20mM melatonin, 0-20mM nicotinamide mononucleotide, 50-200 μ M non-essential amino acids and 0-100 μ M BMSC exosomes.

5. A method for preparing a medicament for treating overactive bladder syndrome is characterized by comprising the following steps:

s1, selecting mesenchymal stem cells separated from organisms;

the prepared mesenchymal stem cells are from bone marrow, umbilical cord, placenta or fat;

the organism is derived from a whole-aged individual, including young individuals or old individuals;

and S2, culturing the mesenchymal stem cells, and adding additives into the culture medium, wherein after the 1 st-2 th generation cells are qualified by flow cytometry, the 3 rd-5 th generation cells are directly applied to the medicine for treating overactive bladder syndrome.

6. The method of claim 5, wherein the step of preparing the medicament for treating overactive bladder comprises the steps of: transferring the mesenchymal stem cells obtained by subculture to a centrifugal tube added with a medicinal solvent for centrifugation, centrifuging at 2000rpm for 5min, discarding supernatant, and adding saline solution to resuspend the cells to obtain the pharmaceutical preparation.

7. A medicament for treating overactive bladder syndrome, characterized by: the medicine comprises mesenchymal stem cells and medicinal solvent;

the medicinal solvent comprises one or more of diluent, excipient, filler, binder, disintegrant, surfactant and lubricant.

8. The medicament for treating overactive bladder syndrome according to claim 7, wherein: the medicine is selected from injection, oral preparation or external preparation, preferably injection; more preferably, the drug is selected from intravenous, intramuscular, subcutaneous or intrathecal injection.

9. The medicament for treating overactive bladder syndrome according to claim 8, wherein: the dosage of intravenous injection is 0.5-5 μ L/kg.

10. The medicament for treating overactive bladder syndrome according to claim 7, wherein: the concentration of mesenchymal stem cells in the pharmaceutical preparation is 1-10 x10⁶one/mL, preferably 1.5-8 x10⁶one/mL, more preferably 2-5 x10⁶one/mL.

11. The medicament for treating overactive bladder syndrome according to claim 7, wherein: the medicinal solvent comprises sodium chloride, the concentration of which is 7-10mg/mL, preferably 7.5-9.5mg/mL, and more preferably 8-9 mg/mL;

and/or calcium pantothenate in a concentration of 0.25 to 5mg/mL, preferably in a concentration of 0.5 to 4mg/mL, more preferably in a concentration of 1 to 3 mg/mL;

and/or ornithine in a concentration of 5-40mg/mL, preferably in a concentration of 10-30mg/mL, more preferably in a concentration of 15-25 mg/mL.

12. Use of a mesenchymal stem cell medicament of any of claims 1-11 in the manufacture of a medicament for the treatment of overactive bladder syndrome.

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