CN115177636A - Composition for treating prostate calcification and preparation method and application thereof - Google Patents
Composition for treating prostate calcification and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a composition for treating prostate calcification, a preparation method and application thereof, and belongs to the technical field of medicines. The composition comprises mesenchymal stem cell extracellular vesicles, mesenchymal stem cells and sodium bicarbonate, wherein 1.5 x 10 of the total weight of the mesenchymal stem cell extracellular vesicles per milliliter 6 ～0.5×10 7 Mesenchymal stem cellStem cells; the volume fraction of the sodium bicarbonate added in the composition is 8-15%. Preparing a composition by mixing mesenchymal stem cells with mesenchymal stem cell extracellular vesicles to prepare a cell-vesicle mixture; and adding sodium bicarbonate into the cell-vesicle mixture to obtain the composition for treating prostate calcification. The composition can solve long-acting antibacterial and reverse tissue fibrosis problem, eliminate calcification focus, and is suitable for treating chronic prostatitis, urethritis or male infertility.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a composition for treating prostate calcification.
Background
Prostate calcification is one of the common conditions in men, and refers to the presence of single or multiple calcium salt deposits in prostate tissue under the influence of chronic prostatitis. Slight calcification has no obvious clinical features, and the calcification causes prostate calculus seriously, and causes symptoms of urgent micturition, incomplete urine, urethral pain, dysuria, etc., and can also induce seminal vesiculitis and orchitis, resulting in impotence and premature ejaculation. Calcification is a precursor of prostate stones and seminal vesiculitis, and is also an important reason for repeated attacks of prostatitis. Patients with prostate calcification are often 41-60 years old, and have high psychological stress, high neurotransmitter catecholamine levels, high adrenergic activity, altered urodynamics, bladder neck and prostatic urethra "spasm", with relaxed external sphincter, high maximum resting urethral occlusive pressure, decreased urine flow rate, resulting in urine reflux, and the urate component of urine becomes the core of calcification. Therefore, inhibition of catecholamine over-release, cutting of nerve conduction and inhibition of urate deposition are one approach to solving calcification.
Prostatitis and calcification are mutually influenced and coexisted, the prostate is stimulated by chronic inflammation for a long time, and prostate tissue degeneration causes prostate fluid retention and prostate gland duct blockage, finally salt crystals cannot be normally discharged, and a calcification focus is gradually formed. With the calcified focus, bacteria are easy to breed to cause prostatitis recurrence, and the process is repeated, so that the radical treatment of prostatitis is the fundamental problem of calcification.
Because of the particularity of the prostate structure, there is no good radical cure method for prostate calcification clinically, and most of them are treated according to prostatitis, and operation treatment is needed in severe cases. No matter the physical therapy of massage and infrared rays or the local antibiotic injection therapy is adopted, only the temporary bacteriostasis purpose is achieved, and the treatment can be repeated within a period of time, so that the problems of long-acting bacteriostasis, tissue fibrosis reversion and calcification focus elimination cannot be fundamentally solved.
Disclosure of Invention
In order to overcome the disadvantages of the prior art, the present invention aims to provide a composition for treating prostate calcification, which solves the problems of long-lasting bacteriostasis and tissue fibrosis reversal, and eliminates calcification foci.
In order to achieve the purpose, the invention adopts the following technical scheme to realize the purpose:
the invention discloses a composition for treating prostate calcification, which comprises a mesenchymal stem cell extracellular vesicle, mesenchymal stem cells and sodium bicarbonate;
wherein 1.5 × 10 per ml of the extracellular vesicles of the mesenchymal stem cells is added 6 ～0.5×10 7 (ii) mesenchymal stem cells; the volume fraction of the sodium bicarbonate added in the composition is 8-15%.
Preferably, the mesenchymal stem cell extracellular vesicles are secreted from one or both of umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, bone marrow mesenchymal stem cells and dental pulp mesenchymal stem cells.
Preferably, the mesenchymal stem cell extracellular vesicle is obtained after induction of mesenchymal stem cells by acetyl hexapeptide-8.
Preferably, the mesenchymal stem cell is one or two of umbilical cord mesenchymal stem cell, adipose mesenchymal stem cell, bone marrow mesenchymal stem cell and dental pulp mesenchymal stem cell.
Preferably, the mesenchymal stem cells are generation P3-P6 mesenchymal stem cells.
Preferably, the sodium bicarbonate is 5% sodium bicarbonate injection.
The invention also discloses a preparation method of the composition for treating prostate calcification, which comprises the steps of mixing the mesenchymal stem cells with the extracellular vesicles of the mesenchymal stem cells to prepare a cell-vesicle mixture; and adding sodium bicarbonate into the cell-vesicle mixture to obtain the composition for treating prostatic calcification.
Preferably, the preparation method of the mesenchymal stem cell extracellular vesicle comprises the following steps: adding 0.5-1 ppm acetyl hexapeptide-8 to mesenchymal stem cells at 37 ℃,5% 2 Co-culturing in incubator, discarding supernatant after 24 hr, washing cells with phosphate buffer, changing serum-free medium, continuing to control CO at 37 deg.C 2 Culturing for 48h in an incubator, collecting cell culture supernatant, and ultracentrifuging to obtain the mesenchymal stem cell extracellular vesicles.
The invention also discloses application of the composition for treating prostate calcification in preparation of a medicinal preparation for treating chronic prostatitis, urethritis or male infertility.
Preferably, the formulation is an injection.
Compared with the prior art, the invention has the following beneficial effects:
the composition for treating prostate calcification, provided by the invention, has the advantages that the adopted mesenchymal stem cells have multidirectional differentiation and unlimited proliferation capabilities, have the effects of regulating immunity, improving microenvironment, inhibiting tissue fibrosis and promoting local tissue repair, act on the prostate, can eliminate pathogenic microorganisms in the prostate through secretion factors generated by paracrine, and repair the microenvironment of the prostate; the adopted extracellular vesicle of the mesenchymal stem cell is a vesicle-shaped structure secreted by the mesenchymal stem cell, contains various bioactive molecules including DNA, RNA, miRNA and the like, has the repairing and regenerating effects of a maternal cell-the mesenchymal stem cell, can promote the differentiation of the mesenchymal stem cell into a regulatory Treg cell, and has the anti-inflammatory effect of the extracellular vesicle of the mesenchymal stem cell, so as to accelerate the repair of the prostate; the adopted sodium bicarbonate can dissolve urate in prostate according to the principle of acid-base neutralization, reduce pain caused by stem cell injection, accelerate extracellular vesicle disintegration in vivo, rapidly release active substances in the vesicle, and shorten repair time. Acute systemic toxicity test and pyrogen detection are carried out on the composition, and the safety and the non-toxicity of the composition are proved; the results of the drug effect verification of the composition show that calcification spots in the inner gland of a tested patient completely disappear, the capsule structure of the prostate inner gland is recovered, and the capsule line is clear, which shows that the composition has good drug effect, can solve the problems of long-acting bacteriostasis and tissue fibrosis reversion, eliminates the calcification spots, and is applicable to the treatment of chronic prostatitis, urethritis or male infertility.
Furthermore, the skin active substance acetyl hexapeptide-8 is applied to the prostate disease repair, and the induced mesenchymal stem cell extracellular vesicle not only can inhibit the expression of inflammatory factors in the prostate, has the anti-inflammatory repair effect, but also can inhibit the deposition of collagen and promote the degradation of formed calcific foci, and on the other hand, the mesenchymal stem cell extracellular vesicle also has the efficacy of the acetyl hexapeptide-8, can inhibit the excessive release of catecholamine in the prostate, cut off the transmission of neurotransmitters and solve the problem of 'spasm' of the prostate and the urethra.
Furthermore, the composition can realize accurate administration, accurate treatment and quick repair by means of ultrasonic guidance during use.
The preparation method of the composition for treating prostate calcification, provided by the invention, is simple in preparation process, the whole preparation period is not more than 4d, and the adopted small molecular polypeptide acetyl hexapeptide-8 is a commercially available raw material which can be produced in a large scale, so that the price is low and the safety is high; the obtained composition has low use frequency, is used once a week, gets rid of the current situation that the existing prostate treatment medicament is taken three times a day, and has more thorough treatment effect.
Drawings
Fig. 1 is an ultrasound image of a subject patient of the present invention before and after injection of a prepared composition for calcification of prostate; wherein, a is an ultrasound image of the prostate of a patient without treatment, B is an ultrasound image of the prostate after injecting the composition of the present invention in example 1 once, C is an ultrasound image of the prostate after injecting the composition of the present invention in example 1 twice, and D is an ultrasound image of the prostate after injecting the composition of the present invention in example 1 three times;
FIG. 2 is an ultrasound image of a subject patient of the present invention before and after injection of a prepared composition for calcification of prostate; wherein, a is an ultrasound image of the prostate of a patient without treatment, B is an ultrasound image of the prostate after injecting the composition of example 2 of the present invention once, and C is an ultrasound image of the prostate after injecting the composition of example 2 of the present invention twice.
Detailed Description
In order to make the technical solutions of the present invention better understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, shall fall within the protection scope of the present invention.
It should be noted that the terms "comprises" and "comprising," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.
The invention is described in further detail below with reference to the accompanying drawings:
the invention provides a composition for treating prostate calcification, which comprises a mesenchymal stem cell extracellular vesicle, a mesenchymal stem cell and sodium bicarbonate;
wherein the mesenchymal stem cell extracellular vesicles are secreted by one or two of umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, bone marrow mesenchymal stem cells and dental pulp mesenchymal stem cells; the extracellular vesicles of the mesenchymal stem cells are obtained by inducing the mesenchymal stem cells by acetyl hexapeptide-8; the mesenchymal stem cell is one or two of umbilical cord mesenchymal stem cell, adipose mesenchymal stem cell, bone marrow mesenchymal stem cell and dental pulp mesenchymal stem cell; adding 1.5 x 10 per milliliter of mesenchymal stem cell extracellular vesicles 6 ～0.5×10 7 Mesenchymal stem cellA cell; the sodium bicarbonate injection with 5 percent of sodium bicarbonate is added into the composition, and the volume fraction of the sodium bicarbonate injection added into the composition is 8 to 15 percent; the composition is administered by perineal injection into prostate gland under ultrasound guidance.
The invention provides a preparation method of a composition for treating prostate calcification, which comprises the step of mixing 1ml of (1.5 multiplied by 10) of mesenchymal stem cell outer vesicles and mesenchymal stem cells in a normal-temperature sterile environment 6 ～0.5×10 7 ) Mixing the components in proportion to prepare a cell-vesicle mixture; adding sodium bicarbonate with the volume fraction of 8-15% into the cell-vesicle mixture to prepare the composition for treating the prostate calcification;
the preparation method of the mesenchymal stem cell extracellular vesicle comprises the following steps: adding 0.5-1 ppm acetyl hexapeptide-8 (purchased from Shenzhen Weiqi organism) into the mesenchymal stem cells of generation P3-P6, 37 deg.C, 5% CO 2 Co-culturing in an incubator, discarding supernatant after 24h, washing cells with phosphate buffer, changing the serum-free medium, continuing at 37 deg.C, 5% 2 Culturing for 48h in an incubator, collecting cell culture supernatant, and ultracentrifuging to obtain the mesenchymal stem cell extracellular vesicles.
Example 1
A composition for treating prostate calcification comprises 2ml of umbilical cord mesenchymal stem cell extracellular vesicles, 3 × 10 6 Umbilical cord mesenchymal stem cells and sodium bicarbonate with volume fraction of 8% and concentration of 5%.
The preparation method of the composition for treating prostate calcification comprises the following specific steps: adding 0.5ppm acetyl hexapeptide-8 to the P3 generation umbilical cord mesenchymal stem cells, standing at 37 deg.C, 5% CO 2 Co-culturing in an incubator, discarding supernatant after 24h, washing cells twice with phosphate buffer, changing the serum-free medium to 37 deg.C, 5% 2 Culturing for 48h in the incubator, and collecting cell culture supernatant. Centrifuging the collected cell supernatant for 5min at 1000rpm, collecting the supernatant, centrifuging the supernatant again through an ultrafiltration tube with the length of 100KD, centrifuging the supernatant for 20min at 2000rpm, and obtaining the umbilical cord mesenchymal stem cell extracellular vesicles at the upper layer of the ultrafiltration tube; then 3X 10 6 Umbilical cord mesenchymal stem cells and 2ml prepared umbilical cord mesenchymal stem cellsMixing extracellular vesicles to obtain a cell-vesicle mixture; and adding sodium bicarbonate injection with the volume fraction of 8% and the concentration of 5% into the cell-vesicle mixture to prepare the composition for treating the prostate calcification.
The performance evaluation of the prepared composition for prostate calcification is carried out, and the specific steps are as follows:
1. acute systemic toxicity test
Selecting 30 Kunming mice with the body weight of 18 +/-2 g and male and female halves, adaptively feeding for 7d, randomly dividing into 3 groups, and setting into a high dose group, a medium dose group and a low dose group, wherein each group comprises 5 male and female mice. The prepared composition is injected into the abdominal cavity of each group of mice, 6uL is injected into a low-dose group (the normal dosage of the mice is about 6uL according to a conversion coefficient table of the dosage of each kilogram of the body weight of animals and human bodies), 12uL is injected into a medium-dose group, 24uL is injected into a high-dose group, and the general symptoms, the poisoning condition and the death quantity of the mice are observed 7d after the injection is finished, wherein the experimental result is shown in a table 1:
TABLE 1 results of acute toxicity test
The results show that the mice are in good mental state, normal diet, free from poisoning symptoms such as curling and prostration and 0 in death number after injection is finished in the high-dose group, the medium-dose group and the low-dose group, and the composition prepared by the invention has no acute toxicity to the mice.
2. Pyrogen detection
Selecting 1.7-3.0kg New Zealand white rabbits as experimental animals, selecting 3 animals with qualified pyrogen, adaptively feeding for 7d, performing intravenous injection on the prepared composition according to 10ml/kg of ear margin, measuring normal body temperature of the animals before injection, and measuring body temperature 1 time and 6 times in total at intervals of 30min after injection according to the previous method. The normal body temperature is subtracted from the highest value in the body temperature of 6 times, and the temperature rise (DEG C) of the body temperature of the rabbit is obtained. If the body temperature of one of the 3 rabbits is increased by 0.6 ℃ or more than 0.6 ℃, or the total body temperature of the 3 rabbits is increased by 1.4 ℃ or more than 1.4 ℃, the pyrogen detection is judged to be unqualified, and the experimental results are shown in the table 2:
TABLE 2 pyrogen test results
The experimental result shows that the temperature rise of the tested 3 animals does not exceed 0.6 ℃, the total temperature rise value of the rabbits is lower than 1.4 ℃, and the pyrogen detection result is qualified, which indicates that the composition prepared by the invention has high safety.
3. Human body efficacy verification
Patients 1, male, 64 years old, chronic prostatitis for years, difficult urination (urgent urination, incomplete urination), many times of getting up at night, and burning sensation of urination. Ultrasonic examination shows that prostate fibrosis has calcified focus and the margin of inner gland envelope is unclear. Under the guidance of ultrasound, the prepared composition is injected into perineum part once a week, and after 3 times of injection, the patient can urinate smoothly without urine exhaustion, and the night times are reduced without burning feeling of perineum part. As shown in fig. 1, the ultrasound image shows that prostate calcification focus is gradually reduced and fibrosis is improved after 2 times of injection treatment; after the injection for the 3 rd time, the calcific foci in the gland completely disappear, the capsule structure of the prostate gland is recovered, and the capsule line is clear.
Example 2
A composition for treating prostate calcification comprises 2ml of umbilical cord mesenchymal stem cell extracellular vesicle and 1 × 10 7 Bone marrow mesenchymal stem cells and sodium bicarbonate with a volume fraction of 15% and a concentration of 5%.
A preparation method of a composition for treating prostate calcification comprises the following specific steps: adding 0.5ppm acetyl hexapeptide-8 to the P3 generation umbilical cord mesenchymal stem cells, standing at 37 deg.C, 5% CO 2 Co-culturing in an incubator, discarding supernatant after 24h, washing cells twice with phosphate buffer, changing serum-free medium to 37 deg.C, 5% 2 Continuously culturing for 48h in the incubator, and collecting umbilical cord mesenchymal stem cell culture supernatant. Centrifuging the collected cell supernatant at 1000rpm for 5min, collecting supernatant, and ultrafiltering with 100KDCentrifuging at 2000rpm for 20min to obtain umbilical cord mesenchymal stem cell extracellular vesicles as the upper layer of the ultrafiltration tube; then 1 × 10 7 Mixing the bone marrow mesenchymal stem cells with 2ml of prepared umbilical cord mesenchymal stem cell extracellular vesicles to prepare a cell-vesicle mixture; and adding sodium bicarbonate injection with the volume fraction of 15% and the concentration of 5% into the cell-vesicle mixture to prepare the composition for treating the prostate calcification.
The human body efficacy verification is carried out on the prepared composition for the prostate calcification, and the specific steps are as follows:
patients 2, male, 45 years old, chronic prostatitis for many years, have sensation of incomplete urination, intermittent pain in the evening and sexual dysfunction after going up to night for 3-5 times. Ultrasonic examination shows that there are calcified focus in prostate, unclear margin of inner line envelope, and tissue degeneration of prostate. Under the guidance of ultrasound, the composition is injected into perineum part once per week, and after 2 times of injection, the patient can eliminate the symptom of incomplete urine, reduce the times of getting up to night, eliminate pain, and recover sexual function. As figure 2, the ultrasound image shows that prostate calcification was reduced after 1 treatment; after 2 times of injection, the prostate calcification focus completely disappears, the inner gland capsule is clear, and the denatured tissue is recovered.
Example 3
A composition for treating prostate calcification comprises 10ml adipose derived mesenchymal stem cell extracellular vesicles, 3 × 10 7 The dental pulp mesenchymal stem cells and sodium bicarbonate with the volume fraction of 10% and the concentration of 5%.
The preparation method of the composition for treating prostate calcification comprises the following specific steps: adding 0.5ppm acetyl hexapeptide-8 into the adipose-derived mesenchymal stem cells for the P6 generation, standing at 37 deg.C, 5% 2 Co-culturing in an incubator, discarding supernatant after 24h, washing cells twice with phosphate buffer, changing serum-free medium to 37 deg.C, 5% 2 Continuously culturing for 48h in the incubator, and collecting the culture supernatant of the adipose-derived mesenchymal stem cells. Centrifuging the collected cell supernatant for 5min at 1000rpm, collecting the supernatant, performing ultrafiltration centrifugation of 100KD again at 2000rpm for 20min, and obtaining the umbilical cord mesenchymal stem cell extracellular vesicles at the upper layer of the ultrafiltration tube; then will be3×10 7 Mixing dental pulp mesenchymal stem cells with 10ml of prepared adipose mesenchymal stem cell extracellular vesicles to prepare a cell-vesicle mixture; and adding sodium bicarbonate injection with the volume fraction of 10% and the concentration of 5% into the cell-vesicle mixture to prepare the composition for treating the prostate calcification.
Example 4
A composition for treating prostate calcification comprises mixed stem cell extracellular vesicles of 4ml and 1.6 × 10 7 Individual mesenchymal stem cells and sodium bicarbonate at a volume fraction of 12% and a concentration of 5%.
The preparation method of the composition for treating the calcification of the prostate comprises the following specific steps: the P4-generation umbilical cord mesenchymal stem cells and the P4-generation dental pulp mesenchymal stem cells were inoculated into the same culture flask at an inoculation ratio of 1 2 After culturing in the incubator for anchorage, 0.5ppm of acetyl hexapeptide-8 was added to the cell culture flask and placed at 37 ℃ 5% 2 Co-culturing in incubator again, discarding supernatant after 24 hr, washing cells twice with phosphate buffer solution, changing serum-free medium at 37 deg.C, and 5% 2 And continuously culturing for 48 hours in the incubator, and collecting the culture supernatant of the mixed culture cells of the two stem cells. Centrifuging the collected cell supernatant for 5min at 1000rpm, collecting the supernatant, and centrifuging again at 100KD for ultrafiltration at 2000rpm for 20min to obtain mixed stem cell extracellular vesicles at the upper layer of the ultrafiltration tube; then 1.6X 10 7 Mixing the mesenchymal stem cells with 4ml of prepared mixed stem cell extracellular vesicles to prepare a cell-vesicle mixture; and adding sodium bicarbonate injection with the volume fraction of 12% and the concentration of 5% into the cell-vesicle mixture to prepare the composition for treating the calcification of the prostate.
The above-mentioned contents are only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited thereby, and any modification made on the basis of the technical idea of the present invention falls within the protection scope of the claims of the present invention.
Claims (10)
1. A composition for treating prostate calcification, comprising a mesenchymal stem cell extracellular vesicle, a mesenchymal stem cell and sodium bicarbonate;
wherein 1.5 × 10 of the total amount of the mesenchymal stem cells is added into each milliliter of extracellular vesicles 6 ～0.5×10 7 (ii) mesenchymal stem cells; the volume fraction of the sodium bicarbonate added in the composition is 8-15%.
2. The composition for treating prostate calcification of claim 1, wherein said mesenchymal stem cell extracellular vesicles are secreted from one or both of umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, bone marrow mesenchymal stem cells and dental pulp mesenchymal stem cells.
3. The composition for treating prostate calcification of claim 1, wherein said mesenchymal stem cell extracellular vesicles are obtained after induction of mesenchymal stem cells by acetyl hexapeptide-8.
4. The composition for treating prostate calcification of claim 1, wherein said mesenchymal stem cells are one or a combination of umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, bone marrow mesenchymal stem cells and dental pulp mesenchymal stem cells.
5. The composition for treating prostate calcification of claim 1, wherein said mesenchymal stem cells are P3-P6 generation mesenchymal stem cells.
6. The composition for treating prostate calcification of claim 1, wherein said sodium bicarbonate is 5% sodium bicarbonate injection.
7. The method of any one of claims 1 to 6, wherein the mesenchymal stem cells are mixed with mesenchymal stem cell extracellular vesicles to prepare a cell-vesicle mixture; and adding sodium bicarbonate into the cell-vesicle mixture to obtain the composition for treating prostate calcification.
8. The method of preparing a composition for treating prostate calcification according to claim 7, wherein the mesenchymal stem cell extracellular vesicles are prepared by: adding 0.5-1 ppm acetyl hexapeptide-8 to mesenchymal stem cells at 37 ℃,5% 2 Co-culturing in incubator, discarding supernatant after 24 hr, washing cells with phosphate buffer, changing serum-free medium, continuing to control CO at 37 deg.C 2 Culturing for 48h in an incubator, collecting cell culture supernatant, and ultracentrifuging to obtain the mesenchymal stem cell extracellular vesicles.
9. Use of a composition according to any one of claims 1 to 6 for the treatment of prostate calcification in the manufacture of a pharmaceutical preparation for the treatment of chronic prostatitis, urethritis or male infertility.
10. The use of claim 9, wherein the formulation is an injection.
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