CN111419877A - Medicine for treating male erectile dysfunction and preparation method thereof - Google Patents
Medicine for treating male erectile dysfunction and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a medicine for treating male erectile dysfunction and a preparation method thereof, the medicine for treating male erectile dysfunction consists of fat EPC and extracellular matrix suspension, the quantity of the fat EPC is 1 × 106~10×106The medicine for treating male erectile dysfunction is used by combining fat EPC with extracellular matrix, the extracellular matrix contains abundant collagen, fibronectin, elastin and proteoglycan, the fat EPC can be fixed, a good microenvironment is provided for the fat EPC, cell proliferation is promoted, the fat EPC is combined with the extracellular matrix, and the reconstruction of the cavernous body structure of the penis is improved, and endothelium and the flatness are increased through the angiogenesis effect and the repair function of endothelial progenitor cellsThe number of smooth muscle cells promotes the recovery of erectile function.
Description
Technical Field
The invention relates to the field of tissue engineering, in particular to a medicine for treating male erectile dysfunction and a preparation method thereof.
Background
Erectile Dysfunction (ED) is one of common diseases affecting male quality of life, and the results of analysis by the International consultation Commission on medical science show that ED prevalence rates of men under 40 years old are about 1% -10%, between 40-49 years old are about 2% -9%, between 60-69 years old are 20% -40%, and more than 70 years old are about 50% -100% of ED with different degrees.
Numerous clinical and preclinical studies have demonstrated that endothelial dysfunction is an important factor in the development of ED. After the cavernous body endothelium of the penis and smooth muscle cells are apoptotic and atrophic, the general drug treatment can not induce the tissue regeneration. Damaged tissue must be regenerated by either replenishing the damaged cells or stimulating the proliferation of residual Stem Cells (SCs) within the tissue. However, if the injection site has sufficient blood flow (such as cavernous sinus of penis), the injected cells will be quickly washed away by the blood flow, thereby affecting the retention rate of the cells in local tissues and also affecting the therapeutic effect of the adipose stem cells (ADSCs). To avoid repeated injections of therapy, the survival rate of the transplanted cells at the injection site is critical to the long-term success of the therapy. Researchers have used methods of cytogenetic engineering, hydrogels and biodegradable membranes, microsphere tissues, etc. to improve the survival rate of cells and the therapeutic effect of cell transplantation. However, most of these methods require complicated preparation procedures and are mostly foreign substances, easily contaminated with viruses and bacteria, and have an immunological rejection effect, and are expensive.
Disclosure of Invention
In view of the above technical problems in the related art, the present invention provides a drug for treating male erectile dysfunction and a method for preparing the same, which can overcome the above disadvantages of the prior art.
In order to achieve the technical purpose, the technical scheme of the invention is realized as follows:
a medicine for treating male erectile dysfunction is composed of fat Endothelial Progenitor Cells (EPCs) and extracellular matrix suspension, wherein the number of the EPCs is 1 × 106～10×106And each m L.
Preferably, the extracellular matrix suspension is a physiological saline suspension of extracellular matrix, and the volume ratio of the extracellular matrix to the physiological saline in the extracellular matrix suspension is 1:5-1: 20.
Preferably, the amount of fat EPC is 5 × 106M L, wherein the volume ratio of the extracellular matrix to the physiological saline in the extracellular matrix suspension is 1: 10.
According to another aspect of the present invention, there is provided a process for the preparation of a medicament for the treatment of male erectile dysfunction, the process comprising the steps of:
1) removing blood vessel tissue and red blood cells in fat: after obtaining fat, repeatedly cleaning the fat by using normal saline, centrifuging, collecting an upper fat tissue layer, and removing lower red blood cells and blood vessel debris;
2) removal of ADSCs from fat: adding collagenase type I into the adipose tissue obtained in the step 1), uniformly mixing, vibrating and digesting, and centrifuging to remove the lower-layer precipitates and ADSCs;
3) fat EPC and extracellular matrix acquisition: collecting the upper-layer adipose tissue obtained in the step 2), adding collagenase type I and trypsin, oscillating and digesting, centrifuging, collecting precipitates, removing upper-layer liquefied fat oil drops and digestive juice, resuspending the precipitates with physiological saline, removing vascular tissue components by using a 2000-micrometer cell sieve to obtain a mixed solution containing extracellular matrix and fat EPC, filtering and collecting the extracellular matrix by using a 100-micrometer cell sieve, wherein the volume ratio of the extracellular matrix to the extracellular matrix is 1:5-1: resuspending with physiological saline solution of 20 deg.C to obtain extracellular matrix suspension, and freezing at-20 deg.C for storage;
4) fat EPC culture: centrifuging the filtrate obtained after filtering the fat extracellular matrix in the step 3), collecting the precipitate, re-suspending the precipitate by using an EPC culture medium, culturing the fat EPC, after the adherent cells form monoclones, respectively selecting each monoclones to continue culturing, and digesting and passaging by using pancreatin when the cell fusion degree reaches 80-90% to obtain the fat EPC;
5) preparation of suspension containing fat EPC and extracellular matrix by thawing the extracellular matrix suspension obtained in step 3) at 37 deg.C, and resuspending the fat EPC obtained in step 4) using the extracellular matrix suspension to keep the amount of fat EPC at 1 × 106～10×106And each m L.
Preferably, thinThe volume ratio of extracellular matrix to physiological saline in the extracellular matrix suspension is 1:10, and the amount of fat EPC in the suspension containing the fat EPC and the extracellular matrix is 5 × 106And each m L.
Preferably, the collagenase type I added in the step 2) has the same volume with the adipose tissue, and the final concentration of the collagenase type I is 0.05%; the collagenase type I and the trypsin which are added in the step 3) have the same volume with the adipose tissue, the final concentration of the collagenase type I is 0.05%, and the final concentration of the trypsin is 0.125%.
Preferably, the condition of the shock digestion in the step 2) is that the shock digestion is carried out for 30min at the rotation speed of 280r/min and the temperature of 37 ℃, and the condition of the shock digestion in the step 3) is that the shock digestion is carried out for 30min at the rotation speed of 250r/min and the temperature of 37 ℃.
Preferably, a volume ratio to extracellular matrix of 1:10 to obtain extracellular matrix suspension.
Preferably, the extracellular matrix collected by filtering the 100 μm cell sieve in the step 3) is resuspended and washed by 10m L physiological saline before being resuspended by physiological saline with the volume ratio of the extracellular matrix being 1:5-1:20, the mixture is added into a 15m L centrifuge tube, the centrifuge is carried out at 300g centrifugal force, the rising speed is 5, the descending speed is 5, the centrifuge is carried out for 8min, the volume of the extracellular matrix is obtained, and the supernatant physiological saline is discarded.
Preferably, the specific conditions of the centrifugation in the step 4) are 400g of centrifugal force, 9 increasing the speed and 9 decreasing the speed, and 10min of centrifugation.
The invention has the beneficial effects that:
1) the preparation method of the medicine for treating male erectile dysfunction is completed by two-step digestion according to different cell traits and tissue internal dispersion degree, the first step is to digest and remove adipose-derived stem cells (ADSCs) to prevent EPC pollution in the later period, and the second step is to digest and obtain adipose EPC cells and extracellular matrix parts;
2) the compound is used in combination with the extracellular matrix, the extracellular matrix contains abundant collagen, fibronectin, elastin and proteoglycan, can fix the fat EPC cells, provides a good microenvironment for the fat EPC cells, and promotes cell proliferation;
3) the fat EPC is combined with the extracellular matrix, so that the reconstruction of the cavernous body structure of the penis is improved, the quantity of endothelial cells and smooth muscle cells is increased, and the recovery of the erectile function is promoted through the angiogenesis function and the repair function of endothelial progenitor cells.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 is a cell morphology map of a single clone of adipose endothelial progenitor cells according to an embodiment of the present invention;
FIG. 2 is a diagram of the morphology of the adipose endothelial progenitor cells with a cell confluency of 90% according to an embodiment of the present invention;
FIG. 3 shows the result of flow cytometry detection of human adipose endothelial progenitor cell surface markers according to the embodiment of the present invention.
Detailed Description
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 that can be derived by one of ordinary skill in the art from the embodiments given herein are intended to be within the scope of the present invention.
Example 1
Removal of blood vessel tissue and red blood cells in fat
1) Transfer adipose tissue from the sampling vials using a 25m L pipette into 50m L centrifuge tubes, 20m L per tube;
2) adding 20m L normal saline into the adipose tissue centrifuge tube;
3) repeatedly beating adipose tissue with 25m L pipette for 10-20 times, washing thoroughly, adding physiological saline to 50m L, capping, repeatedly pouring and mixing;
4)300g of centrifugal force, 9 rising speeds and 9 falling speeds, and 8min of centrifugation;
5) after centrifugation, the liquid is divided into two layers, the upper layer is a fat layer, the lower layer is a washing physiological salt water layer containing the fatty blood vessel tissue sediment, impurities such as the fatty blood vessel tissue, red blood cells and the like are precipitated at the bottom of the centrifuge tube, the upper fat layer is sucked into a new 50m L centrifuge tube by using a 25m L pipette, and 20m L is used for each tube for standby application, the steps 2, 3 and 4 are repeated, and one side is cleaned again;
6) after centrifugation, the adipose layer was collected and the underlying adipose vascular tissue and red blood cells were removed.
Example 2
Removal of ADSCs from fat
1) Adding equal volume of preheated type I collagenase 20m L with the concentration of 0.1% to the adipose tissue of 20m L according to the volume of the adipose tissue, covering, pouring and mixing repeatedly, wherein the final concentration of the type I collagenase is 0.05%;
2) shaking and digesting for 30min at the rotation speed of 280r/min and the temperature of 37 ℃;
3) after digestion, 500g of centrifugal force, 9 acceleration and 9 deceleration, and 10min of centrifugation;
4) after centrifugation, the liquid is divided into three layers, the upper layer is liquefied grease, the middle layer is an adipocyte layer, the digestive juice layer of the ADSCs in the lower layer fat, the upper layer and the lower layer are discarded, and a 25m L pipette is used for absorbing the adipocyte layer.
Example 3
Fat EPC and extracellular matrix acquisition
1) Adding equal volume of collagenase type I and trypsin to the adipose tissue, wherein the final concentration of the collagenase type I is 0.05 percent, and the concentration of the trypsin is 0.125 percent;
2) carrying out shake digestion for 30min at the rotating speed of 250r/min and the temperature of 37 ℃ (the centrifugal tube is horizontally placed so that fat and type I collagenase can be fully mixed in the digestion process);
3) after digestion, 600g of centrifugal force, 9 acceleration and 9 deceleration, and 10min of centrifugation;
4) after centrifugation, the liquid is divided into three layers, the upper layer is liquefied grease, the middle layer is a digestive juice layer, the lower layer is EPC cells and an extracellular matrix layer, and the upper layer and the middle layer are removed;
5) adding physiological saline into the lower layer to 50m L, mixing, centrifuging at 600g, 9 g and 9 g, centrifuging for 10min, and cleaning the digestive juice;
6) resuspending with 30m L physiological saline, precipitating, and removing blood vessel and tissue components with 2000 μm cell sieve to obtain extracellular matrix and mixed solution rich in EPC cells;
7) filtering the mixed solution by using a 100-micron cell sieve, wherein the part on the 100-micron cell sieve is extracellular matrix, and the filtered solution is a suspension rich in EPC cells;
8) collecting extracellular matrix on the cell screen, resuspending with 10m L physiological saline, adding into 15m L centrifuge tube, centrifuging at 300g, 5 rising speed and 5 falling speed, centrifuging for 8min to obtain extracellular matrix volume, and discarding supernatant physiological saline.
9) Using an extracellular matrix and a mixture of 1:10, obtaining extracellular matrix suspension, and freezing and storing at-20 ℃.
10) And (3) carrying out 400g centrifugal force on the EPC-enriched cell suspension filtered in the step (7), increasing the speed by 9, decreasing the speed by 9, and centrifuging for 10min to obtain an EPC-enriched cell layer.
Example 4
Culture of fat EPC
1) Resuspending human Endothelial progenitor cells in complete Medium of Endothelial Cell Medium (scientific-1001) to induce adherence;
2) after 36 hours, changing the liquid in full, removing the non-adherent cells, adding a fresh complete culture medium, and changing the liquid in half every other day;
3) after the adherent cells form monoclonals, respectively selecting each monoclonals to continue culturing, digesting and passaging by 0.05 percent of pancreatin when the cell fusion degree reaches 80-90 percent, continuing culturing, and collecting the P3-P5 generation of adipose-derived endothelial progenitor cells. FIG. 1 is a cell morphology diagram of a single clone of the adipose endothelial progenitor cells, FIG. 2 is a cell morphology diagram of the adipose endothelial progenitor cells with a cell fusion degree of 90%, FIG. 3 is a flow cytometry detection result of the surface markers of the adipose endothelial progenitor cells, and the results of CD31 and CD105 are 98.85% and 99.8%, respectively.
Example 5
Preparation of an extracellular matrix suspension enriched in endothelial progenitor cells
1) Rapidly melting the extracellular matrix at 37 ℃ to obtain an extracellular matrix suspension;
2) obtaining P3-P5 generation adipose-derived endothelial progenitor cells by a digestion method, and cleaning with normal saline to remove pancreatin;
3) in use, the extracellular matrix suspension is used for resuspending adipose derived endothelial progenitor cells, and the number of the endothelial progenitor cells is kept at 5 × 106And each m L.
Example 6
Application of extracellular matrix suspension rich in endothelial progenitor cells
Experimental data were collected for conditioning male erectile dysfunction using autologous adipose-rich endothelial progenitor cell and extracellular matrix suspension prepared in example 5, wherein the volume ratio of extracellular matrix to physiological saline was 1:10, the number of endothelial progenitor cells was maintained at 5 × 106Each m L, the injection is injected by cavernous body of penis, every 60 days, 3 times of injection is a treatment course, 3m L each time, and the total is 6 points.
The effect is shown in table 1 (table designed according to international erection function index), before conditioning with the extracellular matrix suspension rich in endothelial progenitor cells of the invention, the sexual life frequency of the recipient is low, most penis parts can not be erected normally after sexual stimulation, and sexual life is difficult to complete; the intercourse can be partially completed with an average time of less than 2 minutes.
After conditioning after a treatment course, most recipients feed back that morning puffs appear, the sexual life frequency is increased, the autonomous feeling erection strength is obviously enhanced, the size is obviously increased, the sexual intercourse can be normally finished, and the average sexual intercourse time is prolonged.
TABLE 1 Experimental data for autologous fat EPC binding to extracellular matrix for the treatment of male erectile dysfunction
In conclusion, by means of the technical scheme, the fat EPC is combined with the extracellular matrix, the angiogenesis function and the repair function of the endothelial progenitor cells are realized, the reconstruction of the cavernous body structure of the penis is improved, the number of endothelial cells and smooth muscle cells is increased, the recovery of the erectile function is promoted, and the extracellular matrix is self-body, is safe and has no rejection as a whole and is a good fixing material.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (10)
1. A medicine for treating male erectile dysfunction is characterized by consisting of fat EPC and an extracellular matrix suspension, wherein the amount of the fat EPC is 1 × 106~10×106And each m L.
2. The medicament according to claim 1, characterized in that the extracellular matrix suspension is a physiological saline suspension of extracellular matrix, and the volume ratio of extracellular matrix to physiological saline in the extracellular matrix suspension is 1:5-1: 20.
3. The medicament according to claim 2, wherein the fat EPC is present in an amount of 5 × 106M L, wherein the volume ratio of the extracellular matrix to the physiological saline in the extracellular matrix suspension is 1: 10.
4. A method for preparing a medicament for treating male erectile dysfunction, comprising the steps of:
1) removing blood vessel tissue and red blood cells in fat: after obtaining fat, repeatedly cleaning the fat by using normal saline, centrifuging, collecting an upper fat tissue layer, and removing lower red blood cells and blood vessel debris;
2) removal of ADSCs from fat: adding collagenase type I into the adipose tissue obtained in the step 1), uniformly mixing, vibrating and digesting, and centrifuging to remove the lower-layer precipitates and ADSCs;
3) fat EPC and extracellular matrix acquisition: collecting the upper-layer adipose tissue obtained in the step 2), adding collagenase type I and trypsin, oscillating and digesting, centrifuging, collecting precipitates, removing upper-layer liquefied fat oil drops and digestive juice, resuspending the precipitates with physiological saline, removing vascular tissue components by using a 2000-micrometer cell sieve to obtain a mixed solution containing extracellular matrix and fat EPC, filtering and collecting the extracellular matrix by using a 100-micrometer cell sieve, wherein the volume ratio of the extracellular matrix to the extracellular matrix is 1:5-1: resuspending with physiological saline solution of 20 deg.C to obtain extracellular matrix suspension, and freezing at-20 deg.C for storage;
4) fat EPC culture: centrifuging the filtrate obtained after filtering the fat extracellular matrix in the step 3), collecting the precipitate, re-suspending the precipitate by using an EPC culture medium, culturing the fat EPC, after the adherent cells form monoclones, respectively selecting each monoclones to continue culturing, and digesting and passaging by using pancreatin when the cell fusion degree reaches 80-90% to obtain the fat EPC;
5) preparation of suspension containing fat EPC and extracellular matrix by thawing the extracellular matrix suspension obtained in step 3) at 37 deg.C, and resuspending the fat EPC obtained in step 4) using the extracellular matrix suspension to keep the amount of fat EPC at 1 × 106~10×106And each m L.
5. The method according to claim 4, wherein the volume ratio of the extracellular matrix to the physiological saline in the extracellular matrix suspension is 1:10, and the amount of the fat EPC in the suspension containing the fat EPC and the extracellular matrix is 5 × 106And each m L.
6. The method according to claim 4, wherein the collagenase type I added in step 2) has the same volume as that of the adipose tissue, and the final concentration of the collagenase type I is 0.05%; the collagenase type I and the trypsin which are added in the step 3) have the same volume with the adipose tissue, the final concentration of the collagenase type I is 0.05%, and the final concentration of the trypsin is 0.125%.
7. The method according to claim 4, wherein the conditions for the concussion digestion in step 2) are at a rotation speed of 280r/min and a temperature of 37 ℃ for 30min, and the conditions for the concussion digestion in step 3) are at a rotation speed of 250r/min and a temperature of 37 ℃ for 30 min.
8. The method according to claim 4, wherein the ratio of the volume of the extracellular matrix used in step 3) to the volume of the extracellular matrix is 1:10 to obtain extracellular matrix suspension.
9. The method for preparing the extracellular matrix of claim 4, wherein the extracellular matrix collected by filtration using a 100 μm cell sieve in step 3) is resuspended and washed with 10m L physiological saline before being resuspended with physiological saline at a volume ratio of 1:5 to 1:20 to the extracellular matrix, the mixture is added into a 15m L centrifuge tube, centrifuged at 300g and 5 min, centrifuged for 8min to obtain the extracellular matrix volume, and the supernatant physiological saline is discarded.
10. The preparation method according to claim 4, wherein the specific conditions of the centrifugation in the step 4) are 400g of centrifugal force, 9 increasing of speed and 9 decreasing of speed, and 10min of centrifugation.
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| CN104388388A (en) * | 2014-11-11 | 2015-03-04 | 贾瑞鹏 | Endothelial progenitor cell (EPC) culture method |
| CN106074603A (en) * | 2016-07-08 | 2016-11-09 | 中山大学附属第医院 | A kind of stem cell combined preparation for treating Erectile Dysfunction |
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| CN104388388A (en) * | 2014-11-11 | 2015-03-04 | 贾瑞鹏 | Endothelial progenitor cell (EPC) culture method |
| CN106074603A (en) * | 2016-07-08 | 2016-11-09 | 中山大学附属第医院 | A kind of stem cell combined preparation for treating Erectile Dysfunction |
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