Pharmaceutical composition for treating bladder detrusor dysfunction as well as preparation method and application thereof

Abstract

The invention relates to the technical field of stem cell treatment, in particular to a pharmaceutical composition for treating bladder detrusor dysfunction and a preparation method and application thereof. The invention provides a pharmaceutical composition for treating bladder detrusor dysfunction. The pharmaceutical composition is prepared from 4 to 6 * 10 < 10 > pieces / mL of adipose tissue-derived stem cell exosomes, 0.01 to 0.02 mL / mL of platelet-rich plasma, 20 to 30 mg / mL of bamboo leaf flavonoid extract, 25 to 35 mg / mL of fructus rosae laevigatae extract, 30 to 40 mg / mL of human serum albumin, 1.1 to 1.5 mg / mL of carboxymethyl chitosan and the balance of water for injection. The pharmaceutical composition provided by the invention is helpful for increasing the contractility of detrusor muscle, promoting urine emptying of bladder, reducing RUV and reducing urination times, so that the life quality of a patient with detrusor muscle dysfunction is improved, the I-PSS score is reduced, and the pharmaceutical composition has an excellent treatment effect on detrusor muscle dysfunction.

Description

Technical Field

[0001] The present invention relates to the technical field of stem cell therapy, and in particular to a pharmaceutical composition for treating bladder detrusor dysfunction, and a preparation method and application thereof. Background Art

[0002] Prostatic hyperplasia is a common disease in elderly men. According to statistics, 87% of men over 53 years old have varying degrees of dysuria caused by prostatic hyperplasia. It has the characteristics of insidious onset, long course, and serious consequences. Long-term dysuria leads to the inability to completely empty the bladder, which causes hyperplasia and dysfunction of the detrusor muscle. Even after the obstruction caused by prostatic hyperplasia is relieved (TVP surgery), the urination function of the bladder cannot be restored in a short time. In severe cases, there will be no improvement in bladder emptying, causing urinary retention caused by neurogenic bladder.

[0003] Mesenchymal stem cells are important members of the stem cell family. They originate from the early mesoderm of development and are multipotent stem cells. They exist in a variety of tissues (such as bone marrow, umbilical cord blood and umbilical cord tissue, placental tissue, adipose tissue, etc.). Mesenchymal stem cells have multidirectional differentiation potential and are adult cells of non-hematopoietic stem cells. They can differentiate into a variety of mesenchymal series cells (such as osteoblasts, chondrogenic cells, and adipogenic cells, etc.) or non-mesenchymal series cells, and have unique cytokine secretion functions. Mesenchymal stem cells can migrate to the exact site of injury, differentiate into various cell lineages, secrete soluble factors that are essential for cell survival and proliferation, and regulate immune responses. As an adult multipotent stem cell, mesenchymal stem cells can self-renew, multidirectionally differentiate, have a wide range of sources, are easy to isolate and culture in vitro, and have low immunogenicity. They have the functions of tissue damage repair and immune regulation. These characteristics make them potentially valuable in the treatment of detrusor injury.

[0004] Therefore, how to utilize the characteristics of mesenchymal stem cells and apply them in the preparation of drugs for treating bladder detrusor dysfunction is a problem that technical personnel in this field urgently need to solve. Summary of the invention

[0005] The purpose of the present invention is to provide a pharmaceutical composition for treating bladder detrusor dysfunction and its preparation method and application. The present invention utilizes the paracrine function of mesenchymal stem cells to repair the damaged bladder detrusor muscle, and performs multi-point local injection on the detrusor muscle under cystoscopy to achieve the purpose of restoring the bladder emptying function.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a pharmaceutical composition for treating bladder detrusor dysfunction, comprising: 4 to 6×10 10 / mL, platelet-rich plasma 0.01-0.02mL / mL, bamboo leaf flavonoids extract 20-30mg / mL, Rosa laevigata extract 25-35mg / mL, human serum albumin 30-40mg / mL, carboxymethyl chitosan 1.1-1.5mg / mL, and the rest is water for injection.

[0008] Preferably, the water for injection is 0.9% sodium chloride water for injection.

[0009] Preferably, the method for preparing adipose-derived mesenchymal stem cell exosomes comprises the following steps:

[0010] (1) Extraction and separation of primary adipose-derived mesenchymal stem cells:

[0011] (2) inoculating the primary adipose-derived mesenchymal stem cells obtained in step (1) into a culture bottle, supplementing the culture medium and then culturing;

[0012] (3) When the cell density in step (2) reaches 70-80%, the cell culture medium is collected and centrifuged to obtain adipose stem cell exosomes.

[0013] Preferably, the culture medium consists of the following components: 0.3-0.5 g / L L-isoleucine, 1-2 g / L L-valine, 1-2 g / L glycine, 7-9 mM β-glycerophosphate, 7-9 g / L glutathione, 4-8 ng / mL platelet-derived growth factor, 4-6 ng / mL EGF, 9-11 ng / mL bFGF, 4-8 ng / mL fibroblast growth factor, and the rest is basal culture medium.

[0014] Preferably, the basal culture medium is DMEM-F12 culture medium.

[0015] Preferably, the centrifugation method is: centrifuging the cell culture fluid at 200-250×g for 12-16 min, 1900-2100×g for 5-10 min, and 9000-11000×g for 25-35 min to remove cells and cell debris; continuing to centrifuge at 99000-100000×g for 65-75 min, resuspending and washing the precipitate at the bottom, and finally centrifuging at 99000-10000×g for 65-75 min to remove impurities to obtain adipose mesenchymal stem cell exosomes.

[0016] Preferably, the seeding density in step (2) is 4200 to 4400 cells / cm 2 .

[0017] Preferably, the culture temperature in step (2) is 36-38° C., and the carbon dioxide concentration is 4-6%.

[0018] The present invention also provides a method for preparing the pharmaceutical composition, which comprises mixing water for injection, bamboo leaf flavonoids extract, Rosa laevigata extract, and carboxymethyl chitosan, and then sequentially adding platelet-rich plasma, human serum albumin, and adipose-derived mesenchymal stem cell exosomes and mixing.

[0019] The present invention also provides the use of the pharmaceutical composition in preparing a drug for treating bladder detrusor dysfunction.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] Different types of mesenchymal stem cells have different effects, and the exosomes of different types of mesenchymal stem cells also have different immune or repair effects. The present invention applies adipose mesenchymal stem cells to the treatment of bladder detrusor dysfunction. Clinical application experiments have shown that mesenchymal stem cells have a good therapeutic effect in injury repair, which can greatly improve the local state of the patient, making it easier for the patient to recover, and providing a basis for the clinical application of stem cells.

[0022] Detrusor hyperplasia and dysfunction are bladder smooth muscle dysfunction and pathophysiological changes caused by partial bladder outlet obstruction. From a cytological point of view, its pathogenic factors include multiple aspects, including mitochondrial dysfunction, sarcoplasmic reticulum calcium pump dysfunction, changes in cytokines in the bladder wall, and bladder wall denervation. The specific mechanism is: partial bladder outlet obstruction causes bladder overfilling, followed by bladder wall ischemia, while causing bladder wall denervation and changes in cytokine concentrations. Ultimately, it causes bladder smooth muscle hyperplasia and hypertrophy, leading to a series of functional disorders. From the perspective of molecular biological changes, it is mainly caused by the disorder of DNA and RNA synthesis and changes in the intracellular myosin structure.

[0023] The present invention collects the culture fluid during the culture of adipose mesenchymal stem cells, and prepares exosomes by centrifugation. The exosomes prepared by the method carry abundant proteins, RNA, DNA and lipid molecules (including vascular endothelial growth factor and basic fibroblast growth factor, etc.), and have the characteristics of biocompatibility and low immunogenicity. The prepared exosomes can penetrate the physiological barrier of body tissues and participate in information communication and material exchange between cells. The exosomes prepared by the method can improve sarcoplasmic reticulum calcium pump dysfunction, and have the function of balancing cytokines in the bladder wall, avoiding bladder smooth muscle hyperplasia caused by changes in cytokines in the bladder wall, and the use of adipose mesenchymal stem cell exosomes has a certain therapeutic and improvement effect on bladder detrusor dysfunction.

[0024] The bamboo leaf flavonoid extract added by the present invention has carbon glycoside flavonoids as the main components, mainly orientin, isoorientin, vitexin and isovitexin, which have the effects of resisting free radicals, resisting lipid oxidation and protecting DNA oxidative damage. Therefore, the addition of bamboo leaf flavonoid extract can improve the problem of disordered molecular expression from the molecular level. The extract of Rosa laevigata is closely related to M receptors, so it has a certain effect on treating frequent urination and nocturia. In the invention, the bamboo leaf flavonoid extract is used together with the extract of Rosa laevigata, the purpose is to improve the denervation of the bladder wall. Therefore, the pharmaceutical composition for treating bladder detrusor dysfunction provided by the present invention, starting from the cause of bladder detrusor dysfunction, organically cooperates with various components, synergizes and enhances the effect, and achieves the purpose of effectively treating bladder detrusor dysfunction.

[0025] The addition of carboxymethyl chitosan of the present invention can promote the growth of endothelial cells and epithelial cells of the body, and has an inhibitory effect on the growth of fibroblasts, smooth muscle cells, etc. Therefore, the addition of carboxymethyl chitosan can prevent the proliferation and hypertrophy of bladder smooth muscle, and help the exosomes of mesenchymal stem cells to play their role.

[0026] In the composition of the present invention, the addition of human serum albumin can improve the antioxidant capacity of the composition and improve the stability of the composition.

[0027] When culturing adipose mesenchymal stem cells, the L-isoleucine and other amino acids added to the culture medium are all non-polar amino acids. The addition of such amino acids can effectively shorten the hysteresis period of cell growth, thereby enabling rapid expansion and passage. The present invention also adds platelet-derived growth factor and fibroblast growth factor during the adipose mesenchymal stem cell culture process. The addition of the two growth factors plays a role in maintaining the survival and stimulating the proliferation of mesenchymal stem cells.

[0028] The pharmaceutical composition provided by the present invention helps to increase detrusor contractility, promote bladder emptying of urine, reduce RUV, reduce urination frequency, thereby improving the quality of life of elderly patients with detrusor dysfunction, reducing I-PSS scores, and has an excellent therapeutic effect on detrusor dysfunction. DETAILED DESCRIPTION

[0029] The present invention provides a pharmaceutical composition for treating bladder detrusor dysfunction, comprising: 4 to 6×10 10 / mL, platelet-rich plasma 0.01-0.02mL / mL, bamboo leaf flavonoids extract 20-30mg / mL, Rosa laevigata extract 25-35mg / mL, human serum albumin 30-40mg / mL, carboxymethyl chitosan 1.1-1.5mg / mL, and the rest is water for injection; preferably adipose-derived mesenchymal stem cell exosomes 3-7mg / mL, platelet-rich plasma 0.015mL / mL, bamboo leaf flavonoids extract 22-28mg / mL, Rosa laevigata extract 27-33mg / mL, human serum albumin 32-38mg / mL, carboxymethyl chitosan 1.2-1.4mg / mL g / mL; further preferably, adipose-derived mesenchymal stem cell exosomes 4-6 mg / mL, platelet-rich plasma 0.015 mL / mL, bamboo leaf flavonoids extract 24-26 mg / mL, Rosa laevigata extract 29-31 mg / mL, human serum albumin 34-36 mg / mL, and carboxymethyl chitosan 1.3 mg / mL; more preferably, adipose-derived mesenchymal stem cell exosomes 5 mg / mL, platelet-rich plasma 0.015 mL / mL, bamboo leaf flavonoids extract 25 mg / mL, Rosa laevigata extract 30 mg / mL, human serum albumin 35 mg / mL, and carboxymethyl chitosan 1.3 mg / mL.

[0030] In the present invention, the water for injection is 0.9% sodium chloride water for injection.

[0031] In the present invention, the method for preparing adipose-derived mesenchymal stem cell exosomes comprises the following steps:

[0032] (1) Extraction and separation of primary adipose-derived mesenchymal stem cells:

[0033] (2) inoculating the primary adipose-derived mesenchymal stem cells obtained in step (1) into a culture bottle, supplementing the culture medium and then culturing;

[0034] (3) When the cell density in step (2) reaches 70-80%, the cell culture medium is collected and centrifuged to obtain adipose stem cell exosomes.

[0035] In the present invention, the culture medium is composed of the following components: 0.3-0.5 g / L L-isoleucine, 1-2 g / L L-valine, 1-2 g / L glycine, 7-9 mM β-glycerophosphate, 7-9 g / L glutathione, 4-8 ng / mL platelet-derived growth factor, 4-6 ng / mL EGF, 9-11 ng / mL bFGF, 4-8 ng / mL fibroblast growth factor, and the rest is a basic culture medium; preferably 0.4 g / L L-isoleucine, 1.5 g / L L-valine, 1.5 g / L glycine, 8 mM β-glycerophosphate, 8 g / L glutathione, 5-7 ng / mL platelet-derived growth factor, 5 ng / mL EGF, 10 ng / mL bFGF, 5-7 ng / mL fibroblast growth factor, and the rest is a basic culture medium; more preferably 0.4 g / L L-isoleucine, 1.5 g / L L-valine, 1.5g / L glycine, 8mM β-glycerophosphate, 8g / L glutathione, 6ng / mL platelet-derived growth factor, 5ng / mL EGF, 10ng / mL bFGF, 6ng / mL fibroblast growth factor, and the rest are basal culture medium.

[0036] In the present invention, the basal culture medium is DMEM-F12 culture medium.

[0037] In the present invention, the centrifugation method is: centrifuging the cell culture fluid at 200-250×g for 12-16 min, 1900-2100×g for 5-10 min, and 9000-11000×g for 25-35 min to remove cells and cell debris; continuing to centrifuge at 99000-100000×g for 65-75 min, resuspending and washing the sediment at the bottom, and finally centrifuging at 99000-10000×g for 65-75 min to remove impurities to obtain adipose mesenchymal stem cell exosomes; preferably, the centrifugation method is: centrifuging the cell culture fluid at 210-240×g for 13-15 min, 2000×g for 6-9 min, and 10 The mixture was centrifuged at 100000×g for 27 to 33 minutes to remove cells and cell debris; the mixture was centrifuged at 100000×g for 67 to 72 minutes, the sediment at the bottom was resuspended and rinsed, and finally the impurities were removed by centrifugation at 10000×g for 67 to 73 minutes to obtain adipose-derived mesenchymal stem cell exosomes; the centrifugation method was further preferably as follows: the cell culture medium was centrifuged at 230×g for 14 minutes, 2000×g for 8 minutes, and 10000×g for 30 minutes to remove cells and cell debris; the mixture was centrifuged at 100000×g for 70 minutes, the sediment at the bottom was resuspended and rinsed, and finally the impurities were removed by centrifugation at 10000×g for 70 minutes to obtain adipose-derived mesenchymal stem cell exosomes.

[0038] In the present invention, the inoculation density in step (2) is 4200 to 4400 cells / cm 2 ; preferably 4300 pcs / cm 2 .

[0039] In the present invention, the culture temperature in step (2) is 36-38°C, and the carbon dioxide concentration is 4-6%; preferably, the culture temperature is 37°C, and the carbon dioxide concentration is 5%.

[0040] The present invention also provides a method for preparing the pharmaceutical composition, which comprises mixing water for injection, bamboo leaf flavonoids extract, Rosa laevigata extract, and carboxymethyl chitosan, and then sequentially adding platelet-rich plasma, human serum albumin, and adipose-derived mesenchymal stem cell exosomes and mixing.

[0041] The present invention also provides the use of the pharmaceutical composition in preparing a drug for treating bladder detrusor dysfunction.

[0042] In the present invention, the bamboo leaf flavonoid extract (total flavonoids ≥ 40%) is purchased from Jinhua Beikang Biotechnology Development Co., Ltd.

[0043] In the present invention, the preparation of the Rosa laevigata extract can refer to the document "Bian Chen, Liu Xin, Zhang Yang, et al. Protective effect of Rosa laevigata extract on intestinal mucosa of colitis in mice [J]. Journal of Xinjiang Medical University, 2018, 41(5): 5. DOI: 10.3969 / j.issn.1009-5551.2018.05.017.".

[0044] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0045] Example 1

[0046] A method for preparing adipose-derived mesenchymal stem cell exosomes, comprising the following steps:

[0047] (1) Extracting and isolating primary adipose-derived mesenchymal stem cells from the patient’s autologous fat samples:

[0048] (2) The primary adipose-derived mesenchymal stem cells obtained in step (1) were inoculated in a T75 culture flask at a density of 4200 cells / cm 2 , supplement the culture medium and then perform primary culture; the culture temperature is 36°C and the carbon dioxide concentration is 4%.

[0049] The culture medium is composed of the following components: 0.3 g / L L-isoleucine, 1 g / L L-valine, 1 g / L glycine, 7 mM β-glycerophosphate, 7 g / L glutathione, 4 ng / mL platelet-derived growth factor, 4 ng / mL EGF, 9 ng / mL bFGF, 4 ng / mL fibroblast growth factor, and the rest is DMEM-F12 culture medium;

[0050] (3) When the cell density in step (2) reaches 70%, the cell culture medium is collected and centrifuged to obtain adipose stem cell exosomes;

[0051] The centrifugation method is as follows: centrifuging the cell culture medium at 200×g for 12 minutes, 1900×g for 5 minutes, and 9000×g for 25 minutes in sequence to remove cells and cell debris; continuing to centrifuge at 99000×g for 65 minutes, resuspending and washing the sediment at the bottom, and finally centrifuging at 99000×g for 65 minutes to remove impurities to obtain adipose mesenchymal stem cell exosomes.

[0052] Example 2

[0053] A method for preparing adipose-derived mesenchymal stem cell exosomes, comprising the following steps:

[0054] (1) Extracting and isolating primary adipose-derived mesenchymal stem cells from the patient’s autologous fat samples:

[0055] (2) The primary adipose-derived mesenchymal stem cells obtained in step (1) were inoculated in a T75 culture flask at a density of 4400 cells / cm 2 , supplement the culture medium and then perform primary culture; the culture temperature is 38°C and the carbon dioxide concentration is 6%.

[0056] The culture medium is composed of the following components: 0.5 g / L L-isoleucine, 2 g / L L-valine, 2 g / L glycine, 9 mM β-glycerophosphate, 9 g / L glutathione, 8 ng / mL platelet-derived growth factor, 6 ng / mL EGF, 11 ng / mL bFGF, 8 ng / mL fibroblast growth factor, and the rest is DMEM-F12 culture medium;

[0057] (3) When the cell density in step (2) reaches 80%, the cell culture medium is collected and centrifuged to obtain adipose stem cell exosomes;

[0058] The centrifugation method is as follows: centrifuging the cell culture medium at 250×g for 16 min, 2100×g for 10 min, and 11000×g for 35 min to remove cells and cell debris; continuing to centrifuge at 100000×g for 75 min, resuspending and washing the sediment at the bottom, and finally centrifuging at 10000×g for 75 min to remove impurities to obtain adipose mesenchymal stem cell exosomes.

[0059] Example 3

[0060] A method for preparing adipose-derived mesenchymal stem cell exosomes, comprising the following steps:

[0061] (1) Extracting and isolating primary adipose-derived mesenchymal stem cells from the patient’s autologous fat samples:

[0062] (2) The primary adipose-derived mesenchymal stem cells obtained in step (1) were inoculated into a T75 culture flask at a density of 4300 cells / cm 2 , supplement the culture medium and then perform primary culture; the culture temperature is 37°C and the carbon dioxide concentration is 5%.

[0063] The culture medium is composed of the following components: 0.4 g / L L-isoleucine, 1.5 g / L L-valine, 1.5 g / L glycine, 8 mM β-glycerophosphate, 8 g / L glutathione, 6 ng / mL platelet-derived growth factor, 5 ng / mL EGF, 10 ng / mL bFGF, 6 ng / mL fibroblast growth factor, and the rest is DMEM-F12 culture medium;

[0064] (3) When the cell density in step (2) reaches 75%, the cell culture medium is collected and centrifuged to obtain adipose stem cell exosomes;

[0065] The centrifugation method is as follows: centrifuging the cell culture medium at 230×g for 14 min, 2000×g for 8 min, and 10000×g for 30 min to remove cells and cell debris; continuing to centrifuge at 100000×g for 70 min, resuspending and washing the sediment at the bottom, and finally centrifuging at 10000×g for 70 min to remove impurities to obtain adipose mesenchymal stem cell exosomes.

[0066] Example 4

[0067] A pharmaceutical composition for treating bladder detrusor dysfunction, comprising: adipose mesenchymal stem cell exosomes (prepared by the method of Example 1) 4×10 10 / mL, platelet-rich plasma 0.01mL / mL, bamboo leaf flavonoids extract 20mg / mL, Rosa laevigata extract 25mg / mL, human serum albumin 30mg / mL, carboxymethyl chitosan 1.1mg / mL, and the rest is 0.9% sodium chloride water for injection.

[0068] The preparation method of the pharmaceutical composition specifically comprises mixing water for injection, bamboo leaf flavonoids extract, golden cherry fruit extract, and carboxymethyl chitosan, adding platelet-rich plasma and human serum albumin, and finally adding adipose mesenchymal stem cell exosomes.

[0069] Example 5

[0070] A pharmaceutical composition for treating bladder detrusor dysfunction, comprising: adipose mesenchymal stem cell exosomes (prepared by the method of Example 2) 6×10 10 / mL, platelet-rich plasma 0.02mL / mL, bamboo leaf flavonoids extract 30mg / mL, Rosa laevigata extract 35mg / mL, human serum albumin 40mg / mL, carboxymethyl chitosan 1.5mg / mL, and the rest is 0.9% sodium chloride water for injection.

[0071] The preparation method of the pharmaceutical composition specifically comprises mixing water for injection, bamboo leaf flavonoids extract, golden cherry fruit extract, and carboxymethyl chitosan, adding platelet-rich plasma and human serum albumin, and finally adding adipose mesenchymal stem cell exosomes.

[0072] Example 6

[0073] A pharmaceutical composition for treating bladder detrusor dysfunction, comprising: adipose mesenchymal stem cell exosomes (prepared by the method of Example 3) 5×10 10 / mL, platelet-rich plasma 0.015mL / mL, bamboo leaf flavonoids extract 25mg / mL, Rosa laevigata extract 30mg / mL, human serum albumin 35mg / mL, carboxymethyl chitosan 1.3mg / mL, and the rest is 0.9% sodium chloride water for injection.

[0074] The preparation method of the pharmaceutical composition specifically comprises mixing water for injection, bamboo leaf flavonoids extract, golden cherry fruit extract, and carboxymethyl chitosan, adding platelet-rich plasma and human serum albumin, and finally adding adipose mesenchymal stem cell exosomes.

[0075] Comparative Example 1

[0076] The other methods are exactly the same as those in Example 3, except that L-isoleucine and L-valine are replaced by equal amounts of L-threonine and L-alanine in the culture medium.

[0077] Comparative Example 2

[0078] The other methods are exactly the same as those in Example 3, except that β-glycerophosphate and glutathione are not added to the culture medium.

[0079] Experimental Example 1

[0080] Adipose-derived mesenchymal stem cells were cultured according to the methods of Example 3, Comparative Example 1, and Comparative Example 2. When the cell density reached 75%, the cells were subcultured according to the same culture method. When the cells were subcultured to P10, the culture medium was removed, the cells were washed 4 times with PBS, digested with 0.25% trypsin solution, centrifuged at 1000 r / min for 5 min, and the cell concentration was adjusted to prepare a cell culture medium with a concentration of 1×10 5 The cells were incubated at 4°C for 30 min with a single cell suspension of 100 cells / mL and CD105, CD73 and CD90 monoclonal antibodies labeled with phycoerythrin (PE) and CD45, CD34, CD14, CD19 and HLA-DR monoclonal antibodies labeled with fluorescein isothiocyanate (FITC). The cells were washed twice with PBS and centrifuged at 1000 r / min for 5 min. The cells were resuspended with 500 μL of PBS and then detected by flow cytometry. The biological characteristics of adipose-derived mesenchymal stem cells in different experimental groups were determined. Two replicates were set for each experimental group, and the average value of the measurement results was taken, as shown in Table 1.

[0081] Table 1 Expression levels

[0082]

[0083]

[0084] As can be seen from Table 1, the culture method of adipose mesenchymal stem cells provided by the present invention can maintain the biological characteristics of the cells unchanged even after 10 passages, which is conducive to ensuring the activity of exosomes. From the data of Comparative Examples 1 and 2, it can be seen that only the complete culture medium components of the present invention can achieve the best use effect. If the components are replaced or discarded, the biological characteristics of the cells will be significantly affected.

[0085] Experimental Example 2

[0086] 120 patients with impaired bladder detrusor function who came to Xi'an Medical College were selected. They were randomly divided into 6 groups, and each experimental group randomly administered medication to one of the patients. The patients were aged 52 to 78 years old, with an average age of 63.8 years and an average course of disease of 6.5 years. There was no statistically significant difference in age, course of disease, I-PSS score, etc. between the different groups of patients, and they were comparable.

[0087] Diagnostic criteria: referring to the 2009 edition of the "Guidelines for the Diagnosis and Treatment of Urological Diseases in China", patients with clinical manifestations of lower urinary tract symptoms such as dysuria, urinary delay, increased nocturia, and incomplete urination were included. I-PSS score, rectal examination, urodynamic examination, prostate color Doppler ultrasound and other examinations were performed to diagnose benign prostatic hyperplasia.

[0088] Treatment index: efficacy observation index bladder residual urine volume (RUV): calculated according to the formula RUV = 0.52 × left and right diameter (cm) × front and back diameter (cm) × upper and lower diameter (cm); prostate volume (PV), measured by color ultrasound; detrusor contraction force (I-PSS) according to the International Prostate Symptom Score; maximum urine flow rate (Qmax) measured by JDNL-Ⅱ multifunctional urine flowmeter, urine volume of 150 ~ 200ml is effective. All indicators to be measured are measured once before and after treatment.

[0089] Treatment judgment criteria: formulated according to the "Recommendations of the Fifth International Advisory Committee on Benign Prostatic Hyperplasia". Comprehensive efficacy evaluation criteria: markedly effective: detrusor contractility improved by more than grade II or reached normal, RUV reduced by more than 60% and symptoms significantly improved; effective: detrusor contractility improved by more than grade I, RUV reduced by 30% to 59%, and symptoms improved compared with before; ineffective: detrusor contractility did not improve or worsened, RUV reduced by less than 30%, and symptoms did not improve. Bladder detrusor contraction strength improvement evaluation criteria: Improvement grade III: contractility score reduced by 3 points; Improvement grade II: contractility score reduced by 2 points; Improvement grade I: contractility score reduced by 1 point; No improvement: contractility score did not decrease or even increased.

[0090] Treatment groups:

[0091] The medicine obtained in Example 6 was used as the experimental group;

[0092] In Comparative Example 3, other methods were the same as those in Example 6, except that adipose-derived mesenchymal stem cell exosomes were not added.

[0093] In Comparative Example 4, other methods are the same as those in Example 6, except that platelet-rich plasma is not added.

[0094] In Comparative Example 5, other methods are the same as those in Example 6, except that the bamboo leaf flavonoid extract is not added.

[0095] Comparative Example 6: Other methods are the same as those of Example 6, except that the bamboo leaf flavonoids extract and the Rosa laevigata extract are not added.

[0096] The method of using the drugs in Example 6 and Comparative Examples 3 to 6 is as follows: cystoscopic detrusor point injection is used, specifically, the bladder trigone is used as a landmark, 6 points are taken on the left and right sides outside the trigone, each point is 1.5 cm apart, 1 mL is injected into each point, and the needle insertion depth is 3 mm. Once a day, for a total of 2 weeks.

[0097] Prior art group: taking tolterodine tartrate tablets (according to the instructions).

[0098] Before and after treatment, the I-PSS score, Qmax and RUV of the patients in each test group were investigated, and the improvement rate of the bladder detrusor contraction strength was investigated. The results are shown in Tables 2 and 3. After the treatment, the recovery of the patients was investigated, and the results are shown in Table 4.

[0099] Table 2 Treatment indicators before and after treatment

[0100]

[0101] Table 3 Improvement rate of bladder detrusor contraction strength

[0102]

[0103]

[0104] Table 4 Treatment effect questionnaire

[0105] Group Significant effect efficient invalid Total Effectiveness Experimental Group 11 7 2 90％ Comparative Example 3 3 9 8 60％ Comparative Example 4 9 5 6 70％ Comparative Example 5 8 8 4 80％ Comparative Example 6 7 7 6 70％ Existing Technology Group 10 6 4 80％

[0106] From the contents recorded in Tables 2 to 4, it can be seen that after using the drug of the present invention, the I-PSS scores of the patients were all reduced, Qmax was all increased, RUV was all reduced, and the contraction strength of the bladder detrusor muscle was improved, which was significantly different from other control groups. It can also be seen from the final treatment effect survey table that the treatment effect of the drug provided by the present invention is significantly better than that of other control groups and the prior art group.

[0107] From the data obtained from the control group, it can be seen that only by using the complete drug formulation of the present invention can the best therapeutic effect be achieved. If a certain component is discarded, the therapeutic effect of the drug will be significantly affected. In particular, whether adipose-derived mesenchymal stem cell exosomes are added has the most significant effect on the efficacy of the drug.

[0108] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A pharmaceutical composition for treating bladder detrusor dysfunction, characterized in that: include: Adipose-derived mesenchymal stem cell exosomes 4~6×10 10 / mL, platelet-rich plasma 0.01-0.02mL / mL, bamboo leaf flavonoids extract 20-30mg / mL, Rosa laevigata extract 25-35mg / mL, human serum albumin 30-40mg / mL, carboxymethyl chitosan 1.1-1.5mg / mL, and the rest is water for injection.

2. The pharmaceutical composition according to claim 1, characterized in that The water for injection is 0.9% sodium chloride water for injection.

3. The pharmaceutical composition according to claim 1, characterized in that The method for preparing adipose-derived mesenchymal stem cell exosomes comprises the following steps: (1) Extraction and separation of primary adipose-derived mesenchymal stem cells: (2) inoculating the primary adipose-derived mesenchymal stem cells obtained in step (1) into a culture bottle, supplementing the culture medium and then culturing; (3) When the cell density in step (2) reaches 70-80%, the cell culture medium is collected and centrifuged to obtain adipose stem cell exosomes.

4. The pharmaceutical composition according to claim 3, characterized in that The culture medium is composed of the following components: 0.3-0.5 g / L L-isoleucine, 1-2 g / L L-valine, 1-2 g / L glycine, 7-9 mM β-glycerophosphate, 7-9 g / L glutathione, 4-8 ng / mL platelet-derived growth factor, 4-6 ng / mL EGF, 9-11 ng / mL bFGF, 4-8 ng / mL fibroblast growth factor, and the rest is a basic culture medium.

5. The pharmaceutical composition according to claim 4, characterized in that The basic culture medium is DMEM-F12 culture medium.

6. The pharmaceutical composition according to claim 3, characterized in that The centrifugation method comprises: centrifuging the cell culture fluid at 200-250×g for 12-16 min, 1900-2100×g for 5-10 min, and 9000-11000×g for 25-35 min, removing cells and cell debris; continuing to centrifuge at 99000-100000×g for 65-75 min, resuspending and washing the sediment at the bottom, and finally centrifuging at 99000-10000×g for 65-75 min to remove impurities, thereby obtaining adipose mesenchymal stem cell exosomes.

7. The pharmaceutical composition according to claim 3, characterized in that The inoculation density in step (2) is 4200 to 4400 cells / cm 2 .

8. The pharmaceutical composition according to claim 3, characterized in that The culture temperature in step (2) is 36-38° C., and the carbon dioxide concentration is 4-6%.

9. The method for preparing the pharmaceutical composition according to any one of claims 1 to 8, characterized in that: After mixing the water for injection, bamboo leaf flavonoids extract, Rosa laevigata extract, and carboxymethyl chitosan, add platelet-rich plasma, human serum albumin, and adipose-derived mesenchymal stem cell exosomes in sequence and mix.

10. Use of the pharmaceutical composition according to any one of claims 1 to 8 in the preparation of a drug for treating bladder detrusor dysfunction.

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