Recombinant collagen bladder solution with repairing effect as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of pharmaceutical preparations, and provides a recombinant collagen bladder solution with a repairing effect as well as a preparation method and application of the recombinant collagen bladder solution. Comprising 0.1%-0.6% of recombinant human I type collagen, 0.2%-0.3% of recombinant human III type collagen, 0.01%-0.05% of recombinant human X VII type collagen, 0.05%-1.0% of sodium hyaluronate, 0.05%-1.0% of sodium chondroitin sulfate, 0%-0.22% of sodium dihydrogen phosphate, 0%-0.10% of disodium hydrogen phosphate, 0.75%-0.9% of sodium chloride and the balance of water for injection. Wherein the molecular weight range of the sodium hyaluronate is 1,200 kDa to 2,200 kDa. By means of the technical scheme, the problems that in the prior art, the curative effect is limited, the curative effect maintaining time is short, and long-term stable symptom relieving cannot be achieved are solved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of pharmaceutical preparations, and relates to a recombinant collagen bladder solution with a repairing effect as well as a preparation method and application thereof. BACKGROUND

[0002] The bladder mucosa barrier is an important structure for maintaining the normal function of the bladder, and the tight junctions between the bladder mucosa epithelial cells and the special umbrella substance play a key role in blocking bacteria, certain molecules and ions. Specifically, the glycosaminoglycans distributed on the surface of the bladder lamina propria constitute the main component of the bladder barrier. The glycosaminoglycans and proteoglycans synergistically form polysaccharide-protein polymers, which are collectively covered on the surface of the bladder. This unique structure can effectively prevent harmful factors such as crystals and carcinogens in urine from causing damage to the bladder mucosa, and has the function of blocking the adhesion of cells to the bladder mucosa, thereby protecting the bladder mucosa from damage.

[0003] However, when the protective mechanism of the bladder mucosa is damaged due to various reasons, the inflammatory substances in the urine can take advantage of the situation and further aggravate the damage to the bladder mucosa, thereby triggering a series of bladder inflammatory reactions. Among non-bacterial cystitis, there are many chronic bladder inflammatory states such as glandular cystitis, interstitial cystitis, chemical cystitis, radiation cystitis and ketamine-related cystitis. The main clinical manifestations of these diseases include frequent urination, urinary urgency, nocturia, pain in the suprapubic bladder area and relief after urination, and some patients may also have urinary pain, hematuria and other manifestations, but the urine culture result shows no bacterial growth.

[0004] The causes of non-bacterial cystitis are complex and diverse, and the diagnosis process is often difficult. At present, there is no unified treatment standard in clinical practice, and it is very difficult to treat and difficult to achieve a cure. The commonly used conservative treatment method is mainly drug treatment. Patients usually need to take anti-inflammatory and analgesic drugs to relieve symptoms, or through the way of bladder drug perfusion for treatment. Commonly used perfusion drugs include dimethyl sulfoxide, hyaluronic acid, heparin, pentosan polysulfate, lidocaine, etc. Although these drugs and therapies can relieve symptoms such as pain, frequent urination and urinary urgency to some extent, they cannot achieve long-term effective relief, and have limited effect on promoting the repair of bladder mucosa.

[0005] Recent studies have found that sodium hyaluronate and sodium chondroitin sulfate, as an important component of the aminopolysaccharide layer, play a key role in the bladder surface layer of glucosamine glycosaminoglycan layer. Sodium hyaluronate can be used to replace the aminoglucose protective layer of the bladder epithelium, and it can form a protective film to play a barrier lubrication role. Collagen, as an important component widely existing in various tissues and organs of the human body, avoids the risk of virus transmission through recombinant preparation, has low immunogenicity, and has the effect of promoting the repair of damaged tissues and promoting wound healing.

[0006] At present, there is no ideal treatment scheme and corresponding product that can effectively repair the bladder mucosa and long-term relieve symptoms for non-bacterial cystitis and other bladder diseases, which is widely used in clinical practice, which brings new research direction and challenge to researchers and clinicians in the field. For example, a Chinese patent with publication number CN 103861091 A discloses a pharmaceutical composition for treating cystitis, which comprises collagen with a concentration of 0.01-0.5g / mL and an aqueous carrier, and the aqueous carrier is a phosphate buffer saline of sodium dihydrogen phosphate and disodium hydrogen phosphate. The composition can also contain sodium chondroitin sulfate and sodium hyaluronate. The use amount of collagen in the pharmaceutical composition is large, and the collagen is used as a single component of protein, and the curative effect is limited, which can only improve the symptoms in a short term, but the curative effect maintenance time is short, and long-term stable symptom relief cannot be achieved. For example, a Chinese patent application with publication number CN113925959 A discloses a recombinant human collagen composition and its application, which comprises recombinant human collagen, sodium chloride, disodium hydrogen phosphate monohydrate, sodium dihydrogen phosphate, polyhexamethylene biguanide hydrochloride and water for injection. The recombinant human collagen is a recombinant human type III collagen. The composition adds polyhexamethylene biguanide hydrochloride, which is a low-concentration preservative, but it may induce irritant reactions (such as burning sensation) under the condition of bladder mucosa damage, and long-term use may destroy the balance of bladder microenvironment.

[0007] Therefore, it is urgent to study a new collagen bladder solution to improve interstitial cystitis and achieve long-term stable symptom relief. SUMMARY

[0008] The present application provides a recombinant collagen bladder solution with repair effect and its preparation method and application, which solves the problems of limited curative effect, short curative effect maintenance time and inability to achieve long-term stable symptom relief in the prior art.

[0009] The technical scheme of the present application is as follows: A recombinant collagen bladder solution with repairing effect, comprising 0.1-0.6% of recombinant human type I collagen, 0.2-0.3% of recombinant human type III collagen, 0.01-0.05% of recombinant human type XI VII collagen, 0.05-1.0% of sodium hyaluronate, 0.05-1.0% of sodium chondroitin sulfate, 0-0.22% of sodium dihydrogen phosphate, 0-0.10% of disodium hydrogen phosphate, 0.75-0.9% of sodium chloride, and the balance of water for injection, in percentage by mass; Preferably, the sodium hyaluronate has a molecular weight ranging from 1300 kDa to 1800 kDa.

[0010] Preferably, the sodium hyaluronate has a molecular weight ranging from 1300 kDa to 1800 kDa.

[0011] In the present application, the addition of recombinant collagen not only plays a moisturizing role through the water absorption of collagen, but also promotes the adhesion, migration and proliferation of bladder epithelial cells through the synergistic effect of different types of collagen, so as to realize the formation of new epithelial cells and realize the repair of bladder mucosal epithelial cells and the repair of the mucosal layer.

[0012] The use of sodium hyaluronate with a specific molecular weight can utilize its water absorption and moisturizing effect to lock water in the bladder epithelium and form a water-based protective film to protect the bladder epithelium from mechanical flushing of urine and further invasion of harmful substances, thus creating an environment suitable for the repair and regeneration of the bladder epithelium.

[0013] The application of sodium chondroitin sulfate not only exogenously supplements the lost glycosaminoglycan substances after bladder mucosa damage, but also forms a simulated bladder epithelial protective film in combination with sodium hyaluronate with a specific molecular weight, thereby avoiding the chronic evolution of cystitis.

[0014] The solution system in the present application is a phosphate buffer or a sodium chloride solution, the pH value of the solution system ranges from 6.5 to 7.5, and the osmotic pressure ranges from 280 to 320 mOsmol / kg, which is consistent with the acid-base environment and osmotic pressure parameters of the human body.

[0015] Preferably, the solution comprises 0.1-0.6% of recombinant human type I collagen, 0.2-0.3% of recombinant human type III collagen, 0.01-0.05% of recombinant human type XI VII collagen, 0.05-1.0% of sodium hyaluronate, 0.05-1.0% of sodium chondroitin sulfate, 0.12% of sodium dihydrogen phosphate, 0.09% of disodium hydrogen phosphate, 0.84% of sodium chloride, and the balance of water for injection, in percentage by mass. As a preferred embodiment of the present application, by mass percentage, 0.2-0.6% of the recombinant human collagen type I, 0.2-0.3% of the recombinant human collagen type III, 0.01-0.05% of the recombinant human collagen type XVII, 0.3-0.5% of sodium hyaluronate, 0.2-0.5% of sodium chondroitin sulfate, 0.9% of sodium chloride, and the balance of water for injection.

[0016] As a preferred embodiment of the present application, by mass percentage, 0.2% of the recombinant human collagen type I, 0.3% of the recombinant human collagen type III, 0.05% of the recombinant human collagen type XVII, 0.3% of sodium hyaluronate, 0.3% of sodium chondroitin sulfate, 0.9% of sodium chloride, and the balance of water for injection.

[0017] The present application also provides a preparation method of the above-mentioned recombinant collagen vesical solution with repairing effect, comprising the following steps: A. Dividing water for injection into three parts, using the first part of water for injection to pre-dissolve the recombinant human collagen type I, the recombinant human collagen type III and the recombinant human collagen type XVII to obtain a first solution; Using the second part of water for injection to dissolve sodium hyaluronate and sodium chondroitin sulfate to obtain a second solution; Using the third part of water for injection to dissolve sodium chloride or sodium dihydrogen phosphate and disodium hydrogen phosphate to obtain a third solution; B. Adding the first solution and the third solution obtained in step A into the second solution, mixing, and filling to obtain the product.

[0018] As a preferred embodiment of the present application, the first part of water for injection accounts for 20-30% of the total water for injection, the second part of water for injection accounts for 45-55% of the total water for injection, and the balance is the total water for injection.

[0019] The present application also provides the use of the above-mentioned recombinant collagen vesical solution with repairing effect in the preparation of a drug for treating or preventing interstitial cystitis.

[0020] The present application has the following beneficial effects: 1. The recombinant collagen vesical solution provided by the present application is administered by perfusion, so that the drug can be directed to the action site, the effective components in the solution can be uniformly distributed on the surface of the bladder mucosa to form a moist protective film and effectively promote the repair of the bladder mucosal epithelial cells, and the treatment effect is achieved.

[0021] 2、The recombinant human collagen type I, the recombinant human collagen type III and the recombinant human collagen type XVII of the present application all have human gene sequences, have good biocompatibility and avoid the possible immune rejection of animal-derived collagen proteins. Meanwhile, the combination of different types of collagen proteins can better promote the adhesion, migration and proliferation of bladder epithelial cells than single component, and can more effectively repair the bladder epithelial cells and achieve long-term repair and treatment effects.

[0022] 3、The sodium hyaluronate with a molecular weight of 1200kDa-2200kDa used in the present application can form a breathable protective film on the bladder epithelial mucosa surface by its water absorption, and has better film forming property and protection than low molecular weight sodium hyaluronate, which can effectively protect the bladder epithelial mucosa.

[0023] 4、The sodium hyaluronate and sodium chondroitin sulfate used in the present application are both glycosaminoglycan substances existing in the bladder mucosa, which can supplement exogenous nutrients and form a protective film by the strong water absorption of sodium hyaluronate and sodium chondroitin sulfate to protect the bladder epithelium from mechanical erosion of urine and further invasion of harmful substances, and create a suitable environment for bladder epithelial repair and regeneration. BRIEF DESCRIPTION OF DRAWINGS

[0024] The present application will be further described in detail below in combination with the drawings and specific embodiments.

[0025] Figure 1 Cell viability comparison chart of the recombinant collagen bladder solution of the present application and its comparative samples in the cell proliferation test.

[0026] Figure 2 Cell migration rate chart of human bladder epithelial cells of the recombinant collagen bladder solution of the present application and its comparative samples. DETAILED DESCRIPTION

[0027] The technical solutions in the embodiments of the present application will be described clearly and completely below in combination with the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The experimental methods or test methods involved in the embodiments of the present application are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the art, which are very clear and definite in the field of use, and the skilled person in the art can understand the conventional process steps and use the corresponding equipment according to the conventional conditions or the conditions recommended by the manufacturer. The various instruments, equipment, raw materials or reagents used in the embodiments of the present application are not specially limited in terms of source, and are all conventional products that can be purchased through regular commercial channels, or can be prepared according to the conventional methods well known to those skilled in the art.

[0029] In the following examples and comparative examples, the recombinant human collagen type I used is prepared according to the steps of specific embodiments 1-5 of Chinese patent application with authorized announcement number CN119264243B (application number 202411452076.8), and the amino acid sequence of the recombinant human collagen type I is shown in SEQ ID No.1 as recorded in Chinese patent 202411452076.8.

[0030] The recombinant human collagen type III used is prepared according to the steps of specific embodiments 1-5 of Chinese patent application with authorized announcement number CN114671946B (application number 202111598732.1), and the amino acid sequence of the recombinant human collagen type III is shown in SEQ ID No.1 as recorded in Chinese patent 202111598732.1.

[0031] The recombinant human collagen type XVII used is prepared according to the steps of specific embodiments 1-5 of Chinese patent application with authorized announcement number CN117801096B (application number 202410118032.5), and the amino acid sequence of the recombinant human collagen type XVII is shown in SEQ ID No.1 as recorded in Chinese patent 202410118032.5.

[0032] Example 1 A recombinant collagen bladder solution with repairing effect, comprising 0.2% recombinant human collagen type I, 0.3% recombinant human collagen type III, 0.05% recombinant human collagen type XVII, 0.3% sodium hyaluronate (molecular weight 1500kDa), 0.3% sodium chondroitin sulfate, 0.9% sodium chloride, and the balance being water for injection, by mass percentage.

[0033] The preparation method of the recombinant collagen bladder solution comprises the following steps: A. Divide the injection water into three parts, and take 25wt% of the total injection water to pre-dissolve recombinant human type I collagen, recombinant human type III collagen and recombinant human type XI I collagen, and stir until completely dissolved to obtain a first solution; Take 50wt% of the total injection water to swell the sodium hyaluronate and the sodium chondroitin sulfate with injection water until completely dissolved to obtain a second solution; Take 25wt% of the total injection water to dissolve sodium chloride with injection water to obtain a third solution; B. Add the first solution and the third solution obtained in step A to the second solution, mix, stir uniformly, then fill and distribute.

[0034] Example 2 A recombinant collagen bladder solution with repairing effect, comprising, by mass percentage: 0.6% recombinant human type I collagen, 0.2% recombinant human type III collagen, 0.01% recombinant human type XI I collagen, 0.5% sodium hyaluronate (molecular weight 1200kDa), 1.0% sodium chondroitin sulfate, 0.9% sodium chloride, and the balance being injection water.

[0035] The preparation method of the recombinant collagen bladder solution is the same as that in Example 1.

[0036] Example 3 A recombinant collagen bladder solution with repairing effect, comprising, by mass percentage: 0.1% recombinant human type I collagen, 0.2% recombinant human type III collagen, 0.03% recombinant human type XI I collagen, 1.0% sodium hyaluronate (molecular weight 2200kDa), 0.5% sodium chondroitin sulfate, 0.12% sodium dihydrogen phosphate, 0.09% disodium hydrogen phosphate, 0.84% sodium chloride, and the balance being injection water.

[0037] The preparation method of the recombinant collagen bladder solution comprises the following steps: A. Divide the injection water into three parts, and take 25wt% of the total injection water to pre-dissolve recombinant human type I collagen, recombinant human type III collagen and recombinant human type XI I collagen, and stir until completely dissolved to obtain a first solution; Take 50wt% of the total injection water to swell the sodium hyaluronate and the sodium chondroitin sulfate with injection water until completely dissolved to obtain a second solution; Take 25wt% of the total injection water to dissolve sodium dihydrogen phosphate, disodium hydrogen phosphate and sodium chloride with injection water to obtain a third solution; B, the first solution and the third solution obtained in step A are added to the second solution, mixed, stirred uniformly, then filled, and packed.

[0038] Example 4 A recombinant collagen bladder solution with repair effect, comprising 0.2% recombinant human type I collagen, 0.3% recombinant human type III collagen, 0.05% recombinant human type XI I collagen, 0.1% sodium hyaluronate (molecular weight 1300kDa), 0.2% sodium chondroitin sulfate, 0.9% sodium chloride, and the balance being water for injection, in terms of mass percentage.

[0039] The preparation method of the recombinant collagen bladder solution, comprising the following steps: A, the water for injection is divided into three parts, 20wt% of the total water for injection is used to pre-dissolve recombinant human type I collagen, recombinant human type III collagen and recombinant human type XI I collagen, and stirred until completely dissolved to obtain a first solution; 45wt% of the total water for injection is used to swell sodium hyaluronate and sodium chondroitin sulfate with water for injection until completely dissolved to obtain a second solution; 25wt% of the total water for injection is used to dissolve sodium chloride with water for injection to obtain a third solution; B, the first solution and the third solution obtained in step A are added to the second solution, mixed, stirred uniformly, then filled, and packed.

[0040] Example 5 A recombinant collagen bladder solution with repair effect, comprising 0.4% recombinant human type I collagen, 0.3% recombinant human type III collagen, 0.05% recombinant human type XI I collagen, 0.4% sodium hyaluronate (molecular weight 1800kDa), 0.4% sodium chondroitin sulfate, 0.9% sodium chloride, and the balance being water for injection, in terms of mass percentage.

[0041] The preparation method of the recombinant collagen bladder solution, comprising the following steps: A, the water for injection is divided into three parts, 30wt% of the total water for injection is used to pre-dissolve recombinant human type I collagen, recombinant human type III collagen and recombinant human type XI I collagen, and stirred until completely dissolved to obtain a first solution; 45wt% of the total water for injection is used to swell sodium hyaluronate and sodium chondroitin sulfate with water for injection until completely dissolved to obtain a second solution; 25wt% of the total water for injection is used to dissolve sodium chloride with water for injection to obtain a third solution; B, the first solution and the third solution obtained in step A are added to the second solution, mixed, stirred uniformly, then filled, and packed.

[0042] Comparative Example 1 A recombinant collagen bladder solution, comprising by mass percentage: 0.55% recombinant human collagen type III, 0.3% sodium hyaluronate (molecular weight 1500 kDa), 0.3% sodium chondroitin sulfate, 0.9% sodium chloride, and the balance being water for injection.

[0043] The preparation method of the recombinant collagen bladder solution is the same as that in Example 1.

[0044] Comparative Example 2 A recombinant collagen bladder solution, comprising by mass percentage: 0.25% recombinant human collagen type I, 0.3% recombinant human collagen type III, 0.3% sodium hyaluronate (molecular weight 1500 kDa), 0.3% sodium chondroitin sulfate, 0.9% sodium chloride, and the balance being water for injection.

[0045] The preparation method of the recombinant collagen bladder solution is the same as that in Example 1.

[0046] Comparative Example 3 A recombinant collagen bladder solution, comprising by mass percentage: 0.3% recombinant human collagen type III, 0.25% recombinant human collagen type XVII, 0.3% sodium hyaluronate (molecular weight 1500 kDa), 0.3% sodium chondroitin sulfate, 0.9% sodium chloride, and the balance being water for injection.

[0047] The preparation method of the recombinant collagen bladder solution is the same as that in Example 1.

[0048] Comparative Example 4 A recombinant collagen bladder solution, comprising by mass percentage: 0.2% recombinant human collagen type I, 0.3% recombinant human collagen type III, 0.05% recombinant human collagen type XVII, 0.6% sodium hyaluronate (molecular weight 1500 kDa), 0.9% sodium chloride, and the balance being water for injection.

[0049] The preparation method of the recombinant collagen bladder solution is the same as that in Example 1.

[0050] Comparative Example 5 A recombinant collagen bladder solution, comprising by mass percentage: 0.2% recombinant human collagen type I, 0.3% recombinant human collagen type III, 0.05% recombinant human collagen type XVII, 0.6% sodium chondroitin sulfate, 0.9% sodium chloride, and the balance being water for injection.

[0051] The preparation method of the recombinant collagen bladder solution is the same as that in Example 1.

[0052] Comparative Example 6 A kind of recombinant collagen bladder solution, including 0.2% by mass percentage recombinant human collagen type I, 0.3% recombinant human collagen type III, 0.05% recombinant human collagen type XI, 0.3% sodium hyaluronate (molecular weight 800kDa), 0.3% sodium chondroitin sulfate, 0.9% sodium chloride, the balance is water for injection.

[0053] The preparation method of the recombinant collagen bladder solution is same as example 1.

[0054] Comparative example 7 A kind of recombinant collagen bladder solution, by mass percentage, including 1% recombinant human collagen type III, 0.85% sodium chloride, 0.17% sodium dihydrogen phosphate, 0.05% disodium hydrogen phosphate, 0.003% polyhexamethylene biguanide hydrochloride, the balance is water for injection.

[0055] The preparation method of the recombinant collagen bladder solution is same as example 1.

[0056] Example 1 cell proliferation test of recombinant collagen bladder solution Cell proliferation test is carried out using human bladder epithelial cells, and after cell digestion using trypsin, 8×10 3 / hole is used for cell inoculation. After 96 well culture plate is inoculated with cells, it is placed in 37℃, 5% CO2 incubator and is cultured for 24h, and the culture solution in the hole is discarded. Blank control group is added with 100 μL Ham's·F-12K complete culture medium per hole, and sample group is added with 100 μL sample liquid (20 uL recombinant collagen bladder solution+80 uL Ham's F-12K complete culture medium) per hole. 96 well culture plate is placed in 37℃, 5% CO2 incubator and is cultured for 24h. 10 μL of CCK8 reagent is added in each hole, and after being placed in incubator and incubated for 2h, the absorbance value of each hole is detected using enzyme label instrument at wavelength 450nm.

[0057] The test results are as shown in Figure 1 (Example 1-3, comparative example 1-7 in order respectively, sample 1-11): The results show that the three groups of samples of example 1-3 all have obvious effect of promoting bladder epithelial cell proliferation, and especially the proliferation promoting effect of example 1 can reach 130%, which shows that the bladder solution containing protein has obvious potential of promoting bladder epithelial cell proliferation and repair.

[0058] The results of Comparative Examples 1-3 showed that the cell proliferation efficiency of Comparative Example 1 (recombinant human type III collagen single component) was 102%, while the cell proliferation efficiencies of Comparative Example 2 (recombinant human type I + type III collagen dual component) and Comparative Example 3 (recombinant human type XVII + type III collagen dual component) were 106% and 108%, respectively. Compared with Comparative Example 1 (recombinant human type III collagen single component), the cell proliferation efficiency was slightly improved. However, the cell proliferation efficiencies of Comparative Examples 2 and 3 were significantly lower than those of the product in Example 1. The results indicate that the combination of the three recombinant collagens can significantly enhance the cell proliferation-promoting effect of the recombinant collagen bladder solution.

[0059] Comparative samples 4-6 all showed a cell proliferation-promoting effect, with no significant difference in the results.

[0060] Comparative Example 7 is a simulated formulation of Example 1 in the patent application with application number 202111391132.8. The test results show that the cell proliferation efficiency of the formulation of Comparative Example 7 is 98%, which is presumably due to the addition of single-component collagen and polyhexamethylene biguanide hydrochloride preservative affecting the normal growth of bladder epithelial cells.

[0061] Example 2: Cell migration assay of recombinant collagen bladder solution Cell migration assays were performed using human bladder epithelial cells (HBlaEpic). First, three equal divisions were marked horizontally and vertically on the back of each well of a 6-well culture plate using a marker. Cells were then seeded in each well at a density of approximately 1 × 10⁻⁶ cells / well. 6 / well. When cells reached 95% confluence, a 10µL pipette tip was used to create cell scratches. The cells were washed three times with PBS to remove the scratched cells. The area of ​​the scratch at the intersection of the horizontal and vertical scratches was photographed under a microscope and recorded as the 0h scratch area.

[0062] The blank control group was given 100 μL of Ham's F-12K complete medium, while the sample group was given 100 μL of sample solution (20 μL of recombinant collagen bladder solution + 80 μL of Ham's F-12K complete medium), and cultured for a further period. Cell migration was observed under a microscope after 24 h, 48 h, and 72 h of culture.

[0063] The test results are as follows Figure 2 As shown (samples 1-10 are examples 1-3 and comparative examples 1-7 in order): The results show that: All three groups of samples in Examples 1-3 showed significant effects in promoting bladder epithelial cell migration, and all had significant potential to promote bladder epithelial cell adhesion, migration, and repair. Compared with Comparative Example 7, the bladder solutions in Examples 1-3 showed significantly higher effects in promoting bladder epithelial cell migration than Comparative Example 7, and the difference was statistically significant.

[0064] Compared with Example 1, the cell migration promoting rates of Comparative Example 1 (recombinant human collagen type III single component), Comparative Example 2 (recombinant human collagen type I + III double component), and Comparative Example 3 (recombinant human collagen type VII + III double component) are all low, and the results have significant differences, indicating that the synergistic effect of the three types of collagen can effectively enhance the migration and proliferation capacity of bladder epithelial cells.

[0065] Example 1 uses the superposition of chondroitin sulfate and sodium hyaluronate, which has a better cell migration promoting effect compared with Comparative Example 4 and Comparative Example 5. It is expected that chondroitin sulfate and sodium hyaluronate can form a good extracellular matrix environment, providing corresponding media and mediators for cell adhesion and migration. However, compared with Example 1, the solution viscosity of Comparative Example 6 using sodium hyaluronate with a molecular weight of 800 kDa is lower, and the physical film forming property is poorer, which is speculated to be the reason for the slight decrease in cell migration effect of Comparative Example 6 compared with Example 1.

[0066] Example 3 Rat bladder mucosa injury animal test of recombinant collagen bladder solution Establishment of animal model: Adult SD rats were used to establish a rat interstitial cystitis model by a single intraperitoneal injection of cyclophosphamide (CYP). Cyclophosphamide was dissolved in normal saline to a concentration of 30 mg / ml, and a dose of 150 mg / kg was used for a one-time intraperitoneal injection to establish the model. The comparative experiment was performed 48 h after modeling.

[0067] A total of 70 rats were used, divided into seven groups of 10 each, namely blank control group, negative control group, experimental group (Example 1, Comparative Examples 2-3, Comparative Example 6, Comparative Example 7). The blank control group was a normal mucosa group without any treatment, the negative control group was perfused with 0.9% sodium chloride solution, and the experimental group was perfused with the test solution prepared according to the formula, with a perfusion volume of 0.25 mL per rat. The solution was injected into the bladder and kept for 30 min before being withdrawn. The treatment was performed twice a week for a total of 4 weeks. On the 7th day and the 28th day after the treatment, the bladder specimens were taken for gross observation and histopathological examination (HE staining to observe the pathological condition, toluidine blue staining to count the number of mast cells).

[0068] Gross observation results of bladder specimens: The results of sampling and testing 7 days after the experiment show that the blank control group is normal bladder mucosa tissue, the negative control group has extensive necrosis and exfoliation of the bladder mucosa epithelium, and obvious ulcer points, with mucosal edema and swelling, and extensive bleeding points. The bladder mucosa epithelium of Example 1 has less damage, with only a small amount of scattered point-like ulcers, superficial ulcers, and no mucosal edema around the periphery. The bladder mucosa epithelium of Comparative Example 2 and Comparative Example 3 has a small amount of damage, with a small amount of scattered point-like ulcers and scattered bleeding points, and no mucosal edema. The repair effects of the two are not significantly different, but are both worse than that of Example 1, indicating that the repair effect of different types of collagen combinations is better than that of single or double components. Comparative Example 6 has more bladder mucosa epithelium necrosis and ulcer points than Comparative Example 1, but has no mucosal edema, indicating that the film-forming repair effect of low-molecular-weight hyaluronic acid is not as good as that of high-molecular-weight hyaluronic acid. Comparative Example 7 has a small amount of damage to the bladder mucosa epithelium, with a small amount of necrosis and ulcer points, a small amount of scattered bleeding points, and no mucosal edema, indicating that the repair effect of Comparative Example 7 is worse than that of Example 1.

[0069] The results of sampling and testing 28 days after the experiment show that the blank control group is normal bladder mucosa tissue, the negative control group has extensive necrosis and exfoliation of the bladder mucosa epithelium, with no significant improvement in ulcer points and bleeding points, and more severe membrane edema and swelling. The bladder mucosa epithelium of Example 1 has no damage, no superficial ulcers, and no mucosal edema, and tends to be close to normal mucosa tissue. The bladder mucosa epithelium of Comparative Example 2 and Comparative Example 3 still has a small amount of damage, with slight improvement in point-like ulcers and scattered bleeding points, and no mucosal edema. The bladder mucosa epithelium of Comparative Example 6 has no significant change in the damaged area, with a decrease in the number of scattered ulcer points. Comparative Example 7 still has a small amount of damage to the bladder mucosa epithelium, with a small amount of scattered superficial ulcer points and bleeding points.

[0070] Results of histopathological observation of bladder specimens: The results of sampling and testing 7 days after the experiment show that the blank control group has normal mucosa structure, with intact mucosa and submucosa, no bleeding points in the submucosa, and no inflammatory cell infiltration; the negative control group has obvious mucosa damage, with exfoliation of the submucosa, extensive bleeding points and inflammatory edema in the bladder wall tissue, and a large number of neutrophil infiltration; the bladder wall tissue of Example 1 has good mucosa continuity and a thin film covering the surface, with a small amount of mucosal hyperemia and a small amount of inflammatory cell infiltration; the mucosa surface of Comparative Example 2 and Comparative Example 3 has a thin film covering, a small amount of mucosal hyperemia, and a larger number of inflammatory cells than Example 1; the mucosa of Comparative Example 6 has ruptures and exfoliation, with intermittent thin film covering on the surface, a small amount of mucosal hyperemia, and inflammatory cell infiltration. The mucosa of Comparative Example 7 has ruptures and exfoliation, with no thin film covering on the surface, a small amount of mucosal hyperemia, and a number of inflammatory cells comparable to Comparative Example 2.

[0071] The results of the sampling and detection 28 days after the test treatment showed that the blank control group was normal mucosa structure, the mucosa and submucosa were intact, no bleeding points were found in the submucosa, and no inflammatory cell infiltration was observed; the mucosa of the negative control group was obviously damaged, the submucosa was exfoliated, the bleeding points and inflammatory edema of the bladder wall tissue were aggravated, and a large number of neutrophils infiltrated; the mucosa of the bladder wall tissue of Example 1 was continuous and covered with a thin film, the congestion and inflammatory cell infiltration were obviously reduced, and basically tended to be normal bladder mucosa; the mucosa of Comparative Example 2 and Comparative Example 3 was covered with a thin film, but the improvement of the mucosal congestion and the number of inflammatory cells was not obvious; the mucosa of Comparative Example 6 was broken and exfoliated, the surface was covered with an intermittent thin film, and the improvement of the congestion and inflammatory cell infiltration was not obvious; the mucosa of Comparative Example 7 was broken and exfoliated, the surface was not covered with a thin film, and the improvement of the mucosal congestion and inflammatory cell infiltration was not obvious.

[0072] The results of the statistics of the number of mast cells in each square millimeter of the tissue specimen are shown in Table 1: In the bladder tissue of patients with non-bacterial cystitis, a large number of mast cells exist due to long-term stimulation and chronic inflammatory cell infiltration, and present in granular form. The statistics of the number of mast cells can also reflect the inflammation and improvement of the inflammation of the bladder tissue. The number of mast cells in normal tissue is generally less than or equal to 15 / mm 2 The follow-up detection results show that the number of mast cells in the epithelial tissue of Example 1 is slightly higher than the normal value 7 days after the treatment with the bladder repair solution, and is basically within the normal value range in the long-term 28-day observation, indicating that the bladder repair solution of the present application has a good long-term repair effect; the number of mast cells of Comparative Example 2 and Comparative Example 3 is obviously higher than the normal value 7 days after the treatment, and is also not obviously improved 28 days after the treatment; Comparative Example 6 is observed to have a larger number of mast cells than Example 1, and the number of mast cells is not obviously improved in the long-term 28-day observation; the number of mast cells of Comparative Example 7 is obviously reduced compared with the negative control group, indicating that the collagen bladder repair solution has a certain effect on improving interstitial cystitis, but the improvement effect is worse than that of Example 1 7 days after the treatment, has no obvious advantage, and the number of mast cells has a rebounding trend in the long-term 28-day observation, and the long-term improvement effect is not obvious.

[0073] Table 1 According to the animal experiment research on the damage of the bladder mucosa of rats, it is shown that the recombinant collagen protein bladder solution can effectively repair the damaged bladder mucosa tissue, can realize the treatment and relief of interstitial cystitis and bladder pain syndrome and other diseases through the proliferation promotion of the recombinant collagen protein, the replacement and repair of sodium hyaluronate and chondroitin sulfate, can more effectively realize the repair of the bladder epithelial cells, and can achieve the long-term repair treatment effect.

[0074] The above merely provides the preferred embodiment of the present application, and is not used to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall fall into the protection scope of the present application.

Claims

1. A recombinant collagen bladder solution with repairing effects, characterized in that, By weight percentage, it includes 0.1-0.6% recombinant human type I collagen, 0.2-0.3% recombinant human type III collagen, 0.01-0.05% recombinant human type XVII collagen, 0.05-1.0% sodium hyaluronate, 0.05-1.0% sodium chondroitin sulfate, 0-0.22% sodium dihydrogen phosphate, 0-0.10% disodium hydrogen phosphate, 0.75-0.9% sodium chloride, and the balance being water for injection; The molecular weight of the sodium hyaluronate ranges from 1200kDa to 2200kDa.

2. The recombinant collagen bladder solution with repairing effect according to claim 1, characterized in that, The molecular weight range of the sodium hyaluronate is 1300kDa-1800kDa.

3. The recombinant collagen bladder solution with repairing effect according to claim 1, characterized in that, By weight percentage, it includes 0.2-0.6% recombinant human type I collagen, 0.2-0.3% recombinant human type III collagen, 0.01-0.05% recombinant human type XVII collagen, 0.3-0.5% sodium hyaluronate, 0.2-0.5% sodium chondroitin sulfate, 0.9% sodium chloride, and the balance is water for injection.

4. The recombinant collagen bladder solution with repairing effect according to claim 1, characterized in that, By weight percentage, it includes 0.2% recombinant human type I collagen, 0.3% recombinant human type III collagen, 0.05% recombinant human type XVII collagen, 0.3% sodium hyaluronate, 0.3% sodium chondroitin sulfate, 0.9% sodium chloride, and the balance is water for injection.

5. A method for preparing a recombinant collagen bladder solution with repairing effect as described in any one of claims 1-4, characterized in that, Includes the following steps: A. Divide the water for injection into three parts. Use the first part of the water for injection to pre-dissolve recombinant human type I collagen, recombinant human type III collagen, and recombinant human type XVII collagen to obtain the first solution. Sodium hyaluronate and sodium chondroitin sulfate were dissolved in water for injection in the second part to obtain a second solution; Sodium dihydrogen phosphate, disodium hydrogen phosphate, and / or sodium chloride were dissolved in water for injection in the third part to obtain the third solution; B. Add the first and third solutions obtained in step A to the second solution, mix, and then fill into containers.

6. The method for preparing a recombinant collagen bladder solution with repair effect according to claim 5, characterized in that, The first part is water for injection, which accounts for 20-30% of the total water for injection; the second part is water for injection, which accounts for 45-55% of the total water for injection; and the remainder is water for injection in the third part.

7. The use of a recombinant collagen bladder solution with repairing effect as described in any one of claims 1-4 in the preparation of a medicament for treating or preventing interstitial cystitis.

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