Female vaginal mucosa cell maintenance preparation as well as preparation method and application thereof

Abstract

The invention discloses a female vaginal mucosa cell maintenance preparation and a preparation method and application thereof, and belongs to the technical field of biological agents, the female vaginal mucosa cell maintenance preparation is prepared from dental pulp mesenchymal stem cells, portulaca oleracea polysaccharide, lactic acid, quercitrin, glycerin and stem cell carriers, and vaginal injection paste is obtained by evenly mixing the dental pulp mesenchymal stem cells, the portulaca oleracea polysaccharide, the lactic acid, the quercitrin, the glycerin and the stem cell carriers through a proper amount of deionized water. The purslane polysaccharide and the dental pulp mesenchymal stem cells form a unique synergistic effect, through physical wrapping, the survival rate and delivery efficiency of the stem cells in a vaginal cavity are improved, local inflammatory response is inhibited, a microenvironment is optimized to support directional differentiation of the stem cells into epithelial cells, paracrine activity of the stem cells is enhanced, and repairable factors are released to promote cell-level regeneration. The problem of hormone dependence is solved, deep maintenance of targeted mucous membrane cells is achieved, limitation of an existing therapy is broken through by directly nourishing cells, activating and regenerating, enhancing barrier integrity and maintaining an acidic microenvironment, and an ideal treatment effect is achieved.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, specifically to a vaginal mucosal cell maintenance preparation for women, its preparation method, and its application. Background Technology

[0002] The vaginal mucosa, a vital barrier of the female reproductive system, is primarily composed of stratified squamous epithelial cells. Its health directly impacts the vagina's defense function, lubrication, and microecological balance. The surface cells of this mucosa are rich in glycogen, which, through metabolism by lactobacilli, creates an acidic environment, typically between pH 3.8 and 4.5, effectively inhibiting pathogen proliferation. However, various factors can lead to mucosal cell damage and functional decline: such as hormonal fluctuations caused by a sharp drop in estrogen during menopause, resulting in cell atrophy and reduced glycogen synthesis; vaginitis caused by recurrent bacterial / fungal infections, disrupting cell structure and the microecological environment; and iatrogenic procedures and physical / chemical stimuli further exacerbating mucosal barrier damage.

[0003] Current clinical interventions generally suffer from two major shortcomings: first, they rely too much on hormone replacement or surface symptom relief, neglecting the active repair and functional reconstruction of mucosal cells; second, they lack solutions targeting cell metabolic activity, damage repair, and long-term barrier maintenance.

[0004] Therefore, there is an urgent need to develop a non-hormone-dependent preparation that targets the deep maintenance of mucosal cells, which can overcome the limitations of existing therapies and achieve more ideal therapeutic effects by directly nourishing cells, activating regeneration, enhancing barrier integrity, and maintaining an acidic microenvironment. Summary of the Invention

[0005] Therefore, this invention provides a vaginal mucosal cell maintenance preparation for women, its preparation method, and its application, in order to solve the related technical problems existing in the prior art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: According to a first aspect of the present invention, a vaginal mucosal cell care preparation is provided, comprising the following components in weight percentage: Dental pulp mesenchymal stem cells: 0.2-1.8% Purslane polysaccharides: 1.2-3.6% Lactic acid 0.8-1.4% Quercetin 0.01-0.05% 3-5% glycerin Stem cell carrier 1-8% The vaginal injection ointment is obtained by mixing an appropriate amount of deionized water evenly.

[0007] Furthermore, the concentration of the dental pulp mesenchymal stem cells is 1 x 10⁻⁶. 6 -107 per mL.

[0008] Furthermore, the stem cell carrier is chitosan.

[0009] Furthermore, the molecular weight of the purslane polysaccharide is 200-230 kDa.

[0010] Furthermore, the dental pulp mesenchymal stem cells are passaged to the P3-P5 generation.

[0011] According to a second aspect of the present invention, a method for preparing a vaginal mucosal cell maintenance preparation is provided, comprising the following steps: Weigh out glycerol and stem cell carrier according to the proportions and dissolve them in an appropriate amount of deionized water. Stir and mix until homogeneous to form mixture 1 for later use. Weigh out the purslane polysaccharide, lactic acid and quercetin according to the proportions and add them to the above mixture 1. Stir and mix until uniform to form mixture 2 for later use. Cool the above mixture 2 to 5-10℃, weigh out the dental pulp mesenchymal stem cells according to the ratio and add them to the above mixture 2, stir and mix until uniform to form a maintenance preparation, and store at low temperature.

[0012] Furthermore, the preparation steps for dental pulp mesenchymal stem cells are as follows: Tissue preparation: The dental pulp is cut into pieces of 1-3 mm. 3 Small pieces were added to a digestion solution consisting of 2-3 mg / mL type I collagenase and 2-4 mg / mL neutral protease, and digested at 37°C for 15-20 min. The reaction was terminated by adding 3-5 times the volume of serum-containing culture medium, and filtered through a 100 μm sieve to obtain a single-cell suspension. Primary culture: Absorb single-cell suspension and inoculate with 1-5 × 10⁻⁵ cells. 4 The cells were inoculated at a density of 1 / mL in a culture medium consisting of α-MEM, 10–15% FBS and 1% penicillin, and cultured at 37°C and 5% CO2 for 10–15 days to achieve 70–80% confluence. Passaging: Digest with 0.25% trypsin-EDTA for 3-5 minutes, passage at a ratio of 1:2, change the medium every 3-4 days, and culture to P3-5 generation to obtain dental pulp mesenchymal stem cells.

[0013] Furthermore, the dental pulp mesenchymal stem cells are P4 generation cells.

[0014] Furthermore, the preparation steps of purslane polysaccharide are as follows: Pretreatment: Take dried purslane and crush it to 40 mesh. Mix it with petroleum ether at a ratio of 100g: 500mL. Reflux at 60℃ for 2h. Filter and discard the filtrate. Dry the residue at 60℃ to constant weight to obtain defatted raw material for later use. Extraction treatment: Add defatted raw materials to distilled water at a material-to-liquid ratio of 100g:2000mL, extract at 90℃ for 2 hours, stir in a constant temperature water bath at 200rpm, extract twice, and combine the filtrates; Concentration and impurity removal: Evaporate the above filtrate at 60°C to 1 / 5 of its original volume, and add 1 / 4 volume of chloroform:n-butanol solvent in a volume ratio of 4:1 to the concentrate. Shake vigorously for 30 min, centrifuge at 8000 rpm for 15 min, discard the lower organic phase and protein layer, and repeat the impurity removal steps until there are no white flocculent substances at the interface to obtain the concentrate. Purification: Slowly add 4 volumes of 95% ethanol to the concentrate, let stand at 4°C for 12 hours, centrifuge at 8000 rpm for 15 minutes, collect the precipitate to obtain crude polysaccharide, and wash twice with anhydrous ethanol. Dialysis treatment: The crude polysaccharide was redissolved in distilled water, placed in a dialysis bag and dialyzed for 48 hours. The dialysate was collected and freeze-dried for 24 hours to obtain white flocculent refined purslane polysaccharide.

[0015] According to a second aspect of the present invention, the use of the above-described female vaginal mucosal cell maintenance preparation in the preparation of a female vaginal mucosal cell maintenance medicine is provided.

[0016] The present invention has the following advantages: In this application, the high molecular weight of purslane polysaccharide forms a bioadhesive protective layer, effectively reducing frictional damage. Simultaneously, it activates epithelial cell regeneration signaling pathways, promotes tight junction protein expression, accelerates the structural reconstruction and functional recovery of the mucosal barrier, and enhances the ability to resist pathogen invasion. Furthermore, this polysaccharide selectively nourishes the lactobacillus community, synergistically maintaining the acidic environment of the vagina with lactic acid in the formula. Its slow-released sugars provide a continuous substrate for glycogen synthesis in mucosal cells, fundamentally improving the microecological imbalance caused by hormonal imbalance or inflammation. Purslane polysaccharide forms a unique synergistic effect with dental pulp mesenchymal stem cells: on the one hand, it enhances the survival rate and delivery efficiency of stem cells in the vaginal cavity through physical encapsulation; on the other hand, it inhibits local inflammatory responses, optimizes the microenvironment to support the directed differentiation of stem cells into epithelial cells, enhances their paracrine activity, and releases repair factors to promote cell-level regeneration. Combined with glycerin, it constructs a dynamic moisturizing network to maintain mucosal hydration for a prolonged period, while synergistically, quercetin scavenges oxidative stress products, blocks free radical damage to cells, and delays the aging process of the mucosa. Detailed Implementation

[0017] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0018] To address the related technical problems existing in the prior art, according to a first aspect of the present invention, a female vaginal mucosal cell care preparation is provided, comprising the following components by weight percentage: Dental pulp mesenchymal stem cells: 0.2-1.8% Purslane polysaccharides: 1.2-3.6% Lactic acid 0.8-1.4% Quercetin 0.01-0.05% 3-5% glycerin Stem cell carrier 1-8% The vaginal injection ointment is obtained by mixing an appropriate amount of deionized water evenly.

[0019] Furthermore, the concentration of dental pulp mesenchymal stem cells was 1 x 10⁻⁶. 6 -10 7 per mL.

[0020] Furthermore, the stem cell carrier is chitosan.

[0021] Furthermore, the molecular weight of purslane polysaccharide is 200-230 kDa.

[0022] Furthermore, the dental pulp mesenchymal stem cells were passaged to the P3-P5 generation.

[0023] According to a second aspect of the present invention, a method for preparing a vaginal mucosal cell maintenance preparation is provided, comprising the following steps: Weigh out glycerol and stem cell carrier according to the proportions and dissolve them in an appropriate amount of deionized water. Stir and mix until homogeneous to form mixture 1 for later use. Weigh out the purslane polysaccharide, lactic acid and quercetin according to the proportions and add them to the above mixture 1. Stir and mix until uniform to form mixture 2 for later use. Cool the above mixture 2 to 5-10℃, weigh out the dental pulp mesenchymal stem cells according to the ratio and add them to the above mixture 2, stir and mix until uniform to form a maintenance preparation, and store at low temperature.

[0024] Furthermore, the preparation steps for dental pulp mesenchymal stem cells are as follows: Tissue preparation: The dental pulp is cut into pieces of 1-3 mm. 3 Small pieces were added to a digestion solution consisting of 2-3 mg / mL type I collagenase and 2-4 mg / mL neutral protease, and digested at 37°C for 15-20 min. The reaction was terminated by adding 3-5 times the volume of serum-containing culture medium, and filtered through a 100 μm sieve to obtain a single-cell suspension. Primary culture: Absorb single-cell suspension and inoculate with 1-5 × 10⁻⁵ cells. 4The cells were inoculated at a density of 1 / mL in a culture medium consisting of α-MEM, 10–15% FBS and 1% penicillin, and cultured at 37°C and 5% CO2 for 10–15 days to achieve 70–80% confluence. Passaging: Digest with 0.25% trypsin-EDTA for 3-5 minutes, passage at a ratio of 1:2, change the medium every 3-4 days, and culture to P3-5 generation to obtain dental pulp mesenchymal stem cells.

[0025] Furthermore, the dental pulp mesenchymal stem cells were P4 generation cells.

[0026] Furthermore, the preparation steps of purslane polysaccharide are as follows: Pretreatment: Take dried purslane and crush it to 40 mesh. Mix it with petroleum ether at a ratio of 100g: 500mL. Reflux at 60℃ for 2h. Filter and discard the filtrate. Dry the residue at 60℃ to constant weight to obtain defatted raw material for later use. Extraction treatment: Add defatted raw materials to distilled water at a material-to-liquid ratio of 100g:2000mL, extract at 90℃ for 2 hours, stir in a constant temperature water bath at 200rpm, extract twice, and combine the filtrates; Concentration and impurity removal: Evaporate the above filtrate at 60°C to 1 / 5 of its original volume, and add 1 / 4 volume of chloroform:n-butanol solvent in a volume ratio of 4:1 to the concentrate. Shake vigorously for 30 min, centrifuge at 8000 rpm for 15 min, discard the lower organic phase and protein layer, and repeat the impurity removal steps until there are no white flocculent substances at the interface to obtain the concentrate. Purification: Slowly add 4 volumes of 95% ethanol to the concentrate, let stand at 4°C for 12 hours, centrifuge at 8000 rpm for 15 minutes, collect the precipitate to obtain crude polysaccharide, and wash twice with anhydrous ethanol. Dialysis treatment: The crude polysaccharide was redissolved in distilled water, placed in a dialysis bag and dialyzed for 48 hours. The dialysate was collected and freeze-dried for 24 hours to obtain white flocculent refined purslane polysaccharide.

[0027] According to a second aspect of the present invention, the use of the above-described female vaginal mucosal cell maintenance preparation in the preparation of a female vaginal mucosal cell maintenance medicine is provided.

[0028] To better illustrate the technical effects of this application, the following embodiments are also provided.

[0029] Preparation Example 1 This preparation example provides the preparation steps for dental pulp mesenchymal stem cells, as follows: Tissue processing: Collect healthy deciduous teeth extracted during treatment, and mince the pulp into 1-3mm pieces. 3Small pieces were added to a digestion solution consisting of 3 mg / mL type I collagenase and 4 mg / mL neutral protease, and digested at 37°C for 20 min. The reaction was terminated by adding 5 times the volume of serum-containing culture medium, and filtered through a 100 μm sieve to obtain a single-cell suspension from the filtrate.

[0030] Primary culture: Absorb single-cell suspension and incubate at 3 × 10⁻⁶ cells / mL. 4 Cells were inoculated at a density of 1 cell / mL in a culture medium consisting of α-MEM, 15% FBS and 1% penicillin. The cells were cultured at 37°C and 5% CO2 for 12 days until 80% confluence was achieved, and then the cells were harvested.

[0031] Passaging: Digest with 0.25% trypsin-EDTA for 5 min, passage at a ratio of 1:2, change medium every 4 days, and culture to P3-5 generation to obtain dental pulp mesenchymal stem cells. In this preparation example, dental pulp mesenchymal stem cells were cultured to P4 generation and the cells were harvested for use.

[0032] Preparation Example 2 This preparation example provides the preparation steps for purslane polysaccharides, as follows: Pretreatment: Take dried purslane and crush it to 40 mesh. Mix it with petroleum ether at a ratio of 100g: 500mL. Reflux at 60℃ for 2 hours. Filter and discard the filtrate. Dry the residue at 60℃ to constant weight to obtain defatted raw material for later use.

[0033] Extraction treatment: Add defatted raw materials to distilled water at a material-to-liquid ratio of 100g:2000mL, extract at 90℃ for 2 hours, stir in a constant temperature water bath at 200rpm, extract twice, and combine the filtrates.

[0034] Concentration and impurity removal: Evaporate the above filtrate at 60°C to 1 / 5 of its original volume, and add 1 / 4 volume of chloroform:n-butanol solvent in a volume ratio of 4:1 to the concentrate. Shake vigorously for 30 min, centrifuge at 8000 rpm for 15 min, discard the lower organic phase and protein layer, and repeat the impurity removal steps until there are no white flocculent substances at the interface to obtain the concentrate.

[0035] Purification: Slowly add 4 volumes of 95% ethanol to the concentrate, let stand at 4°C for 12 hours, centrifuge at 8000 rpm for 15 minutes, collect the precipitate to obtain crude polysaccharide, and wash twice with anhydrous ethanol.

[0036] Dialysis treatment: The crude polysaccharide was redissolved in distilled water, placed in a dialysis bag, and dialyzed for 48 hours. The dialysate was collected and freeze-dried for 24 hours to obtain white flocculent refined purslane polysaccharide for later use.

[0037] Example 1 This embodiment provides a method for preparing a vaginal mucosal cell maintenance preparation, including the following steps: Weigh out 3g of glycerin and 1g of chitosan according to the proportion and dissolve them in an appropriate amount of deionized water. Heat to 40℃ and stir at 200rpm until uniform to form a mixture 1 for later use. Weigh 1.2g of purslane polysaccharide, 0.8g of lactic acid and 0.01g of quercetin collected in Preparation Example 1 according to the proportions and add them to the above mixture 1. Heat to 60°C and stir at 200rpm until uniform to form mixture 2 for later use. Cool the above mixture 2 to 5-10℃. Wash the stem cells collected in Preparation Example 1 with PBS buffer and centrifuge to collect them. Weigh 0.2g of dental pulp mesenchymal stem cells according to the ratio and add them to the above mixture 2. Make up the volume to 100mL with deionized water and stir until homogeneous, so that the concentration of dental pulp mesenchymal stem cells is 1×10⁻⁶. 7 The concentration of each sample was increased to 100 mg / mL to obtain a vaginal injection ointment, which was then stored at low temperature for later use.

[0038] Example 2 This embodiment provides a method for preparing a vaginal mucosal cell maintenance preparation, including the following steps: Weigh out 4g of glycerol and 4.5g of chitosan according to the ratio and dissolve them in an appropriate amount of deionized water. Heat to 40℃ and stir at 200rpm until homogeneous to form a mixture 1 for later use. Weigh 2.4g of purslane polysaccharide, 1.1g of lactic acid and 0.03g of quercetin collected in Preparation Example 1 according to the proportions and add them to the above mixture 1. Heat to 60°C and stir at 200rpm until uniform to form mixture 2 for later use. Cool the above mixture 2 to 5-10℃. Wash the stem cells collected in Preparation Example 1 with PBS buffer and centrifuge to collect them. Weigh 1.0g of dental pulp mesenchymal stem cells according to the ratio and add them to the above mixture 2. Make up the volume to 100mL with deionized water and stir until homogeneous, so that the concentration of dental pulp mesenchymal stem cells is 1×10⁻⁶. 7 The concentration of each sample was increased to 100 mg / mL to obtain a vaginal injection ointment, which was then stored at low temperature for later use.

[0039] Example 3 This embodiment provides a method for preparing a vaginal mucosal cell maintenance preparation, including the following steps: Weigh out 5g of glycerin and 8g of chitosan according to the proportion and dissolve them in an appropriate amount of deionized water. Heat to 40℃ and stir at 200rpm until uniform to form a mixture 1 for later use. Weigh out 3.6g of purslane polysaccharide, 1.4g of lactic acid and 0.05g of quercetin collected in Preparation Example 1 according to the proportions and add them to the above mixture 1. Heat to 60°C and stir at 200rpm until uniform to form mixture 2 for later use. Cool the above mixture 2 to 5-10℃. Wash the stem cells collected in Preparation Example 1 with PBS buffer and centrifuge to collect them. Weigh 1.8g of dental pulp mesenchymal stem cells according to the ratio and add them to the above mixture 2. Make up the volume to 100mL with deionized water and stir until homogeneous, so that the concentration of dental pulp mesenchymal stem cells is 1×10⁻⁶. 7 The concentration of each sample was increased to 100 mg / mL to obtain a vaginal injection ointment, which was then stored at low temperature for later use.

[0040] Comparative Example 1 Comparative Example 1 uses the preparation method of Example 2, the difference being that purslane polysaccharide is not added.

[0041] Comparative Example 2 Comparative Example 2 uses the preparation method of Example 2, the difference being that no dental pulp mesenchymal stem cells are added.

[0042] Comparative Example 3 Comparative Example 3 uses the preparation method of Example 2, the difference being that it does not add purslane polysaccharide and dental pulp mesenchymal stem cells.

[0043] Test Example 1 Vaginal injury modeling Female 6-month-old SD rats, weighing 220-280g, were selected. They were fasted for 12 hours before surgery but allowed free access to water. Sodium pentobarbital was injected intraperitoneally at a concentration of 45mg / kg. The surgical area of ​​the abdomen was shaved and disinfected alternately with iodine and alcohol. An incision of about 1.5-2cm was made along the midline of the abdomen from the pubic symphysis to 1cm below the xiphoid process. The muscle layer was bluntly dissected to enter the abdomen. The uterine horn was gently lifted to both sides, revealing the fat mass surrounding the ovary and the "cauliflower-like" ovary. The fallopian tube-uterus junction was double-ligated. The ovary and part of the fallopian tube were removed. The muscle layer and skin were sutured layer by layer. Vancomycin 80,000 units were injected into the peritoneum to prevent infection.

[0044] Keep warm until awake, house individually for 24 hours, and inject penicillin for 3 consecutive days at a concentration of 40,000 U / kg / day to prevent infection. The recovery period is 4 weeks.

[0045] Acetic acid damage modeling On day 29 after oophorectomy and partial fallopian tube removal, rats were fixed in a supine position with the vagina exposed. 50 μL of 4% acetic acid solution was injected using a microsyringe, retained for 2 minutes, and then rinsed three times with physiological saline to remove residual acetic acid. This procedure was repeated for one week.

[0046] After successful model creation, verification will be conducted according to the following plan: Rats that successfully developed the model were randomly divided into four groups: control group, Example 1 group, Example 2 group, Example 3 group, Comparative Example 1 group, Comparative Example 2 group, and Comparative Example 3 group, with 8 rats in each group. Injection dosage: 0.1 mL was administered vaginally daily for 14 consecutive days. The control group was treated with physiological saline instead.

[0047] Fourteen days after the above treatment, mucosal thickness, glycogen content, tight junction proteins, and inflammatory factors were measured. The results are shown in Table 1 below: Group Mucosal thickness (μm) Glycogen content (IOD) Tight junction protein (Claudin-1,%) Inflammatory factor (IL-6, pg / mL) Blank group 28.1±2.2 12.6±1.5 17.5±2.6 186±9 Example 1 Group 59.3±3.5 34.2±1.7 64.1±3.6 85±6 Example 2 group 72.2±4.8 51.5±3.1 82.4±5.1 62±8 Example 3 Group 67.5±3.4 43.7±2.9 76.8±4.5 69±6 Comparative Example 1 42.5±2.9 21.4±1.5 34.7±2.6 144±10 Comparative Example 2 43.9±2.1 22.9±1.4 41.6±2.7 129±12 Comparative Example 3 Groups 33.6±1.8 16.6±2.1 22.3±2.1 175±8 As shown in Table 1 above, compared with the blank group, all three groups of Examples 1-3 showed better repair effects, especially Example 2, which achieved the best results, with a mucosal thickness 2.5 times that of the blank group. In Comparative Example 1, the lack of purslane polysaccharide led to a 59% decrease in glycogen synthesis, and in Comparative Example 2, the lack of stem cells reduced the repair capacity by 40%, thus proving the core role of stem cells in regeneration. In Comparative Example 3, since neither purslane polysaccharide nor dental pulp mesenchymal stem cells were added, the effect was no different from that of the blank group, proving the synergistic necessity of purslane polysaccharide and dental pulp mesenchymal stem cells.

[0048] Based on the above experimental analysis, the vaginal mucosal cell maintenance preparation provided in this application has shown good efficacy and has promising application prospects in the maintenance of vaginal mucosal cells.

[0049] Test Example 2 To evaluate the efficacy and safety of the formulation of this invention in repairing the vaginal mucosa, restoring the microecological balance, and improving symptoms in postmenopausal atrophic vaginitis patients, the following experimental procedure was set up.

[0050] 1. Inclusion criteria: Requirements: Age 45-60 years, natural menopause ≥1 year, meeting the diagnostic criteria for atrophic vaginitis: clinical symptoms (dryness, burning pain, dyspareunia, itching, etc.) and signs (pale, thin mucosa, pinpoint bleeding or congestion); Vaginal Health Index (VHI) ≤15 points (out of 25). Ninety qualified participants were recruited and randomly divided into three groups of 30 each using a random number table.

[0051] 2. Dosing regimen: Treatment group: Administered the formulation prepared in Example 2 of this invention.

[0052] Positive control group: administered commercially available single-component lactobacillus gel.

[0053] Blank control group: Administered placebo gel (containing only matrix, without stem cells and other active ingredients).

[0054] All patients received vaginal administration once daily before bedtime for 14 consecutive days, followed by twice weekly for 4 weeks (total treatment duration 6 weeks).

[0055] 3. Testing standards: The following indicators were measured before treatment (T0) and 6 weeks after treatment (T1), as shown in Table 2: Evaluation indicators Detection methods Purpose Subjective symptom score Visual analog scale (VAS) is used to score dryness, burning, and itching. Assess the degree of improvement in the patient's perceived symptoms. Objective physical signs score (VHI) The doctor assesses elasticity, secretions, pH, mucosal integrity, and moisture. Quantitative assessment of the recovery of mucosal appearance and function Vaginal microecological testing Gram staining microscopy, Nugent score Assess the balance of vaginal flora (Nugent score 0-3: normal; 4-6: intermediate; 7-10: bacterial vaginosis). Core indicators of mucosal repair ELISA method for detecting sIgA and IL-6 levels in vaginal douches sIgA (mucosal barrier function), IL-6 (inflammation level) Safety indicators Record adverse events (AEs), complete blood count, and liver and kidney function tests. Evaluate the clinical safety of the product The test results are shown in Table 3 below: Group VHI score (points) Symptom VAS score (points) Nugent rating (points) sIgA (μg / mL) IL-6 (pg / mL) Treatment group T0 12.1±1.5 7.5±1.2 6.8±1.0 45.2±10.1 35.6±8.7 Treatment group T1 21.5±1.8 2.1±0.8 3.2±0.8 95.8±15.3 12.3±4.1 Positive control group T0 11.8±1.7 7.3±1.4 6.5±1.2 47.5±11.2 36.8±9.2 Positive control group T1 17.2±1.6 4.2±1.1 4.5±0.9 68.4±12.5 20.5±5.6 Blank control group T0 12.3±1.6 7.6±1.3 6.7±1.1 46.1±10.5 35.1±8.9 Blank control group T1 13.5±1.8 6.8±1.5 6.5±1.0 49.5±11.8 32.8±7.5 The data analysis is as follows: Treatment group: 25 cases (83.3%) showed significant improvement, 5 cases (16.7%) showed improvement, and 0 cases were ineffective, with a total effective rate of 100%.

[0056] Positive control group: 12 cases (40.0%) showed significant improvement, 13 cases (43.3%) showed improvement, and 5 cases (16.7%) showed no effect, with a total effective rate of 83.3%.

[0057] Blank control group: 0 cases with significant improvement (0%), 5 cases with improvement (16.7%), 25 cases with no effect (83.3%), and a total effective rate of 16.7%.

[0058] Two patients in the treatment group experienced a mild warming sensation after the first dose, which resolved spontaneously within a short period and did not affect continued treatment. No abnormalities related to the investigational drug were found in blood routine tests or liver and kidney function tests in any group.

[0059] The formulation of this invention is significantly more effective than single lactobacillus products and placebo in improving patients' subjective symptoms, repairing vaginal mucosa (significantly improving VHI score), rebuilding healthy microecology (reducing Nugent score), enhancing mucosal immune barrier (increasing sIgA) and anti-inflammatory (reducing IL-6) effects (P<0.05).

[0060] Clinical data have confirmed the dual mechanism of "stem cell-polysaccharide" synergistic repair of mucosal barrier and regulation of microenvironment described in this invention, which not only replaces traditional hormone therapy, but also achieves long-term regulation that "treats the root cause".

[0061] This product is easy to use, has high patient compliance, no serious adverse reactions, and is safe and reliable for clinical application.

[0062] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A vaginal mucosal cell maintenance preparation for women, characterized in that, Includes the following components by weight percentage: Dental pulp mesenchymal stem cells: 0.2-1.8% Purslane polysaccharides: 1.2-3.6% Lactic acid 0.8-1.4% Quercetin 0.01-0.05% 3-5% glycerin Stem cell carrier 1-8% The vaginal injection ointment is obtained by mixing an appropriate amount of deionized water evenly.

2. The vaginal mucosal cell maintenance preparation as described in claim 1, characterized in that, The concentration of the dental pulp mesenchymal stem cells was 1 x 10⁻⁶. 6 -10 7 per mL.

3. The vaginal mucosal cell maintenance preparation as described in claim 1, characterized in that, The stem cell carrier is chitosan.

4. The vaginal mucosal cell maintenance preparation as described in claim 1, characterized in that, The molecular weight of the purslane polysaccharide is 200-230 kDa.

5. The vaginal mucosal cell maintenance preparation as described in claim 1, characterized in that, The dental pulp mesenchymal stem cells were passaged to the P3-P5 generation.

6. A method for preparing a vaginal mucosal cell maintenance preparation, characterized in that, Includes the following steps: Weigh out glycerol and stem cell carrier according to the proportions and dissolve them in an appropriate amount of deionized water. Stir and mix until homogeneous to form mixture 1 for later use. Weigh out the purslane polysaccharide, lactic acid and quercetin according to the proportions and add them to the above mixture 1. Stir and mix until uniform to form mixture 2 for later use. Cool the above mixture 2 to 5-10℃, weigh out the dental pulp mesenchymal stem cells according to the ratio and add them to the above mixture 2, stir and mix until uniform to form a maintenance preparation, and store at low temperature.

7. The method for preparing the vaginal mucosal cell maintenance preparation as described in claim 6, characterized in that, The preparation steps for dental pulp mesenchymal stem cells are as follows: Tissue preparation: The dental pulp is cut into pieces of 1-3 mm. 3 Small pieces were added to a digestion solution consisting of 2-3 mg / mL type I collagenase and 2-4 mg / mL neutral protease, and digested at 37°C for 15-20 min. The reaction was terminated by adding 3-5 times the volume of serum-containing culture medium, and filtered through a 100 μm sieve to obtain a single-cell suspension. Primary culture: Absorb single-cell suspension and inoculate with 1-5 × 10⁻⁵ cells. 4 The cells were inoculated at a density of 1 / mL in a culture medium consisting of α-MEM, 10–15% FBS and 1% penicillin, and cultured at 37°C and 5% CO2 for 10–15 days to achieve 70–80% confluence. Passaging: Digest with 0.25% trypsin-EDTA for 3-5 minutes, passage at a ratio of 1:2, change the medium every 3-4 days, and culture to P3-5 generation to obtain dental pulp mesenchymal stem cells.

8. The method for preparing the vaginal mucosal cell maintenance preparation as described in claim 7, characterized in that, The dental pulp mesenchymal stem cells are P4 generation cells.

9. The method for preparing the female vaginal mucosal cell maintenance preparation as described in claim 6, characterized in that, The preparation steps of purslane polysaccharide are as follows: Pretreatment: Take dried purslane and crush it to 40 mesh. Mix it with petroleum ether at a ratio of 100g: 500mL. Reflux at 60℃ for 2h. Filter and discard the filtrate. Dry the residue at 60℃ to constant weight to obtain defatted raw material for later use. Extraction treatment: Add defatted raw materials to distilled water at a material-to-liquid ratio of 100g:2000mL, extract at 90℃ for 2 hours, stir in a constant temperature water bath at 200rpm, extract twice, and combine the filtrates; Concentration and impurity removal: Evaporate the above filtrate at 60°C to 1 / 5 of its original volume, and add 1 / 4 volume of chloroform:n-butanol solvent in a volume ratio of 4:1 to the concentrate. Shake vigorously for 30 min, centrifuge at 8000 rpm for 15 min, discard the lower organic phase and protein layer, and repeat the impurity removal steps until there are no white flocculent substances at the interface to obtain the concentrate. Purification: Slowly add 4 volumes of 95% ethanol to the concentrate, let stand at 4°C for 12 hours, centrifuge at 8000 rpm for 15 minutes, collect the precipitate to obtain crude polysaccharide, and wash twice with anhydrous ethanol. Dialysis treatment: The crude polysaccharide was redissolved in distilled water, placed in a dialysis bag and dialyzed for 48 hours. The dialysate was collected and freeze-dried for 24 hours to obtain white flocculent refined purslane polysaccharide.

10. The use of the female vaginal mucosal cell maintenance preparation according to any one of claims 1-5 in the preparation of a female vaginal mucosal cell maintenance drug.