Method for preparing and use of sPL gel for endometrial lesion repair

Abstract

The application relates to a preparation method and application of sPL gel for endometrial injury repair, relates to the field of endometrial repair drugs, and aims at solving the problems of short treatment time and poor treatment effect of existing platelet-rich plasma. Method: 1. preparing a platelet lysate PL; 2. diluting placental chorionic trophoblast cells with complete culture medium to obtain a cell suspension, culturing the cell suspension by adding bisabolene, collecting a culture solution, filtering the culture solution to obtain a conditioned medium, and mixing the platelet lysate PL and the conditioned medium to prepare sPL; 3. adding a collagen solution and carbomer to the sPL, uniformly mixing the sPL, collagen solution and carbomer to prepare sPL gel. The sPL gel prepared by the method can be solidified by temperature rise, effectively improves the internal microenvironment of the uterine cavity, and is used for promoting the repair of endometrial injury.

Description

Technical Field

[0001] This invention relates to the field of endometrial repair drugs, specifically to a method for preparing and applying an sPL gel for repairing endometrial damage. Background Technology

[0002] Endometrial damage is a group of diseases caused by factors such as endocrine and immune disorders, local trauma and infection resulting from intrauterine procedures, leading to varying degrees of decline in endometrial function. Local uterine infection and aseptic inflammation cause endometrial damage, disrupting the microenvironment of stem cells, hindering the regeneration and repair of epithelial and stromal cells, obstructing angiogenesis, and resulting in fibrosis, scarring, and intrauterine adhesions. Ultimately, this leads to the uterine cavity being covered with only a small amount of endometrium, glandular atrophy, and loss of normal uterine cavity shape and function. Damaged endometrium cannot fully proliferate and transform into the secretory phase during a normal menstrual cycle, hindering implantation of the fertilized egg and seriously affecting human reproductive health. According to the World Health Organization, 60 to 80 million people worldwide are infertile, accounting for 15% to 20% of the reproductive-age population. The main treatment methods include surgery combined with estradiol hormone drugs and intrauterine devices. Although these treatments can achieve certain therapeutic effects, the high recurrence rate and poor prognosis due to intrauterine sclerosis necessitate the development of new treatment methods.

[0003] Studies have found that while platelet-rich plasma (PRP) can provide cytokines and play a certain role in tissue repair, the appropriate factors for repairing damage vary from tissue to tissue. Even with high concentrations of PRP, the factors cannot be fully released due to insufficient platelet lysis, resulting in poor repair efficacy. Furthermore, as a liquid, PRP is highly fluid, causing it to flow out of the vagina quickly after injection into the uterus, failing to remain at the site of injury for an extended period, thus shortening the overall treatment time. Summary of the Invention

[0004] The present invention aims to address the problems of short treatment time and poor treatment effect of existing platelet-rich plasma therapy, and provides a method for preparing and applying sPL gel for endometrial injury repair.

[0005] The present invention discloses a method for preparing sPL gel for endometrial injury repair, comprising the following steps:

[0006] I. Preparation of platelet lysis buffer PL;

[0007] 2. Dilute placental chorionic trophoblast cells with complete culture medium to obtain cell suspension, add 1~10 μg / mL of myrrh-angelicone for culture, collect the culture medium, filter, and obtain conditioned medium.

[0008] sPL was prepared by mixing platelet lysis buffer (PL) with conditioned medium.

[0009] 3. Add collagen solution and carbomer to sPL, mix well, and prepare sPL gel.

[0010] Furthermore, the preparation method of the platelet lysis buffer PL mentioned in step one is as follows:

[0011] A. Centrifuge the blood in a sodium citrate anticoagulant tube and collect the plasma layer and the upper 1 / 3 volume of liquid from the red blood cell layer;

[0012] B. Place the collected plasma layer and the upper 1 / 3 volume of liquid from the red blood cell layer into a venous blood collection container, centrifuge, and collect the upper platelet-poor plasma layer and the platelet-rich plasma layer from the separating gel.

[0013] C. Dilute platelet-rich plasma with platelet-poor plasma to a platelet concentration of 0.5 × 10⁻⁶. 9 ~2×10 9 Platelet-rich plasma was prepared by collecting platelets per mL.

[0014] D. Centrifuge the concentrated platelet plasma, collect the supernatant, filter, and obtain platelet lysis buffer PL.

[0015] Furthermore, the complete culture medium described in step two is Knockout DMEM / F12 medium containing 10% FBS.

[0016] Furthermore, in step two, platelet lysis buffer PL is mixed with conditioned medium at a volume ratio of (1~3):1.

[0017] Furthermore, the concentration of the cell suspension mentioned in step two is 0.5 × 10⁻⁶. 5 ~1.5×10 5 per mL.

[0018] Furthermore, in step two, myrrh-angelicone is added and cultured for 36-60 hours.

[0019] Furthermore, the sPL gel described in step three contains 90% to 95% sPL by mass, 2% to 3% collagen, 0.5% to 1.0% carbomer, and the balance being water.

[0020] The application of sPL gel prepared by the above method in the preparation of drugs for treating and repairing endometrial damage.

[0021] The beneficial effects of this invention are:

[0022] 1. In this invention, myrrh-angelicone is added during the culture of placental chorionic trophoblast cells, which can effectively promote the secretion of interleukin-4 (IL-4), interleukin-10 (IL-10), insulin-like growth factor (IGF), epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) in the cells, so that the conditioned medium has nutritional and anti-inflammatory effects.

[0023] 2. A conditioned medium containing myrrh and angelicone to stimulate placental chorionic trophoblast cells, mixed with PL, was prepared as sPL. sPL can effectively stimulate the proliferation and migration of functional cells, thereby improving the intrauterine microenvironment. Furthermore, sPL promotes endometrial angiogenesis, increases endometrial thickness, and thus promotes the repair of endometrial damage.

[0024] 3. The gel prepared by this invention has no irritating odor and is neutral. It has good adhesion. When injected into the uterine cavity, it becomes solid due to the increase in temperature and can adhere well to the surface of the endometrium. It releases sPL for a long time to treat the damaged endometrium.

[0025] 4. In this invention, the gel material is directly fused with sPL to prepare sPL gel, wherein the volume percentage of sPL exceeds 90%, which can fully maintain the activity and content of cytokines. Furthermore, the raw materials used in this invention are readily available, biodegradable, safe, non-toxic, and non-irritating, offering advantages for clinical translation.

[0026] The sPL gel of this invention can repair endometrial damage, treat intrauterine adhesions and thin endometrium, and solve the problems of infertility and recurrent miscarriage caused by endometrial damage. Attached Figure Description

[0027] Figure 1 The statistical results of cell proliferation rate of rESCs in each treatment group;

[0028] Figure 2 The statistical results of cell proliferation rate of rVECs in each treatment group;

[0029] Figure 3 Images of cell scratches obtained from scratch experiments;

[0030] Figure 4 The statistical results of cell migration rate for each treatment group;

[0031] Figure 5 The sustained release of VEGF factor from sPL gel;

[0032] Figure 6 Images of H&E staining results of the uterus in each treatment group;

[0033] Figure 7 Endometrial thickness in each treatment group;

[0034] Figure 8 IHC staining results of vimentin in each treatment group. Detailed Implementation

[0035] The technical solution of the present invention is not limited to the specific embodiments listed below, but also includes any combination of the specific embodiments.

[0036] Specific Implementation Method 1: The preparation method of sPL gel for endometrial injury repair in this implementation method includes the following steps:

[0037] I. Preparation of platelet lysis buffer PL;

[0038] 2. Dilute placental chorionic trophoblast cells with complete culture medium to obtain cell suspension, add 1~10 μg / mL of myrrh-angelicone for culture, collect the culture medium, filter, and obtain conditioned medium.

[0039] sPL was prepared by mixing platelet lysis buffer (PL) with conditioned medium.

[0040] 3. Add collagen solution and carbomer to sPL, mix well, and prepare sPL gel.

[0041] Specific Implementation Method Two: The preparation method of platelet lysis buffer PL in step one of this implementation method is as follows:

[0042] A. Centrifuge the blood in a sodium citrate anticoagulant tube and collect the plasma layer and the upper 1 / 3 volume of liquid from the red blood cell layer;

[0043] B. Place the collected plasma layer and the upper 1 / 3 volume of liquid from the red blood cell layer into a venous blood collection container, centrifuge, and collect the upper platelet-poor plasma layer and the platelet-rich plasma layer from the separating gel.

[0044] C. Dilute platelet-rich plasma with platelet-poor plasma to a platelet concentration of 0.5 × 10⁻⁶. 9 ~2×10 9 Platelet-rich plasma was prepared by collecting platelets per mL.

[0045] D. Centrifuge the concentrated platelet plasma, collect the supernatant, filter, and obtain platelet lysis buffer (PL). Other steps and parameters are the same as in Specific Implementation Method 1.

[0046] Specific Implementation Method 3: The complete culture medium mentioned in step 2 of this implementation method is Knockout DMEM / F12 medium containing 10% FBS. Other steps and parameters are the same as in Specific Implementation Method 1 or 2.

[0047] Specific Implementation Method Four: In step two of this implementation method, platelet lysis buffer (PL) and conditioned medium are mixed at a volume ratio of (1~3):1. Other steps and parameters are the same as in any of Specific Implementation Methods One to Three.

[0048] Specific Implementation Method Five: The concentration of the cell suspension mentioned in step two of this implementation method is 0.5 × 10⁻⁶. 5 ~1.5×10 5 The number of cells / mL is the same as in any of the specific implementation methods one to four.

[0049] Specific Implementation Method Six: In step two of this implementation method, the culture time after adding myrrh and angelicone is 36-60 hours. Other steps and parameters are the same as in any of Specific Implementation Methods One to Five.

[0050] Specific Implementation Method Seven: The sPL gel described in step three of this implementation method contains 90%~95% sPL, 2%~3% collagen, 0.5%~1.0% carbomer, and the balance water. Other steps and parameters are the same as in any one of Specific Implementation Methods One to Six.

[0051] Specific Implementation Method 8: Application of sPL gel in the preparation of drugs for treating and repairing endometrial damage in this implementation method.

[0052] The embodiments of the present invention will be described in detail below. The following embodiments are implemented based on the technical solution of the present invention, and detailed implementation schemes and specific operation processes are given. However, the protection scope of the present invention is not limited to the following embodiments.

[0053] Example 1:

[0054] The preparation method of sPL gel for endometrial injury repair in this embodiment includes the following steps:

[0055] I. Preparation of rat platelet lysate (PL)

[0056] A. Collect 10-20 mL of rat blood aseptically using sodium citrate anticoagulant tubes. Centrifuge at 1000 rpm for 30 min. The blood is separated into a plasma layer and a cell layer. Remove the lower 2 / 3 volume of cells from the cell layer and collect the plasma layer and the upper 1 / 3 volume of cells from the cell layer.

[0057] B. Transfer the liquid collected in step A into a sodium citrate separating gel blood collection tube and centrifuge at 3000 rpm for 10 minutes. After centrifugation, the plasma will be separated from top to bottom into a platelet-poor plasma layer, a platelet-rich plasma layer, a separating gel, and a blood cell layer. Collect the platelet-poor plasma layer and the platelet-rich plasma layer separately, and discard the red blood cells in the lower layer of the separating gel.

[0058] C. Count the cells in the collected platelet-rich plasma, and dilute the platelet-rich plasma with platelet-poor plasma to a concentration of approximately 1 × 10⁻⁶ cells per milliliter. 9 Platelets were collected to prepare concentrated platelet plasma with a standard platelet concentration, which was then stored at -80℃ for 24 hours.

[0059] D. Take out the concentrated platelet plasma of standard platelet concentration stored at -80℃, thaw it at 4℃, vortex mix it for 30 s, store it at -80℃ again for 24 h, thaw it at 4℃, sonicate it for 10 min, centrifuge it at 1500 g for 5 min, collect the supernatant and filter it with a 0.22 μm filter membrane to remove cell membrane debris and other impurities to obtain PL solution.

[0060] II. Preparation of Super-Active Platelet Lysis Buffer (sPL)

[0061] A. Dilute P1 generation rat placental chorionic trophoblast cells with complete culture medium to obtain a concentration of 1×10⁻⁶. 5 A cell suspension of 10 mL cells / mL was seeded at a 75 cm⁻¹ plate. 2 The culture medium was placed in a culture flask and 5 μg / mL of bisabolangelone was added. After 48 h of culture, the culture medium was collected and filtered through a 0.22 μm filter membrane to obtain the conditioned medium (CM). The complete medium was Knockout DF12 medium containing 10% FBS.

[0062] P1 generation rat placental chorionic trophoblast cells were diluted with complete culture medium to obtain a concentration of 1×10⁻⁶. 5 A cell suspension of 10 mL cells / mL was seeded at a 75 cm⁻¹ plate. 2 After 48 h of incubation in the culture flask, the culture medium was collected, filtered through a 0.22 μm filter membrane, and used as the control conditioned medium (CCM).

[0063] B. Mix PL with conditioned medium at a volume ratio of 1:1 to prepare sPL.

[0064] In this embodiment, myrrh-angelonone was added during the culture of rat placental chorionic trophoblast cells. Myrrh-angelonone is a sesquiterpene compound, mainly extracted from Angelica sinensis, and its structure contains a ketone group and a double bond, possessing potential for anti-inflammatory, antioxidant, and cell signaling regulation. Currently, research on this compound mainly focuses on neurological diseases (such as Alzheimer's disease) and tumors; its application in endometrial therapy has not yet been directly reported.

[0065] III. Gel Preparation

[0066] A. Weigh 100 mg of collagen, add 10 mL of 0.1 mol / L acetic acid, stir with a glass rod until the collagen is completely dissolved, slowly add 2 mol / L NaOH solution dropwise to adjust the pH to 7.5, add sterile deionized water to adjust the collagen concentration to 4 mg / mL, and store at 4℃ for later use.

[0067] B. Weigh 0.5 g of carbomer, add 9.5 mL of sterile deionized water, vortex mix for 30 s, centrifuge at 1000 g for 3 min, let stand for 1 h, then mix again, let stand overnight to allow the carbomer to fully dissolve, vortex to remix the solid and liquid to prepare a 10% carbomer solution, sterilize and store at 4℃ for later use.

[0068] C. Add 4 mg / mL collagen solution and 10% carbomer solution to the sPL prepared in step 2. Mix the sPL solution and collagen solution completely in a vortex mixer to prepare an sPL gel containing 95% sPL mixed solution, 3% collagen, 1.5% water and 0.5% carbomer.

[0069] The CCM, CM, PL and sPL obtained in this embodiment were detected by ELISA to evaluate the contents of IL-4, IL-10, IGF, EGF and VEGF.

[0070] IL-4 and IL-10 are important immunomodulatory cytokines with significant anti-inflammatory effects. VEGF is a key regulator of angiogenesis, promoting the proliferation and migration of vascular endothelial cells, inducing angiogenesis in damaged tissues, and ensuring adequate nutrition and oxygen supply to the damaged site. EGF and IGF-1 can stimulate mitosis of endometrial epithelial cells and stromal cells, increasing cell number; they can also regulate extracellular matrix (ECM) synthesis, providing structural support for endometrial repair; in addition, EGF and IGF-1 can synergistically enhance the angiogenic effect of VEGF, promoting neovascularization at the site of endometrial injury and accelerating nutrient and oxygen supply.

[0071] The contents of anti-inflammatory and repair factors in CCM, CM, PL, and sPL are shown in Table 1. Compared with CCM, the concentrations of inflammatory control factors IL-4 and IL-10 in CM were significantly increased (P<0.01), indicating that Bisabolangelone can significantly enhance the expression of anti-inflammatory repair factors related to endometrial repair in placental chorionic trophoblast cells. sPL is a combination of CM and PL. Comparison revealed that the concentrations of anti-inflammatory factors (IL-4 and IL-10) in CM were significantly higher than those in PL (P<0.05), but the concentrations of repair factors (IGF, EGF, and VEGF) were significantly lower than those in PL (P<0.01). Furthermore, the concentrations of anti-inflammatory factors in sPL were significantly higher than those in PL (P<0.05), and the concentrations of repair factors were significantly higher than those in CM (P<0.01). This suggests that sPL, obtained by combining CM and PL, contains a more balanced range of anti-inflammatory and repair factors, which may be more suitable for endometrial repair.

[0072] Table 1. Content of anti-inflammatory repair factors in CCM, CM, PL and sPL

[0073]

[0074] Table 1 The comparison between CM and CCM shows P < 0.01. This represents a comparison between sPL and PL, with P < 0.05; This represents a comparison between sPL and PL, with P < 0.01. The comparison between sPL and CM shows P < 0.01. The comparison between PL and CM showed P < 0.05. The comparison between PL and CM shows P < 0.01.

[0075] Example 2: Study on the effects of CM, PL and sPL on the proliferation of rat endometrial stem cells induced by lipopolysaccharide (LPS) inflammation and on the proliferation and migration of rat vascular endothelial cells.

[0076] Endometrial stem cells (ESCs) are self-renewing and multi-lineage differentiated stem cells in the basal layer, capable of differentiating into epithelial cells and stromal cells, playing a crucial role in the repair and regeneration of the functional layer. Vascular endothelial cells (VECs) form the inner walls of arterioles, veins, and capillaries in the basal layer, responsible for vascular exchange and blood flow. The vessels of the basal layer are the origin of the vessels in the functional layer, and the regeneration of vessels in the functional layer depends on the extension of vessels in the basal layer.

[0077] 1. Detection of the regulatory effect of sPL on the proliferation of rat endometrial stem cells (rESCs) induced by LPS inflammation.

[0078] rESCs were resuspended in α-MEM medium containing 10% FBS, 1% penicillin-antibody, 10 ng / mL bFGF, 20 ng / mL EGF, and 1% insulin-transferrin-selenium (ITS), and cultured at a concentration of 1×10⁻⁶. 4 cells / cm 2 Cells were seeded at a density of [insert density here] in Marigold-coated culture flasks and cultured at 37°C with 5% CO2. When cell confluence reached approximately 80%, the cells were digested with 0.25% trypsin (containing 1 mMEDTA) and incubated at 37°C. Digestion was stopped once the cells became rounded, followed by centrifugation at 1000 rpm for 5 min. The cell pellet was resuspended in fresh culture medium and cultured at 1×10⁻⁶ [insert concentration here]. 4 Cells were seeded at a density of 1 / well in 96-well plates and cultured for 24 h until adherence.

[0079] Cells were divided into 5 groups, with 3 replicates per group. The control group was treated with α-MEM basal medium for 48 h; the LPS group was treated with α-MEM basal medium containing 1 μg / mL LPS for 48 h; the 10% CM+LPS group was treated with α-MEM basal medium containing 10% CM and 1 μg / mL LPS for 48 h; the 10% PL+LPS group was treated with α-MEM basal medium containing 10% PL and 1 μg / mL LPS for 48 h; and the 10% sPL+LPS group was treated with α-MEM basal medium containing 10% sPL and 1 μg / mL LPS for 48 h. Cell-free media served as blank controls for their respective groups. Cell count was detected using a CCK-8 assay kit, and absorbance (OD value) was measured at 450 nm using a microplate reader. Cell proliferation rate was calculated.

[0080] Proliferation rate (%) = (OD value of experimental group - OD value of corresponding blank group) / (OD value of control group - OD value of corresponding blank group) × 100%.

[0081] The cell proliferation rate results for each group are as follows: Figure 1 As shown, Figure 1 middle This indicates that P < 0.05; P < 0.01. The results showed that, compared with the control group, LPS treatment significantly reduced rESC activity (P < 0.01). When LPS stimulated rESCs, the addition of CM, PL, or sPL significantly increased cell activity, with all differences reaching a highly significant level (P < 0.01). Furthermore, the repair effect of sPL was significantly higher than that of CM and PL (P < 0.05). This indicates that the higher levels of inflammatory regulatory factors and cell repair factors in sPL contribute to regulating rESC activity.

[0082] 2. Detection of the regulatory effect of sPL on the proliferation and migration of rat vascular endothelial cells (rVECs) induced by LPS inflammation.

[0083] A. CCK8 assay to detect the effect of sPL on LPS-induced rVEC proliferation activity.

[0084] rVECs were resuspended in M199 basal medium containing 10% FBS, counted, and analyzed at 1×10⁻⁶. 4 cells / cm 2 Cells were seeded at a density suitable for gelatin-coated culture flasks and cultured at 37°C with 5% CO2. When cell confluence reached approximately 80%, the cells were digested with 0.25% trypsin (containing 1 mM EDTA) and incubated at 37°C for 10 min. Digestion was stopped once the cells became rounded, followed by centrifugation at 1000 rpm for 5 min. The cell pellet was resuspended in fresh culture medium, counted, and cultured at 5 × 10⁻⁶ cells / mL. 3 Cells were seeded at a density of 96 cells / well in 96-well plates and cultured for 24 h until adherence.

[0085] Cells were divided into 5 groups, with 3 replicates per group. The control group was treated with M199 basal medium for 48 h; the LPS group was treated with M199 basal medium containing 1 μg / mL LPS for 48 h; the 10% CM+LPS group was treated with M199 basal medium containing 10% CM and 1 μg / mL LPS for 48 h; the 10% PL+LPS group was treated with M199 basal medium containing 10% PL and 1 μg / mL LPS for 48 h; and the 10% sPL+LPS group was treated with M199 basal medium containing 10% sPL and 1 μg / mL LPS for 48 h. Cell-free media served as blank controls for their respective groups. Cell count was detected using a CCK-8 assay kit, and absorbance (OD value) was measured at 450 nm using a microplate reader. Cell proliferation rate was calculated.

[0086] Proliferation rate (%) = (OD value of experimental group - OD value of corresponding blank group) / (OD value of control group - OD value of corresponding blank group) × 100%.

[0087] The cell proliferation rate results for each group are as follows: Figure 2 As shown, Figure 2 middle P < 0.01. The results showed that, compared with the control group, LPS treatment significantly reduced rVEC activity (P < 0.01). When LPS stimulated rVECs, the addition of CM, PL, or sPL significantly increased cell activity, with all differences reaching highly significant levels (P < 0.01). Furthermore, the repair effect of sPL was significantly higher than that of CM and PL (P < 0.01). This indicates that the higher levels of inflammatory regulatory factors and cell repair factors in sPL help regulate rVEC activity, and the results are basically consistent with those of rESCs.

[0088] B. Scratch test to detect the effect of sPL on the migration ability of LPS-induced inflammation-induced rVECs.

[0089] rVECs were cultured in M199 basal medium containing 10% FBS. When the cell confluence reached 80%–90%, the cells were digested, counted, and seeded into 24-well plates at a density of 1.2 × 10⁻⁶. 5Cells were cultured in wells at 37 °C for 24 h. After 24 h of culture, a 200 μL sterile tip was used to make a scratch on the bottom of the wells from top to bottom. The floating cells were then washed once with physiological saline, and M199 basal medium was added to infiltrate the wells. The wells were observed and photographed under a microscope, and this was recorded as 0 h. After photographing, the cell culture wells were grouped and the corresponding culture media were added: Control group: M199 basal medium was added to the cell culture wells; 10% CM group: M199 basal medium containing 10% CM was added to the cell culture wells; 10% PL group: M199 basal medium containing 10% PL was added to the cell culture wells; 10% sPL group: M199 basal medium containing 10% sPL was added to the cell culture wells. 1 μg / mL LPS was added to each group. The cells were cultured for another 24 h, and then observed and photographed, and this was recorded as 24 h. Calculate the mobility Rn using the following formula.

[0090] Rn (%) = (S0 - Sn) / S0×100%

[0091] In the formula, S0 is the scratch area at 0 h, and Sn is the scratch area at 24 h.

[0092] Current research reports that low concentrations of LPS (1-100 ng / mL) can promote the migration of human umbilical vein endothelial cells. However, high concentrations of LPS (≥1 μg / mL) can inhibit cell migration and induce damage. Anti-inflammatory factors such as IL-4 and IL-10 can regulate the inflammatory state and endothelial cell activity induced by high concentrations of LPS, while growth factors such as VEGF, EGF, and IGF can promote the proliferation and migration of vascular endothelial cells. The binding and balance of anti-inflammatory factors and growth factors can effectively increase angiogenesis in damaged tissues, thereby achieving a repair effect.

[0093] Cell scratch images obtained from scratch assays are as follows: Figure 3 As shown, the cell migration rate statistics for each group are as follows: Figure 4 As shown (where This indicates that P < 0.05; (Indicates P < 0.01). From Figure 3 and Figure 4The results showed that scratch healing was significant at 24 h. The cell migration rates in the 10% CM, 10% PL, and 10% sPL groups were significantly higher than those in the basal culture medium control group (P<0.01). Furthermore, the cell migration rates in the 10% PL and 10% sPL groups were significantly higher than those in the 10% CM group (P<0.01), with all differences reaching highly significant levels. In addition, the scratches in the 10% sPL group were essentially healed, with a significantly higher migration rate than that in the 10% PL group (P<0.05). This indicates that CM, PL, and sPL can all accelerate scratch healing, and the sPL mixture (CM and PL) showed the best scratch healing effect. After the anti-inflammatory factors in CM regulate the inflammatory state, the growth factors in PL further promote the proliferation and migration of vascular endothelial cells, effectively increasing the formation of blood vessels in the damaged tissue, thereby achieving a repair effect.

[0094] Example 3: Study on sustained-release effect of sPL gel

[0095] 1. Gel water loss rate detection

[0096] 1 mL of the sPL gel prepared in Example 1 was added to a vial. The gel solidification was observed every 1 min under the conditions of 35% humidity and 37°C. The gel water loss rate at 24 h, 48 h and 72 h was calculated. The results are shown in Table 2.

[0097] Gel loss rate = (Initial sample weight - Real-time sample weight) / Initial sample weight × 100%

[0098] As can be seen from the data in Table 2, the water loss rate of the sPL gel gradually increases with the extension of time, indicating that the sPL gel of the present invention has a sustained-release effect and can continuously release sPL for more than 72 hours.

[0099] Table 2 Gel water loss rate

[0100]

[0101] 2. Study on the release rate of factors

[0102] Assay for the sustained-release effect of sPL gel: 2 mL of sPL liquid gel was added to a centrifuge tube and allowed to solidify at 37 ℃. Then, 1 mL of PBS buffer was slowly added along the tube wall, and the tube was placed in a 37 ℃ water bath at 50 rpm (simulating a mild in vivo environment to promote mixing of the release solution). Samples were taken at 0.5 days, 1 day, 2 days, 3 days, 4 days, and 5 days. The sampling method was as follows: 100 μL of the supernatant was aspirated using a sterile pipette and immediately stored at -20 ℃. An equal volume of fresh PBS buffer was added simultaneously to ensure a constant volume of the release system. After sampling at each time point, the VEGF content in the supernatant was determined using an ELISA kit, and the cumulative VEGF release was calculated.

[0103] VEGF release generally showed an increasing trend, with a cumulative release rate exceeding 65%, indicating that the sPL gel has a certain controlled-release effect. The results are as follows... Figure 5 As shown.

[0104] Example 4: sPL gel promotes endometrial repair

[0105] 1. Rat endometrial model

[0106] After anesthetizing the female rats, an incision of approximately 1 cm was made 0.5–1 cm to the right of the midline of the lower abdomen using a scalpel, exposing the uterus. The connections between the uterus and the ovary, and the proximal vagina, were sealed with hemostatic clips. 150 μL of anhydrous ethanol was injected using a 0.03 mm needle syringe. After 5 minutes, the anhydrous ethanol was aspirated from the uterus, and the uterine cavity was flushed three times with physiological saline. The clips were removed, and the rat's abdomen was sutured with 7-0 absorbable sutures.

[0107] 2. Animal treatment

[0108] Rats were divided into a normal group (no modeling), a model group (Model), and an sPL gel treatment group (sPL). Six to eight hours after modeling, 300 μL of sPL-free gel was injected into the uterine injury site of the model group rats, and 300 μL of sPL gel was injected into the uterine injury site of the sPL gel treatment group rats. The rat abdomens were sutured layer by layer with 7-0 absorbable sutures. Rats received daily penicillin injections starting three days post-surgery.

[0109] 3. Material selection and effect testing

[0110] Twelve days after treatment, the treated uterus was cut into two parts. One part was placed in formalin, and the other was stored at -80°C. The formalin-preserved tissue was sectioned in paraffin and stained with hematoxylin and eosin (HE) to detect endometrial thickness and cell distribution. Immunohistochemical staining (IHC) was also performed to detect vimentin expression in rat endometrial stromal cells.

[0111] The endometrial thickness of rats in each group is as follows: Figure 6 and Figure 7 As shown in the figure, the endometrial thickness in the model group was significantly lower than that in the sPL gel treatment group, while the endometrial thickness in the sPL gel treatment group was close to that in the model group, indicating that sPL gel can promote an increase in endometrial thickness. No uterine adhesions occurred in either the model group or the sPL gel treatment group, suggesting that the gel or sPL gel can prevent uterine adhesions, and that sPL has a significant effect on endometrial cell proliferation.

[0112] IHC staining results of vimentin in each treatment group are as follows: Figure 8As shown, IHC staining results indicate that vimentin (…) is present in the endometrial stromal cells of rats treated with sPL gel. Figure 8 The number of cells expressing vimentin (in the brown staining area) increased significantly, indicating that after the endometrial structure of rats was destroyed, sPL promoted the number of cells expressing vimentin in the rat endometrium. This suggests that sPL can promote the proliferation of basal stem cells in the basal layer of the endometrium and transform them into stromal cells expressing vimentin to supplement the missing cells in the functional layer and achieve endometrial regeneration.

Claims

1. A method for preparing sPL gel for repairing endometrial damage, characterized in that, The method includes the following steps: I. Preparation of platelet lysis buffer PL; 2. Dilute placental chorionic trophoblast cells with complete culture medium to obtain cell suspension, add 1~10 μg / mL of myrrh-angelicone for culture, collect the culture medium, filter, and obtain conditioned medium. sPL was prepared by mixing platelet lysis buffer (PL) with conditioned medium.

3. Add collagen solution and carbomer to sPL, mix well, and prepare sPL gel.

2. The method for preparing sPL gel for endometrial injury repair according to claim 1, characterized in that, The preparation method of platelet lysis buffer PL in step one is as follows: A. Centrifuge the blood in a sodium citrate anticoagulant tube and collect the plasma layer and the upper 1 / 3 volume of liquid from the red blood cell layer; B. Place the collected plasma layer and the upper 1 / 3 volume of liquid from the red blood cell layer into a venous blood collection container, centrifuge, and collect the upper platelet-poor plasma layer and the platelet-rich plasma layer from the separating gel. C. Dilute platelet-rich plasma with platelet-poor plasma to a platelet concentration of 0.5 × 10⁻⁶. 9 ~2×10 9 Platelet-rich plasma was prepared by collecting platelets per mL. D. Centrifuge the concentrated platelet plasma, collect the supernatant, filter, and obtain platelet lysis buffer PL.

3. The method for preparing sPL gel for endometrial injury repair according to claim 2, characterized in that, The complete culture medium described in step two is Knockout DMEM / F12 medium containing 10% FBS.

4. The method for preparing sPL gel for endometrial injury repair according to claim 3, characterized in that, In step two, platelet lysis buffer PL is mixed with conditioned medium at a volume ratio of (1~3):

1.

5. The method for preparing sPL gel for endometrial injury repair according to claim 4, characterized in that, The concentration of the cell suspension mentioned in step two is 0.5 × 10⁻⁶. 5 ~1.5×10 5 per mL.

6. The method for preparing sPL gel for endometrial injury repair according to claim 5, characterized in that, In step two, myrrh and angelica ketone are added and cultured for 36-60 hours.

7. The method for preparing sPL gel for endometrial injury repair according to claim 6, characterized in that, The sPL gel described in step three contains 90% to 95% sPL, 2% to 3% collagen, 0.5% to 1.0% carbomer, and the remainder water.

8. The use of the sPL gel prepared by the method according to any one of claims 1 to 7 in the preparation of a medicament for treating and repairing endometrial damage.

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