A biological agent for regulating abnormal placental development

Abstract

The present invention provides a biological agent for regulating abnormal placental development, belonging to the technical field of obstetrics and gynecology. The core active ingredient of the biological agent is a combination of Lutjanus argentimaculatus fish bones, fish scales and fish meat in a mass ratio of 5:3:2. Through steps such as desalting treatment, ultrasonic-assisted degreasing, double-enzyme synergistic enzymatic hydrolysis and double ultrafiltration purification, a Lutjanus argentimaculatus mixed polypeptide with a molecular weight in the range of 2-10 kDa is prepared. Experimental results show that the Lutjanus argentimaculatus mixed polypeptide has a significant protective effect on the human trophoblast cell line HTR-8 / SVneo under high-glucose conditions, can effectively promote cell proliferation and migration, and reduce high-glucose-induced cell damage. Therefore, the biological agent provides a new and effective intervention means for abnormal placental development caused by gestational diabetes and has good application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of obstetrics and gynecology, and particularly relates to a biological agent for regulating abnormal placental development. Background Art

[0002] Gestational diabetes is a state of abnormal glucose metabolism that occurs during pregnancy, characterized by elevated maternal blood glucose levels. The hyperglycemic environment not only has an adverse effect on maternal health but also has a negative impact on fetal growth and development by affecting the normal development of the placenta. The placenta is a key organ constructed by trophoblast cells, responsible for nutrient, gas exchange, and metabolic waste excretion between the mother and the fetus. The normal function of trophoblast cells is crucial for maintaining the structure and function of the placenta.

[0003] Under a hyperglycemic environment, trophoblast cells will show a decrease in proliferation ability and dysfunction. Specifically, cell viability is reduced, and migration and invasion abilities are weakened. These changes will interfere with the normal invasion of trophoblast cells into the maternal endometrium and the remodeling of spiral arteries, resulting in abnormal placental angiogenesis and reduced material exchange efficiency.

[0004] Due to the impaired function of trophoblast cells, abnormal placental development may lead to a series of pregnancy complications, such as fetal growth restriction, preterm birth, preeclampsia, and neonatal complications, etc. Existing research and treatment methods mainly focus on controlling maternal blood glucose levels, but there is less treatment for the damage of trophoblast cells directly caused by the hyperglycemic environment. Therefore, developing new pharmaceutical preparations to protect the function of trophoblast cells is of great significance.

[0005] In recent years, studies have shown that polypeptides derived from marine organisms have various biological activities, such as antioxidant, anti-inflammatory, antibacterial, etc., and thus have attracted much attention in the biomedical field. However, the research on Lutjanus argentimaculatus polypeptides is still relatively scarce at present, and their specific biological functions and mechanisms of action have not been fully revealed. In particular, whether Lutjanus argentimaculatus polypeptides can be used to intervene in trophoblast cell damage caused by high glucose remains a problem to be explored. In-depth study of the biological activities of Lutjanus argentimaculatus polypeptides is expected to provide new ideas and strategies for the prevention and treatment of abnormal placental development caused by gestational diabetes. Summary of the Invention

[0006] The purpose of the present invention is to provide a biological agent for regulating abnormal placental development, so as to provide a new option for abnormal placental development caused by gestational diabetes.

[0007] To achieve the above purpose, the present invention provides the following technical solutions:

[0008] First, the present invention provides a biological agent for regulating abnormal placental development, characterized in that every 100 ml of the biological agent is prepared according to the following preparation method:

[0009] Weigh 2 - 10 g of mannitol, dissolve the mannitol with normal saline to obtain a mannitol stabilizer;

[0010] Weigh 5 - 40 mg of Lutjanus mixed polypeptide and dissolve it to obtain a polypeptide solution;

[0011] Mix the polypeptide solution and the mannitol stabilizer evenly, and make up the volume to 100 ml with normal saline;

[0012] Filter through a 0.22 - μm sterile filter membrane to obtain the said biological preparation.

[0013] Preferably, the Lutjanus mixed polypeptide is prepared according to the following preparation method

[0014] Select fresh and pollution - free Lutjanus fish scales, fish bones and fish meat, and rinse them clean with water;

[0015] Soak the fish bones in dilute hydrochloric acid, after the soaking treatment, wash them repeatedly with distilled water until the pH is neutral;

[0016] After mixing the desalted fish bones, fish scales and fish meat according to the mass ratio of 5:3:2, soak them in 95% ethanol, and carry out ultrasonic - assisted degreasing at room temperature to obtain a mixed raw material;

[0017] Place the mixed raw material in an oven to dry, and then use a grinder to crush it into powder to obtain a mixed extraction raw material;

[0018] Add the mixed extraction raw material to distilled water according to the mass - to - volume ratio of 1:10, stir evenly, and homogenize it twice under a pressure of 80 MPa;

[0019] Add 1% alkaline protease and 0.5% flavor protease based on the mass of the mixed extraction raw material, adjust the pH to 8.0, and the temperature to 50 °C, and carry out enzymatic hydrolysis for 3 hours to obtain an enzymatic hydrolysate;

[0020] Heat the enzymatic hydrolysate to inactivate the alkaline protease and flavor protease;

[0021] Collect the supernatant by centrifugation to obtain a Lutjanus mixed polypeptide solution;

[0022] Ultrafilter the Lutjanus mixed polypeptide solution with a 10 - kDa molecular weight cut - off ultrafiltration membrane, and collect the permeate;

[0023] Ultrafilter the permeate with a 2 - kDa molecular weight cut - off ultrafiltration membrane, and collect the retentate;

[0024] Carry out freeze - drying on the retentate to obtain Lutjanus mixed polypeptide.

[0025] Preferably, the regulation of abnormal placental development is to regulate the abnormal placental development caused by gestational diabetes.

[0026] Second, the present invention provides an application of the Lutjanus mixed polypeptide in the preparation of a biological agent for treating placental dysplasia caused by gestational diabetes mellitus, and the Lutjanus mixed polypeptide is prepared by the method for preparing the Lutjanus mixed polypeptide described above.

[0027] Preferably, in the biological agent, the use concentration of the Lutjanus mixed polypeptide is 50 μg / mL - 400 μg / mL.

[0028] Preferably, when the use concentration of the Lutjanus mixed polypeptide is 200 μg / mL, the biological agent achieves the best therapeutic effect.

[0029] Preferably, the placental dysplasia caused by gestational diabetes mellitus is placental dysplasia caused by the decrease in the proliferation ability of trophoblast cells and the decrease in the migration ability of trophoblast cells caused by high glucose.

[0030] Third, the present invention provides an application of the Lutjanus mixed polypeptide in the preparation of a biological agent for treating the decrease in the proliferation ability and migration ability of trophoblast cells caused by gestational diabetes mellitus, and the Lutjanus mixed polypeptide is prepared by the method for preparing the Lutjanus mixed polypeptide described above;

[0031] In the biological agent, the use concentration of the Lutjanus mixed polypeptide is 200 μg / mL.

[0032] The present invention provides a biological agent for regulating placental dysplasia, namely Lutjanus mixed polypeptide Ⅰ. Through specific raw material ratios and optimized preparation processes, the present invention significantly improves the biological activity of the Lutjanus mixed polypeptide, making it have a good protective and therapeutic effect on trophoblast cell damage caused by a high-glucose environment. The present invention has the following specific beneficial effects:

[0033] The high-glucose environment caused by gestational diabetes mellitus can lead to inhibited proliferation and reduced migration ability of trophoblast cells. Experimental results show that the Lutjanus mixed polypeptide Ⅰ prepared by the present invention can repair the damaged trophoblast cell function in a high-glucose environment, which is of great significance for maintaining normal placental development.

[0034] The present invention combines fish bones, fish scales and fish meat in a ratio of 5:3:2, fully utilizes the polypeptide active ingredients of each raw material, enables the polypeptides prepared from the three to produce a synergistic effect, and enhances the biological activity of the polypeptides. It has achieved unexpected technical effects compared with the polypeptides prepared by other ratios.

[0035] The present invention adopts optimized process steps such as desalting treatment, double-enzyme synergistic enzymatic hydrolysis and double ultrafiltration purification, and successfully prepares a Lutjanus mixed polypeptide with a molecular weight of 2 - 10 kDa, significantly improving its effect.

[0036] Meanwhile, the raw materials of the present invention are taken from natural snapper by-products and are subjected to strict pretreatment and purification, ensuring the safety and biocompatibility of the preparation. Therefore, this biological preparation is expected to be applied to the prevention and treatment of placental dysplasia caused by gestational diabetes, as well as other pregnancy complications related to trophoblast cell dysfunction. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 It is a bar graph showing the effect of snapper mixed polypeptide Ⅰ on the proliferation of trophoblast cells;

[0038] Figure 2 It is a bar graph showing the therapeutic effect of different snapper mixed polypeptides on high glucose-induced damage of trophoblast cells;

[0039] Figure 3 It is a result graph and statistical graph showing the therapeutic effect of different snapper mixed polypeptides on the damage of migration ability of trophoblast cells induced by high glucose;

[0040] Figure A is the result graph and Figure B is the statistical graph. DETAILED DESCRIPTION OF THE INVENTION

[0041] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0042] Example 1

[0043] 1. Raw material collection and pretreatment

[0044] (1) Select fresh and pollution-free snapper scales, fish bones and fish meat, and rinse them clean with water;

[0045] (2) Immerse the fish bones in 0.5M dilute hydrochloric acid, soak them at room temperature for 12 hours, and then wash them repeatedly with distilled water until the pH is neutral;

[0046] (3) Immerse the defatted fish bones (50g), scales (30g) and fish meat (20g) after desalting treatment in 95% ethanol, and perform ultrasonic-assisted degreasing at room temperature for 2 times to remove the grease and obtain the mixed raw materials;

[0047] (4) Place the mixed raw materials in an oven at 60°C to dry, then use a grinder to crush them into powder, and pass through an 80-mesh sieve to obtain the mixed extraction raw materials;

[0048] 2. Preparation of snapper mixed polypeptide

[0049] (1) Add the mixed extraction raw materials to distilled water at a mass - to - volume ratio of 1:10. After stirring evenly, homogenize twice at a pressure of 80 MPa;

[0050] (2) Add alkaline protease at 1% of the mass of the mixed extraction raw materials and flavor protease at 0.5%. Adjust the pH to 8.0 and the temperature to 50 °C, and enzymatically hydrolyze for 3 hours to obtain an enzymatic hydrolysate;

[0051] (3) Heat the enzymatic hydrolysate to 95 °C and maintain for 10 minutes to inactivate the enzyme;

[0052] (4) Collect the supernatant by centrifugation to obtain a Lutjanus mixed polypeptide solution;

[0053] 3. Polypeptide purification

[0054] (1) Ultrafilter the Lutjanus mixed polypeptide solution using an ultrafiltration membrane with a molecular weight cut - off of 10 kDa, and collect the permeate;

[0055] (2) Ultrafilter the permeate using an ultrafiltration membrane with a molecular weight cut - off of 2 kDa, and collect the retentate;

[0056] (3) Freeze - dry the retentate to obtain Lutjanus mixed polypeptide I.

[0057] Example 2

[0058] 1. Raw material collection and pretreatment

[0059] (1) Select fresh and pollution - free Lutjanus fish scales, fish bones and fish meat, and rinse them clean with water;

[0060] (2) Immerse the fish bones in 0.5 M dilute hydrochloric acid, soak at room temperature for 12 hours, and then wash repeatedly with distilled water until the pH is neutral;

[0061] (3) Immerse the desalted fish bones (20 g), fish scales (50 g) and fish meat (30 g) in 95% ethanol, and perform ultrasonic - assisted degreasing at room temperature, repeat 2 times to remove the grease and obtain a mixed raw material;

[0062] (4) Place the mixed raw material in an oven at 60 °C to dry, then use a grinder to crush it into powder, and pass through an 80 - mesh sieve to obtain a mixed extraction raw material;

[0063] 2. Preparation of Lutjanus mixed polypeptide

[0064] (1) Add the mixed extraction raw materials to distilled water at a mass - to - volume ratio of 1:10. After stirring evenly, homogenize twice at a pressure of 80 MPa;

[0065] (2) Add alkaline protease at 1% of the mass of the mixed extraction raw materials and flavor protease at 0.5%. Adjust the pH to 8.0 and the temperature to 50 °C, and enzymatically hydrolyze for 3 hours, obtaining an enzymatic hydrolysate;

[0066] (3) Heat the enzymatic hydrolysate to 95 °C and keep it for 10 minutes to inactivate the enzyme;

[0067] (4) Collect the supernatant by centrifugation to obtain Lutjanus sanguineus mixed polypeptide solution 1;

[0068] 3. Polypeptide purification

[0069] (1) Ultrafilter the Lutjanus sanguineus mixed polypeptide solution using a 10 kDa molecular weight cut-off ultrafiltration membrane and collect the permeate;

[0070] (2) Ultrafilter the permeate using a 2 kDa molecular weight cut-off ultrafiltration membrane and collect the retentate;

[0071] (3) Lyophilize the retentate to obtain Lutjanus sanguineus mixed polypeptide II.

[0072] Example 3

[0073] 1. Raw material collection and pretreatment

[0074] (1) Select fresh and pollution-free Lutjanus sanguineus fish scales, fish bones and fish meat and rinse them clean with water;

[0075] (2) Immerse the fish bones in 0.5 M dilute hydrochloric acid and soak them at room temperature for 12 hours, then wash them repeatedly with distilled water until the pH is neutral;

[0076] (3) Immerse the defatted fish bones (40 g), fish scales (40 g) and fish meat (20 g) after desalting treatment in 95% ethanol, and perform ultrasonic-assisted degreasing at room temperature for 2 times to remove the grease and obtain the mixed raw materials;

[0077] (4) Place the mixed raw materials in an oven at 60 °C to dry, then use a grinder to crush them into powder and pass through an 80-mesh sieve to obtain the mixed extraction raw materials;

[0078] 2. Preparation of Lutjanus sanguineus mixed polypeptide

[0079] (1) Add the mixed extraction raw materials to distilled water according to a mass-to-volume ratio of 1:10, stir evenly, and homogenize 2 times at a pressure of 80 MPa;

[0080] (2) Add 1% alkaline protease and 0.5% flavor protease based on the mass of the mixed extraction raw materials, adjust the pH to 8.0 and the temperature to 50 °C, and perform enzymatic hydrolysis for 3 hours to obtain the enzymatic hydrolysate;

[0081] (3) Heat the enzymatic hydrolysate to 95 °C and keep it for 10 minutes to inactivate the enzyme;

[0082] (4) Collect the supernatant by centrifugation to obtain Lutjanus sanguineus mixed polypeptide solution;

[0083] 3. Polypeptide purification

[0084] (1) Ultrafilter the Lutjanus sanguineus mixed polypeptide solution using a 10 kDa molecular weight cut-off ultrafiltration membrane, and collect the permeate;

[0085] (2) Ultrafilter the permeate using a 2 kDa molecular weight cut-off ultrafiltration membrane, and collect the retentate;

[0086] (3) Freeze-dry the retentate to obtain Lutjanus sanguineus mixed polypeptide Ⅲ.

[0087] Comparative example

[0088] 1. Raw material collection and pretreatment

[0089] (1) Select fresh and pollution-free Lutjanus sanguineus fish scales, fish bones and fish meat, and rinse them clean with water;

[0090] (2) Immerse fish bones (50 g), fish scales (30 g) and fish meat (20 g) in 95% ethanol, and perform ultrasonic-assisted degreasing at room temperature for 2 times to remove grease and obtain a mixed raw material;

[0091] (4) After drying the mixed raw material in an oven at 60 °C, use a grinder to crush it into powder, and pass through an 80-mesh sieve to obtain a mixed extraction raw material;

[0092] 2. Preparation of Lutjanus sanguineus mixed polypeptide

[0093] (1) Add the mixed extraction raw material to distilled water according to a mass-volume ratio of 1:10, and stir evenly;

[0094] (2) Add alkaline protease at 1.5% of the mass of the mixed extraction raw material, adjust the pH to 8.0, the temperature to 50 °C, and enzymatically hydrolyze for 3 hours to obtain an enzymatic hydrolysate;

[0095] (3) Heat the enzymatic hydrolysate to 95 °C and keep it for 10 minutes to inactivate the enzyme;

[0096] (4) Collect the supernatant by centrifugation to obtain Lutjanus sanguineus mixed polypeptide solution;

[0097] (5) After freeze-drying the Lutjanus sanguineus mixed polypeptide solution, obtain Lutjanus sanguineus mixed polypeptide Ⅳ.

[0098] Example 4

[0099] Screen the optimal concentration of the polypeptide pair for the proliferation effect of trophoblast cells through Lutjanus sanguineus mixed polypeptide Ⅰ

[0100] (1) Cell culture

[0101] Cell line: Human trophoblast cell line HTR-8 / SVneo.

[0102] Culture medium: High-glucose DMEM (i.e., complete DMEM medium) containing 10% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 μg / mL streptomycin.

[0103] Culture conditions: 37 °C, 5% CO2, saturated humidity.

[0104] Experimental procedures:

[0105] 1. Inoculate HTR-8 / SVneo cells into a culture flask and add an appropriate amount of complete DMEM medium.

[0106] 2. Place the cells in an incubator for culture, change the medium every 2 - 3 days to maintain the normal growth of the cells.

[0107] 3. When the cells grow to the logarithmic phase, use them for subsequent experiments.

[0108] (2) Preparation of polypeptide working solution

[0109] Experimental procedures:

[0110] 1. Dissolve the lyophilized powder of Lutjanus mixed polypeptide Ⅰ in complete DMEM medium to prepare a high-concentration stock solution of 10 mg / mL.

[0111] 2. Prepare working solutions of Lutjanus mixed polypeptide Ⅰ at 50 μg / mL, 100 μg / mL, 150 μg / mL, 200 μg / mL, and 400 μg / mL respectively.

[0112] (3) Cell seeding

[0113] Experimental procedures:

[0114] 1. Digest and collect HTR-8 / SVneo cells in the logarithmic growth phase.

[0115] 2. Resuspend the cells with complete DMEM medium and adjust the cell concentration to 1×10 4 cells / 100 μL.

[0116] 3. Add 100 μL of cell suspension to each well and seed them in a 96-well cell culture plate.

[0117] 4. Place the culture plate in an incubator and culture at 37 °C, 5% CO2 for 24 hours to allow the cells to adhere and recover.

[0118] (4) Polypeptide treatment

[0119] Experimental procedures:

[0120] 1. After 24 hours, carefully aspirate the original medium in each well, gently wash the cells once with a small amount of PBS to remove the residual medium.

[0121] 2. Group treatment:

[0122] Control group: Add 100 μL of DMEM complete medium without polypeptide to each well.

[0123] Treatment group: Add 100 μL of working solutions of Lutjanus mixed polypeptide Ⅰ with different concentrations (50, 100, 150, 200, 400 μg / mL) to each well respectively.

[0124] Put the culture plate back into the incubator and continue culturing for 24 hours.

[0125] (5) Cell proliferation detection (CCK-8 method)

[0126] Experimental steps:

[0127] 1. After culturing for 24 hours, add 10 μL of CCK-8 reagent to each well, gently tap the edge of the culture plate to make the reagent evenly distributed, and avoid generating bubbles.

[0128] 2. Place the culture plate in the incubator, incubate at 37 °C in the dark for 2 hours. After the incubation, gently mix the culture plate.

[0129] 3. Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance (OD value) of each well at a wavelength of 450 nm.

[0130] The results obtained in this example are as Figure 1 and shown in Table 1:

[0131] Table 1 Absorbance differences among different treatment groups

[0132]

[0133] From Figure 1 and the results in Table 1, it can be seen that compared with the group without adding the working solution of Lutjanus mixed polypeptide Ⅰ (0 μg / mL group), adding 50 μg / mL of Lutjanus mixed polypeptide Ⅰ can effectively promote the proliferation ability of trophoblast cells, and the cell proliferation rate is significantly increased. With the further increase of the polypeptide concentration, the promoting effect shows a continuous increasing trend. When the concentration reaches 200 μg / mL, the cell proliferation rate reaches the highest value, indicating that the promoting effect of the polypeptide is the most significant at this concentration.

[0134] When the concentration continues to increase to 400 μg / mL, although Lutjanus mixed polypeptide Ⅰ still has a significant effect on promoting cell proliferation, the overall effect is lower than that of the 200 μg / mL group. Therefore, in subsequent detections, the present invention selects 200 μg / mL as the detection concentration of the present invention.

[0135] Example 5

[0136] Detect the therapeutic effect of different Lutjanus mixed polypeptides on high glucose-induced trophoblast cell injury

[0137] (1) Cell culture

[0138] Cell line: Human trophoblast cell line HTR-8 / SVneo.

[0139] Culture medium: High-glucose DMEM (i.e., complete DMEM medium) containing 10% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 μg / mL streptomycin.

[0140] Culture conditions: 37 °C, 5% CO2, saturated humidity.

[0141] Experimental procedure:

[0142] 1. Inoculate HTR-8 / SVneo cells into a culture flask and add an appropriate amount of complete DMEM medium.

[0143] 2. Place the cells in an incubator for culture and change the medium every 2 - 3 days to maintain the normal growth of the cells.

[0144] 3. When the cells grow to the logarithmic phase, use them for subsequent experiments.

[0145] (2) Preparation of polypeptide working solution

[0146] Experimental procedure:

[0147] 1. Dissolve the freeze-dried powder of Lutjanus mixed polypeptide Ⅰ, Lutjanus mixed polypeptide Ⅱ, Lutjanus mixed polypeptide Ⅲ, and Lutjanus mixed polypeptide Ⅳ in complete DMEM medium to prepare a high-concentration stock solution of 10 mg / mL.

[0148] 2. Combine the high-concentration stock solutions of Lutjanus mixed polypeptide Ⅰ, Lutjanus mixed polypeptide Ⅱ, Lutjanus mixed polypeptide Ⅲ, and Lutjanus mixed polypeptide Ⅳ with the glucose stock solution to obtain working solutions containing 200 μg / mL of Lutjanus mixed polypeptide Ⅰ, Lutjanus mixed polypeptide Ⅱ, Lutjanus mixed polypeptide Ⅲ, or Lutjanus mixed polypeptide Ⅳ and 25 mmol / L glucose respectively.

[0149] (3) Cell seeding

[0150] Experimental procedure:

[0151] 1. Digest and collect HTR-8 / SVneo cells in the logarithmic growth phase.

[0152] 2. Resuspend the cells with complete DMEM medium and adjust the cell concentration to 1×10 4 cells / 100 μL.

[0153] 3. Add 100 μL of the cell suspension to each well and inoculate it into a 96-well cell culture plate.

[0154] 4. Place the culture plate in an incubator and culture at 37°C and 5% CO2 for 24 hours to allow the cells to adhere to the plate and recover.

[0155] (4) Polypeptide treatment

[0156] Experimental procedure:

[0157] 1. After 24 hours, carefully aspirate the original culture medium from each well, gently wash the cells once with a small amount of PBS to remove the residual culture medium.

[0158] 2. Group treatment:

[0159] Control group: Add 100 μL of DMEM complete medium without polypeptide to each well.

[0160] High-glucose group: Add 100 μL of DMEM complete medium containing 25 mmol / L glucose to each well respectively.

[0161] Treatment group I: Add 100 μL of working solution containing 200 μg / mL snapper mixed polypeptide I and 25 mmol / L glucose to each well respectively.

[0162] Treatment group II: Add 100 μL of working solution containing 200 μg / mL snapper mixed polypeptide II and 25 mmol / L glucose to each well respectively.

[0163] Treatment group III: Add 100 μL of working solution containing 200 μg / mL snapper mixed polypeptide III and 25 mmol / L glucose to each well respectively.

[0164] Treatment group IV: Add 100 μL of working solution containing 200 μg / mL snapper mixed polypeptide IV and 25 mmol / L glucose to each well respectively.

[0165] (5) Cell proliferation detection (CCK-8 method)

[0166] Experimental procedure:

[0167] 1. After culturing for 24 hours, add 10 μL of CCK-8 reagent to each well, gently tap the edge of the culture plate to make the reagent evenly distributed, and avoid generating bubbles.

[0168] 2. Place the culture plate in an incubator and incubate at 37°C in the dark for 2 hours. After incubation, gently mix the culture plate.

[0169] 3. Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance of each well at a wavelength of 450 nm and calculate the cell survival rate.

[0170] The results obtained are shown in Table 2 and Figure 2 as follows:

[0171] Table 2 Therapeutic effects of different Lutjanus mixed polypeptides on high glucose-induced trophoblast cell damage

[0172]

[0173]

[0174] As can be seen from the results in Table 2, the cell survival rate in the high glucose group decreased to 55.32%, indicating that the high glucose environment significantly inhibited the proliferation of trophoblast cells and induced cell damage. The cell survival rate in Treatment Group I (the polypeptide of Example 1) recovered to 88.55%, showing the best effect. The cell survival rates in Treatment Group II (the polypeptide of Example 2) and Treatment Group III (the polypeptide of Example 3) were 72.06% and 76.48% respectively, inferior to Treatment Group I. The cell survival rate in Treatment Group IV (the polypeptide of the comparative example) was 62.76%, with the worst effect, but still higher than that in the high glucose group.

[0175] As can be seen from the above results, the Lutjanus mixed polypeptide IV prepared in the comparative example had the worst therapeutic effect on high glucose-induced trophoblast cell damage, indicating that desalting treatment, double enzyme synergistic enzymatic hydrolysis, and double ultrafiltration purification are the key steps to improve the biological activity of Lutjanus mixed polypeptides. The protective effect of Lutjanus mixed polypeptides prepared without these steps in cell damage caused by high glucose will be significantly reduced;

[0176] At the same time, it can be seen that the ratio difference among fish bones, fish scales, and fish meat also has a huge impact on the performance of the prepared polypeptides. Only when the ratio of fish bones, fish scales, and fish meat is 5:3:2 can the Lutjanus mixed polypeptide composed of fish bone, fish scale, and fish meat polypeptides exert a synergistic therapeutic effect on high glucose-induced trophoblast cell damage. At other ratios, the effect will be significantly reduced. This may be because the protein types and active ingredients contained in different raw materials are different, and the optimal ratio can make the polypeptide rich in effective active peptide segments, producing a synergistic effect.

[0177] Example 6

[0178] (1) Cell culture

[0179] Cell line: Human trophoblast cell line HTR-8 / SVneo.

[0180] Culture medium: High glucose type DMEM (i.e., DMEM complete medium) containing 10% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 μg / mL streptomycin.

[0181] Culture conditions: 37 °C, 5% CO2, saturated humidity.

[0182] Experimental steps:

[0183] 1. Inoculate HTR-8 / SVneo cells into a culture flask and add an appropriate amount of complete DMEM medium.

[0184] 2. Place the cells in an incubator for culture, change the medium every 2 - 3 days to maintain the normal growth of the cells.

[0185] 3. When the cells grow to the logarithmic phase, use them for subsequent experiments.

[0186] (2) Preparation of polypeptide working solution

[0187] Experimental steps:

[0188] 1. Dissolve the lyophilized powder of Lutjanus mixed polypeptide Ⅰ, Lutjanus mixed polypeptide Ⅱ, Lutjanus mixed polypeptide Ⅲ, and Lutjanus mixed polypeptide Ⅳ in DMEM basal medium to prepare a high-concentration stock solution of 10 mg / mL.

[0189] 2. Combine the high-concentration stock solutions of Lutjanus mixed polypeptide Ⅰ, Lutjanus mixed polypeptide Ⅱ, Lutjanus mixed polypeptide Ⅲ, and Lutjanus mixed polypeptide Ⅳ with the glucose stock solution to obtain working solutions containing 200 μg / mL of Lutjanus mixed polypeptide Ⅰ, Lutjanus mixed polypeptide Ⅱ, Lutjanus mixed polypeptide Ⅲ, or Lutjanus mixed polypeptide Ⅳ and 25 mmol / L of glucose respectively.

[0190] (3) Cell inoculation

[0191] Experimental steps:

[0192] 1. Collect HTR-8 / SVneo cells in the logarithmic growth phase, resuspend the cells with serum-free DMEM basal medium, and adjust the cell concentration to 1×10 5 cells / mL.

[0193] 2. Place an 8-μm pore size Transwell chamber in a 24-well culture plate.

[0194] Cell inoculation:

[0195] Add 200 μL of cell suspension (about 2×10 4 cells) to the upper chamber of each Transwell, and set up 3 replicate wells for each treatment group.

[0196] Lower chamber medium:

[0197] Add 600 μL of complete DMEM medium containing 10% FBS to the lower chamber as a chemical inducer to promote cell migration.

[0198] Pre-culture:

[0199] Place the culture plate in an incubator and culture at 37°C and 5% CO2 for 30 minutes to allow the cells to adapt to the environment.

[0200] (4) Polypeptide treatment

[0201] Experimental procedure:

[0202] 1. Upper chamber treatment:

[0203] Control group: Add 200 μL of serum-free DMEM basal medium without polypeptide and glucose to each well of the upper chamber.

[0204] High-glucose group: Add 200 μL of serum-free DMEM basal medium containing 25 mmol / L glucose to each well of the upper chamber.

[0205] Treatment group I: Add 200 μL of working solution containing 200 μg / mL of Lutjanus mixed polypeptide I and 25 mmol / L glucose to each well of the upper chamber.

[0206] Treatment group II: Do the same as above, using Lutjanus mixed polypeptide II.

[0207] Treatment group III: Do the same as above, using Lutjanus mixed polypeptide III.

[0208] Treatment group IV: Do the same as above, using Lutjanus mixed polypeptide IV.

[0209] 2. Lower chamber treatment:

[0210] Keep the original medium in the lower chamber unchanged (DMEM complete medium containing 10% FBS).

[0211] 3. Put the culture plate back into the incubator and culture at 37 °C and 5% CO2 for 24 hours.

[0212] (5) Cell migration detection

[0213] 1. After culturing for 24 hours, take out the Transwell plate, carefully aspirate the medium in the upper chamber, and gently wipe the inner side of the upper chamber membrane with a cotton swab to remove the non-migrated cells.

[0214] 2. Take out the small chamber inserted in the Transwell, gently wash the upper chamber 2 times with PBS, and then put it into a new culture plate.

[0215] 3. Add 600 μL of 4% paraformaldehyde to the lower chamber and fix at room temperature for 20 minutes;

[0216] 4. After fixation, discard the paraformaldehyde solution, wash 3 times with PBS, add 0.1% crystal violet staining solution to the lower chamber, and stain for 15 minutes;

[0217] 5. After staining, wash 3 times with PBS to remove the excess dye,

[0218] 6. Under the microscope, randomly select 5 fields of view on each membrane and count the number of cells migrated to the lower side of the membrane.

[0219] The obtained results are as Figure 3 shown. From Figure 3 it can be seen that the overall trend of the results obtained from the cell migration assay is basically consistent with those from the cell proliferation assay. The difference is that the Lutjanus mixed polypeptide Ⅳ prepared in the comparative example did not effectively protect against the decrease in the migration ability of trophoblast cells caused by high glucose. This may be because the ability of this polypeptide to regulate migration is inferior to its effect on cell proliferation.

[0220] Generally speaking, only the Lutjanus mixed polypeptide Ⅰ prepared in Example 1 can achieve a very significant protective effect on the decrease in the migration ability of trophoblast cells caused by high glucose, while the effects of Lutjanus mixed polypeptide Ⅱ and Lutjanus mixed polypeptide Ⅲ are significantly reduced. This result further proves that Lutjanus mixed polypeptide Ⅰ can effectively protect against the decrease in the migration ability of trophoblast cells caused by high glucose.

[0221] Based on the above results, it can be seen that the Lutjanus mixed polypeptide Ⅰ prepared in the present invention can effectively protect against the decrease in the proliferation ability and migration ability of trophoblast cells caused by high glucose. Therefore, Lutjanus mixed polypeptide Ⅰ can be used as a drug for placental dysplasia caused by gestational diabetes.

[0222] Example 7

[0223] Preparation method of the Lutjanus mixed polypeptide Ⅰ biological agent at a concentration of 200 μg / mL

[0224] 1. Weigh 5 g of mannitol on an electronic balance, put the mannitol into a beaker, add 80 mL of normal saline, and stir with a magnetic stirrer until the mannitol is completely dissolved to obtain a mannitol stabilizer.

[0225] 2. Weigh 20 mg of Lutjanus mixed polypeptide Ⅰ, place it in a small beaker, add 15 ml of normal saline, and stir with a magnetic stirrer until it is completely dissolved to obtain a polypeptide solution.

[0226] 3. Mix the polypeptide solution and the mannitol stabilizer evenly, and make up the volume to 100 ml with normal saline.

[0227] 4. Filter through a 0.22 μm sterile filter membrane to ensure that the solution is sterile and remove particles. The filtered solution is collected in a sterile vial.

[0228] 5. After sealing and sub-packaging on a sterile operating table, store it frozen at -20 °C.

Claims

1. A biological agent for regulating abnormal placental development caused by gestational diabetes, characterized in that, Every 100 ml of the biological preparation is prepared according to the following preparation method: Weigh 2 - 10 g of mannitol, dissolve the mannitol with physiological saline to obtain a mannitol stabilizer; Weigh 5 - 40 mg of the mixed Lutjanus polynucleotides and dissolve them to obtain a polypeptide solution; Mix the polypeptide solution and the mannitol stabilizer evenly, and make up the volume to 100 ml with physiological saline; Filter through a 0.22 μm sterile filter membrane to obtain the biological preparation; The mixed Lutjanus polynucleotides are prepared according to the following preparation method Select fresh and pollution-free Lutjanus scales, fish bones and fish meat, and rinse them clean with water; Soak the fish bones in dilute hydrochloric acid. After the soaking treatment, wash them repeatedly with distilled water until the pH is neutral; After mixing the desalted fish bones, fish scales and fish meat according to the mass ratio of 5:3:2, soak them in 95% ethanol and carry out ultrasonic-assisted defatting at room temperature to obtain a mixed raw material; Dry the mixed raw material in an oven and then crush it into powder with a grinder to obtain a mixed extraction raw material; Add the mixed extraction raw material to distilled water according to the mass-volume ratio of 1:10, stir evenly, and homogenize it twice at a pressure of 80 MPa; Add 1% alkaline protease and 0.5% flavor protease based on the mass of the mixed extraction raw material, adjust the pH to 8.0, and the temperature to 50 °C, and carry out enzymatic hydrolysis for 3 hours to obtain an enzymatic hydrolysate; Heat the enzymatic hydrolysate to inactivate the alkaline protease and flavor protease; Collect the supernatant by centrifugation to obtain a mixed Lutjanus polynucleotide solution; Ultrafilter the mixed Lutjanus polynucleotide solution with a 10 kDa molecular weight cut-off ultrafiltration membrane, and collect the permeate; Ultrafilter the permeate with a 2 kDa molecular weight cut-off ultrafiltration membrane, and collect the retentate; Carry out freeze-drying on the retentate to obtain the mixed Lutjanus polynucleotides; The placental dysplasia caused by gestational diabetes is the placental dysplasia caused by the decrease in the proliferation ability and migration ability of trophoblast cells caused by high glucose.

2. Use of Lutjanus mixed polypeptide in the preparation of a biological agent for treating placental dysplasia caused by gestational diabetes, characterized in that, The mixed Lutjanus polynucleotides are prepared by the preparation method described in claim 1; The placental dysplasia caused by gestational diabetes is the placental dysplasia caused by the decrease in the proliferation ability and migration ability of trophoblast cells caused by high glucose.

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