Biological preparation for regulating placenta dysplasia

Abstract

The invention provides a biological preparation for regulating placenta dysplasia, and belongs to the technical field of gynaecology and obstetrics. Core effective components of the biological preparation are lutjanus mixed polypeptide with the molecular weight ranging from 2 kDa to 10 kDa, and the lutjanus mixed polypeptide is prepared by combining lutjanus fish bones, fish scales and fish flesh according to the mass ratio of 5: 3: 2 through the steps of desalting treatment, ultrasonic-assisted degreasing, double-enzyme synergistic enzymolysis, double ultrafiltration purification and the like. Experimental results show that the lutjanus mixed polypeptide has a remarkable protection effect on a human trophoblast cell line HTR-8 / SVneo in a high-glucose environment, and can effectively promote cell proliferation and migration and reduce high-glucose-induced cell damage. Therefore, the biological preparation provides a novel effective intervention means for placenta dysplasia caused by gestational diabetes mellitus, and has a good application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of obstetrics and gynecology, and in particular relates to a biological preparation 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. A hyperglycemic environment not only has an adverse effect on maternal health, but also has a negative effect on fetal growth and development by affecting the normal development of the placenta. The placenta is a key organ constructed by trophoblast cells, which are responsible for nutrition, gas exchange and metabolic waste discharge between the mother and the fetus. The normal function of trophoblast cells is essential for maintaining the structure and function of the placenta.

[0003] Under high-sugar conditions, trophoblasts will experience decreased proliferation and dysfunction. Specifically, they will show decreased cell viability, and weakened migration and invasion abilities. These changes will interfere with the normal invasion of trophoblasts into the maternal endometrium and the transformation of spiral arteries, leading to abnormal placental angiogenesis and reduced material exchange efficiency.

[0004] Due to impaired trophoblast function, abnormal placental development may lead to a series of pregnancy complications, such as fetal growth restriction, premature birth, preeclampsia and neonatal complications. Existing research and treatment methods mainly focus on controlling maternal blood sugar levels, but there are fewer treatments for trophoblast damage directly caused by a high-sugar environment. Therefore, it is of great significance to develop new drug preparations to protect trophoblast function.

[0005] Studies in recent years have shown that peptides derived from marine organisms have a variety of biological activities, such as antioxidant, anti-inflammatory, and antibacterial, and therefore have attracted much attention in the field of biomedicine. However, there are still relatively few studies on snapper peptides, and their specific biological functions and mechanisms of action have not yet been fully revealed. In particular, whether snapper peptides can be used to intervene in trophoblast cell damage caused by high sugar is still an issue to be explored. In-depth research on the biological activity of snapper peptides is expected to provide new ideas and strategies for the prevention and treatment of placental abnormalities caused by gestational diabetes. Summary of the invention

[0006] The purpose of the present invention is to provide a biological preparation for regulating placental development abnormalities, thereby providing a new option for placental development abnormalities caused by gestational diabetes.

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

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

[0009] Weigh 2-10 g of mannitol, and dissolve the mannitol in physiological saline to obtain a mannitol stabilizer;

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

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

[0012] The biological preparation was obtained by filtering through a 0.22 μm sterile filter membrane.

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

[0014] Choose fresh, uncontaminated snapper and rinse the scales, bones and meat with water;

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

[0016] The desalted fish bones, fish scales and fish meat are mixed in a mass ratio of 5:3:2, immersed in 95% ethanol, and subjected to ultrasonic-assisted defatting at room temperature to obtain a mixed raw material;

[0017] The mixed raw material is placed in an oven to dry, and then crushed into powder using a grinder to obtain a mixed extraction raw material;

[0018] The mixed extraction raw materials were added with distilled water at a mass volume ratio of 1:10, stirred evenly, and homogenized twice at a pressure of 80 MPa;

[0019] Add 1% alkaline protease and 0.5% flavor protease by weight of the mixed extraction raw materials, adjust the pH to 8.0, the temperature to 50° C., and perform enzymolysis for 3 hours to obtain an enzymolysis solution;

[0020] The enzymatic hydrolysate is heated to inactivate alkaline protease and flavor protease;

[0021] Collect the supernatant by centrifugation to obtain a mixed peptide solution of snapper;

[0022] The mixed peptide solution of snapper was ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa, and the permeate was collected;

[0023] Ultrafiltration was performed using an ultrafiltration membrane with a molecular weight cutoff of 2 kDa, and the retentate was collected;

[0024] The retentate was freeze-dried to obtain the Lutjanus bream mixed polypeptides.

[0025] Preferably, the regulation of placental developmental abnormalities is regulation of placental developmental abnormalities caused by gestational diabetes.

[0026] Second, the present invention provides an application of a Lutjanus bream mixed polypeptide in the preparation of a biological preparation for treating placental dysplasia caused by gestational diabetes, wherein the Lutjanus bream mixed polypeptide is prepared by the aforementioned Lutjanus bream mixed polypeptide preparation method.

[0027] Preferably, in the biological preparation, the Lutjanus mixed polypeptide is used at a concentration of 50 μg / mL-400 μg / mL.

[0028] Preferably, when the used concentration of the snapper mixed polypeptide is 200 μg / mL, the biological preparation achieves the best therapeutic effect.

[0029] Preferably, the placental development abnormality caused by gestational diabetes is placental development abnormality caused by decreased trophoblast cell proliferation ability and decreased trophoblast cell migration ability caused by high sugar.

[0030] Thirdly, the present invention provides an application of a mixed peptide of snapper in the preparation of a biological preparation for treating decreased proliferation and migration abilities of trophoblastic cells caused by gestational diabetes, wherein the mixed peptide of snapper is prepared by the aforementioned mixed peptide preparation method of snapper;

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

[0032] The present invention provides a biological preparation for regulating placental developmental abnormalities, namely, Lutjanus bream mixed polypeptide I. Through a specific raw material ratio and an optimized preparation process, the present invention significantly improves the biological activity of Lutjanus bream mixed polypeptide, so that it has a good protective and therapeutic effect on trophoblast cell damage caused by a high sugar environment. The present invention specifically has the following beneficial effects:

[0033] The high-sugar environment caused by gestational diabetes can inhibit the proliferation of trophoblast cells and reduce their migration ability. The experimental results show that the Lutjanus mixed polypeptide I prepared by the present invention can repair the damaged trophoblast cell function in a high-sugar environment, which is of great significance for maintaining the normal development of the placenta.

[0034] The present invention combines fish bones, fish scales and fish meat in a ratio of 5:3:2, making full use of the polypeptide active ingredients of each raw material, so that the polypeptide prepared by the three produces a synergistic effect and enhances the biological activity of the polypeptide. Compared with the polypeptide prepared by other ratios, it produces unexpected technical effects.

[0035] The present invention adopts optimized process steps such as desalting, double enzyme synergistic enzymolysis and double ultrafiltration purification to successfully prepare the mixed snapper polypeptide with a molecular weight of 2-10 kDa, thereby significantly improving its effect.

[0036] At the same time, the raw materials of the present invention are taken from natural snapper byproducts, and after strict pretreatment and purification, the safety and biocompatibility of the preparation are guaranteed. Therefore, the biological preparation is expected to be used in the prevention and treatment of placental dysplasia caused by gestational diabetes and other pregnancy complications related to trophoblast dysfunction. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is a bar graph showing the effect of Lutjanus bream mixed polypeptide Ⅰ on trophoblast cell proliferation;

[0038] Figure 2 It is a bar graph showing the therapeutic effects of different mixed snapper peptides on trophoblast cell damage induced by high glucose;

[0039] Figure 3 The result graphs and statistical graphs are the therapeutic effects of different mixed snapper peptides on the damage of trophoblast cell migration ability induced by high glucose;

[0040] A is the result graph, and B is the statistical graph. DETAILED DESCRIPTION

[0041] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0042] Example 1

[0043] 1. Raw material collection and pretreatment

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

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

[0046] (3) soaking the desalted fish bones (50 g), fish scales (30 g) and fish meat (20 g) in 95% ethanol, and performing ultrasonic-assisted degreasing at room temperature, repeating the process twice to remove fat and obtain a mixed raw material;

[0047] (4) drying the mixed raw material in an oven at 60° C., crushing it into powder using a grinder, and passing it through an 80-mesh sieve to obtain a mixed extraction raw material;

[0048] 2. Preparation of Snapper Mixed Peptide

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

[0050] (2) adding 1% alkaline protease and 0.5% flavor protease by weight of the mixed extraction raw material, adjusting the pH to 8.0, the temperature to 50° C., and performing enzymolysis for 3 hours to obtain an enzymolysis solution;

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

[0052] (4) collecting the supernatant by centrifugation to obtain a mixed polypeptide solution of snapper;

[0053] 3. Peptide Purification

[0054] (1) ultrafiltration of the mixed peptide solution of snapper using an ultrafiltration membrane with a molecular weight cut-off of 10 kDa, and collecting the permeate;

[0055] (2) ultrafiltration of the permeate using an ultrafiltration membrane with a molecular weight cutoff of 2 kDa, and collecting the retentate;

[0056] (3) freeze-drying the retentate to obtain the snapper mixed polypeptide I.

[0057] Example 2

[0058] 1. Raw material collection and pretreatment

[0059] (1) Select fresh, uncontaminated snapper scales, bones and fish meat and rinse them with water;

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

[0061] (3) soaking the desalted fish bones (20 g), fish scales (50 g) and fish meat (30 g) in 95% ethanol, and performing ultrasonic-assisted degreasing at room temperature, repeating the process twice to remove fat and obtain a mixed raw material;

[0062] (4) drying the mixed raw material in an oven at 60° C., crushing it into powder using a grinder, and passing it through an 80-mesh sieve to obtain a mixed extraction raw material;

[0063] 2. Preparation of Snapper Mixed Peptide

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

[0065] (2) adding 1% alkaline protease and 0.5% flavor protease by weight of the mixed extraction raw material, adjusting the pH to 8.0, the temperature to 50° C., and performing enzymolysis for 3 hours to obtain an enzymolysis solution;

[0066] (3) heating the enzymatic hydrolysate to 95°C for 10 minutes to inactivate the enzyme;

[0067] (4) collecting the supernatant by centrifugation to obtain a snapper mixed polypeptide solution 1;

[0068] 3. Peptide Purification

[0069] (1) ultrafiltration of the mixed peptide solution of snapper using an ultrafiltration membrane with a molecular weight cut-off of 10 kDa, and collecting the permeate;

[0070] (2) ultrafiltration of the permeate using an ultrafiltration membrane with a molecular weight cutoff of 2 kDa, and collecting the retentate;

[0071] (3) freeze-drying the retentate to obtain the snapper mixed polypeptide II.

[0072] Example 3

[0073] 1. Raw material collection and pretreatment

[0074] (1) Select fresh, uncontaminated snapper scales, bones and fish meat and rinse them with water;

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

[0076] (3) soaking the desalted fish bones (40 g), fish scales (40 g) and fish meat (20 g) in 95% ethanol, and performing ultrasonic-assisted degreasing at room temperature, repeating the process twice to remove fat and obtain a mixed raw material;

[0077] (4) drying the mixed raw material in an oven at 60° C., crushing it into powder using a grinder, and passing it through an 80-mesh sieve to obtain a mixed extraction raw material;

[0078] 2. Preparation of Snapper Mixed Peptide

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

[0080] (2) adding 1% alkaline protease and 0.5% flavor protease by weight of the mixed extraction raw material, adjusting the pH to 8.0, the temperature to 50° C., and performing enzymolysis for 3 hours to obtain an enzymolysis solution;

[0081] (3) heating the enzymatic hydrolysate to 95°C for 10 minutes to inactivate the enzyme;

[0082] (4) collecting the supernatant by centrifugation to obtain a mixed polypeptide solution of snapper;

[0083] 3. Peptide Purification

[0084] (1) ultrafiltration of the mixed peptide solution of snapper using an ultrafiltration membrane with a molecular weight cut-off of 10 kDa, and collecting the permeate;

[0085] (2) ultrafiltration of the permeate using an ultrafiltration membrane with a molecular weight cutoff of 2 kDa, and collecting the retentate;

[0086] (3) freeze-drying the retentate to obtain the snapper mixed polypeptide III.

[0087] Comparative Example

[0088] 1. Raw material collection and pretreatment

[0089] (1) Select fresh, uncontaminated snapper scales, bones and fish meat and rinse them with water;

[0090] (2) soaking fish bones (50 g), fish scales (30 g) and fish meat (20 g) in 95% ethanol, and performing ultrasonic-assisted degreasing at room temperature, repeating twice to remove fat and obtain a mixed raw material;

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

[0092] 2. Preparation of Snapper Mixed Peptide

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

[0094] (2) adding alkaline protease in an amount of 1.5% by weight of the mixed extraction raw material, adjusting the pH to 8.0, the temperature to 50° C., and performing enzymolysis for 3 hours to obtain an enzymolysis solution;

[0095] (3) heating the enzymatic hydrolysate to 95°C for 10 minutes to inactivate the enzyme;

[0096] (4) collecting the supernatant by centrifugation to obtain a mixed polypeptide solution of snapper;

[0097] (5) The Lutjanus bream mixed polypeptide solution is freeze-dried to obtain Lutjanus bream mixed polypeptide IV.

[0098] Example 4

[0099] Screening the optimal concentration of peptides for the proliferation of trophoblastic cells using mixed peptides from snapper 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 steps:

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

[0106] 2. Place the cells in an incubator and culture them. Replace the culture medium every 2 to 3 days to maintain normal cell growth.

[0107] 3. When the cells grow to the logarithmic phase, they are used for subsequent experiments.

[0108] (2) Preparation of peptide working solution

[0109] Experimental steps:

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

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

[0112] (3) Cell seeding

[0113] Experimental steps:

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

[0115] 2. Resuspend the cells in DMEM complete 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 inoculate into a 96-well cell culture plate.

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

[0118] (4) Peptide treatment

[0119] Experimental steps:

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

[0121] 2. Group processing:

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

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

[0124] Return the culture plate to the incubator and continue incubation for 24 hours.

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

[0126] Experimental steps:

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

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

[0129] 3. Use an 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 follows Figure 1 As shown in Table 1:

[0131] Table 1 Absorbance differences of untreated groups

[0132]

[0133] from Figure 1 The results in Table 1 show that compared with the group without adding the Lutjanus mixed polypeptide I working solution (0 μg / mL group), the addition of 50 μg / mL of Lutjanus mixed polypeptide I can effectively promote the proliferation ability of trophoblastic cells, and the cell proliferation rate is significantly improved. With the further increase of the polypeptide concentration, the promotion effect shows a trend of continuous enhancement. When the concentration reaches 200 μg / mL, the cell proliferation rate reaches the highest value, indicating that the promotion effect of the polypeptide at this concentration is most significant.

[0134] When the concentration continued to increase to 400 μg / mL, although the mixed snapper polypeptide I still had a significant effect of promoting cell proliferation, the overall effect was lower than that of the 200 μg / mL group. Therefore, in subsequent tests, the present invention selected 200 μg / mL as the detection concentration of the present invention.

[0135] Example 5

[0136] Detecting the therapeutic effects of different mixed snapper peptides on trophoblast cell damage caused by high sugar

[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 steps:

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

[0143] 2. Place the cells in an incubator and culture them. Replace the culture medium every 2 to 3 days to maintain normal cell growth.

[0144] 3. When the cells grow to the logarithmic phase, they are used for subsequent experiments.

[0145] (2) Preparation of peptide working solution

[0146] Experimental steps:

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

[0148] 2. The high-concentration stock solutions of Lutjanus mixed polypeptide I, Lutjanus mixed polypeptide II, Lutjanus mixed polypeptide III and Lutjanus mixed polypeptide IV were combined with the glucose stock solution to obtain working solutions containing 200 μg / mL 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 steps:

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

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

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

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

[0155] (4) Peptide treatment

[0156] Experimental steps:

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

[0158] 2. Group processing:

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

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

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

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

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

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

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

[0166] Experimental steps:

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

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

[0169] 3. Use an ELISA reader to measure the absorbance of each well at a wavelength of 450 nm and calculate the cell viability.

[0170] The results are shown in Table 2 and Figure 2 As shown:

[0171] Table 2 The therapeutic effects of different mixed peptides from snapper on trophoblast cell damage caused by high sugar

[0172]

[0173]

[0174] From the results in Table 2, it can be seen that the cell survival rate of the high sugar group dropped to 55.32%, indicating that the high sugar environment significantly inhibited the proliferation of trophoblast cells and induced cell damage. The cell survival rate of treatment group I (polypeptide of Example 1) recovered to 88.55%, with the best effect. The cell survival rates of treatment group II (polypeptide of Example 2) and treatment group III (polypeptide of Example 3) were 72.06% and 76.48%, respectively, which were lower than those of treatment group I. The cell survival rate of treatment group IV (polypeptide of the comparative example) was 62.76%, which was the worst effect, but still higher than that of the high sugar group.

[0175] From the above results, it can be seen that the peptide snapper mixed polypeptide IV prepared in the comparative example has the worst therapeutic effect on trophoblast cell damage caused by high sugar, indicating that desalting treatment, dual enzyme synergistic enzymolysis and double ultrafiltration purification are key steps to improve the biological activity of the snapper mixed polypeptide. The protective effect of the snapper mixed polypeptide prepared without these steps in cell damage caused by high sugar will be greatly reduced;

[0176] At the same time, it can be seen that the difference in the ratio of fish bones, fish scales and fish meat also has a huge impact on the performance of the prepared polypeptide. Only when the ratio of fish bones, fish scales and fish meat is 5:3:2, can the mixed snapper polypeptide composed of fish bones, fish scales and fish meat polypeptides play a synergistic therapeutic effect on trophoblast cell damage caused by high sugar. At other ratios, the effect will be greatly reduced. This may be due to the different types of proteins and active ingredients contained in different raw materials. The optimal ratio can make the polypeptide rich in effective active peptides and produce 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 DMEM (i.e., complete DMEM 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 DMEM complete medium.

[0184] 2. Place the cells in an incubator and culture them. Replace the culture medium every 2 to 3 days to maintain normal cell growth.

[0185] 3. When the cells grow to the logarithmic phase, they are used for subsequent experiments.

[0186] (2) Preparation of peptide working solution

[0187] Experimental steps:

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

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

[0190] (3) Cell seeding

[0191] Experimental steps:

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

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

[0194] Cell seeding:

[0195] Add 200 μL of cell suspension (about 2×10 4 Each treatment group had 3 replicate wells.

[0196] Lower chamber culture medium:

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

[0198] Pre-culture:

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

[0200] (4) Peptide treatment

[0201] Experimental steps:

[0202] 1. Upper chamber processing:

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

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

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

[0206] Treatment group II: Same as above, using Lutjanus bream mixed polypeptide II.

[0207] Treatment group III: Same as above, using Lutjanus bream mixed polypeptide III.

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

[0209] 2. Lower chamber processing:

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

[0211] 3. Return the culture plate to the incubator and culture at 37°C, 5% CO2 for 24 hours.

[0212] (5) Cell migration assay

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

[0214] 2. Remove the Transwell insert, gently wash the upper chamber twice with PBS, and then place it in 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 with PBS three times, 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 excess dye.

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

[0219] The results are as follows Figure 3 As shown, from Figure 3 It can be seen that the overall trend of the results obtained from the cell migration test is basically consistent with the results obtained from the cell proliferation test. The difference is that the mixed snapper polypeptide IV prepared in the comparative example does not effectively protect the reduced migration ability of trophoblast cells caused by high sugar. This may be because the ability of the polypeptide to regulate migration is inferior to the effect of cell proliferation.

[0220] In general, only the Lutjanus mixed polypeptide I prepared in Example 1 can achieve a very significant protective effect against the reduced migration ability of trophoblast cells caused by high sugar, 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 the reduced migration ability of trophoblast cells caused by high sugar.

[0221] Based on the above results, it can be seen that the Lutjanus bream mixed polypeptide I prepared by the present invention can effectively protect the decreased proliferation and migration abilities of trophoblast cells caused by high sugar, so that the Lutjanus bream mixed polypeptide I can be used as a drug for placental development abnormalities caused by gestational diabetes.

[0222] Example 7

[0223] Preparation method of Lutjanus bream mixed polypeptide I biological preparation taking 200 μg / mL as an example

[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 snapper mixed polypeptide I, place it in a small beaker, add 15 ml of physiological saline, and stir with a magnetic stirrer until it is completely dissolved to obtain a polypeptide solution;

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

[0227] 4. Filter through a 0.22 μm sterile filter membrane to ensure the sterility of the solution and remove particles. Collect the filtered solution in a sterile vial;

[0228] 5. After packaging and sealing on a sterile operating table, store in a freezer at -20℃.

Claims

1. A biological agent for regulating placental developmental abnormalities, characterized in that: Each 100 ml of the biological preparation is prepared according to the following preparation method: Weigh 2-10 g of mannitol, and dissolve the mannitol in physiological saline to obtain a mannitol stabilizer; Weigh 5-40 mg of the mixed snapper polypeptide and dissolve it to obtain a polypeptide solution; Mix the polypeptide solution and mannitol stabilizer evenly, and dilute to 100 ml with normal saline; The biological preparation was obtained by filtering through a 0.22 μm sterile filter membrane.

2. The biological agent according to claim 1, characterized in that The mixed polypeptide of snapper is prepared according to the following preparation method Choose fresh, uncontaminated snapper and rinse the scales, bones and meat with water; Soak the fish bones in dilute hydrochloric acid, and then wash them repeatedly with distilled water until the pH is neutral; The desalted fish bones, fish scales and fish meat are mixed in a mass ratio of 5:3:2, immersed in 95% ethanol, and subjected to ultrasonic-assisted defatting at room temperature to obtain a mixed raw material; The mixed raw material is placed in an oven to dry, and then crushed into powder using a grinder to obtain a mixed extraction raw material; The mixed extraction raw materials were added with distilled water at a mass volume ratio of 1:10, stirred evenly, and homogenized twice at a pressure of 80 MPa; Add 1% alkaline protease and 0.5% flavor protease by weight of the mixed extraction raw materials, adjust the pH to 8.0, the temperature to 50° C., and perform enzymolysis for 3 hours to obtain an enzymolysis solution; The enzymatic hydrolysate is heated to inactivate alkaline protease and flavor protease; Collect the supernatant by centrifugation to obtain a mixed peptide solution of snapper; The mixed peptide solution of snapper was ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa, and the permeate was collected; Ultrafiltration was performed using an ultrafiltration membrane with a molecular weight cutoff of 2 kDa, and the retentate was collected; The retentate was freeze-dried to obtain the Lutjanus bream mixed polypeptides.

3. The biological agent according to claim 1, characterized in that The regulating placental development abnormality is regulating placental development abnormality caused by gestational diabetes.

4. Use of a mixed peptide from snapper in the preparation of a biological preparation for treating placental dysplasia caused by gestational diabetes, characterized in that: The snapper mixed polypeptide is prepared by the preparation method described in claim 2.

5. The use according to claim 4, characterized in that: In the biological preparation, the use concentration of the Lutjanus mixed polypeptide is 50 μg / mL-400 μg / mL.

6. The use according to claim 5, characterized in that: When the use concentration of the snapper mixed polypeptide is 200 μg / mL, the biological preparation achieves the best therapeutic effect.

7. The use according to claim 6, characterized in that: The placental development abnormality caused by gestational diabetes is the placental development abnormality caused by the decreased proliferation ability and migration ability of trophoblast cells caused by high sugar.

8. Use of a mixed snapper polypeptide in the preparation of a biological preparation for treating decreased proliferation and migration abilities of trophoblastic cells caused by gestational diabetes, characterized in that: The snapper mixed polypeptide is prepared by the preparation method described in claim 2; In the biological preparation, the use concentration of the Lutjanus mixed polypeptide is 200 μg / mL.

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