CN114796278A - Application of fat active protein in preparing medicine for treating intrauterine adhesion - Google Patents
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- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
- A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
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Abstract
The invention relates to an application of a fat active protein in preparing a medicament for treating intrauterine adhesion. The fat cell-free active protein is used for treating an IUA mouse model, has the main treatment effects of increasing endometrial cells and blood vessels, increasing endometrial receptivity and improving fertility, and provides a new treatment method for the clinical treatment of IUA.
Description
Technical Field
The invention belongs to the field of biological medicine, and particularly relates to application of a fat active protein in preparation of a medicine for treating intrauterine adhesion.
Background
Intrauterine Adhesions (IUA) are the basement of the endometrium caused by a variety of causesThe layers are damaged, causing the walls of the uterine muscles to adhere to each other, so that the cervical canal and the uterine cavity are partially or totally occluded. Clinically, the traditional Chinese medicine can cause symptoms such as hypomenorrhea, amenorrhea, infertility and repeated abortion, and seriously jeopardize the reproductive health of women of childbearing age [1,2] . Repeated abortions and uterine curettage are considered risk factors for intrauterine adhesions. A review of the system and meta-analysis showed that about 1/5 of women who had been treated with curettage during midday labor would have had IUA [3] 。
There is no unified consensus on the exact mechanism of IUA generation at home and abroad, and it is currently believed that multiple factors participate together [4] : 1) the endometrium basal layer is damaged, and endometrium can not normally proliferate to repair the wound surface, so that a large amount of single-layer epithelium and fibrous tissues are substituted. Endometrium atrophy, phenanthrene thinness, inactive glands, low response to hormone stimulation and fuzzy boundary between a functional layer and a basal layer; 2) bacteria or viruses and the like invade endometrium to cause uterine cavity inflammatory reaction to cause immune imbalance and wound surface adhesion; 3) angiogenesis is reduced, resulting in hypoxic injury.
Currently, hysteroscopic adhesion release is considered the primary method of treating IUA [5] However, a systematic review and meta-analysis showed that hysteroscopic lysis had poor fertility improvement in infertility patients with IUA [6] The underlying mechanisms may be related to sperm transport, poor embryo migration, or failure of implantation of the embryo due to decreased endometrial receptivity [7] . Therefore, in order to restore normal regeneration of endometrium after operation and improve endometrial receptivity, hormone therapy is often combined with hysteroscopic adhesion release in clinic [7] . In addition, other bioactive substances such as stem cells have also been reported to restore endometrial structure and function [8] . However, currently, the best management for clinically preventing postoperative adhesion reformation of IUA is not well known, and further research and exploration are needed.
The current therapy of jointly applying estrogen after hysteroscopic adhesion release has the following defects [9] : 1. adverse reactions of large doses of estrogen, such as nausea, vomiting, breast distending pain, etc.; 2. the dosage of the estrogen is not known in common,the control is difficult (2mg-12mg is different), the effect is unstable, and a certain proportion of estrogen resistant patients exist; 3. studies have suggested that this therapy is not effective in improving female fertility. In view of the above problems, some researchers have tried stem cell transplantation methods to improve the outcome of IUA treatment, and have achieved some success in animal experiments. However, stem cell repair is accompanied by immune rejection and neoplastic risk [10,11] . Meanwhile, the activity and culture of stem cells are not beneficial to the quality control and batch production of products, and the popularization and application of the technology are limited. Based on the above current therapeutic situation, it is urgent to find new strategies for improving the fertility function of IUA patients.
Reference documents:
1.Bosteels J,Weyers S,D'Hooghe TM,et al.Anti-adhesion therapy following operative hysteroscopy for treatment of female subfertility.Cochrane Database Syst Rev.2017；11(11):CD011110.Published 2017Nov 27.
2.Kou L,Jiang X,Xiao S,Zhao YZ,Yao Q,Chen R.Therapeutic options and drug delivery strategies for the prevention of intrauterine adhesions.J Control Release.2020；318:25-37.
3.Hooker AB,Lemmers M,Thurkow AL,et al.Systematic review and meta-analysis of intrauterine adhesions after miscarriage:prevalence,risk factors and long-term reproductive outcome.Hum Reprod Update.2014；20(2):262-278.
4.Lee WL,Liu CH,Cheng M,Chang WH,Liu WM,Wang PH.Focus on the Primary Prevention of Intrauterine Adhesions:Current Concept and Vision.Int J Mol Sci.2021；22(10):5175.Published2021May 13.
5.Salazar CA,Isaacson K,Morris S.A comprehensive review of Asherman's syndrome:causes,symptoms and treatment options.Curr Opin Obstet Gynecol.2017；29(4):249-256.
6.Di Spiezio Sardo A,Di Carlo C,Minozzi S,et al.Efficacy of hysteroscopy in improving reproductive outcomes of infertile couples:a systematic review and meta-analysis.Hum Reprod Update.2016；22(4):479-496.
7.AAGL Advancing Minimally Invasive Gynecology Worldwide.AAGL practice report:practice guidelines for management of intrauterine synechiae.J Minim Invasive Gynecol.2010；17(1):1-7.
8.Song YT,Liu PC,Tan J,et al.Stem cell-based therapy for ameliorating intrauterine adhesion and endometrium injury.Stem Cell Res Ther.2021；12(1):556.Published 2021Oct 30.
9.Liu L,Huang X,Xia E,Zhang X,Li TC,Liu Y.A cohort study comparing 4mg and 10mg daily doses of postoperative oestradiol therapy to prevent adhesion reformation after hysteroscopic adhesiolysis.Hum Fertil(Camb).2019；22(3):191-197.
10.Lin J,Wang Z,Huang J,et al.Microenvironment-Protected Exosome-Hydrogel for Facilitating Endometrial Regeneration,Fertility Restoration,and Live Birth of Offspring.Small.2021；17(11):e2007235.
11.Chen JM,Huang QY,Zhao YX,Chen WH,Lin S,Shi QY.The Latest Developments in Immunomodulation of Mesenchymal Stem Cells in the Treatment of Intrauterine Adhesions,Both Allogeneic and Autologous.Front Immunol.2021；12:785717.Published 2021 Nov 15.
disclosure of Invention
The invention aims to solve the technical problem of providing an application of a fat-activated protein in preparing a medicine for treating intrauterine adhesion, so as to overcome the defects of adverse reaction, poor treatment, unstable effect and the like of the medicine for treating intrauterine adhesion in the prior art.
The invention provides an application of a fat active protein in preparing a medicament for treating intrauterine adhesion.
Preferably, the amount of the lipoactive protein is 25-30 ug/time, and the administration period is a single administration after operation without continuous administration.
Preferably, the fat active protein is: active protein complex extracted by removing cell components from adipose tissue.
Preferably, the medicine takes the fat active protein as an active component, and is prepared into a preparation by matching with pharmaceutically acceptable auxiliary materials or auxiliary components.
Preferably, the preparation is selected from one of tablets, powders, granules, capsules, liquid preparations and sustained release preparations.
Advantageous effects
The invention uses the fat decellularization active protein for the treatment of an IUA mouse model, and the main treatment effects comprise endometrial cells and blood vessels increase, endometrial receptivity increase and fertility improvement. The fat decellularization active protein provides a new treatment method for the clinical treatment of IUA.
In addition, the fat decellularization active protein can be extracted from autologous fat of a patient, so that the biological active protein is retained, and meanwhile, cell components are removed, and immune rejection and ethical disputes brought by the treatment of the cell factors from foreign body tissues can be avoided.
Drawings
FIG. 1 is a flow chart of the preparation of CEFFE of the present invention.
FIG. 2 is a flow chart of IUA animal model construction and detection.
FIG. 3 shows the morphology evaluation results of mouse uterus at 14 days after IUA single-sided modeling of mice of the present invention, wherein (A) is a typical photograph of mouse uterus at 14 days after IUA modeling of mice (in the figure, #1, #2, #3 is a representative image of 3 mouse uterus), (B) statistics of mouse uterus angle effusion at 14 days after IUA modeling of mice.
FIG. 4 is a graph showing pathological evaluation results of mouse uterus at 14 days after IUA single-sided modeling of mice according to the present invention, wherein (A) is a typical graph of HE staining, Masson staining, vimentin IHC analysis and CD31IHC analysis of mouse uterus at 14 days after IUA modeling of mice, and (B) is analysis of endometrial thickness, collagen level and intensity of vimentin and CD31 staining.
FIG. 5 shows scanning electron micrographs of mouse endometrium (A) and statistics of pinocytosis numbers (B) at day 4 after IUA unilaterally modeling and mating with male mice in mice of the present invention.
Fig. 6 shows the results of evaluating the pregnancy of the mice on the 10 th day after the mice are IUA-modeled and mated with females, wherein (A) is a typical image of the uterus of the mice on the 10 th day after the mice are IUA-modeled and mated with males (# 1, #2, #3 is a representative image of the pregnant uterus of 3 mice in the figure), and (B) is the statistics of the number of uterine horns of pregnancy.
FIG. 7 shows the morphology of the uterus of mice in IUA group and treatment group (in the figure, #1, #2, #3 is a representative image of the uterus of 3 mice) and the statistics of fluid accumulation in uterine horn (B) at 14 days after treatment according to the present invention.
Fig. 8 shows pathological evaluation results of uterus of mice in IUA group and IUA treated group on day 14 in the present invention, wherein (a) is typical pictures of HE staining, Masson staining, vimentin IHC analysis and CD31IHC analysis of uterus of IUA group and IUA treated group, and (B) is analysis of endometrial thickness, collagen level, vimentin and CD31 staining intensity.
FIG. 9 shows scanning electron micrographs of uterus (A) and statistics of pinocytosis counts (B) 4 days after mating in IUA mice and IUA treated groups of the present invention.
Fig. 10 shows the pregnancy evaluation results of mice of IUA group and IUA treatment group 10 days after mating according to the present invention, wherein (a) is a typical image of uterus of mice of IUA group and 10 days after mating of the treatment group and male mice (in the figure, #1, #2, #3 is a representative image of the uterus of 3 mice in pregnancy), and (B) is the statistics of the number of uterine horns in pregnancy.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.
Example 1
Fat decellularization active protein (CEFFE) extraction:
liposuction is performed on fat-rich sites such as the abdomen and the inner thigh of a patient, blood and tissue debris in adipose tissue are removed by flushing with physiological saline in a sterile environment, and the mixture is divided into three layers by centrifugation at 1200g for 3 minutes. Only the middle fat layer was left and subjected to emulsification treatment at normal temperature for 30min by a mechanical method. The emulsified fat was then stored at-80 ℃ and rapidly thawed at 37 ℃ to disrupt the cell membrane. After one freeze-thaw cycle, the sample was centrifuged at 1200g for 5 minutes and the centrifuged mixture was separated into four layers. Only the third layer of liquid was collected and filtered through a 0.22 μm filter membrane to remove bacteria and other debris, the product being CEFFE. The CEFFE was protein quantified by BCA method to 1.25. mu.g/. mu.l before it was frozen at-80 ℃ for further experiments.
Constructing and treating an animal model of intrauterine adhesion:
breeding the mice: c57BL/6N female mice 8 weeks old are bred in an SPF laboratory, kept at constant temperature (25 +/-2 ℃), kept at constant humidity of 45-55% and adjusted for one week in a day-night regular manner (12 hours of light every day).
Constructing an animal model of intrauterine adhesion: anesthetizing the animals with 1% amobarbital at a dose of 5ul/g, and fixing the mice supinely in a clean bench; sterilizing the abdomen of the mouse with 75% ethanol, and sequentially cutting the abdominal tissues with a sterile surgical instrument to expose the uterus; scratching the left side by using a metal brush (with the diameter of 0.5-2cm) to form a die set; right uterus was cut only as a control group; the abdominal region is sutured with surgical thread to supplement sufficient drinking water and food supply. The mice after molding are randomly divided into three parts for detection so as to judge whether the IUA model is successfully constructed: 1) after injecting 10IU PMSG into abdominal cavity 12 days after model building, killing mice 2 days later to observe the gross morphology of uterus, and counting the uterine volume liquid rate, and carrying out HE staining to detect the thickness of endometrium, Masson staining to detect the fibrosis of endometrium and immunohistochemical detection to detect the blood vessel density; 2) after 12 days 10IU PMSG was injected, after 2 days hCG was injected and mixed with the male mice at a ratio of 1: 1, and 4 days after mating, killing the mice, observing pinocytosis by an electron microscope, and presuming the endometrial receptivity of the mice; 3) after 12 days 10IU PMSG was injected, after 2 days hCG was injected and mixed with the male mice at a ratio of 1: 1, and observing the implantation of the mouse embryo 10 days after the mating. The specific detection is as follows:
and (3) observing the morphology of the uterus:
killing the mouse by a cervical dislocation method, taking the supine position, splitting a skin layer and a peritoneal muscle layer of the mouse after sterilizing with 75% ethanol, fully exposing the abdominal cavity, separating adipose tissues, taking out the uterus, rapidly putting the uterus into PBS for cleaning, stripping the residual adipose tissues, observing the shape of the uterus, taking a picture for recording, and counting the uterine fluid deposition rate.
And (3) carrying out HE staining on uterine tissues:
tissue fixation: the uterine tissue is cut off (0.5-1 cm/section), and the uterus of the uterine adhesion part of the mouse at the molding side is fixed in 4% PFA. The corresponding position of the uterus of the control group or the treatment group is taken for fixing.
Tissue dehydration embedding: tissues that had been infiltrated in 4% PFA for over 2 days were fixed embedded sequentially with different concentrations of alcohol and alkylbenzenes, xylene and paraffin.
Tissue section dewaxing: and (3) slicing a paraffin block with the thickness of 7.5 mu m by using a paraffin slicer, and sequentially passing through dimethylbenzene and ethanol with different concentrations until the double distilled water is washed.
Dyeing: staining with hematoxylin for 5min, washing with tap water, differentiating with hydrochloric acid and ethanol, washing with clear water, and staining with eosin solution.
Dehydrating, transparent, sealing: dehydrating with ethanol of different concentrations, transparent xylene, and performing microscopic examination on a neutral resin mounting.
Uterine tissue Masson staining:
the steps of tissue fixation, dehydration embedding, slicing and dewaxing are the same as the above.
And (5) washing after hematoxylin staining solution staining for 60 s. Adding acid fuchsin dye liquor for dyeing and then washing.
Color separation is carried out on phosphomolybdic acid color separation liquid, aniline blue complex dye liquid is added for dyeing, and then the color separation liquid is washed by absolute ethyl alcohol and dried, and the mounting piece is subjected to microscopic examination.
Immunohistochemical staining of uterine tissue:
the steps of tissue fixation, dehydration embedding, slicing and dewaxing are the same as the above.
Antigen retrieval and non-specific signal blocking: antigen retrieval and non-specific signal blocking were performed by boiling citrate buffer (10 mM; pH6.0), incubation with 3% hydrogen peroxide, and blocking 5% goat serum at room temperature.
Antibody incubation: after three TBS washes, the corresponding antibody or isotype control antibody was added for incubation, and the antibody information is shown in the following table.
| Antibodies for IHC | Dilution ratio | Goods number |
| Anti-Vim | 1:100 | ab92547,Abcam,CA,USA |
| anti-CD31 | 1:2000 | ab182981,Abcam,CA,USA |
| Rabbit IgG | 1:200 | Biyuntian, A7016, China |
Secondary antibody incubation and hematoxylin counterstaining: after three TBS washes, the cells were incubated with the corresponding secondary antibody and counterstained with hematoxylin.
And (3) observing by a scanning electron microscope:
mice were sacrificed by cervical dislocation, the uterus was removed (same procedure as above), and endometrial tissue (tissue mass volume) was obtained with a sharp surgical blade<3mm 2 ) The tissue is washed by PBS for 1 time, fixed in the fixing liquid of an electron microscope for 2 hours at room temperature and then stored at 4 ℃. And (5) dehydrating, freeze-drying and spraying gold, and observing the result by using a scanning electron microscope.
Mouse pregnancy test:
mice are treated by IUA modeling for 12 days, then 10IU PMSG is injected into the abdominal cavity, 48 hours later, hCG is injected into the abdominal cavity, and the ratio of the hCG to male mice is 1: mating was performed at a ratio of 1.
The next day pessaries were examined and female and male mice were separated into cages. The Day of vaginal embolus is defined as Day1, Day10 mice were sacrificed, uteri were exposed, and the number of implanted embryos was observed for each group.
FIG. 3 shows: approximately 80% of the uterine horn on the molding side (IUA group) formed distal hydrops due to intratubal obstruction and poor internal circulation.
FIG. 4 shows that: compared to the control (Ctrl group) uterus, HA staining showed a decrease in intimal thickness in the modelled (IUA group) mice, Masson staining showed an increase in collagen content, IHC staining showed a decrease in vimentin-labeled stromal cells, and IHC staining showed a decrease in CD 31-labeled vascular staining.
FIG. 5 shows that: the number of pinocytosis after molding is obviously reduced compared with that of a control group.
FIG. 6 shows that: compared with the control group, the uterus number of the IUA group mice is obviously reduced, and the unilateral pregnancy number is also obviously reduced.
Animal model treatment of intrauterine adhesion: exposing both uterine sides after anesthesia (same procedure as above); cutting the upper edges of uterine horn at both sides, scratching both sides with a metal brush (diameter 0.5-2 cm); injecting 20 μ l PBS through incision by using a micro-syringe, injecting 20 μ l fat decellularization active protein (containing 25 μ g of active protein) at the right side, and administrating once without repeated administration; the abdominal region is sutured with surgical thread to supplement sufficient drinking water and food supply. The detection indexes are as above.
FIG. 7 shows that: about 40% of uterine horns on the treated side of CF (IUA + CF) formed distal hydrocele due to intrafallopian obstruction and poor internal circulation, significantly lower than the untreated side of molding (IUA).
FIG. 8 shows that: compared to the IUA untreated group (IUA group) uterus, HA staining showed increased intimal thickness in the treated group (IUA + CF group) mice, Masson staining showed decreased collagen content, IHC staining showed increased numbers of vimentin-labeled stromal cells, and IHC staining showed increased intensity of CD 31-labeled vascular staining.
FIG. 9 shows that: the number of pinocytosis after treatment was significantly increased compared to the modelled untreated group.
FIG. 10 shows: compared with the IUA untreated group, the number of pregnant uteri of the treated mice is remarkably increased, and the number of unilateral pregnant embryos is also remarkably increased.
Claims (5)
1. Application of a lipoactive protein in preparing medicine for treating intrauterine adhesion is disclosed.
2. The use according to claim 1, wherein the amount of the lipoactive protein is 25-30 ug/dose and the administration period is a single post-operative administration without continuous administration.
3. The use according to claim 1, wherein the lipoactive protein is: active protein complex extracted by removing cell components from adipose tissue.
4. The use of claim 1, wherein the medicament is prepared into a preparation by using the fat active protein as an active component and adding pharmaceutically acceptable auxiliary materials or auxiliary components.
5. The use according to claim 4, wherein the formulation is selected from one of a tablet, a powder, a granule, a capsule, a liquid, and a sustained release formulation.
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