CN120060478A - Application of parabacteroides johnsonii in ovarian aging - Google Patents
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Abstract
本申请涉及生物技术领域，尤其涉及一种约氏副拟杆菌在卵巢衰老中的应用；所述应用包括：将低丰度的约氏副拟杆菌作为卵巢衰老的生物标志物；所述应用包括将约氏副拟杆菌用于延缓卵巢衰老的药剂中；所述应用还包括将约氏副拟杆菌的发酵产物用于延缓卵巢衰老的药剂中；通过实验发现在约氏副拟杆菌在不同卵巢衰老程度的小鼠内存在显著的差异，并且通过后续实验明确了约氏副拟杆菌具有显著增加卵母细胞数量、并可以上调抗缪勒氏管激素、上调雌二醇水平和下调促卵泡生成激素水平的效果，这提示约氏副拟杆菌可以作为延缓卵巢衰老的药剂中。
The present application relates to the field of biotechnology, and in particular to an application of Parabacteroides johnsonii in ovarian aging; the application includes: using low-abundance Parabacteroides johnsonii as a biomarker of ovarian aging; the application includes using Parabacteroides johnsonii in a drug for delaying ovarian aging; the application also includes using a fermentation product of Parabacteroides johnsonii in a drug for delaying ovarian aging; experiments have shown that there are significant differences in Parabacteroides johnsonii in mice with different degrees of ovarian aging, and subsequent experiments have shown that Parabacteroides johnsonii has the effect of significantly increasing the number of oocytes, upregulating anti-Mullerian hormone, upregulating estradiol levels and downregulating follicle-stimulating hormone levels, which suggests that Parabacteroides johnsonii can be used as a drug for delaying ovarian aging.
Description
Technical Field
The application relates to the technical field of biology, in particular to application of parabacteroides johnsonii in ovarian aging.
Background
Female reproductive aging refers to the decline in ovarian fertility function, whereas decline in female ovarian function generally begins at 30 years and accelerates after 35 years. The reserve function in the ovary is critical for fertility, and the accelerated decrease of the reserve function of the ovary leads to premature aging or dysfunction of the ovary, due to the fact that the reserve of germ cells and follicles at birth determines the reproductive life and the menopausal time of mammals, and at the same time the reserve function of the ovary is accelerated to be depleted with age, the main manifestation is more follicular locking and apoptosis, and besides the decrease of the reserve of the follicles, the quality of oocytes is also reduced with the age of females. The quality of oocytes generally begins to deteriorate gradually after age 30, which coincides with the time of reduced oocyte count, and this also partly explains the fact that women have reduced fertility before menopause and endocrine abnormalities in women with reproductive age. The aging of oocytes is mainly characterized by lectin dysfunction, low cross maturation efficiency, chromosome dislocation, meiosis spindle damage, telomere abrasion, DNA damage, mitochondrial dysfunction and the like, and the ovum dysplasia caused by the quality reduction of the oocytes becomes one of the main reasons of female reproductive disorders with the increase of age.
In addition to reproductive function, the ovaries can also maintain female hormone secretion, which is important in maintaining female health, and current ovarian aging can trigger hypothalamic-pituitary-ovarian axis hormone secretion regulation to be disturbed, so that the level of Follicle Stimulating Hormone (FSH) is increased and the level of anti-mullerian hormone (AMH) is reduced, and the excessive FSH in serum can directly act on hippocampus and cortical neurons to accelerate the deposition of amyloid beta and tau, so that cognitive ability is damaged and the organism can also display the characteristic of Alzheimer's disease. At the same time, follicular depletion in the ovaries can trigger a decrease in estrogen secretion levels in the body, up to menopause. The estrogen is a steroid hormone with wide and important physiological functions, has obvious benefits on other systems of the organism, such as endocrine, bone, nerve and cardiovascular, and has proved by related researches that the estrogen also has potential therapeutic value on various pulmonary diseases, such as asthma, pulmonary arterial hypertension, pulmonary fibrosis and the like, and even has a certain protective effect on respiratory tract virus infection. In conclusion, the ovarian failure is a complex process and involves a plurality of factors, so that the deep understanding of the ovarian failure plays an important role in the aging process of human health and brings about wider social benefits.
The marked characteristic of ovarian senescence is a decrease in the number and quality of oocytes, and the senescence process is often accompanied by a change in the composition of intestinal microorganisms, which also have an effect on senescence. In recent years, studies have found that there may also be interactions between ovarian aging and gut microbiota, while gut microbiota may play a role in the progression of ovarian aging. In addition, in cohort studies involving premature ovarian dysfunction (POI) females and healthy females, researchers have found that the intestinal microbiota of POI females is altered compared to healthy females and that these alterations are closely related to changes in hormone levels associated with ovarian aging. These suggest that the progression of ovarian aging may be related to intestinal microorganisms.
At present, the research on related mechanisms of small molecule for delaying ovarian aging suggests that microorganisms possibly participate in the regulation and control process of drug for delaying ovarian aging, for example, research shows that resveratrol can remarkably reduce the influence of oxidative stress induced by tBHP on spawning rate and follicular number, and activates SIRT1/FoxO1 and Nrf2 pathways, thereby enhancing antioxidant gene expression, and research shows that intestinal flora plays a vital role in maintaining tryptophan metabolism in ovarian oxidative stress through kynurenine pathway. However, there is currently no report on the aging of ovaries by Paramethobacterium johnsonii (Parabacteroides johnsonii) among intestinal microorganisms.
Disclosure of Invention
The application provides an application of parabacteroides johnsonii in ovarian aging, which fills the blank of the correlation between the parabacteroides johnsonii and the ovarian aging in intestinal microorganisms in the prior art.
In a first aspect, the application provides an application of parabacteroides johnsonii in an ovarian aging biomarker, the application comprising:
Low abundance parabacteroides johnsonii was used as biomarker for ovarian aging.
Alternatively, the abundance of Paramycolatopsis johnsonii is positively correlated with the extent of ovarian aging.
Optionally, the abundance of parabacteroides johnsonii is less than 0.77 times the abundance of normal parabacteroides johnsonii.
Optionally, when the abundance of the parabacteroides johnsonii is lower than 0.77 times of the abundance of the normal parabacteroides johnsonii, judging that the ovary is in an aging state at the moment.
In a second aspect, the application provides an agent for screening or aiding in screening for ovarian aging, the agent comprising an agent that detects the relative abundance of Paralopecuroides johnsonii.
In a third aspect, the application provides an agent for evaluating or aiding in the evaluation of the prognostic effect of ovarian aging, the agent comprising an agent that detects the relative abundance of Paralopecuroides johnsonii.
In a fourth aspect, the application provides an application of parabacteroides johnsonii in delaying ovarian aging, wherein the application comprises the application of the parabacteroides johnsonii in medicaments for delaying ovarian aging.
Optionally, the delaying ovarian aging includes at least one of:
Increasing oocyte numbers, up-regulating anti-mullerian hormone, up-regulating estradiol levels, and down-regulating follicle stimulating hormone levels.
Optionally, the use further comprises using the fermentation product of parabacteroides johnsonii in an agent for delaying ovarian aging.
Alternatively, the fermentation product of Paramycolatopsis johnsonii comprises a supernatant from culturing Paramycolatopsis johnsonii.
Compared with the prior art, the technical scheme provided by the embodiment of the application has the following advantages:
According to the application of the parabacteroides johnsonii in the ovarian aging, a large number of experiments show that the parabacteroides johnsonii is high in abundance in young mice with smaller ages, the abundance of the parabacteroides johnsonii in the mice with larger ages is obviously reduced, the possibility that the parabacteroides johnsonii is used as an ovarian aging biomarker can be clearly determined according to the difference, and the follow-up experiments show that the parabacteroides johnsonii has the effects of obviously increasing the number of oocytes, up-regulating the anti-mullerian hormone, up-regulating the estradiol level and down-regulating the follicle-stimulating hormone level, so that the parabacteroides johnsonii can be used as an agent for delaying the ovarian aging, and the blank of the parabacteroides johnsonii in the intestinal microorganisms in the aspect of the ovarian aging is filled.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the application and together with the description, serve to explain the principles of the application.
In order to more clearly illustrate the embodiments of the application or the technical solutions of the prior art, the drawings which are used in the description of the embodiments or the prior art will be briefly described, and it will be obvious to a person skilled in the art that other drawings can be obtained from these drawings without inventive effort.
FIG. 1 is a schematic diagram of transcriptome sequencing results of 3-month-old and 10-month-old mice according to an embodiment of the present application, wherein FIG. 1A is a PCA result graph of two groups of mice, FIG. 1B is a schematic diagram of differential expression gene analysis results of two groups of mice, and FIG. 1C is a KEGG function enrichment result graph of two groups of mice;
Fig. 2 is a schematic diagram of a fecal fungus transplantation result of an SPF young mouse and an ovarian failure mouse provided by the embodiment of the present application, wherein fig. 2A is a schematic diagram of a fecal fungus transplantation flow of an SPF young mouse and an ovarian failure mouse, fig. 2B is a schematic diagram of an ovarian HE staining result of two groups of mice, fig. 2C is a graph of a counting result of each stage of follicles of two groups of mice, fig. 2D is a schematic diagram of a staining result of Ki-67 cell proliferation, fig. 2E is a graph of a statistics result of Ki-67 cell proliferation positivity, fig. 2F is a schematic diagram of a color result of TUNEL apoptosis, and fig. 2G is a schematic diagram of a statistics result of TUNEL cell apoptosis positivity;
FIG. 3 is a schematic diagram of metagenome detection results of 3-month-old and 10-month-old C57BL/6 mice provided by the embodiment of the application, wherein FIG. 3A is an alpha diversity analysis result graph of two groups of mice, FIG. 3B is a beta diversity analysis result graph of two groups of mice, and FIG. 3C is a strain abundance graph of two groups of mice;
Fig. 4 is a graph showing the effect of paracasei johnsonii and its culture medium supernatant on ovarian aging, in which fig. 4A shows the effect of paracasei johnsonii and its culture medium supernatant on the number of oocytes, fig. 4B shows the effect of paracasei johnsonii and its culture medium supernatant on mullerian hormone, fig. 4C shows the effect of paracasei johnsonii and its culture medium supernatant on estradiol, and fig. 4D shows the effect of paracasei johnsonii and its culture medium supernatant on follicle stimulating hormone.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present application more apparent, the technical solutions of the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present application, and it is apparent that the described embodiments are some embodiments of the present application, but not all embodiments of the present application. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.
Unless otherwise specifically indicated, the various raw materials, reagents, instruments, equipment and the like used in the present application are commercially available or may be prepared by existing methods.
The inventive thinking of the application is:
The ovarian aging is characterized by the reduction of the number and quality of oocytes, the aging process is often accompanied by the change of the composition of intestinal microorganisms, and the intestinal microorganisms also have an effect on the ovarian aging, and in recent years, research has found that interaction between the ovarian aging and the intestinal microorganisms is possible. For example, it was studied that vaginal atrophy was observed in ovariectomized mice with a concomitant change in intestinal microbiota, whereas transplantation of intestinal microbiota from ovariectomized female mice into ovariectomized female mice revealed a marked relief of vaginal epithelial atrophy, which suggests that normal ovarian function is conducive to modulating intestinal microbiota in mice, which may have a regulatory effect on other parts of the body.
The gut microbiota may play a role in the progression of ovarian aging. For example, by transplanting intestinal microbiota of young (5 week old) female mice into the body of an ovarian aging mouse (42 week old), it was found that "young" intestinal microbiota remodelled the intestinal microbiota of the ovarian aging mouse via fecal transplantation and slowed the loss of ovarian function of the ovarian aging mouse by reducing follicular occlusion and apoptosis, increasing granulosa cell proliferation, and improving the immune microenvironment of the ovary. In addition, in cohort studies involving premature ovarian dysfunction (POI) females and healthy females, researchers have found that the intestinal microbiota of POI females is altered compared to healthy females and that these alterations are closely related to changes in hormone levels associated with ovarian aging. Both studies suggest that the progression of ovarian aging may be related to intestinal microorganisms.
The current research on the related mechanism of small molecule for delaying ovarian aging suggests that microorganisms may participate in the regulation and control process of drugs for delaying ovarian aging. For example, resveratrol has been found to significantly reduce the effects of tBHP-induced oxidative stress on egg laying rate and follicular number and activate SIRT1/FoxO1 and Nrf2 pathways, thereby enhancing antioxidant gene expression, while intestinal flora has been found to play a critical role in ovarian oxidative stress by maintaining tryptophan metabolism via the kynurenine pathway. However, there is currently no report on the aging of ovaries by Paramethobacterium johnsonii (Parabacteroides johnsonii) among intestinal microorganisms.
The embodiment of the application provides an application of parabacteroides johnsonii in an ovarian aging biomarker, which comprises the following steps:
Low abundance parabacteroides johnsonii was used as biomarker for ovarian aging.
In some alternative embodiments, the abundance of parabacteroides johnsonii is positively correlated with the extent of ovarian aging.
In the embodiment of the application, the abundance of the parabacteroides johnsonii and the degree of ovarian aging are defined to be positively correlated, and the specific abundance of the parabacteroides johnsonii can be definitely determined, so that the accuracy of the parabacteroides johnsonii serving as the biomarker of ovarian aging can be definitely determined.
In some alternative embodiments, the parabacteroides johnsonii is less than 0.77 times the normal parabacteroides johnsonii abundance.
In some alternative embodiments, when the abundance of parabacteroides johnsonii is less than 0.77 times the abundance of normal parabacteroides johnsonii, then the ovary is judged to be in a senescent state at that time.
In the embodiment of the application, the specific abundance of the parabacteroides johnsonii is limited to be higher than the specific multiple of the abundance of the normal parabacteroides johnsonii, so that the minimum abundance of the parabacteroides johnsonii can be clear, and the specificity and sensitivity of the parabacteroides johnsonii serving as a biomarker can be improved.
Based on one general inventive concept, embodiments of the present application provide a reagent for screening or aiding in screening for ovarian aging, the reagent comprising an agent for detecting the relative abundance of Paralopecuroides johnsonii.
The reagent is realized based on the application, and the specific principle of the application can refer to the embodiment, and because the reagent adopts part or all of the technical schemes of the embodiment, the reagent has at least all the beneficial effects brought by the technical schemes of the embodiment, and the description is omitted here.
Based on one general inventive concept, embodiments of the present application provide an agent for evaluating or aiding in the evaluation of the prognostic effect of ovarian aging, the agent comprising an agent that detects the relative abundance of Paralogue bacillus.
The reagent is realized based on the application, and the specific principle of the application can refer to the embodiment, and because the reagent adopts part or all of the technical schemes of the embodiment, the reagent has at least all the beneficial effects brought by the technical schemes of the embodiment, and the description is omitted here.
Based on one general inventive concept, the embodiment of the application provides an application of parabacteroides johnsonii in delaying ovarian aging, wherein the application comprises the application of the parabacteroides johnsonii in medicaments for delaying ovarian aging.
The application of the parabacteroides johnsonii in delaying the ovarian aging is realized based on the application of the parabacteroides johnsonii in the ovarian aging biomarker, the specific principle of the application of the parabacteroides johnsonii in the ovarian aging biomarker can be referred to the embodiment, and the reagent adopts part or all of the technical schemes of the embodiment, so that the reagent has at least all the beneficial effects brought by the technical schemes of the embodiment and is not repeated herein.
In some alternative embodiments, the delaying ovarian aging comprises at least one of:
Increasing oocyte numbers, up-regulating anti-mullerian hormone, up-regulating estradiol levels, and down-regulating follicle stimulating hormone levels.
In the embodiment of the application, the specific mode for slowing down the ovarian aging is limited, so that the parabacteroides johnsonii can influence the number of oocytes, the level of anti-mullerian hormone, the level of estradiol and the level of follicle stimulating hormone, and the specific mode of action of the parabacteroides johnsonii in slowing down the ovarian aging can be clarified.
In some alternative embodiments, the use further comprises using the fermentation product of parabacteroides johnsonii in an agent that delays ovarian aging.
In some alternative embodiments, the fermentation product of Paramycolatopsis johnsonii comprises a supernatant from culturing Paramycolatopsis johnsonii.
In the embodiment of the application, the supernatant of the parabacteroides johnsonii can be used for delaying the ovarian aging by influencing the number of oocytes, the level of anti-mullerian hormone, the level of estradiol and the level of follicle stimulating hormone, so that the parabacteroides johnsonii and a fermentation product thereof can be used as a medicament for delaying the ovarian aging.
The application will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present application and are not intended to limit the scope of the present application. The experimental procedures, which are not specified in the following examples, are generally determined according to industry standards. If there is no corresponding industry standard, it is carried out according to the general international standard, the conventional conditions, or according to the conditions recommended by the manufacturer.
Example 1
1. Experimental materials and methods:
1. test materials:
(1) Test animals C57BL/6J wild type mice of different ages were purchased from the university of agriculture animal experiment center, and all animal manipulations followed the university of agriculture animal study-related regulations.
(2) The main instruments and equipment are shown in table 1.
Table 1 instrument and apparatus
(3) The main reagents and kits are shown in table 2.
TABLE 2 Main reagents and kit
| Primary reagent name | Manufacturer or unit |
| Chloroform (chloroform) | University of agricultural equipment department of China |
| Isopropyl alcohol | University of agricultural equipment department of China |
| Absolute ethyl alcohol | University of agricultural equipment department of China |
| Paraffin wax | University of agricultural equipment department of China |
| 10% Formalin solution | University of agricultural equipment department of China |
| Fetal bovine serum | Gibco Co Ltd |
| Hematoxylin dye liquor | Beijing China fir gold bridge biotechnology Co.Ltd |
| Eosin liquid | Beijing China fir gold bridge biotechnology Co.Ltd |
| Ki-67 monoclonal antibodies | Abcam Co |
| ZO-1 monoclonal antibody | Abcam Co |
| Fecal DNA kit | OMEGA Co., ltd |
| Trizol Reagent | Company Applied Biosystems U.S |
| Taq DNA polymerase | Promega Co Ltd |
| Taq DNA Buffer | Promega Co Ltd |
| DNTP mixed liquor | Finnish HYTEST LTD Co Ltd |
| Primer(s) | Newly created Wuhan engine science Co.Ltd |
| DNA marker | TaKaRa Co Ltd |
| Reverse transcription kit | Thermo Scientific company |
| Neutral resin | Beijing China fir gold bridge biotechnology Co.Ltd |
| DAB chromogenic reagent kit | Beijing China fir gold bridge biotechnology Co.Ltd |
| AxyPrepDNA gel recovery kit | OMEGA Co., ltd |
| Transfer buffer solution | Gibco Co Ltd |
2. The experimental method comprises the following steps:
(1) Raising mice:
the mice of each group are fed with standard diet and drinking water for one week, and the feed fed to the mice is sterilized by strict radiation, and the drinking water bottle, the feeding cage and the padding are sterilized by high-pressure steam for use.
The raising environment is SPF level, the setting environment temperature is 24 ℃ plus or minus 1 ℃, and the automatic lighting device is adjusted according to the daytime. All procedures followed guidelines approved by the ethical committee of animal experiments at agricultural university in China.
(2) Hematoxylin-eosin (H & E) staining:
1) Selecting paraffin sections with intact sections, and drying the sections in a 65 ℃ oven for 50min;
2) Dewaxing the slices until water is xylene I for 15min, xylene II for 15min,100% alcohol I for 5min,100% alcohol II for 5min,95% alcohol for 5min,90% alcohol for 5min,80% alcohol for 5min,70% alcohol for 5min and distilled water for 5min;
3) H & E staining, namely hematoxylin 8min, tap water flushing 15min,70% alcohol 4min and eosin 1min;
4) Dehydrating 95% alcohol I for 3min,95% alcohol II for 3min,100% alcohol I for 3min, and 100% alcohol II for 3min;
5) Transparent xylene I for 5min and xylene II for 5min;
6) Sealing the neutral resin, covering with cover glass, and air drying at room temperature.
(3) Tissue immunofluorescence
1) Selecting paraffin sections with intact sections, and drying the sections in a 65 ℃ oven for 50min;
2) Dewaxing paraffin slice until water contains xylene I20 min, xylene II 20min,100% alcohol I5 min,100% alcohol II 5min,95% alcohol 5min,90% alcohol 5min,80% alcohol 5min,70% alcohol 5min, and distilled water 5min;
3) Antigen retrieval, namely placing a tissue slice into a retrieval box filled with 0.01mol/L sodium citrate buffer solution (pH is 7.0), performing antigen retrieval in a microwave oven, cutting off power for 10min after medium fire is boiled, and then using medium low fire to boil again, wherein the process is to prevent excessive evaporation of the buffer solution and prevent a piece from being dried;
4) BSA blocking, namely after spin-drying the slice, circling the tissue by a histochemical pen (preventing antibody from flowing away), dripping 3% BSA in the circle to cover the tissue, and blocking for 30min at room temperature;
5) Adding a primary antibody, namely lightly throwing off a sealing solution, dropwise adding the primary antibody prepared by PBS according to a certain proportion on a slice, horizontally placing the slice into a wet box, and incubating overnight at 4 ℃ (a small amount of water is added into the wet box to prevent the antibody from evaporating);
6) Adding a secondary antibody, namely placing a slide in PBS (pH 7.4), shaking and washing on a decolorizing shaking table for 3 times, each time for 5min, dripping a secondary antibody covering tissue corresponding to the primary antibody in a circle after slicing and spin-drying, and incubating for 50min at room temperature in a dark place;
7) The cell nucleus is counterstained by DAPI, the slide is placed in PBS (pH 7.4) and washed 3 times in 5min on a decolorizing shaker, after DAPI is added dropwise for incubation for 10min, the slide is washed 3 times in PBS (pH 7.4) for 5 min;
8) Spin-drying the slide, sealing the slide with an anti-quenching agent, and photographing.
(4) EdU staining:
EdU staining kit was purchased from Sharp organism Inc. of Guangzhou, and specific experimental procedures were performed with reference to kit (C10310) instructions.
(5) TUNEL staining:
Apoptosis was detected by TUNEL method by taking testes of wild type and knockout mice, fixing, dehydrating and transparentizing, embedding into paraffin sections, and performing TUNEL staining according to the following procedure:
1) Baking slices, namely picking paraffin slices with intact cut surfaces, and baking the slices in a 65 ℃ baking oven for 50min;
2) Dewaxing the slices until water is xylene I for 15min, xylene II for 15min,100% alcohol I for 5min,100% alcohol II for 5min,95% alcohol for 5min,90% alcohol for 5min,80% alcohol for 5min,70% alcohol for 5min and distilled water for 5min;
3) Washing, namely washing once by adopting 0.85% NaCl for 5min, and washing once by adopting PBS for 5min;
4) Fixing 4% paraformaldehyde for 15min;
5) Washing with PBS for 5min for three times;
6) Penetration proteinase K (volume ratio 1:500, PBS dilution) incubation for 10min at room temperature;
7) Washing with PBS for 5min for three times;
8) Fixing by 4% paraformaldehyde for 5min;
9) Washing with PBS for 5min for three times;
10 Equilibration, namely dripping rTdT reaction equilibration liquid, and incubating for 10min at room temperature;
11 Wiping off the balance liquid, dripping TdT reaction liquid (balance liquid: rTdT enzyme: dUTP substrate=98:1:1) prepared in advance, and reacting for 1.5h at 37 ℃;
12 Terminating the reaction by incubating in 2 XSSC for 15 min;
13 Washing with PBS for three times, each time for 5min;
14 Inactivating, namely, adopting H 2O2 with the treatment concentration of 3% to incubate for 5min at room temperature so as to inactivate endogenous peroxidase;
15 Washing with PBS for three times, each time for 5min;
16 Dripping HRP reactant (volume ratio 1:500, PBS dilution), and incubating for 30min at room temperature;
17 Washing with PBS for three times, each time for 5min;
18 Dripping DAB color development liquid (water: substrate: dilution liquid: H 2O2 = 17:1:1), immediately observing under a microscope to master proper color development degree, and immediately stopping;
19 Terminating the development by washing with distilled water;
20 Counterstaining, namely dripping hematoxylin dye solution for 30sec, and flushing with tap water for 10min;
21 Differentiation, using 1% hydrochloric acid alcohol, 5sec;
22 Distilled water washing for 5min;
23 dehydration, 50% alcohol 3min,70% alcohol 3min,80% alcohol 3min,90% alcohol 3min,100% alcohol I3 min,100% alcohol II 3min;
24 Transparent, wherein the xylene I is 3min, and the xylene II is 3min;
25 Sealing with neutral resin, and observing and photographing under microscope.
(6) Superovulation and mating:
For the superovulation experiments in mice, 21d size female mice were intraperitoneally injected with pregnant equine gonadotropins (PREGNANT MARE serum gonadotropin, PMSG) for 44h, hCG was injected for 16h, COC complex cell mass was removed from the ampulla of the oviduct, and cumulus cells were fully digested in hyaluronic acid (1:10 in M2). Female mice at 8 weeks of age were naturally mated with male mice, and the vaginal suppositories of the female mice were checked the next morning to see the suppositories as signs of successful mating.
(7) Collecting and culturing bare eggs
1) Preparation and equilibration of microdroplets (about 100. Mu.L each of M16 broth, 1ng/mL rh-AMHM broth, 10ng/mL rh-AMH M16 broth, and 100ng/mL rh-AMH M16 broth) were prepared 30min before incubation, and after the microdroplets were covered with paraffin oil, they were placed in an incubator for pH equilibration. Meanwhile, adding a little M16 culture solution into a 1.5mL centrifuge tube, and putting into an incubator for balancing so as to clean oocytes;
2) Culturing bare eggs, namely taking 5 female mice of 3 weeks old, rapidly killing the female mice after neck breakage, taking out ovaries on two sides, placing the ovaries in PBS for cleaning for a plurality of times to remove residual blood stains and fat tissues on the surfaces of the ovaries, placing the ovaries in a disposable culture dish (35 mm) at the center, chopping the ovaries by a blade to release the oocytes from the follicles, adding a proper amount of bare egg picking liquid to suspend ovary tissue fragments, picking the oocytes in GV stage by a self-made oral suction tube under a stereoscopic microscope, and then transferring the cleaned oocytes into droplets prepared in advance for culturing.
3. Experimental results:
1. SPF mice of 3M and 10M were selected and the aging changes of ovarian tissue were characterized using transcriptome:
SPF grade C57BL/6 mice 3 months of age and 10 months of age, respectively, were selected to represent young and ovarian aging status, respectively, and the ovaries of both groups of mice were transcriptomically sequenced.
The PCA results are shown in fig. 1A, with the 3 month old and 10 month old mouse ovarian samples being clearly distinguishable at PC1, indicating significant changes in the ovarian expression profile during aging.
The results of KEGG functional enrichment are shown in FIG. 1C, the pathways of B cell receptor signals, NF- κB signals, MAPK signals and the like are up-regulated along with aging, which suggests that the inflammatory and stress reactions caused by the aging of ovaries are increased, while the pathways of cell cycle, oocyte meiosis, PI3K-Akt signals and the like are down-regulated along with aging, which show that the functions of ovaries are reduced, the aging of cells and the reduction of follicular reserves, and the results are consistent with the results of previous researches.
2. The influence of intestinal microorganisms on ovarian aging is explored through a fecal transplantation experiment:
In order to explore whether intestinal microorganisms can influence the ovarian aging, the application adopts a fecal fungus transplanting technology, the fecal fungus of SPF young mice and ovarian aging mice is transplanted into a sterile mouse body (as shown in figure 2A) at the same time, and the relevant phenotype of the ovarian aging is detected after the stomach irrigation is finished. The results showed that the ovaries of recipient mice (senescent group) transplanted with the ovarian failure mice fecal bacteria were more senescent than those of recipient mice (young group) transplanted with the young mice fecal bacteria, the young group mice were found to have a greater number of luminal follicles and a lesser number of occluded follicles by staining the ovaries HE and counting the follicles at each stage (as shown in fig. 2B and 2C), in addition to the Ki-67 cell proliferation experiments, which showed that the young group mice had significantly higher ovarian granulosa cell proliferation rate than the senescent group (as shown in fig. 2D and 2E), and the TUNEL cell apoptosis experiments showed that the young group mice had significantly lower ovarian granulosa cell apoptosis rate than the senescent group (as shown in fig. 2F and 2G). The above results indicate that intestinal microorganisms can influence the ovarian aging process.
3. Detecting the change of intestinal microorganism composition in the ovarian aging process, and screening strains related to ovarian aging:
Wild type C57BL/6 mice were selected as a study model, and feces from young (3 months old) and ovaries aged (10 months old) mice were collected for metagenomic detection. After standard flow treatment, the alpha diversity and the beta diversity are evaluated and the strain abundances of the two groups of samples are compared respectively, and the results are shown in a figure 3, which shows that intestinal microorganisms of young and ovary-attenuated mice have no great difference in flora structure, but part of the strain abundances have obvious differences (shown in figures 3A, 3B and 3C), wherein the common bacteroides shows high abundance at 10 months, and the current research shows that the bacteria can influence the normal functions of ovaries and induce polycystic ovary syndrome, while the Acremodelling bacteria and the parabacteroides johnsonii (Parabacteroides johnsonii) show high abundance at 3 months, and the research shows that the two strains are consumed after female menopause, and the parabacteroides johnsonii is obviously reduced in the intestinal tracts of the aged mice at 10 months.
The above results indicate that there is a difference in intestinal microorganisms in young and in ovariectomized mice, and that this difference may be related to ovarian function.
4.Parabacteroides johnsonii slow down ovarian aging in mice:
Parabacteroides johnsonii (experimental group) and normal saline (control group), and supernatant (experimental group) and blank medium (control group) in which Parabacteroides johnsonii medium was cultured were respectively perfused into six month-old C57 female mice, and the respective groups of mouse oocytes were collected by continuous intragastric administration for 12 weeks, and subjected to number statistics, and serum of each group of mice was collected to detect hormone levels in blood. The results are shown in fig. 4, which demonstrates that Parabacteroides johnsonii and supernatant from its culture medium significantly increased oocyte numbers compared to the control group (as shown in fig. 4A), and up-regulated anti-mullerian hormone and estradiol levels (as shown in fig. 4B and 4C), down-regulated Follicle Stimulating Hormone (FSH) levels (as shown in fig. 4D).
In summary, the application of the parabacteroides johnsonii in the ovarian aging biomarker provided by the embodiment of the application finds that the parabacteroides johnsonii has obvious differences in mice with different ovarian aging degrees through experiments, and further confirms that the parabacteroides johnsonii has the effects of obviously increasing the number of oocytes, up-regulating anti-mullerian hormone, up-regulating estradiol level and down-regulating follicle-stimulating hormone level through subsequent experiments, which indicates that the parabacteroides johnsonii can be used as a medicament for delaying ovarian aging.
Various embodiments of the application may exist in a range format, it being understood that the description in a range format is merely for convenience and brevity and should not be construed as a rigid limitation on the scope of the application, as the range format described above specifically disclosing all possible sub-ranges and individual values within the range. For example, it should be considered that a description of a range from 1 to 6 has specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the range, such as 1,2, 3,4, 5, and 6, wherever applicable. In addition, whenever a numerical range is referred to herein, it is meant to include any reference number (fractional or integer) within the indicated range.
In the present application, unless otherwise specified, terms such as "upper" and "lower" are used specifically to refer to the orientation of the drawing in the figures. In addition, in the description of the present specification, the terms "include", "comprising" and the like mean "including but not limited to". Relational terms such as "first" and "second", and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Herein, "and/or" describes an association relationship of an association object, which means that there may be three relationships, for example, A and/or B, and may mean that A exists alone, while A and B exist together, and B exists alone. Wherein A, B may be singular or plural. Herein, "at least one" means one or more, and "a plurality" means two or more. "at least one", "at least one" or the like refer to any combination of these items, including any combination of single item(s) or plural items(s). For example, "at least one (a), b, or c)", or "at least one (a, b, and c)", may each represent a, b, c, a-b (i.e., a and b), a-c, b-c, or a-b-c, wherein a, b, c may be single or multiple, respectively.
The foregoing is only a specific embodiment of the application to enable those skilled in the art to understand or practice the application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the application. Thus, the present application is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (10)
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