CN112126671B

CN112126671B - Application of streptococcus agalactiae streptococcus agalactiae in treating endometriosis

Info

Publication number: CN112126671B
Application number: CN202010833844.XA
Authority: CN
Inventors: 何欢欢; 曾发敏; 严志祥; 刘长青; 谢冰帆; 何建忠; 庄圆
Original assignee: Fifth Affiliated Hospital of Sun Yat Sen University
Current assignee: Fifth Affiliated Hospital of Sun Yat Sen University
Priority date: 2020-08-18
Filing date: 2020-08-18
Publication date: 2021-08-31
Anticipated expiration: 2040-08-18
Also published as: CN112126671A

Abstract

The invention belongs to the field of biotechnology, and identifies that streptococcus agalactiae of streptococcus is abnormally increased in cervical orifice definite value of a patient with endometriosis by sequencing. Through clinical samples and construction of an endometriosis mouse model, researches show that streptococcus agalactiae regulates and controls Ang-2 to promote angiogenesis, and further, the fixed value and the invasion capacity of ectopic tissues in an abdominal cavity are enhanced. After the teicoplanin is added to inhibit streptococcus agalactiae, the number and the size of ectopic disease focuses in the abdominal cavity of the mouse model with endometriosis are obviously inhibited, so that a new way for treating endometriosis is confirmed, and a new strategy is provided for treating endometriosis.

Description

Application of streptococcus agalactiae streptococcus agalactiae in treating endometriosis

Technical Field

The invention relates to the technical field of gynecology, in particular to application of streptococcus agalactiae streptococcus pneumoniae agalactiae in treating endometriosis.

Background

Endometriosis is a common gynecological disease caused by the implantation of active endometrial cells outside uterine cavities, and has the characteristics of wide lesion, various tissue forms, invasiveness, relapse and the like. The disease affects about 10% to 15% of women of childbearing age, and the proportion is up to 50% among infertility women, and the incidence rate rises year by year in recent years, causing great harm to the health of women. The typical clinical symptoms are chronic pelvic pain, congestive dysmenorrhea, heavy menstrual bleeding, advanced dyspareunia and fatigue, which are associated with a great deal of suffering in endometriosis patients due to a lack of effective treatment modalities. The current treatment means mainly comprises surgical treatment and drug treatment, and is assisted by traditional Chinese medicine treatment and assisted reproduction technology, the obtained treatment effect is limited, and the postoperative recurrence rate is high.

With respect to the cause of endometriosis, it is currently widely accepted by the Morpson theory that endometriosis is caused by the retrograde movement of menstrual blood to bring endometrial tissue sloughed off during the menstrual cycle outside the uterine cavity and implant. In the process, the fallen retrograde endometrium can be planted, grown and formed into the focus of endometriosis through the processes of adhesion, invasion, angiogenesis and the like. Studies have shown that after endometrioid epithelial cells enter the pelvic cavity and colonize other tissues, ectopic endometrial tissues rapidly establish their own blood supply, a prerequisite for their successful colonization and long-term survival. Angiogenesis factor-regulated neovascularization is considered to be a key factor in the progression of endometriosis. Among many growth factors regulating angiogenesis, Ang-2 expression is a reinforcing factor for angiogenesis initiation and is closely related to neovascularization and enhancement.

In addition to the pathogenic factors causing the occurrence and development of endometriosis such as menstrual blood reflux, hormones, immunogenetics and epigenetics, the genital tract microorganisms of endometriosis patients play an important role in promoting the process of endometriosis. Further research shows that the colonization of intrauterine microorganisms is closely related to the cascade reaction of the innate immune system, which jointly promotes the formation of endometriosis. Although studies have shown that microorganisms of the female genital tract play an important role in the development and progression of endometriosis, the specific molecular mechanisms are still unclear. Therefore, it is necessary to provide a new solution for clinical treatment by undoubtedly elucidating the biological behavior and molecular mechanism of the female genital tract microorganisms during endometriosis.

Disclosure of Invention

The technical problem to be solved by the present invention is to overcome the lack of effective diagnostic or/and therapeutic means for endometriosis in the prior art, and firstly to provide a method for diagnosing endometriosis.

It is a second object of the present invention to provide a medicament for the treatment of endometriosis.

The third purpose of the invention is to provide the application of streptococcus agalactiae streptococcus agalactiae in preparing a diagnostic reagent for endometriosis.

The purpose of the invention is realized by the following technical scheme:

a medicament comprising streptococcus agalactiae for use in the diagnosis of endometriosis.

According to the invention, firstly, 16SrRNA of cervical secretion of a patient suffering from endometriosis is sequenced, and a bioinformatics analysis result shows that the abundance of streptococcus agalactiae is abnormally increased and the streptococcus agalactiae is abnormally planted in the cervical of the patient. Therefore, Streptococcus agalactiae in the cervix becomes a molecular marker of endometriosis, and the possibility of endometriosis can be determined by detecting the number of Streptococcus agalactiae in the cervix of a patient.

Secondly, QPCR and ELISA experiments show that the colonization abundance of the streptococcus agalactiae in the cervix of the endometriosis patient is obviously and positively correlated with the expression level of Ang-2 in serum, and the combination of the two can obviously improve the accuracy of diagnosing the endometriosis.

Therefore, the method for diagnosing endometriosis according to the present invention further comprises a substance for detecting the expression level of Ang-2 in serum.

The invention also discovers that the quantity and the size of the endometriosis ectopic focus can be obviously reduced by inhibiting the streptococcus agalactiae through constructing an endometriosis mouse model, wherein the change of the red ectopic focus is particularly obvious.

Accordingly, the present invention also provides a medicament for the treatment of endometriosis comprising a substance for use against streptococcus agalactiae. Preferably, the drug is teicoplanin.

Previous studies have demonstrated that the formation of new blood vessels is a prerequisite for successful colonization of the endometrium in ectopic sites. According to the invention, firstly, the discovery that the streptococcus agalactiae planted in ectopic cervical orifices promotes the formation of endometriosis is firstly carried out, and on the basis of the discovery, the relationship between the streptococcus agalactiae and the endometriosis is further explored, so that the new discovery that the abundance of the streptococcus agalactiae in the cervical orifices is obviously and positively correlated with the expression level of Ang-2 in serum is obtained, and previous researches show that Ang-2 plays an important role in mediating the formation of new blood vessels. Later, the research of inhibiting streptococcus agalactiae by adding teicoplanin shows that the quantity and the size of endometriosis are obviously reduced. Therefore, the invention emphasizes that the formation of endometriosis is influenced by streptococcus agalactiae in the cervical orifice, the permeability of the streptococcus agalactiae can up-regulate Ang-2 to mediate the formation of new blood vessels, promote the occurrence and the development of endometriosis, and can achieve the effect of inhibiting the occurrence and the development of endometriosis by inhibiting the streptococcus agalactiae.

Therefore, it is preferable that the above-mentioned medicament for treating endometriosis according to the present invention further comprises a substance for reducing the expression level of Ang-2 in serum.

The invention also provides the application of streptococcus agalactiae in preparing a diagnostic reagent for endometriosis.

Compared with the prior art, the invention has the following beneficial effects:

the sequencing of the invention identifies that the quantitative value of streptococcus agalactiae of the streptococcus genus is abnormally increased in the cervical orifice of a patient with endometriosis. Through clinical samples and construction of an endometriosis mouse model, researches show that streptococcus agalactiae regulates and controls Ang-2 to promote angiogenesis, and further, the fixed value and the invasion capacity of ectopic tissues in an abdominal cavity are enhanced. After the teicoplanin is added to inhibit streptococcus agalactiae, the number and the size of ectopic disease focuses in the abdominal cavity of the mouse model with endometriosis are obviously inhibited, so that a new way for treating endometriosis is confirmed, and a new strategy is provided for treating endometriosis.

Drawings

FIG. 1 is a bioinformatics analysis diagram of cervical secretions of a patient with endometriosis, wherein EMS is a sample of the patient with endometriosis;

FIG. 2 is a schematic illustration of standard solution preparation dilution;

FIG. 3 is an Alpha diversity analysis diagram, in which, Chaol reaction flora abundance, Shannon represents diversity index (quantification index of flora abundance and equilibrium, more sensitive to abundance, larger value, larger sample diversity), pielou _ e represents the measure of flora uniformity, higher value, larger flora uniformity (less dominant species), Simpson represents diversity index (qualitative measure of flora abundance. the range is between 0-1, larger value, larger sample diversity);

FIG. 4 is an Alpha and Beta analysis chart, wherein FIG. 4A is a cervical microbial genus diversity significance analysis; FIG. 4B is a PLS-DA analysis (left panel, supervised classification, for predicting actual differences in samples or sample groupings) and a PCoA analysis (right panel, principal coordinates analysis, for determining variations between samples);

FIG. 5 is a differential enrichment genus analysis diagram; wherein FIG. 5A is a LEfSe analysis chart, FIG. 5B is a phylogenetic tree chart, and FIG. 5C is a Streptococcus agalactiae abundance scattergram;

FIG. 6 is the expression of Ang-2 in the serum of a patient with endometriosis, wherein FIG. 6A is the expression curve of Ang-2 in different stages of endometrium, and FIG. 6B is the expression level statistical chart of Ang-2;

FIG. 7 is a graph showing the relationship between the genus related to endometriosis patients and the expression of Ang-2 in serum, FIG. 7A is a correlation analysis between the genus closely related to endometriosis and Ang-2, FIG. 7B is a ROC analysis, and FIG. 7C is a PICRUSt function prediction analysis graph;

FIG. 8 is a test of the effect of Streptococcus agalactiae on the development and onset of endometriosis; wherein, the left lower side paper is a focus of mouse abdominal cavity endometrium ectopic focus;

FIG. 9 is a graph of the effect of Streptococcus agalactiae on red and white foci of endometriosis.

Detailed Description

The following further describes the embodiments of the present invention. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

The test methods used in the following experimental examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.

All subjects in the trial signed informed consent, and the general data differences between patients were not statistically significant.

Example 1 analysis of microbial diversity in the cervical opening of a patient with endometriosis

Firstly, collecting cervical oral secretions of 10 cases of endometriosis patients and cervical secretions of 10 cases of normal-examination people, performing OUT characteristic analysis, phylogenetic tree construction, Alpha and Beta diversity analysis, PCoA and NNMDS analysis, PLS-DA, LEfSe analysis and PICRUSt function prediction analysis by combining 16SrRNA sequencing and bioinformatics, and obtaining results shown in figures 1, 3 and 4. As can be seen from fig. 1, 3 and 4A to 5C, the endometriosis patients have a more abundant microbial diversity in the cervical orifice compared to the normal control group; as can be seen from FIG. 4B, the microbial diversity in the cervical orifice of endometriosis patients was more similar than that in the normal control group. As indicated above, patients with endometriosis have an increased microbial diversity in the cervical orifice and similar microbial characteristics.

Secondly, analyzing the colonization condition of streptococcus agalactiae at the cervix of a patient with endometriosis

LDA analysis and a cladogram show that the microbial genera are differentially enriched in endometriosis, and as can be seen from FIG. 5, the abundance of Streptococcus in the cervical opening of a patient with endometriosis is increased, with the Streptococcus agalactiae being the most obvious.

Thirdly, analysis of the expression of Ang-2 in serum of patients with endometriosis

1. ELISA procedure [ R & D, Human Angiopoietin-2Immunoassay (Catalog Number DANG20) ]:

1.1 sample Collection and storage, all reagents are left at room temperature

(1) Clinical serum samples were processed at the early stage and stored at-80 ℃ in aliquots.

(2) The samples were thawed on ice using an ice box prior to the experiment.

1.2 preparation of reagents

Wash buffer Wash: if crystals form in the concentrate, warm to room temperature and mix gently until the crystals are completely dissolved. 20mL of the wash buffer concentrate was added to deionized or distilled water to prepare 500mL of the wash buffer. 480ml of distilled water +20ml of concentrated washing solution were 500ml of wash buffer.

Substrate solution: the colour reagents a and B should be mixed together in the same volume within 15 minutes after use. And (4) avoiding light. 200 μ L of the resulting mixture was required per well.

1.3 preparing gradient standard substance

(1) Human Angiopoietin-2Standard was diluted-please see sample vial label.

The human angiopoietin 2standard is prepared by deionized water. Stock solutions of 30,000pg/mL were prepared and gently stirred for at least 15 minutes prior to dilution.

(2) Using a polypropylene tube, a stock solution of 30,000pg/mL was diluted at a factor of 30,000pg/mL with the standard diluent RD5-5 to give a concentration of: 3000pg/mL, 1500pg/mL, 750pg/mL, 375pg/mL, 187.5pg/mL, 93.7pg/mL, 1500pg/mL, 46.9pg/mL, 0pg/mL, and the like.

1.4 first add 100. mu.L of assay diluent RD1-76 to each well

1.5, 50. mu.L of the standard solution diluted in the step 1.3(2) and the serum sample were added to each well. Incubate at room temperature for 2 hours on a shaker at (maximum speed) 500rpm ± 50 rpm.

1.6, aspirate each well and wash, repeat the procedure three times for a total of four washes. Each well was filled with wash buffer (400. mu.L) for washing. Complete removal of liquid in each step facilitates thorough washing. After the last wash, any remaining wash buffer is removed by aspiration or decantation. The plate was inverted and wiped dry with a clean paper towel.

1.7, 200. mu.L of human angiopoietin 2 conjugate was added to each well, covered with a fresh tape, and incubated on a shaker at room temperature for 2 hours.

1.8, repeat pumping/washing as in step 1.6.

1.9 Add 200. mu.L of substrate solution per well and incubate on the bench for 30 min at room temperature, protected from light.

1.10, adding 50 mu L of stop solution into each hole, wherein the color of each hole changes from blue to yellow; if the wells were green in color or the color variation was not uniform, please tap the plate gently to ensure thorough mixing.

1.11, determining the optical density of each well within 30 minutes using a microplate reader set at 450 nm; if wavelength correction is possible, set to 540nm or 570 nm; subtracting the reading at 540nm or 570nm from the reading if there is no wavelength correction, 450 nm; this subtraction will correct for optical defects in the plate. Readings taken directly at 450nm without correction may be higher and less accurate.

1.12 calculation of results

Duplicate readings for each standard, control and sample were averaged and the average zero standard Optical Density (OD) was subtracted.

And calculating a standard curve according to the concentration of the standard solution diluted by multiple times and the corresponding OD value, and further calculating the concentration of the sample to be detected according to the standard curve and the OD value of the sample to be detected.

Through ELISA experiments, the serum Ang-2 expression level of the endometriosis patient is increased compared with that of a normal control group, wherein the increase is particularly obvious in the secretion period (shown in figure 6).

Fourth, correlation analysis of cervical Streptococcus agalactiae and serum Ang-2 concentration of endometriosis patients

The experiment included the following procedures:

(1) extracting the abundance value of the streptococcus agalactiae in the sequencing result of the cervical secretion of the patient with the endometriosis at the early stage;

(2) screening a blood sample corresponding to a prophase sequencing patient, and detecting the expression level of Ang-2 according to the ELISA experiment steps;

(3) SPSS performs a relevant statistical analysis.

Correlation analysis shows that the abundance of Streptococcus agalactiae in the cervix of a patient with endometriosis is obviously and positively correlated with the expression of serum Ang-2, and the combination of the two can obviously improve the accuracy of diagnosing endometriosis (shown in figure 7A/B); PICRUSt function prediction shows that the abnormal cervical orifice colonizing bacteria mainly relate to metabolism-related passages.

Fifth, study of the Effect of Streptococcus agalactiae on endometriosis

Construction of mouse model of endometriosis: c57BL/65 female mice were serially smeared with vaginal secretions and mice with a complete oestrus cycle were selected as donor and recipient mice. The donor mice were injected with estradiol, and were all in estrus. One week later, the endometrium is cut into pieces of about 1mm³Injected into the abdominal cavity of a receptor mouse, and dissected on the 7 th day and the 14 th day by estradiol intramuscular injection; the result of HE staining of suspicious lesions is taken to show that the forming rate of ectopic lesions is 100 percent after one week, a typical HE graph is shown in figure 8, and the specific experimental steps are as follows:

the experimental steps are as follows:

(one) screening mice with oestrus cycle

Continuously performing mouse vaginal cell smear for 5 days, observing the oestrus cycle change of the mouse by HE staining, and screening the mouse with relatively normal oestrus cycle;

HE staining procedure was as follows:

(1) after the mouse vaginal cell routine smear is slightly dried, fixing the mouse vaginal cell routine smear for 10-15min by 95% ethanol;

(2) dewaxing: xylene I, II and III are respectively added for 10 min;

(3) hydration: 100%, 90%, 80%, 70% ethanol for 5min each;

(4) washing with tap water for 5min/3 times;

(5) staining with hematoxylin for 5 min;

(6) differentiation with 5% acetic acid;

(7) returning blue by PBS for 3 min;

(8) staining with eosin for 1 min;

(9) and (3) dehydrating: 70%, 80%, 90% and 100% ethanol for 10s each, and 1min xylene;

(10) and (5) sealing the neutral gum.

(II) modeling

(1) Weighing the weight of the mouse, and calculating the dosage of the estradiol for injection; (molecular weight: 272.382; density: 1.17 g/cm)³；1cm³＝1ml；1mg＝1000ug＝1*10⁶ng)

(2) Injecting 98% estradiol 100ng subcutaneously every day continuously three days before modeling, so that the mice are uniformly in a uniform estrus cycle-estrus;

(3) carrying out mouse vaginal smear before modeling, observing whether the mouse is in estrus, and selecting the mouse in estrus as a donor mouse;

(4) dissecting 15 mice in estrus, carefully separating out uterus, peeling off endometrium, placing in 6cm dish filled with PBS/normal saline, washing off blood stain, and cutting into small pieces of 1mm x 1 mm;

(5) sucking 1ml of the mixture by a No. 16 needle, injecting the mixture into the abdominal cavity of the mouse by intraperitoneal injection;

(6) subcutaneous injection of recipient mice the next day after modelingEstradiol, 0.1mg/kg^—1/d^-1Every 3 days to promote the growth of endometrium;

(7) mice were dissected at 21 days each;

(8)CO₂mice were sacrificed, dissected, and observed for lesions and mouse endometrium (for comparison);

(9) taking the suspicious focus, taking a picture, and fixing the suspicious focus in 4% formaldehyde/paraformaldehyde.

(10) HE staining, and observing whether the suspicious part has the subtopic morphology of endometrium under a microscope and whether the suspicious part is consistent with the normal endometrial cycle of the mouse.

(III) treatment with bacterial liquid and antibiotics

(1) Streptococcus agalactiae strains purchased from ATCC (ATCC No. 13813) and Streptococcus agalactiae strains isolated from human genital tract were inoculated on blood agar plates at 37 ℃ with 5% CO₂After 24h of culture, a milky single colony which is unique to the streptococcus agalactiae grows out, and beta hemolytic ring exists.

(2) Selecting the single colony, shake culturing with brain heart infusion Broth (BHI) culture medium, taking the amplified bacterial liquid as stock solution after 24 hr, diluting the stock solution by 10 times, and sequentially diluting to 1 × 10¹Multiple, 1 × 10²Multiple, 1 × 10³Multiple, 1 × 10⁴Multiple, 1 × 10⁵Multiple, 1 × 10⁶Multiple, 1 × 10⁷Multiple, 1 × 10⁸Double, take each dilution concentration and plate. 37 ℃ and 5% CO₂After 24h incubation, photographs were taken and colonies were counted. The bacteria count of the original bacterial liquid was calculated from the colony count.

(3) Three days after molding, ATCC standard strain and clinical strain of Streptococcus agalactiae were treated at 1X 10 per mouse⁵Intraperitoneal injection is carried out one/only/week.

(4) The antibiotic treatment can be carried out on the third day of the bacterial liquid injection, the intramuscular injection is carried out according to the amount of 8mg/kg, the injection is carried out twice in the morning and at night every day in the first three days, the injection is changed into once every day, and the injection lasts until the final sacrifice.

As can be seen from FIG. 8, the standard Streptococcus agalactiae strain (ATCC) and the clinical Streptococcus agalactiae strain isolated from the clinical patients significantly increased the number and size of endometriosis ectopic foci compared with the Control group (Control); teicoplanin inhibition of streptococcus agalactiae significantly reduced the number and size of endometriosis ectopic foci. The results show that the streptococcus agalactiae can promote the occurrence and the development of endometriosis, and the inhibition of the streptococcus agalactiae by teicoplanin can reduce the number and the size of endometriosis ectopic foci, so that the effect of treating endometriosis is achieved.

Sixth, the Effect of Streptococcus agalactiae on the number and size of ectopic Red lesions

The experimental process comprises the following steps:

(1) 5 days of mouse vaginal cell smear is carried out through the experimental steps to observe the oestrus cycle change of the mice, and 30 mice are preliminarily judged to have relatively normal oestrus cycles;

(2) modeling

2.1, weighing the weight of the mouse, and calculating the dosage of the estradiol for injection; (molecular weight: 272.382; density: 1.17 g/cm)³；1cm3＝1ml；1mg＝1000ug＝1*10⁶ng)；

2.2, injecting 98% estradiol and 100ng subcutaneously every day continuously from three days before modeling, so that 15 mice are uniformly in a uniform estrus cycle-estrus;

2.3, carrying out mouse vaginal smear before modeling, observing whether the mouse is in estrus, and selecting the mouse in estrus as a donor mouse;

2.4, taking 15 mice in estrus for dissection, carefully separating out the uterus, stripping the endometrium, putting into 6cm dish filled with PBS/normal saline, washing off blood stains, and cutting into small blocks of about 1mm x 1 mm;

2.5, sucking 1ml of the mixture by using a No. 16 needle, injecting the mixture into the abdominal cavity of the mouse through intraperitoneal injection;

2.6 subcutaneous injection of estradiol, 0.1mg/kg, into recipient mice the next day after modeling^—1/d^-1Every 3 days to promote the growth of endometrium;

2.7, dissecting one mouse at 14 days and 21 days respectively;

2.8、CO₂mice sacrificed by methodsDissecting, observing whether there is focus and mouse endometrium (comparing);

2.9, taking the suspicious lesion, and fixing in 4% formaldehyde/paraformaldehyde.

2.10 HE staining, and observing whether the suspicious part has the subtopic morphology of endometrium under a microscope and whether the suspicious part is consistent with the normal endometrium cycle of the mouse.

As can be seen in FIG. 9, Streptococcus agalactiae promoted more marked changes in the red ectopic lesions than the control group; while teicoplanin significantly inhibited the number and size of red ectopic lesions. The red ectopic focus is obviously related to angiogenesis, so the result is combined to indicate that the streptococcus agalactiae in the cervical orifice promotes the generation and the development of the endometriosis, the endometriosis is predicted by the great potential of the streptococcus agalactiae combined with Ang-2, and the targeted streptococcus agalactiae in the cervical orifice provides a new strategy for treating the endometriosis.

It will be appreciated by those skilled in the art that the use of the present invention is not limited to the specific applications described above. The invention is also not limited to the preferred embodiments thereof with respect to the specific elements and/or features described or depicted herein. It should be understood that the invention is not limited to the disclosed embodiment or embodiments, but is capable of numerous rearrangements, modifications and substitutions without departing from the scope of the invention as set forth and defined by the following claims.

Claims

1. Used for resisting streptococcus agalactiae (I) in cervical orificeStreptococus agalactiae) The use of the substance of (1) for the manufacture of a medicament for the treatment of endometriosis, said medicament for the treatment of endometriosis further comprising a substance for reducing the level of Ang-2 expression in serum.

2. Use according to claim 1, for combating Streptococcus agalactiae (S.agalactiae) of the cervical orificeStreptococus agalactiae) The substance of (1) is teicoplanin.