CN113209313A

CN113209313A - Application of Tgfbr2 in preparation of ovarian function protection medicine

Info

Publication number: CN113209313A
Application number: CN202110713595.5A
Authority: CN
Inventors: 王世宣; 张金金; 马菱蔚
Original assignee: Tongji Medical College of Huazhong University of Science and Technology
Current assignee: Tongji Medical College of Huazhong University of Science and Technology
Priority date: 2021-06-25
Filing date: 2021-06-25
Publication date: 2021-08-06

Abstract

The invention discloses an application of Tgfbr2 in preparing an ovarian function protection medicine, and belongs to the technical field of tumor fertility protection. Specifically, a gene therapy method is adopted to inject a certain dose of AdV-Tgfbr2 into the ovary of a tested mammal body in situ, and the overexpression level of Tgfbr2 is detected. The damaged part is repaired to realize the purpose of rebuilding the ovary function. Therefore, the AdV-Tgfbr2 recombinant vector and the pharmaceutical preparation thereof can protect the ovarian function without influencing the anti-cancer effect, and play an important role in repairing ovarian damage and/or resisting and delaying ovarian senescence and/or improving the ovarian reserve function and/or reconstructing the ovarian function and the like as novel medicaments.

Description

Application of Tgfbr2 in preparation of ovarian function protection medicine

Technical Field

The invention relates to an application of Tgfbr2 in an ovarian function protection medicine, belongs to the technical field of tumor fertility protection, and particularly relates to an application of Tgfbr2 in the aspect of preparing an ovarian function protection medicine.

Background

Ovarian function, including endocrine and reproductive ability, is an important factor affecting female quality of life. The existing factors influencing the female ovarian function comprise: age, genetics, immunity, iatrogenicity, infectivity, environment, ethology, psychology, etc. The iatrogenic ovarian damage caused by chemotherapy is common and urgently needed to be improved in clinic.

The chemotherapy can cause the damage of the ovarian function, the induced reproductive endocrine disorder can be manifested as amenorrhea, infertility, perimenopause symptoms, psychological disorder and the like, and the damage of the chemotherapy to the follicle loss and the ovarian function is irreversible, so that the incidence rate of premature ovarian failure and irreversible infertility of women caused by the chemotherapy or the whole body radiotherapy is up to 80-100 percent, and the physical and psychological health of patients is seriously influenced.

Therefore, it is important to search for measures of chemotherapy that simultaneously protect ovarian function, reduce ovarian damage, and perform post-treatment. Various researchers have carried out a plurality of researches aiming at the problems of follicle injury, accelerated follicle-pool exhaustion, ovary reserve reduction and the like caused by chemotherapy. The current major birth function protection measures are as follows. 1. The embryo is frozen and preserved, and has the advantages of perfect and mature technology and high success rate; the disadvantage is that oocyte stimulation and maturation takes 4-6 weeks, which results in delayed chemotherapy, and may not be suitable for patients requiring urgent chemotherapy and male partners or sperm donors, and ovarian hormone stimulation is not suitable for female breast cancer patients who are estrogen receptor positive. 2. The mature oocytes are preserved by freezing, and the advantage is that sperm donors are not needed; the disadvantages are that the success rate is currently lower than for embryo use, that delayed chemotherapy may not be appropriate for invasive cancer, and that hormonal stimulation may not be appropriate for breast cancer. 3. The ovarian tissue is frozen, preserved and implanted again, the success rate is not clear, and the method has the advantages that the chemotherapy time does not need to be delayed, and can be used for patients with inappropriate oocyte stimulation delay; the disadvantage is that at the current experimental stage, secondary malignancies may result.

The TGFBR2 gene encodes a member of the serine/threonine protein kinase family and the TGFB receptor subfamily, encoding a transmembrane protein with a protein kinase domain. It forms a heterodimeric complex with another protein receptor protein and binds to TGF-. beta.s. Mutations in this gene have been associated with Marfan's syndrome, Loeys-Deitz aortic aneurysm syndrome, Osler-Weber-Rendu syndrome, and the development of various types of tumors. It maps to human chromosome 3p24.1 and is a highly conserved gene. The gene is about 87.54kb in length. The cDNA open reading frame of TGFBR2 is about 1779nt long, contains 11 exons and 10 introns, and can encode 592 amino acids of protein with relative molecular weight of about 70/80 kDa. The basic composition structure of TGFBR 2: mainly consists of a C-terminal protein kinase structural domain and an N-terminal extracellular structural domain. The outer domain comprises a compact fold comprising 9 beta strands and a network-stabilized single helix composed of 6 intrachain disulfide bonds. The specific structure of TGFBR2 determines its biological functions, including participation in Notch pathway, aging, protein kinase activation, vascular development and other biological processes.

The document Kim, K.K., D.Shepard, and H.A.Chapman, TGF-beta 1 signalling and Tissue fibers, Cold Spring Harbor perspectives in biology,2018.10(4): p.1-34. Tgfb1 is reported to be a pro-fibrotic factor, closely related to extracellular matrix (ECM) synthesis, and capable of inducing fibrotic progression in various organs.

The literature Zhou, F., L. -B.Shi, and S. -Y.Zhang, Ovarian fibers: A Phenomenon of concern. Chinese medical journal,2017.130(3): p.365-371. it is reported that abnormal elevation of Tgfb1 levels in the ovary leads to follicular dysplasia.

The document Davidsohn, N.et al, A single combination gene therapy programs multiple-related diseases of the National Academy of Sciences, 2019.116(47): p.23505-23511, reports that Tgfbr2 specifically binds to Tgfb1, and that exogenous administration of Tgfbr2 has been reported to reduce Tgfb1 binding to receptors on cell membranes and subsequent signaling cascades, thereby halting the progression of fibrosis.

Gene therapy is a novel treatment for diseases caused by gene defects or gene expression abnormalities, and a novel treatment means for correcting or compensating for diseases caused by gene defects or gene expression abnormalities by introducing exogenous genes into cells. At present, gene therapy not only has great development in the field of cancer, but also has relatively extensive clinical research in the direction of rare diseases, particularly monogenic diseases and rare genetic diseases, but is limited in the research in the field of non-monogenic deletion non-neoplastic diseases, and no report on ovarian injury caused by chemotherapy is provided.

Disclosure of Invention

In order to solve the technical problems, the invention discloses application of Tgfbr2 in preparing an ovarian function protection medicament. In particular, the gene up-regulation in the ovary is realized by injecting the adenovirus vector in situ through the ovary, and the purpose of rebuilding the ovary function is realized by repairing the damaged part. Therefore, the AdV-Tgfbr2 recombinant vector and the pharmaceutical preparation thereof can protect the ovarian function without influencing the anti-cancer effect, and play an important role in repairing ovarian damage and/or resisting and delaying ovarian senescence and/or improving the ovarian reserve function and/or reconstructing the ovarian function and the like as novel medicaments.

In order to achieve the aim, the invention discloses application of Tgfbr2 in preparing a medicine for protecting ovarian function, wherein the protection of ovarian function comprises repairing ovarian damage and/or resisting and delaying ovarian senescence and/or improving ovarian reserve function and/or rebuilding ovarian function.

Further, the ovarian function protection is against delayed ovarian senescence.

Further, the ovarian function protection is the improvement of ovarian reserve function.

Further, the ovarian function protection is to reestablish ovarian function.

Further, the ovarian function protection is to repair ovarian damage, and the ovarian damage comprises any one of ovarian damage caused by aging, ovarian damage caused by genetic or immune function deficiency or environment, iatrogenic ovarian damage or pathological ovarian damage.

Further, the ovarian damage is iatrogenic ovarian damage.

Further, the ovarian damage is ovarian interstitial fibrosis.

Preferably, the iatrogenic ovarian injury is ovarian interstitial fibrosis caused by chemotherapy. The invention further selects the symptoms of ovarian interstitial fibrosis caused by chemotherapy, such as the reduction of ovarian endocrine function, the reduction of ovarian reserve function and the like, thereby influencing the reduction of reproductive capacity.

Preferably, the ovarian functional protection is the alleviation of ovarian interstitial fibrosis due to iatrogenic origin.

The invention also discloses an application of Tgfbr2 in preparing a medicament for relieving ovarian interstitial fibrosis caused by chemotherapy.

Specifically, the invention discloses an application of Tgfbr2 in preparing a medicine for relieving ovarian interstitial fibrosis caused by adriamycin.

Further, the medicament is a recombinant nucleic acid construct for expressing the Tgfbr2 protein, and an expression vector of the recombinant nucleic acid construct is any expression vector suitable for gene therapy, and the expression vector comprises a viral vector or a non-viral vector.

Further, the viral vector is an adenoviral vector.

Further, the dosage form of the medicine is an injection type.

Further, the method specifically comprises the steps of injecting a certain dose of AdV-Tgfbr2 into the ovary of a tested mammal body in situ by adopting a gene therapy measure, and detecting the overexpression level of Tgfbr 2.

Furthermore, the tested mammal body comprises a control group and an experimental group, wherein the experimental group is a chemotherapeutic ovarian injury experimental group.

Further, the construction process of the AdV-Tgfbr2 is as follows:

1) construction of pENTER vector for mammalian subjects: constructing a Tgfbr2 gene fragment of a tested mammal body on a shuttle vector pENTER in a PCR mode;

2) obtaining a certain titer of adenovirus: transfecting the pENTER vector constructed in the step 1) into HEK293 cells containing an adenovirus skeleton vector pAD-FH through a lipo2000 transfection system, screening and collecting positive cells successfully recombined, cracking the cells, collecting virus supernatant, concentrating and purifying to obtain adenovirus with a certain titer;

3) adenovirus purification: purifying the adenovirus in the step 2) by adopting an iodixanol density gradient ultracentrifugation method;

4) adenovirus titer determination: the adenovirus titer is 7.0 x 10¹⁰PFU/mL。

Further, the subject mammalian body includes, but is not limited to, rat, mouse, non-human primate, human, dog, cat, horse, cow, sheep, pig, goat, and preferably mouse.

In addition, the invention also discloses an ovarian function protection medicine, which comprises AdV-Tgfbr2 as a main raw material and an indispensable medicine carrier.

Has the advantages that:

1. the invention discloses a method for realizing gene up-regulation in an ovary by injecting an adenovirus vector into a tested mammal body in situ through the ovary, and realizing the purpose of rebuilding the ovary function by repairing a damaged part. The ovary in-situ injection method is minimally invasive, the surgical incision is small, the survival of a mouse is not obviously influenced, the defect of lack of targeting of gene expression in a tail vein injection method or an intraperitoneal injection method is avoided, and the problem that the adenovirus genome is not integrated into a host cell genome to cause the phagophily to other organs, such as hepatotoxicity caused by the phagophily, is also avoided.

2. The AdV-Tgfbr2 recombinant vector and the pharmaceutical preparation thereof designed by the invention can protect the ovarian function without influencing the anticancer effect, and play an important role in the aspects of repairing ovarian injury, resisting and delaying ovarian senescence, improving the ovarian reserve function, reconstructing the ovarian function and the like as a novel medicament.

Drawings

FIG. 1 is a test chart of the results of detecting the fibrosis damage of ovaries caused by adriamycin and the protein level of Tgfbr 2;

FIG. 2 is a graph showing the results of measuring the levels of mRNA and protein after AdV-Tgfbr2 was injected into mouse ovaries in situ;

FIG. 3 shows the overexpression of Sirt1 and Tgfbr2 proteins over time following in situ injection of AdV-Sirt1 and AdV-Tgfbr2 into the ovary. Wherein, FIG. 3A shows the protein expression of Sirt1 of the AdV-Sirt1 group at different time points compared with the AdV-GFP group, and FIG. 3B shows the protein expression of Tgfbr2 of the AdV-Tgfbr2 group at different time points compared with the AdV-GFP group; FIG. 3C is a semi-quantitative statistical analysis of Sirt1 protein expression; figure 3D is a semi-quantitative statistical analysis of Tgfbr2 protein;

FIG. 4 is a graph of ovarian endocrine function decline caused by ovarian gene therapy against chemotherapy. Wherein, fig. 4A is the estrogen level changes in the groups of mice; FIG. 4B shows the change in progestogen levels in various groups of mice; figure 4C shows FSH level changes in various groups of mice; fig. 4D shows changes in AMH levels in each group of mice, and in fig. 4, P <0.05, P <0.01, P <0.001, P <0.0001 compared to the Dox group.

FIG. 5 is a graph of ovarian reserve function reduction caused by anti-chemotherapy by ovarian gene therapy, wherein FIG. 5A is a picture of H & E staining of the ovaries of various groups of mice, and FIG. 5B is the body weight, ovarian mass and ovarian index of various groups of mice; fig. 5C shows the follicle count of each group of mice. And in fig. 5, P <0.05, P <0.01, P <0.001, P <0.0001, compared to the Dox group. PMF primordial follicles, primordial follicle; PF primary folliculules; SF, secondary follicles; ANF, anti folliculules, antral follicles; THF, total heathy folliculules, total healthy follicles; ATF, atretic folliculles, atretic follicles.

Detailed Description

At present, because many factors such as age, heredity, immunity, iatrogenic, pathological, environmental, ethology, and psychology may cause damage to the ovary and/or aging of the ovary of a mammal, how to solve the technical problem to protect the ovary function is a technical problem to be solved by those skilled in the art.

Definition and use of terms

Chemotherapy: is the abbreviation of chemical drug therapy, and achieves the purpose of treatment by using chemical therapeutic drugs to kill cancer cells. Clinically, chemotherapeutic drugs also have different types, and can be classified into alkylating agents, antimetabolites, antibiotics, antitumor drugs, plant antitumor drugs, hormone antitumor drugs, miscellaneous drugs and the like according to different action mechanisms. Most patients need to be treated by more than two chemotherapeutics in combination, including oral chemotherapeutics, intravenous chemotherapeutics and the like, and in addition, part of patients can also be infused with the drugs through arteries in an interventional mode.

Ovarian damage: the invention mainly refers to injuries caused by various factors such as age, heredity, immunity, iatrogenic, pathological, environment, ethology, psychology and the like, and specific injury results comprise tumor cancer, inflammation, amenorrhea, infertility, perimenopause symptoms and the like.

Ovarian damage caused by chemotherapy: the invention mainly refers to ovarian function damage caused by destroying follicle and ovarian tissues in drug chemotherapy, menstrual disorder, infertility and premature ovarian failure even appearing in different degrees. Wherein, alkylating agents such as cyclophosphamide and the like have toxicity on mitotically active large follicles, increase primordial follicle recruitment, and generate delayed damage on primordial follicles, which results in reduction of ovarian primordial follicles and primary follicles, ovarian fibrosis, ovarian medulla destruction and ovarian volume reduction, and finally ovarian failure. The anticancer drug can inhibit the function of granulosa cells, and can cause hypofunction of ovary or premature ovarian failure by reducing the number of FSH and LH receptors. Cisplatin and other therapeutic drugs can inhibit granulosa cells and induce apoptosis of the granulosa cells, and the apoptosis of the granulosa cells directly influences the development of follicles, so that follicular dysplasia, anovulation and even infertility are caused.

Ovarian senescence: the premature ovarian failure of the patients mainly comprises the premature ovarian failure phenomenon of the patients caused by DNA and epigenetic modification change, telomere shortening and telomerase activity reduction, change of ovarian microenvironment, accumulation of oxidative damage products, apoptosis and aging of ovarian cells, blood vessel factors and the like.

Improving or increasing ovarian reserve function: the ovarian reserve function of the invention refers to the ability of primordial follicles in the ovarian cortex region to develop into fertilized oocytes, and the current indexes for clinically evaluating the ovarian reserve function mainly include age, basic endocrine hormone, cytokine, basic antral follicle number (AFC) and the like.

And (3) rebuilding ovarian function: the patient's ovary is allowed to recover normal physiological functions, such as normal menstrual period or normal fertility.

A drug carrier: the drug delivery system is a system which can change the mode of entering a human body and the distribution of the drug in the human body, control the release speed of the drug and deliver the drug to a target organ, and specifically comprises microcapsules, microspheres, nanoparticles, liposome and the like.

Adriamycin: an antibiotic drug of formula C₂₇H₂₉NO₁₁The compound has a wide antitumor spectrum, and is suitable for acute leukemia (lymphocytic and granulocytic), malignant lymphoma, breast cancer, bronchogenic carcinoma (undifferentiated small cell and non-small cell), ovarian cancer, soft tissue sarcoma, osteogenic sarcoma, rhabdomyosarcoma, Ewing's sarcoma, blastoma, neuroblastoma, bladder cancer, thyroid cancer, prostate cancer, head and neck squamous cell carcinoma, testicular cancer, gastric cancer, liver cancer and the like.

The invention mainly researches a strategy for repairing ovaries after the ovaries are damaged due to chemotherapy. The current measures for repairing the damaged ovary and protecting the ovarian reproductive function are invasive operations and have limited application range, some of the measures can delay chemotherapy, some of the measures have low success rate, and some of the measures can cause secondary cancer; at the same time, therapeutic approaches that primarily aim at preserving fertility are unlikely to be useful for long-term maintenance or for reestablishing ovarian function.

The specific technical scheme is as follows：

The invention discloses an application of Tgfbr2 in preparing a medicine for protecting ovarian function, wherein the protection of the ovarian function comprises repairing ovarian damage and/or resisting and delaying ovarian senescence and/or improving ovarian reserve function and/or rebuilding the ovarian function.

Further, the ovarian function protection is against delayed ovarian senescence.

Further, the ovarian function protection is to reestablish ovarian function.

Further, the ovarian function protection is repair of ovarian damage.

Further, the ovarian damage includes any one of ovarian damage caused by aging, ovarian damage caused by genetic or immune function deficiency or environment, iatrogenic ovarian damage or pathological ovarian damage.

Further, the viral vector is an adenoviral vector.

Further, the dosage form of the medicine is an injection type.

Furthermore, the tested mammal body comprises a control group and an experimental group, wherein the experimental group is a chemotherapeutic ovarian injury experimental group. The present invention preferably employs doxorubicin to cause fibrotic damage to the ovaries of the mammalian subject. And the control group is a physiological saline intervention group.

Meanwhile, the AdV-Tgfbr2 designed by the invention is obtained by adopting the following recombinant vector mode:

1. the sequence of the coding region of mouse Tgfbr2 gene transcript (NM-009371.3, 1179bp) was obtained and analyzed at the GenBank website, and PCR primers were designed with the sequences as shown in Table 1 below:

TABLE 1 primer sequence List

	Primer sequence (5 '-3')
		Upstream primer (F)	GACTGTCCACTTGCGACAAC
Downstream primer (R)	GGCAAACCGTCTCCAGAGTAA

Constructing a target gene Tgfbr2 gene fragment on a shuttle vector pENTER in a PCR (polymerase chain reaction) mode; the multiple cloning sites comprise Asisl and Mlul;

2. transfecting the constructed pENTER vector into HEK293 cells containing an adenovirus skeleton vector pAD-FH through a lipo2000 transfection system, carrying out homologous recombination on 2 vectors in the cells, and recombining a target gene fragment into the adenovirus skeleton vector; after homologous recombination, screening a positive cell which is successfully recombined, namely a cell containing an adenovirus skeleton vector inserted with a target gene sequence through amp resistance, completing virus coating of the skeleton vector under the assistance of a packaging cell 293, and releasing the cell; collecting cells, cracking the cells, collecting virus supernatant, concentrating and purifying to obtain adenovirus reaching a certain titer;

3. purifying adenovirus by using an iodixanol density gradient ultracentrifugation method, and prefabricating an iodixanol density gradient layer; adding the adenovirus crude liquid above the iodixanol density gradient layer; centrifuging at 268000g for 2.5h at 18 ℃; absorbing adenovirus of 40% iodixanol layer, ultrafiltering with 50K ultrafiltering concentration centrifuge tube at 4000g and 10 deg.C for about 1 hr; then suspending the adenovirus liquid in PBS;

4. adenovirus titer determination: 1 block of 96-well plate is taken, and 293T cells 10 are added into each well⁴Adding 200 mu L of liquid, placing in an incubator for 24h, and diluting the virus to be detected to 1-10 by using DMEM (DMEM) full culture medium^-8And (4) concentration. Discarding the culture medium of crude oil in 96-well plate, and sequentially diluting the virusAir was added at 200. mu.L per well, and two duplicate wells were made at each concentration, with media controls (without adenovirus) being included. Then placing at 37 ℃ and 5% CO₂The culture was continued for 36h under the conditions. Counting the fluorescence positive cells with a fluorescence microscope, counting the number of the fluorescence cells in the last two wells, summing the total number of the 2 replicate wells and calculating the average, assuming a (average number of fluorescence cells in the second last well) and B (average number of fluorescence cells in the first last well), as shown in table 2;

TABLE 2 fluorescent cell number List

1

2

3

4

5

6

7

8

9

10

A

10μl

10⁰

10^-1

10^-2

10^-3

10^-4

10^-5

10^-6

10^-7

10^-8

B

10μl

10⁰

10^-1

10^-2

10^-3

10^-4

10^-5

10^-6

10^-7

10^-8

Viral titers were calculated using the formula (BT ═ PFU/ml, transducing units):

the adenovirus titer obtained by amplification was about 7.0X 10 as calculated by the above formula¹⁰PFU/mL。

5. Sequencing and verifying: verifying whether Tgfbr2 is inserted into the pADM-FH framework vector, and sending the extracted recombinant vector to Beijing Optimalaceae New Biotechnology Co.

In addition, the invention also discloses an ovarian function protection medicine, which comprises AdV-Tgfbr2 as a main raw material and an indispensable medicine carrier. Meanwhile, the ovarian function protection medicine can be prepared into any one of granules, powder, paste, pills, oral liquid, injection or capsules, and the injection is preferred in the invention.

In order to better explain the technical scheme disclosed by the invention, the following detailed description is combined with specific embodiments.

EXAMPLE ovarian in situ injection overexpression experiments

Experimental animals and materials:

1. experimental animals: a mouse;

source, species, strain: beijing Weitongli Hua Biotech Co., inbred line, C57 BL/6;

and (4) breeding age: 8 weeks in size;

2. experimental materials:

chloral hydrate: shanghai Lingfeng Chemicals, Inc.; AdV-GFP: a control adenovirus containing a green fluorescent protein gene; the titer of the invention is preferably 1.0X 10¹⁰pfu/mL；

A stereoscopic microscope: olympus, model: SZ 61;

microsyringe needle: shanghai Pigeon worker and trade Co., Ltd 10 μ L (tip) model: a124019;

step one, anesthesia of mice: the mice were fasted for 12 hours before surgery, and after weighing the mice, the mice were given an intraperitoneal injection by aspirating a corresponding volume of 4% chloral hydrate according to a dose of 10 μ L/g. Then placing the mouse on a sterile gauze pad with the back of the mouse facing upwards, stretching and fixing the limbs on a wood board, and binding the mouse;

step two, exposing the operation visual field: shaving the back of the mouse by using a shaver, wherein an incision is positioned above the left ovary and the right ovary, disinfecting the incision point by using 75% alcohol, lifting the skin on the back of the mouse by using tweezers, making a small incision on the inner side of the back and the position right above an ovarian fat pad by using scissors, sequentially cutting the abdominal membrane wall, placing a sterile saline gauze pad near the incision, finding the ovarian fat pad, slightly pulling the ovarian fat pad outwards, and placing the ovarian fat pad on gauze;

step three, inserting a micro-injection needle: under a stereomicroscope, a microsyringe (30gauge, G) sterilized by alcohol is gently inserted into the oviduct bend of the ovarian cyst, and the needle head is seen below the ovarian cyst;

step four, injection: sucking 4-5 μ L of prepared AdV-Tgfbr2, injecting into ovarian cyst as experimental group, and AdV-GFP as control group. After the slow injection is finished, the needle head is quickly taken out to seal the puncture point, the action is soft, and the ovarian cyst is not required to be torn. If the injection is appropriate, a slight expansion of the ovarian cysts should be seen;

step five, discussing experimental results: the ovary is returned to the original anatomical position, and the peritoneal wall, the muscular layer and the skin are sutured by absorbable suture lines; after suturing, the incision is disinfected by iodophor. The mice after the operation are placed on the heating pad to avoid hypothermia, so that the mice can have a rest and recover, and the recovery is accelerated. The respiratory rate and comfort of the mice and the ability to move autonomously were monitored to assess post-operative recovery. Specifically, after 1 week of operation, ovaries of an experimental group and an ovarian of a control group are respectively taken, fat tissues around the ovaries are separated under a stereoscopic microscope, on one hand, total RNA and protein of the tissues are extracted, qPCR and WB are utilized to detect the overexpression level of Tgfbr2, and on the other hand, frozen section immunofluorescence and paraffin section immunohistochemical detection is carried out on the ovaries.

And sixthly, repeating the operation, and performing intraperitoneal injection of adriamycin (10mg/kg) to the control group and the experimental group about 2 weeks after the ovary is injected with the virus in situ to cause an ovary damage model. The specific operation is described in the following documents;

1、Ben-Aharon,I.,et al.,Doxorubicin-induced ovarian toxicity.Reproductive biology and endocrinology:RB&E,2010.8:p.20.

2、Bar-Joseph,H.,et al.,In vivo bioimaging as a novel strategy to detect doxorubicin-induced damage to gonadal blood vessels.PloS one,2011.6(9):p.e23492.

meanwhile, fig. 1 shows the results of the detection of fibrosis damage caused by doxorubicin to the mouse ovary and the level of Tgfbr2 protein, and it can be seen from fig. 1A that the ovarian damage induced by doxorubicin includes an increase in the degree of fibrosis, and from fig. 1B and fig. 1C that the expression level of Tgfbr2 in mRNA and protein is decreased. . For the overexpression effect of Tgfbr2 in ovary, we performed qPCR and WB experiments, and examined the overexpression effect of AdV-Tgfbr2 after in situ ovarian injection at mRNA and protein levels as shown in fig. 2. The expression of the polypeptide is obviously up-regulated.

In order to better embody the beneficial effect of Tgfbr2 designed by the invention in the protection of ovarian function, the invention also describes the using effect of AdV-Sirt1 under the same experimental conditions, and the specific effect is as follows:

(1) in terms of the maintenance time of overexpression of the target gene:

the ovarian tissues of mice in the AdV-GFP group, the AdV-Tgfbr2 group and the AdV-Sirt1 group were taken at each time point of 2 weeks, 4 weeks, 6 weeks and 8 weeks, respectively, for protein overexpression detection. And extracting the ovarian tissue proteins of each group, carrying out Western blot experiment, and detecting the expression of the proteins of Sirt1 and Tgfbr2 at each time point. The protein expression of Sirt1 was found to be significantly improved after 2 weeks of intervention with AdV-Sirt1 and 4 weeks, as shown in FIGS. 3A and 3C, respectively; and no statistical difference is obvious by 6-8 weeks; however, it was found that the protein expression of Tgfbr2 was significantly increased after 2, 4 and 6 weeks of AdV-Tgfbr2 intervention, but did not have a significant upward trend at 8 weeks, as shown in fig. 3B and 3D, respectively. This indicates that the designed AdV-Tgfbr2 can last longer than the target gene of AdV-Sirt1, which also indirectly indicates that the gene therapy effect of AdV-Tgfbr2 may be better if the control experiment conditions are the same or similar.

(2) In terms of the decline in ovarian endocrine function caused by anti-doxorubicin chemotherapy:

the detection of the level of hormones such as serum estrogen and progestogen finds that: the levels of estrogen in the AdV-Tgfbr2+ Dox group were significantly increased compared to the AdV-GFP + Dox group as shown in FIGS. 4A and 4B, respectively, and AMH as depicted in FIG. 4D.

As shown in FIG. 4C, there was no significant statistical difference in FSH levels among the groups, but there was a tendency to decline in the AdV-Tgfbr2+ Dox group.

And the estrogen and AMH levels of the AdV-Sirt1+ Dox group also tended to increase compared to the AdV-GFP + Dox group, but there was no significant statistical difference. This indicates that AdV-Tgfbr2 has a stronger improving ability on the ovarian endocrine function decline caused by doxorubicin chemotherapy than AdV-Sirt 1.

(3) In terms of the reduced ovarian reserve function caused by anti-doxorubicin chemotherapy:

as can be seen from FIG. 5B, by detecting the ovaries of the mice, the quality of the ovaries of the mice in the AdV-Tgfbr2+ Dox group is obviously increased compared with the AdV-GFP + Dox group, the ovarian indexes of the AdV-Sirt1+ Dox group and the AdV-Tgfbr2+ Dox group are obviously increased, but the increase of the ovaries of the AdV-Tgfbr2+ Dox group is more obvious. Continuing to perform serial section H & E staining of mouse ovaries, as shown in FIG. 5A, and performing follicle counting, as shown in FIG. 5C, it was found that the number of atretic follicles was significantly decreased in the AdV-Sirt1+ Dox group and the AdV-Tgfbr2+ Dox group, as compared to the AdV-GFP + Dox group, and that AdV-Tgfbr2 decreased the number of atretic follicles more significantly, and was more potent in improving atretic follicles than AdV-Sirt 1.

In conclusion, AdV-Tgfbr2 disclosed in the present application advantageously ameliorates the diminished ovarian endocrine function or ovarian reserve function caused by doxorubicin. This is because AdV-Tgfbr2 disclosed in the present application can compensate for the decrease in Tgfbr2 expression caused by ovarian interstitial injury caused by doxorubicin through gene supplementation effect, and its possible action mechanism is to reduce the degree of ovarian interstitial fibrosis caused by chemotherapy, thereby facilitating the restoration of the endocrine and reserve functions of the ovary. In addition, since ovarian interstitial fibrosis may also cause ovarian senescence or cause the ovaries to lose response function, it is speculated that AdV-Tgfbr2 also has an effect on delaying ovarian senescence and/or improving ovarian reserve function and/or reestablishing ovarian function.

As those skilled in the art will appreciate, Sirt1 plays a key role in follicular development and also delays ovarian senescence. In the application, AdV-Tgfbr2 is adopted to supplement related genes, compared with AdV-Sirt1, although the supplement of the respective related genes can also realize the protection of ovarian functions, the action mechanism is different, as described above, AdV-Tgfbr2 can reduce the degree of ovarian interstitial fibrosis caused by chemotherapy, AdV-Sirt1 can protect the development of follicles or a follicle microenvironment, and AdV-Tgfbr2 is superior to AdV-Sirt1 in the aspects of protein expression maintenance time, ovarian reserve and function recovery, so that the two genes still have great difference.

The above examples are merely preferred examples and are not intended to limit the embodiments of the present invention. In addition to the above embodiments, the present invention has other embodiments. All technical solutions formed by adopting equivalent substitutions or equivalent transformations fall within the protection scope of the claims of the present invention.

Claims

Use of Tgfbr2 in the manufacture of a medicament for the protection of ovarian function, wherein the protection of ovarian function comprises the repair of ovarian damage and/or the fight against the delay of ovarian senescence and/or the improvement of ovarian reserve function and/or the reconstitution of ovarian function.
2. The use of Tgfbr2 in the preparation of a medicament for the protection of ovarian function according to claim 1, wherein the protection of ovarian function is the repair of ovarian damage.
3. The use of Tgfbr2 in the preparation of a medicament for the protection of ovarian function according to claim 2, wherein the ovarian damage comprises chemotherapy-induced interstitial ovarian fibrosis.
4. The use of Tgfbr2 in the preparation of a medicament for protecting ovarian function according to any one of claims 1 to 3, wherein the medicament is a recombinant nucleic acid construct expressing Tgfbr2 protein, and the expression vector of the recombinant nucleic acid construct is any expression vector suitable for gene therapy, including viral vectors or non-viral vectors.
5. The use of Tgfbr2 in the preparation of a medicament for protecting ovarian function according to claim 4, wherein the viral vector is an adenoviral vector.
6. The use of Tgfbr2 in the preparation of a medicament for protecting ovarian function according to any one of claims 1 to 3, wherein the medicament is in the form of an injection.
7. The use of Tgfbr2 in the manufacture of a medicament for the protection of ovarian function according to any one of claims 1 to 3, comprising administering a dose of AdV-Tgfbr2 to the ovaries of a mammalian subject in situ by gene therapy, and detecting the level of Tgfbr2 over-expression.
8. The use of Tgfbr2 in the manufacture of a medicament for protecting ovarian function according to claim 7, wherein the mammalian subject comprises a control group and an experimental group, and the experimental group is a chemotherapeutic ovarian injury experimental group.
9. The use of Tgfbr2 in the preparation of a medicament for protecting ovarian function according to claim 7, wherein AdV-Tgfbr2 is constructed as follows:

1) construction of pENTER vector for mammalian subjects: constructing a Tgfbr2 gene fragment of a tested mammal body on a shuttle vector pENTER in a PCR mode;

2) obtaining a certain titer of adenovirus: transfecting the pENTER vector constructed in the step 1) into HEK293 cells containing an adenovirus skeleton vector pAD-FH through a lipo2000 transfection system, screening and collecting positive cells successfully recombined, cracking the cells, collecting virus supernatant, concentrating and purifying to obtain adenovirus with a certain titer;

3) adenovirus purification: purifying the adenovirus in the step 2) by adopting an iodixanol density gradient ultracentrifugation method;

4) adenovirus titer determination: the adenovirus titer is 7.0 x 10¹⁰PFU/mL。
10. An ovarian function protection medicine is characterized by comprising AdV-Tgfbr2 serving as a main raw material and an indispensable medicine carrier.