小型猪模型,异位卵巢移植 z-bo 2020-12-04 574

　　Background: Ovarian tissue transplantation can be used to preserve the fertility and ovarian function of women experiencing premature ovarian failure and infertility. Every year, more and more women suffer from cancer. Therefore, people are paying more and more attention to the effects of various cytotoxic treatments on gonadal function. The current fertility preservation alternative, ovarian tissue transplantation is different from cryopreservation of embryos and oocytes. Because it is the only way that adolescent girls can provide and realize any delay that is not dealt with. This technique can use fresh or cryopreserved tissues transplanted in situ or heterotopic sites. In general, autologous ovarian transplantation is more common than allogeneic transplantation due to immunosuppressive problems. However, transplant rejection, through apoptosis or necrosis, can even occur in autologous transplants. The mechanism of apoptosis rejection is still largely unknown. The research progress of apoptosis in ovarian transplantation shows that this process is involved in transplantation. Our research evaluates the use of fresh and frozen tissues at different implant sites to evaluate cell apoptosis. The research on ovarian transplantation is mainly experimental and involves a short evaluation period. In addition, these studies use a series of different technologies to be carried out on small animal models. Although ovarian tissue transplantation is a relatively new technology, it has great development prospects. Further research is needed to standardize the technology and broaden the clinical application prospects. . In view of the fact that the macroscopic characteristics of miniature pigs are similar to human physiology, this animal model is used to evaluate the feasibility of ovarian implants, whether fresh or cryopreserved, orthotopic or heterotopic transplantation. We speculate that the quality and viability of the transplanted tissue will vary according to the transplant site. Therefore, the purpose of this study was to evaluate the expression of three markers of apoptosis (Bcl-2, Bax and Bcl-2, Bax and Bcl-2, Bax and Bcl-2). caspase-3).

　　Method: Experimental animals: 40 female miniature pigs, the animals are 6 months to 1 year old, and the average weight is 24.5 kg. Animals have gone through at least one cycle of estrus to ensure that they are in the reproductive development stage. Miniature pigs maintain a 12-hour light-dark cycle and provide free water and food. After three days of acclimatization, each animal received a synthetic progestin cycle for 18 days. After 5 days, the animals were then randomized. Study group: The animals were randomly divided into five groups (n = 8): GI, the animals only underwent laparoscopic bilateral ovariectomy, in which the ovarian tissue was removed as a control group; GII, the animals underwent bilateral oophorectomy and fresh ovarian tissue Autologous transplantation to subcutaneous tissue immediately (heterotopic transplantation); GIII, animals underwent bilateral oophorectomy and ovarian tissue autotransplantation, fresh immediately surrounding the ovarian area of the intestinal abdominal cavity (orthotopic transplantation); GIV, animals underwent bilateral oophorectomy and The subsequent autologous frozen ovarian tissue was transplanted into the subcutaneous tissue (heterotopic transplantation); GV, animals underwent bilateral ovariectomy and the subsequent autologous frozen ovarian tissue was transplanted to the ovarian region of the peri-funnel abdominal cavity (orthotopic transplantation).

　　 Ovariectomy: Laparoscopic bilateral oophorectomy, using Stryker endoscopy equipment to identify ovarian pedicles and uterine ovarian ligaments, clamping, cauterizing and cutting. The ligature is placed in a bipolar sealed container system. Preparation of ovarian fragments: remove the ovaries, dissect each ovarian cortical area from adjacent tissues and medullary area, and use Leibovitz L-15 medium supplemented with 0.1% bovine serum albumin (BSA) in a sterile environment at room temperature. The pieces were cut into 2mm×4mm×4mm thick pieces under aseptic conditions. Ovarian cortical fragments are freshly transplanted in the same operation (GII and GIII) or after a slow freezing process, stored in liquid nitrogen for seven days, and then transplanted (GIV, GV). Use a heterotopic graft of GII and GIV and an orthotopic graft of GIII and GV.

　　 Cryopreservation: After ovariectomy, the intact ovaries are transferred to a petri dish containing cryoprotectant to cool (4°C) (Leibovitz L-15). The ovarian strips were dissected from adjacent tissues, the cortical area was stripped, and placed in Leibovitz L-15 medium containing 0.1% bovine serum albumin, NaCl solution (10%) and 0.1 mol/ml sucrose and mannitol. The fragments were placed in a 1.2-ml cryotube with 1.5 M DMSO as the cryoprotectant.

　　 Thaw: The vial is taken out of liquid nitrogen, placed at room temperature for 40 seconds, and then immersed in a 37°C water bath for two minutes until the ice melts. The ovarian cortex fragments were washed with a series of 1000 mg/L glucose and 36 mg/L pyruvate phosphate buffered saline (PBS).

　　Subcutaneous transplantation: The groin fold is selected as the subcutaneous area for autologous transplantation of ovarian tissue due to its ease of operation. In addition, this time it is not easy to be harmed by itself and other animals. After dissection, six ovarian tissue sections were inserted, the subcutaneous area was sutured with 3.0 VICRYL suture, and the skin was sutured with 3.0 nylon suture. In addition to stripping the tissue and preparing the ovarian strips, the average time required for subcutaneous transplantation is 2.2 minutes.

　　 Intra-abdominal transplantation: laparoscopic examination of the pelvic cavity. The chosen location in the abdominal cavity is the mural peritoneal fold near the uterine tube. Six ovarian tissue strips were initially secured to a hemostatic gauze with 4.0 VICRYL sutures and then passed through an 11 mm cannula into the abdominal cavity. The parietal peritoneal fold was sutured with the ovarian strip, close to the left fallopian tube, using 3.0 VICRYL suture. In addition to stripping the tissue and preparing the ovarian strips, the average time required for intra-abdominal transplantation is 5.8 minutes.

　　Tissue collection and histological evaluation: Ovarian strips were collected 30 days after GII and GIII autotransplantation (fresh) and 37 days after GV autotransplantation (frozen). A synthetic progestin was used for 18 days after the recovery period of 7 days after surgery. The samples were fixed with formalin, embedded in paraffin, and cut into 5 μm sections for histological preparation. These tissue samples are then transferred to a 70% ethanol solution and stored at room temperature until processing. According to the ovarian germinal epithelium, cortex and stroma, a detailed histological description of the ovarian tissue is made.

　　 HE staining evaluates the quality of ovarian tissue implants and follicle density, general aspects of tissue and vascularization. And carry out follicle count and classification. Ovarian follicles are divided into the following categories: primitive follicles, antral healthy follicles, atretic follicles, corpus luteum and white body. Then, each tissue area determines the number of each follicle type. A primordial follicle is defined as a follicle with only one oocyte and a single flat squamous cell layer. Follicle growth Follicles are formed from the primordial follicular cavity, but no antrums are formed. Preantral follicles are divided into the following groups: primary follicles, one oocyte and monolayer cubic epithelial cells; secondary follicles, one oocyte and two to eight layers of granular cells and no antral cavity; third (antral) follicles , More than eight layers of granular cells, no sinus cavity. The follicular cavity, regardless of size, is considered an antral follicle. In all these cases, an oocyte must be visible. The corpus luteum presents typical corpus luteum cells, surrounded by large nuclei and blood vessels. White body is the product of luteal degradation. The follicles were counted in each group, and computer images were used for analysis. Use an optical microscope to couple to a high-resolution camera and a color video monitor for image acquisition and measurement.

　　Immunohistochemical analysis: use standard histochemical techniques for dewaxing and washing. Tissue sections were incubated overnight at 4°C in a dark room with anti-Bcl-2, anti-Bax or anti-Caspase-3 primary antibody diluted 1:400 in PBS. The tissue sections were washed repeatedly in TBS containing 0.1% Tween 20, and the secondary antibody was mouse anti-IgG antibody (1:400) against Bcl-2 antibody and rabbit anti-IgG antibody (1:400) against Bax and caspase-3 protein. Subsequently, the sample was incubated with AB reagent (avidin-biotin complex) for 30 minutes at room temperature, and incubated with DAB for 5 minutes at room temperature. Then the sections were counterstained with hematoxylin. The above-mentioned apoptosis markers were evaluated with the 10× and 40× objective lenses of an optical microscope. The relative amount of cell staining in each area was analyzed by visualizing ten different sections of each section. 10 slices of each animal, the immunohistochemical data was evaluated using Image-Pro Plus image capture software, and ImageJ software was used to image the number of follicles, the number of follicles per tissue area, and the intensity of staining.

　　Result: In the control group (G1), the ovary is covered with a single layer of cubic or columnar epithelium, with good medulla and cortical areas formed, and a large number of follicles are shown at all stages of development. The morphology of ovarian strips (GII and GIV) in the heterotopic transplantation group was similar to that of the control group. A lower concentration of primordial follicles and a larger area of connective tissue were observed in all transplantation groups, especially in orthotopic transplantation groups (GIII and GV).

　　Immunohistochemical analysis: Follicles in the transplantation group, especially GIII, showed weak Bcl-2 staining. In addition, the expression of Bax in the transplanted follicles was more frequent and more intense than the control group (GI). This observation is most obvious in the orthotopic transplantation group with fresh ovaries (GIII). No lysed caspase-3 staining was observed in the interstitial follicular cells of the GI group, although there was slightly stained corpus luteum. The matrix of GII group was not stained, and the follicular cells were moderately stained. In GIII, moderate staining is seen, and staining also appears in the inner membrane; on the contrary, the matrix is weakly positive. In GIV, stained and unstained follicles were observed, although all clusters and stroma were slightly or moderately stained, and the staining was weaker than that of GII. In GV, there are stained and unstained follicles and mild or moderate chromatin.

　　 Conclusion: Our data show that fresh ovarian tissue transplanted into the subcutaneous area shows a lower rate of apoptosis. Therefore, this transplantation method is beneficial to maintain ovarian viability. The potential clinical significance involves the use of heterotopic sites in human ovarian transplantation. These findings have broad implications for the clinical application of ovarian transplantation technology, and will strengthen the discussion of heterotopic ovarian tissue transplantation.