All the animals were purchased from the experimental animal center of Ningxia Medical University. The 3-week-old female New Zealand white rabbits were used for the isolation of BMSCs and the rabbits aged 12 weeks were used for the establishment of the IUA model. The experimental protocols and animal handling procedures were approved by the Ethics Committee of General Hospital of Ningxia Medical University.
Isolation, identification and differentiation of BMSCs
BMSCs were isolated from the tibia and femur of 3-week-old female New Zealand white rabbits using the method of adherent culture of whole bone marrow. Briefly, under complete aseptic conditions, bone marrow cells were harvested and cultured with low glucose Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco, Gainesville, MD, USA) containing 10% fetal bovine serum (FBS) (Gibco, Australia) and 1% penicillin–streptomycin (Gibco, USA) at 37℃ in a humidified atmosphere with 5% CO2. The medium was refreshed every 2 days until the cell confluence reached 90%. Then, the cells were digested with 0.25% trypsin (Hyclone, USA) and subcultured to the third generation for following experiment. Subsequently, BMSCs were washed twice with PBS and fixed in 4% para formaldehyde (PFA), and then the related cell-surface markers such as CD29, CD44, CD34 and CD45 (Bioss, USA) were evaluated by flow cytometer.
Next, the osteogenesis and adipogenesis differentiation experiments of BMSC were performed. Briefly, When the degree of cell confluence reached 80%, the cells were digested with 0.25% trypsin and seeded into a 0.1% gelatin-coated culture dish, then add 2 mL of commercial osteogenic or adipogenic differentiation medium of induction kits according to the manufacturer’s instructions (RBXMX-90021 and RBXMX-90031, Cyagen, Santa Clara, CA). The medium was changed every 3 days and observe the cell morphology and growth for 3 weeks. The mineral components are stained with Alizarin Red and the lipid accumulation are stained with Oil Red O. Observe the effect of osteogenic or adipogenic induction under the microscope.
PKH26 fluorescent dye-labeled BMSCs
To track the distribution of BMSCs after transplantation, cells were harvested and labeled with PKH26, a lipophilic red fluorescent linker dye (Cat. # MINI26, Sigma Aldrich, USA). Add culture medium containing 1 ml Diluent C and 4ul PKH26 reagent when BMSCs reach 80% confluency after centrifuged and washed. Then, the mixture was mixed in a centrifuge tube and incubated at room temperature for 5 min. Subsequently, 2 mL of FBS was added to stop the staining. The supernatant was discarded and cells were resuspended in fresh medium. The PKH26-labeled BMSCs were examined under fluorescent microscope (Olympus, Tokyo, Japan). The cells were maintained in the growth medium prior to being transplantated into the uterine cavity of rabbits.
Animal IUA model and experimental protocol
The IUA rabbit model was generated by a dual damage method of mechanical curettage and lipopolysaccharide (LPS, 6 mg/L, Sigma) infection. Briefly, After anesthesia with 3% sodium pentobarbital (1 mL/kg), the abdominal wall and cavity was surgically opened with a vertical incision to expose the bilateral uterus under sterile conditions. A 2 mm transverse incision at the bilateral uterus junctions was cut with ophthalmic scissors, and a micro endometrial curette was inserted into the uterine cavity through the incision and rotated repeatedly until the uterine wall appeared rough and grainy. Subsequently, the end of the LPS-soaked cotton thread was placed into the uterine cavity through the uterine incision, leaving about 5 cm of the other end outside, and remove the cotton thread after 48 h. Finally, the abdominal cavity were stitched in layers and injected penicillin to prevent infection.
The study comprised of 75 female New Zealand white rabbits aged 12 weeks, which were randomly assigned to the following five groups (15 in each group): sham operation group (sham), rabbits merely had laparotomy without any treatment. IUA model group (IUA), rabbits underwent induction of previously described double injury. estrogen treated group (IUA + E2), intramuscular injections of estrogen (0.1 mg/kg) for 21 days. BMSCs treated group (IUA + BMSCs), 7 × 106 PKH26-labeled BMSCs were resuspended in 200μL medium and orthotopic injected into the bilateral injured uterine segment. BMSCs plus estrogen group (IUA + BMSCs + E2). All the study rabbits were euthanized on the 1 week, 2 weeks, 3 weeks and bilateral uterus were collected for the next experiment.
Hematoxylin–eosin (H&E) staining and Masson staining
At the appointed time, rabbits were sacrificed and bilateral uterine were resected. The endometrial tissue samples were fixed in 4% paraformaldehyde (Sigma-Aldrich, Cat# P6148) for 24 h and then embedded into paraffin blocks after dehydration and hyalinization. The paraffin-embedded sections were cut into 4-μm serial sections and subjected to HE and Masson staining according to routine procedures. Sections were taken using an orthostatic microscope (Olympus, Tokyo, Japan). Choose three different high-magnification fields, and calculate the number of endometrial glands and the degree of fibrosis by HE and Masson staining respectively. Image J (Image in Java, USA) software was used for statistical analysis of the average proportion of each group.
Samples were fixed in 4% paraformaldehyde and embedded in paraffin. The transverse paraffin sections were deparaffinized using xylene, rehydrated through a series of alcohol gradients. Then, the sections were incubated in 3% hydrogen peroxide for 30 min to inactivate endogenous peroxidase activity and incubated with the following primary antibodies: anti-p-Keratin (1:200, Abcam, USA,) and anti-a-SMA (1:200, Abcam, USA) for 2 h. Subsequently, the sections were washed with PBS treated with anti-rabbit or anti-mouse immunoglobulinG (IgG) secondary antibodies. Protein expression was visualized with diaminobenzidine (Dako Cytomation, USA) staining. The number of positively stained cells were observed under a high-power microscope and quantified at five randomly selected fields.
To further verify the effects of BMSC combined with estrogen therapy on the growth of endometrial epithelial cells and endometrial stromal cells, we detected the expression of epithelial specific marker CK7 and interstitial specific marker Vimentin by Immunofluorescence staining. Uterine tissues were fixed with 4% paraformaldehyde for 24 h and then was dehydrated in 30% sucrose solution at 4 °C. The dehydrated tissues were embedded with OCT compound (Tissue-Tek; Sakura) for rapid freezing, and cut into 6-μm slices with a microtome. Sections were incubated with blocking solution consisting of 0.3% Triton-X 100 and 5% donkey serum, followed by incubation with antibodies against CK7 (1:200, Abcam, USA) and Vimentin (1:200, Abcam, USA) at 4 °C overnight. After the slices was washed with PBS solution and incubated with Alexa fluorochrome-conjugated antibodies (1:200, Life Technologies, USA) at room temperature for 2 h. The nucleus was visualized with DAPI staining (1:500, Sigma, USA) for 15 min in the dark. Images were taken under a fluorescence microscope (Olympus, Japan).
Western blot analysis
The total protein samples was extracted and lysed from rabbit uterine tissues using RIPA lysis buffer (Solarbio, China), supplemented with the protease inhibitors (1 μg/mL) and PMSF (100 mM) and then homogenized using sterile scissor. The extract was centrifuged at 12,000 rpm for 20 min after lysising on ice for 1 h, and then the supernatant was collected and the total protein concentration was determined using the BCA protein assay kit (Thermo Scientific, MA, USA). Protein samples (30 μg) was separated on a 10% SDS-PAGE gel and transferred to PVDF membranes (Millipore, MA, USA). The membranes were blocked with 5% defatted milk for 1 h at room temperature and incubated overnight with the following primary antibodies at 4 °C: anti-N-cadherin (1:1000, Abcam), anti-Collagen I (1:1000, Abcam), anti-TGF-β1 (1:1000, CST), anti-a-SMA (1:2000, Abcam), anti-E-cadherin (1:1000, CST), anti-Vimentin (1:2000, Abcam), anti-ZEB1 (1:1000, Santa Cruz), anti-β-catenin (1:1000, NOVUS), anti-C-myc (1:1000, Abcam), anti-CyclinE (1:1000, Abcam), anti-Axin2 (1:2000, Abcam), anti-GAPDH (1:5000, Abcam). Membranes were washed three or four times with PBS and then incubated with the secondary antibody at 37℃ for 1 h in the dark. Finally, the proteins were visualized using ECL in the BioImaging System (BIO-RAD, USA). GAPDH was used as an internal control to normalize the relative expression of each protein of interest. The density of the bands was analyzed using ImageJ software.
Statistical analysis was performed using SPSS version 20.0 and GraphPad Prism version 6.0. All results were repeated 3 times and presented as the mean ± SD. Two samples were compared using independent sample two-tailed t-test. Statistical differences among multiple groups were determined by one-way analysis of variance (ANOVA). P < 0.05 was considered to be statistically significant.