In this experiment, 32 SD rats, SPF grade, 8–9 weeks old, weighing 160–220 g, were provided by the Animal Experimental Center of Shanghai University of Traditional Chinese Medicine. Animal were maintained in cages (4/cage),at an ambient temperature of 26 °C, humidity of 50%-60%, 12 hours of light and darkness daily, and given free access to food and drinking water. This experimental study was carried out in accordance to the "Guidelines for the Protection and Application of Experimental Animals" issued by the National Institute of Health in the United States. The use of laboratory animals strictly follow the corresponding regulations of the Animal Experimental Management Committee of the Shanghai University of Traditional Chinese Medicine.
Culture of hUCMSCs
The hUCMSCs used in this experiment were obtained as a gift from from the Shandong Stem Cell Group. After thawing the hUCMSCs, the cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, GIBCO) medium containing 10% fetal bovine serum(FBS, GIBCO) ,in an incubator at 37℃, 5% CO2 and 40%-60% saturated humidity. Thereafter, the culture medium was refreshed every 2–3 days, until cells formed a confluent monolayer. Serial passage of hUCMSCs was carried out with 0.25% (w/v) trypsin (0.02% EDTA).
hUMSCs were phenotypically characterized by flow Cytometry (BECKMAN COULTER). Briefly, the cells in each tube (1.0 × 105) were labelled with the following monoclonal antibodies: CD146, CD140, CD105, CD29 and CD44. The results of flow cytometry were analysed by the FlowJo® software.
Rat Model of Severe Endometrial Damage
The model of severe endometrial injury in rats was made with reference to Jing . Intrauterine perfusion of absolute ethanol caused severe damage to the endometrium. Endometrial injury surgery is performed under anesthesia. The operation of abdominal anesthesia is simple and safe. The anesthesia time can be maintained for one hour. The choice of narcotic drugs is sodium pentobarbital, the dosage is small, and the mortality from anaesthesia is low. On the morning of surgery day,rats were anesthetized with 3% (w/v) pentobarbital sodium at a dose of 40 mg/kg. The lower abdomen was cut longitudinally and the skin was layered into the abdominal cavity. The rat uterus is Y-shaped, and the arterial clip is clamped at the right uterine horn and the right uterine cavity (the arterial clip is toothless, no damage to the uterus). The needle (1 ml) is extended into the uterine cavity and 95% (v/v) absolute ethanol was injected into the uterine cavity and maintained the filling state of the uterine cavity for 6 min. After 6 min, the ethanol in the uterine cavity was aspirated. The left uterus is treated in the same way. After flushing the abdominal cavity with 0.9% sodium chloride solution, the abdominal cavity and skin were sutured layer by layer.The entire operation takes about 20–25 minutes.After the operation, the rats were placed in an incubator to maintain their body temperature. After waking up, they were returned to their cages and fed with high-energy feed for 1 day and kept free to take water. On the day of surgery, 80,000 units of penicillin were injected intramuscularly into each rat.
Transplantation of hUCMSCs
Thirty-two SD rats were randomly divided into 4 groups of 8 rats each: normal group(NG), injury control group (ICG), MSC1 group (MG1) and MSC2 group (MG2) (Fig. 1). The normal group did not undergo any treatment. In the ICG, a severe model of rat endometrial damage was established by the above method. In the MG1, under isoflurane inhalation anesthesia,the hUCMSCs were injected into the tail vein within 24 hours after the model was established ( 1 × 107 cells ). In the MG2 within 24 hours after the model was established each rat was treated in the same way as the MG1.Then, on the 7th day after the model was established, rats in MG2 were again subjected to laparotomy under abdominal anesthesia. During the operation, hUCMSCs were injected into the uterine cavity with a 1 ml syringe (1 × 107 cells per side of the uterine cavity). On the 15th day after modeling, 32 rats were sacrificed by cervical dislocation.
Rat uterus were fixed in 4% (w/v) formaldehyde, and these uterus were cut into tissue sections with a thickness of about 4 mm. The cut surface was a cross section and were placed vertically in an embedding cassette. The water in the tissue block was removed with an alcohol gradient at a concentration of 50%→70%→80%→90% →95%. Then, the tissue block was placed in the clearing agent xylene I, II for 2 h, followed by dissolved paraffin, and then in an embedded wax block, prior to being cut into thin slices (thickness, 4 µm), and subjected to the process of Hematoxylin-Eosin Staining.
The sections were immersed in xylene to remove paraffin from the sections, and then rehydrated by immersion in high concentration to low concentration alcohol. For antigen retrieval, the sample was immersed in citrate buffer (0.01M, pH 6.0), boiled for 10 min, and naturally cooled. After rinsing 3 times with PBS for 2 minutes each time, the samples were incubated with primary antibody at room temperature for 60 minutes, and rinsed 3 times with PBS for 2 minutes each time. Then the samples were incubated with detection reagent (Beijing Jinqiao Biological) at room temperature for 30 minutes, rinse again with PBS for 3 times, and color was developed vy exposure to horseradish peroxidase (DAB, Shanghai Shenggong Biological Company) for 5–10 minutes, followed by counterstaining with hematoxylin, dehydration, and sealing with neutral resin.
Real-time PCR (q-PCR)
RNA was extracted from excised uterines using the RNAiso Plus kit (BID-RAD) according to the manufacturer’s instructions. As previously described, cDNA was prepared and amplified by SYBR Green-based relative quantification PCR using the ABI StepOne machine. We processed one excised uterine sample at a time, utilizing GAPDH as an internal reference. Results of the q-PCR showed that the relative variation of gene expression between groups, which was analyzed by the 2−ΔΔCT method. Data was computed from three different experiments. The primer sequences used in this study are as follows:
Primers are designed using Primer 5.0 software(Shanghai Biosynthesis).
Forward primer 5’-CCTCTTCTCATTCCTGCTCGT-3’
Reverse Primer 5’-GGCCATGGAACTGATGAGAGG-3’
Forward primer 5’-CCCACACGTCAAACTACAGCTC-3’
Reverse Primer 5’-TAACACACTTAGAAGCCAGCAG-3’
Forward primer 5’-AATGCCTCGTGCTGTCTGACC-3’
Reverse Primer 5’-GCCACAGGGATTTTGTCGTT-3’
Forward primer 5’-CAGCCAGTTGCCTTCTTGGGA-3’
Reverse Primer 5’-TGCACAACTCTTTTCTCATTTCCA-3’
Forward primer 5’-ACCACAGTCCATGCCATCAC-3’
Reverse Primer 5’-TCCACCACCCTGTTGCTGTA-3’
The uterus of all 32 rats were subjected to Hematoxylin-Eosin staining and endometrial cell marker protein expression analysis.Seven samples from NG(7/8), eight samples from ICG (8/8), seven samples from MG1 (7/8), and eight samples from MG2 (8/8). Two samples were abandoned due to specimen preservation issues.A total of 30 samples were analyzed by q-PCR. All data was statistically processed using the SPSS 21.0 software package. One-way ANOVA was applied by comparing multiple sets of samples. P < 0.05 was considered statistically significant.