Restorative Effects Of BMSCs On Ovaries In POF Rats

ObjectiveThe purpose of the present study is to evaluate the homing and distribution of BMSCs in ovaries injured by cisplatin after intravenous injection, and to evaluate its restorative effects on ovarian structure and function.Methods1. Isolation, phenotype characterization and labeling of BMSCs. BMSCs were isolated from Sprague-Dawley rats with adherent method, and then cultured with DMEM/F12 media supplemented with 10% fetal bovine serum. The expression of CD29、CD34、CD45 and CD90 in cellular surface and cell cycle was assessed by flow cytometer(FCM). BMSCs were labeled by the remombinant adenovirus vector with enhanced green fluorescent protein gene(Ad-EGFP). BMSCs were exposed to Ad-EGFP with a multiplicity of infection(MOI) of 50, 100, 150, 200, and 400 for 12 h. The viability and transfection efficiency of BMSCs was evaluated by an inverted fluorescence microscope after 24 h, 48 h, and 72 h.2. Effects of cisplatin and BMSCs on granulosa cells in vitro. GCs were isolated from immature female Sprague-Dawley rats. The follicle stimulating hormone receptor(FSHR) of the GCs was assessed by immunocytochemistry. GCs were exposed to various cisplatin concentrations(0, 1.0, 5.0 and 7.5 mg/L). Toxicity of cisplatin to GCs was assessed by Cell Counting Kit-8. GCs were co-cultured with BMSCs in the GCs/BMSCs ratio of 1:1 after optimal cisplatin was added. After 48 h, the GCs were collected and assessed by flow cytometry with an Annexin V/PI apoptosis detection kit. The migration of BMSCs was evaluated by Transwell Permeable Supports with 8.0 μm pore filters.3. The homing and distribution of BMSCs in ovaries in POF rats, and its restorative effects on ovarian function. The animals were divided into three groups(10 rats/group): normal control group(group 1), cisplatin-induced POF group(group 2) and BMSCs treatment group(group 3). To establish the POF model, the rats were intraperitoneally injected with a daily dose of cisplatin at 2 mg/kg of body weight for six days. BMSCs(4 × 106 cells/rat) labeled with EGFP in 0.6 ml PBS were injected via the tail vein on the seventh day. At day 15 and 30, five rats from each group were narcotized; the blood and organs were collected. The serum was isolated, and stored at-80°C for hormone test. The ovaries and other organs were dissected immediately, and were fixed in 4% paraformaldehyde for follicle counting, BMSCs migration and distribution were evaluated in ovarian sections using immunohistochemical staining. Apoptosis of GCs in ovaries was investigated with TUNEL analysis.Results1. After isolating from the bone marrow and 24 h of adherent growth, the BMSCs morphologically resembled fibroblasts. Morphology of third passage BMSCs was consensus. Most of the BMSCs were CD29+, CD90+, CD34-,and CD45-. Most of BMSCs were in still and DNA presynthetic phase(G0/G1 phase), and only few were in mitotic phase and DNA synthetic phase. After transfection, the green fluorescence protein started to express after 48 h; and highly expressed after 72 h. When the BMSCs were transfected with an MOI of 200, 99% of the BMSCs expressed green fluorescence proteins2. After isolating the GCs from the ovarian follicles, the cells showed adherent growth and morphologically resembled round or oval cells. Round or oval cells expressed as intracellular follicle stimulating hormone receptor(FSHR), the polygonal cells were negative. Compared with the control group, the viability of GCs with high doses of cisplatin(5.0 and 7.5 mg/L) significantly decreased. When co-cultured with BMSCs, apoptosis was reduced in GCs. An abundant number of BMSCs migrated to GCs that were injured by cisplatin.3. Following the transplantation of BMSCs, ovaries displayed EGFP-positive BMSCs. The number of BMSCs in the ovarian hilum and ovarian medulla was greater than in the ovarian cortex. We also found BMSCs surrounding the follicles; but there were no BMSCs in the ovarian follicle and corpus lutea. In the BMSCs treatment group, a significant increase in health antral follicles and corpuslutea count and estradiol level was detected, compared with the POF group after 30 days. The number of healthy antral follicles in the BMSCs group was greater than in the POF group after 30 days.Conclusions1. BMSCs were uniform and stable, which can be used for molecular biology researches and tissue engineering. BMSCs labeled with Ad-EGFP were stable and secure for long time tracing.2. BMSCs had protective effects on GCs injured be cisplatin in vitro.3. BMSCs home to the ovaries and restore follicular development. Therefore, BMSCs may be an efficient treatment method for POF.