| Part Ⅰ:Isolation, culture and characterization of mouse bone marrow mesenchymal stem cellsObjectiveTo explore the isolation of mouse bone marrow mesenchymal stem cells (BMSCs) using adherence-culture of compact bone fragments, and the comparisons of cell yields, proliferative potential, immune phenotypes and differentiation capacity into osteoblasts and adipocytes between primary BMSCs from compact bones and whole bone marrow.Methods1. The fragments and whole bone marrow of mouse femur and tibia were incubated to isolate BMSCs respectively;2. The comparisons of cell yields (cell counting), proliferative potential ([3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide], MTT;[colony forming unit-fibroblast], CFU-F assays), immune phenotypes (flow cytometry) and differentiation capacity (induction assays) between primary BMSCs from fragments and whole bone marrow were performanced;3. Passage3of BMSCs were obtained using the better isolation, and characterized by typical morphology, immune phenotypes and differentiation capacity.Results.1. Cell yield from cultured fragments was slightly fewer than that from cultured bone marrow using the same bone quantity. However, the trypsinized cells from cultured fragments exhibited significantly higher proliferation and were accompanied with more CD90and CD44expressions and fewer CD45expression. The osteogenic and adipogenic differentiation capacity of cells from cultured fragments were better than that of cells from bone marrow (P<0.05); 2. Passsge3cells from cultured fragments showed the BMSCs phenotypes by expressing CD90and CD44, negatively expressing CD45and CD34, and differentiating into osteoblast and adipocytes.ConclusionsThe directly adherent culture of compact bone is suitable for mouse BMSC isolation, and more BMSCs with potentially improved proliferation capacity can be obtained in the primary culture. Part Ⅱ:Secreted factors of bone marrow mesenchymal stem cells delay mouse spermatogenesis impairment induced by busulfanObjectiveTo explore the effect of BMSC secreted factors on busulfan-treated spermatogenesis, and to analyze the potential mechanisms, incuding cell adhesion and apoptosis.Methods.1. BMSC secreted factors (also termed as conditioned medium, CM) were obtained by ultrafiltration of BMSCs (passage3) serum-free culture for48h (as MSC-CM). HEK293cell secreted factors with similar protein concentration (0.60mg/mL) were obtained similarly except for cells (as293-CM);2. BALB/c mice received injection of40mg/kg busulfan (busulfan group), saline and DMSO (control groups) respectively. Testicular ultrastructures were analyzed by transmission electron microscope at day4. Testis were weighed and stained by hematoxylin-eosin (HE) to analyze the seminiferous tubules at weeks1,2,3,4after the injection. Expressions of cell junction genes, including N-cadherin, P-cadherin, occludin, zonula occludens-1(ZO-1), connexin43, intercelluler cell adhesion molecule-1(ICAM-1), and apoptosis in testis were detected using real-time polymerase chainreaction (PCR), western blot and TdT-mediated dUTP nick end labeling (TUNEL) assays at week2;3. After receiving injection of busulfan BALB/c mice were divided into two groups: MSC-CM and293-CM. CM were injected via tail vein on the next day after busulfan injection (200μL per mouse, every3days for2weeks). N-cadherin, P-cadherin, occludin, ZO-1, connexin43and ICAM-1expressions and apoptosis were analyzed at week2. Seminiferous tubules were observed by HE staining at weeks2and4;4. TM4were cultured in Dulbecco modified Eagle medium/F12(DMEM/F12) containing10%MSC-CM,10%293-CM, and10%fetal blood serum (FBS) respectively for16h. Then cells were subcultured at same density for2h for cell adherence detection (MTT assay), and CD44and CD54expressions (flow cytometry);5. GC-1were cultured in DMEM/F12containing2%FBS,10%MSC-CM+20mmol/lL cyclophosphamide (CP)+2%FBS,10%293-CM+20mmol/L CP+2%FBS or16h. GC-1apoptosis was analyzed using Annexin V/Prodium Iodide staining.Results1. Degeneration of spermatogenesis during4-week busulfan treatment was observed, including decrease of testicular and epididymal weights, separation of germ cell from the basal epitheliem and loss of germ cells in histological morphology. N-cadherin, P-cadherin, occludin, ZO-1, connexin43and ICAM-1expressions in busulfan testis significantly decreased at week2, while apoptosis increased (P<0.05). Electron microscope showed disintegration of tight, gap and adhesion junctions at day4;2. Compared with293-CM testis, spermatogenesis degeneration in MSC-CM testis delayed, including less separation of germ cells and vacuoles; N-cadherin, P-cadherin and ICAM-1expressions significantly increased; Spermatogenic cell apoptosis also decreased (P<0.05); 3. Compared with10%FBS and10%293-CM groups,10%MSC-CM group of TM4showed obviously more adherent cells, and more CD54and CD44expressions by flow cytometry (P<0.05);4. Compared with10%293-CM+20mmol/L CP group,10%MSC-CM+20mmol/l CP group of GC-1showed decreased apoptosis (P<0.05).Conclusions1. BMSCs secreted factors delay spermatogenic damage induced by busulfan, associated with reduced apoptosis and enhancement of N-cadherin, P-cadherin and ICAM-1expressions potentially;2. BMSCs secreted factors promote TM4adhesion through increasing expressions of adhesion molecules CD54and CD44in vitro;3. BMSCs secreted factors protecte GC-1apoptosis induced by cyclophosphamide in vitro. Part Ⅲ:Restoration of bone marrow mesenchymal stem cells secrected factors on azoospermia testisObjectiveTo explore the effect of BMSCs secrected factors on mouse azoospermia testis and to analyze potential factors of meiosis-specific genes and cell adhesion.Methods1. Secreted factors of BMSCs (MSC-CM) and293cells (293-CM) were obtained using the same methods of Part Ⅱ;2. BALB/c mice received single injection of40mg/kg busulfan (busulfan group). Azoospermia model was assessed by HE stain of seminiferous tubules4weeks later; mice receiving DMSO injection were served as control. Junction gene (N-cadherin, P-cadherin, ZO-1, occludin, connexin43) expressions were detected in both busulfan and control groups at week5;3. Azoospermia mice were divided into3groups:control (buslfan), MSC-CM and293-CM groups. CM were injected via tail vein on week5(200μL per mouse, every3days for3weeks). Meiosis-specific genes, including CyclinAl, synaptonemal comlex protein3(Scp3), deletedin azoospermia-like (Dazl), piwi-like protein1(Miwi), phosphoglycerate kinase2(Pgk2), stimulated by retinoic acid8(Stra8) and DEAD box polypeptide4(Vasa), and junction gene (N-cadherin, P-cadherin, ZO-1, occludin, connexin43) expressions were detected;4. TM4cells grown to confluent monolayers, scratch was made using200μL pipette scratches, and DMEM/F12containing10%MSC-CM,10%293-CM,10%FBS were added for24h respectively. Scratch area was recorded and analyzed at initial time (0h),6h and24h later;5. TM4were cultured in DMEM/F12containing10%MSC-CM,10%293-CM,10%FBS for24h respectively, and viability was detected by MTT assay.Results1. Azoospermia mice were conformed by testis HE stains:most of seminiferous tubules showed cell vacuolization, and spermatogonia, spermatocytes, spermatids and sperm almost entirely disappeared;2. CyclinAl, Dazl, Miwi, Pgk2, Stra8, Scp3, Vasa expressions in MSC-CM testis were significantly higher than those in293-CM group at week8;3. N-cadherin, connexin43, ZO-1, occludin expressions were inhibited in azoospermia testis at week5compared with DMSO group; N-cadherin expression in MSC-CM testis were significantly higher than that in293-CM group at week8;4. Scratch repair in10%MSC-CM group was significantly higher than that in10%293-CM and10%FBS group at6h, and obviously higher than that in10%293-CM group at24h. Compared with10%293-CM cells, TM4proliferation in10%MSC- CM group obviously increased.Conclusions1. BMSCs secrected factors promote the expressions of CyclinAl, Dazl, Miwi, Pgk2, Stra8, Scp3, Vasa and N-cadherin in azoospermia testis in vivo.2. BMSCs secrected factors improve TM4migration and proliferation in vitro. |
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