| According to the epidemiology statistics, each year about 40 thousand leukemia patients were founded in China, more than 20 thousand of them were children. Childhood leukemia disease was first on the list of children malignant tumor, and especially in male children, increasing year by year. Traditional chemotherapy drugs of busulfan has been widely applied to the pretreatment of hematopoietic stem cell transplantation, but this kind of non-specific cell cycle alkylating agent used for long-term can lead to male sterility. Following the development of the treatment, many children and adolescents with leukemia can survive to adulthood and then have the fertility requirements. At present, for different doses of busulfan in testicular result in production problems and lack the recovery data’s after discontinuation of busulfan in short and long-term researchBusulfan in the experiment has been widely used at the preparation of spermatogonial stem cell transplant recipients. Busulfan can removed the endogenous of sperm cells, and then transplant exogenous spermatagonial stem cells to the receptors in the testicles, a series of experiment was to study spermatogenesis. Different species using busulfan have a large different dose, rats was about 15 mg/kg; mice approximately 30 mg/kg; the monkey was 8 to 12 mg/kg, but that might have an unrecoverable reproduction damage. After intraperitoneal injection of busulfan, the body through the circulation and sent to the testicular tissue, it lead to testicular germ cells in seminiferous tubule piece of damage process. If the original spermatagonial stem cells don’t apoptosis, after a period of time, seminiferous tubule of germ cell can start the regeneration of spermatogenesis. So it‘s important for us to understand the mechanism of the Busulfan in testicular germ cell damage and find a way to reduce the effects are necessary.As the research object, this study applied the ICR mice celiac injection of busulfan, infertility disease mice model was established, and use it to found the mechanism of busulfan in reproductive toxicology. Firstly, through the morphological, histological, immunofluorescence staining, CCK-8 cell vitality testing to confirm the busulfan first influence spermatagonial stem cells in the testis, followed by spermatocyte, and this toxicity was time- and dose- dependence. Further we applied the TUNEL staining, flow cytometry detection the cell apoptosis, flow cytometry analysis the cell cycle, WB and PCR to determine the busulfan, p38 signaling pathways were activated, and lead to cell apoptosis. In the mechanism research, we tested the intracellular ROS levels, and found that the intracellular H2O2 and O2- level was raised, and the expression of MnSOD, SIRT1 significantly suppressed after busulfan treatment. Detection the GSH/GSSG ratio, we found that busulfan could devastate consumption the intracellular GSH, greatly destroy the balance of ROS in the cell. JC-1 testing found that imbalance of ROS could affect the balance of membrane potential in mitochondrial, triggering cell apoptosis. We used experiments in vivo to confirmed that melatonin can effectively alleviate cell damage caused by busulfan, but it was timedependence; In vitro experiments confirmed that melatonin could effectively alleviate the busulfan lead to intracellular ROS imbalance, thus reducing the ratio of apoptosis.This preliminary experiment proves that the mechanism of busulfan cause no refined disease in mice, and determine that busulfan can cause germ stem cell apoptosis mainly through destroing the balance of intracellular ROS, and it would be activated the p38 signaling pathway, instead of p53 signaling pathway. Also found the melatonin can effectively alleviate the cell apoptosis by which busulfan treatment, and reduce the risk of the azoospermia disease caused by busulfan. |
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