Cell-specific Expression Analysis Of Mouse Testis Gene Fancd2os And Its Pre Liminary Function

Objectives: Real-time PCR and Western blotting were used to analyze the cell specific expression patterns of mouse testis gene fancd2 os on m RN A and protein levels, The cellular localization of FANCD2 OS protein in testis were detected b y immunohistochemistry and immunofluorescence. HEK293 T cell model with over-expression of fancd2 os was establishedand were applied to explore the potential function of fancd2 os gene from the cellular level.Methods: 1. The expression of fancd2 os m RNA in sperm cell(GC1-spg), sperm cell(GC2-spd), Leydig cells(TM3) and Sertoli cells(TM4) were analyzed by real-time PCR; 2. The expression of FANCD2 OS protein in GC1-spg, GC2-spd TM3 and TM4 were detected by Western blotting; 3. Cellular localization of FANCD2 OS protein intestis were demonstrated by immunohistochemical and immunofluorescence staining; 4. HEK293 T cell model with over-expression of fancd2 os was established and the expression levels of fancd2 os gene were detected by reverse transcription polymerase chain reaction(RT-PCR) and Western blotting; 5. The effect of fancd2 os on the proliferation of HEK293 T cells were detected by CCK8; 6. The cleaved Caspase3 protein levels in HEK293 T cells were detected with Western blotting;7. Cell apoptosis of HEK293 T cells induced by ultraviolet light were analyzed by using Caspase3 fluorescent staining kit; 8. Cell apoptosis, and mitochondrial membrane potential were detected by flow cytometry in model HEK293 T cells.Results: 1. Real-time PCR analysis showed that fancd2 os m RNA were expressed in four kinds different cells, but the expression in TM3 was relative higher; 2. Western blotting results showed that FANCD2 OS protein were expressed in four kinds cells, with higherexpression in TM3; 3. Immunohistochemical and immunofluorescence staining demonstrated that FANCD2 OS protein was mainly localized in TM3; 4. RT-PCR and Western blotting revealed that mouse fancd2 os gene were over-expressed in transfected HEK293 T cells; 5. CCK8 assay results demonstrated that the proliferation of HEK293 T cells with over-expressed fancd2 os were decreased obviously; 6. Western blotting results showed that the cleaved Caspase3 level in model cells was significantly higher than the other two groups; 7. Caspase3 fluorescence staining indicated that the positive red fluorescence signal in HEK293 T with fancd2 os was significantly higher than that of the empty group and the control group; 8. With flow cytometry analysis,We found that cell apoptosis in transfected group was significantly higher than that of the no- load group and null cell group(P < 0.01), and the percentage of mitochondrial membrane potential decrease in transfected group was significantly higher than that of no- load group and null cell group(P < 0.05);Conclusions: 1. fancd2 os gene was expressed in GC1-spg, GC2-spd, TM3 and TM4, but therelative expression of gene in TM3 was higher; 2. fancd2 os gene can inhibit the proliferation of HEK293 T cells. It can aslo decrease the mitochondrial membrane potential. fancd2 os could promote the apoptosis of HEK293 T cells through increasing the level of cleaved Caspase3 in HEK293 T cells.