D-半乳糖刺激小鼠睾丸TM4支持细胞分泌功能的衰退及其机制

Decline of secretory function of TM4 Sertoli cells stimulated by D-galactose in mice and its mechanism

目的采用D-半乳糖(D-Gal)刺激小鼠TM4睾丸支持细胞,建立衰老所致TM4细胞分泌功能衰退模型。方法将小鼠TM4睾丸支持细胞分为正常对照组、(25、50、100、150、200、250)mmol/L D-Gal刺激组。MTT法检测TM4细胞活力、流式细胞术检测细胞周期、光镜观察细胞形态、衰老相关β-半乳糖苷酶(SA-β-Gal)染色观察衰老细胞百分率;反转录PCR检测P21、P16、胶质细胞源性神经营养因子(GDNF)和干细胞因子(SCF)的mRNA水平;Western blot法检测GDNF、SCF、红系2样核因子2(nuclear factor,erythroid 2 like 2,NRF2)、血红素加氧酶1(HO-1)、醌氧化还原酶1(NQO-1)的蛋白水平。结果与正常对照组比较,D-Gal刺激组细胞活力显著下降;处于G1期的细胞比例增高、S期细胞比例降低,细胞阻滞于G0/G1期;SA-β-Gal染色阳性率明显增多,衰老基因P21 mRNA表达上调,GDNF和SCF mRNA及蛋白水平均显著下调;氧化应激相关蛋白NRF2、HO-1和NQO-1的蛋白水平显著降低。结论成功建立D-Gal刺激的TM4睾丸支持细胞分泌功能衰退模型,衰老机制可能与下调NRF2信号通路有关。

Objective To establish a model of decline in secretion of senescent TM4 cells in vitro induced by D-galactose（ D-gal）. Methods Different concentrations of D-Gal（ 25,50,100,150,200 and 250 mmol/L） were used to induce TM4 cell senescence. The viability of TM4 cells was detected by MTT assay. The cell cycle was analyzed by flow cytometry. The cell morphology was observed by light microscopy and the percentage of senescent cel s was observed by senescence-associatedβ-galactosidase（ SA-β-Gal） staining. The mRNA expression levels of P21,P16,glial-derived neurotrophic factor（ GDNF）and stem cell factor（ SCF） were detected by reverse transcription PCR. The protein expression levels of GDNF,SCF,nuclear factor erythroid 2 like 2（ NRF2）,NAD（ P） H dehydrogenase [quinone]1（ NQO-1） and heme oxygenase-1（ HO-1）were detected by Western blot analysis. Results Compared with normal control group,D-Gal stimulation significantly decreased the cell viability in a concentration-dependent manner. The arrest of D-Gal-treated cells in G1 phase of cell cycle significantly increased,while it significantly decreased in S phase,and D-Gal induced cell cycle arrest at G0/G1 phase in TM4 cells. The percentages of SA-β-Gal positive cells increased significantly. The expression levels of P21 and P16 mRNAs were significantly up-regulated. The mRNA and protein levels of GDNF and SCF-1 were significantly down-regulated.Furthermore,the expression levels of oxidative stress-related protein NRF2,HO-1 and NQO-1 were significantly reduced.Conclusion The model of declined secretion function of senescent TM4 cells induced by D-Gal we established is stable and reliable. Its mechanism may be related to the down-regulation of NRF2 signaling pathway.