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目的:观察rhTNF-a在体外对人正常精子线粒体功能和质膜完整性的影响。方法:40例健康自愿者正常精液,Peroll梯度离心将精子密度调整为10×106/mL,分别与终浓度0 pg/mL(对照组)、30 pg/mL、60 pg/mL、90 pg/mL、270 pg/mL的rhTNF-a孵育。在0.5 h、1 h、2 h、3 h、4 h时间段取样,用终浓度为10 mg/mL 的Rh123和PI双染色精子,流式细胞仪检测精子线粒体膜电位及质膜完整性的变化。结果:与对照组相比较,60 pg/mL、90 pg/mL、270 pg/mL各组线粒体功能良好的精子明显减少,这种变化趋势与TNF-a浓度和孵育时间呈正相关(P <0.01)。30 pg/mL组的精子线粒体功能与对照组相比无统计学差异(P >0.05);30 pg/mL、60 pg/mL、90 pg/mL、270 pg/mL各组的PI和Rh123双染色均阳性的精子数较对照组多,且与rhTNF-a的浓度和孵育时间呈正相关(P <0.01)。结论: rhTNF-a对人精子线粒体功能有一定的影响,提示有可能干扰精子的能量代谢,降低精子受精能力,并有可能通过线粒体凋亡途径,促进精子凋亡;同时影响精子质膜的完整性。
Objective: To observe the effects of rhTNF-a on the mitochondrial function and plasma membrane integrity of human normal sperm in vitro. Methods: Forty healthy volunteers with normal semen and Peroll gradient centrifugation were used to adjust the sperm density to 10 × 106 / mL, respectively. The final sperm concentrations were 0 pg / mL, 30 pg / mL, 60 pg / mL and 90 pg / mL, 270 pg / mL of rhTNF-a. Samples were taken at 0.5 h, 1 h, 2 h, 3 h, and 4 h. Rh123 and PI double staining at a final concentration of 10 mg / mL was used to detect sperm mitochondrial membrane potential and plasma membrane integrity by flow cytometry Variety. Results: Compared with the control group, the mitochondrial functionally sperm counts of 60 pg / mL, 90 pg / mL and 270 pg / mL groups decreased significantly, which was positively correlated with the concentration of TNF-a and the incubation time (P <0.01 ). The sperm mitochondrial function in 30 pg / mL group was not significantly different from that in control group (P> 0.05). The PI and Rh123 double groups in 30 pg / mL, 60 pg / mL, 90 pg / mL and 270 pg / The number of sperm positive staining was more than that of control group, and was positively correlated with the concentration of rhTNF-a and the incubation time (P <0.01). CONCLUSION: rhTNF-a has a certain influence on human sperm mitochondrial function, suggesting that rhTNF-a may interfere with the energy metabolism of sperm and reduce sperm fertilization ability, and may promote apoptosis of sperm through mitochondrial apoptosis pathway; at the same time, affect the integrity of sperm plasma membrane Sex.