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目的:观察SET蛋白对GC-2 spd精母细胞株增殖和组蛋白乙酰化的影响,探讨SET蛋白调节精子形成的作用。方法:体外培养GC-2 spd细胞,通过精母细胞标志物乳酸脱氢酶C(LDHC)和睾丸特异激酶1(TESK1)鉴定。琼脂糖珠免疫共沉淀试剂盒检测SET蛋白与组蛋白H4的结合。分别转染空载腺病毒(Ad H1-si RNA/NS,对照组)和SET干涉腺病毒(Ad H1-si RNA/SET,干涉组),比较细胞增殖情况,荧光显微镜观察GC-2spd细胞中SET蛋白的表达;用实时定量逆转录聚合酶链反应(Real time RT-PCR)和蛋白质印迹(Western blot)检测m RNA以及蛋白的表达。结果:免疫荧光显示精母细胞株GC-2 spd阳性表达LDHC和TESK1;SET蛋白表达于GC-2细胞的胞核和胞质中,免疫共沉淀提示SET蛋白与组蛋白H4结合。转染SET干涉腺病毒后,核内和胞质中SET蛋白表达明显降低(P<0.01)。与对照组相比,干涉组细胞增殖明显降低,组蛋白H4乙酰化水平明显增高(P<0.05);但组蛋白乙酰化酶(HATs)和去乙酰化酶(HDACs)m RNA表达水平差异无统计学意义(P>0.05)。结论:精母细胞核内和胞浆中均表达SET蛋白;SET蛋白降调,则抑制GC-2spd细胞增殖。SET蛋白可能通过与组蛋白H4相互作用调节其乙酰化,参与调节精子形成。
OBJECTIVE: To observe the effect of SET protein on the proliferation and histone acetylation of GC-2 spd cell lines and to explore the role of SET protein in the regulation of spermatogenesis. Methods: GC-2 spd cells were cultured in vitro and identified by the spermatocyte markers LDH and TESK1. The agarose beads co-immunoprecipitation kit detects the binding of SET protein to histone H4. The transfected A549 cells were transfected with Ad H1-si RNA / NS, control group and SET interference RNA interference group respectively. The proliferation of GC-2 spd cells was observed by fluorescence microscopy SET protein expression was detected by real-time reverse transcription-polymerase chain reaction (Real time RT-PCR) and Western blot (Western blot) detection of m RNA and protein expression. Results: Immunofluorescence showed that positive expression of LDHc and TESK1 were detected by immunofluorescence in GC-2 spd. SET protein was expressed in the nucleus and cytoplasm of GC-2 cells. Co-immunoprecipitation suggested that SET protein binds to histone H4. The expression of SET protein in nucleus and cytoplasm was significantly decreased after transfection of SET interference adenovirus (P <0.01). Compared with the control group, the proliferation of the intervention group was significantly decreased, and the histone H4 acetylation level was significantly increased (P <0.05). However, there was no difference in the expression levels of histamine acetylase (HATs) and sAC (HDACs) m RNA Statistical significance (P> 0.05). Conclusion: The SET protein is expressed both in nucleus and cytoplasm of spermatocytes. When SET protein is down-regulated, the proliferation of GC-2 spd cells is inhibited. SET protein may regulate its acetylation by interacting with histone H4, which is involved in the regulation of spermatogenesis.