雌激素对人子宫内膜样癌细胞中P57kip2表达及形态变化影响的研究

Effects of estrogen on P57 kip2 expression and morphological changes in human endometrial carcinoma cells

目的研究雌激素(Estrogen,E)对人子宫内膜样癌(Endometrioid carcinoma,EC)Ishikawa细胞(高分化)和JEC细胞(中分化)中P57kip2蛋白表达及细胞形态变化的影响。方法用等量含低、中、高浓度雌二醇(β-Estradiol,E2)(10-6、10-8、10-10mol/L)的培养基干预Ishikawa和JEC细胞作为实验组,等量不含E2的完全培养基培养作为对照组。细胞培养24、48、72 h后,MTT法检测细胞增殖情况;培养24、72 h后,Western blot法检测P57kip2蛋白表达情况,光镜、电镜观察细胞形态变化。结果①MTT结果:与对照组比较,在Ishikawa细胞中,E2低、中浓度组增殖明显(P<0.05),高浓度E2组细胞增殖不明显(P>0.05);在JEC细胞中,E2各浓度组均随药物浓度增高,促细胞生长的作用更明显(P<0.05)。②形态结构:与对照组比较,在低、中浓度组生长密度均增大,但低浓度组部分细胞内线粒体、内质网肿胀较轻微,中浓度组Ishikawa细胞部分线粒体肿胀较明显,JEC细胞损伤加重,可见核袋、核孔。高浓度组Ishikawa细胞生长密度减少,可见线粒体肿胀、内质网扩张囊泡化更明显,可见凋亡小体;JEC细胞生长密度则持续增大,线粒体高度肿胀并出现絮状沉积物,核内染色质均匀化,核孔增多。两株EC细胞核质比均较对照组增大。③Western blot结果:随着E2作用时间延长、作用浓度增加,P57kip2蛋白在Ishikawa细胞中表达逐渐增加,在JEC细胞中表达逐渐降低(P<0.05)。结论随着E浓度增加,P57kip2蛋白在Ishikawa细胞中表达呈递增趋势,发挥其抑制细胞生长的作用;P57kip2蛋白在JEC细胞中表达呈递减趋势,使其负向调控细胞周期的能力减弱,致细胞增殖明显;较高浓度则使EC细胞水肿明显、结构破坏,甚至造成不可逆性损伤,还可能诱导EC细胞往分化更差的方向发展。

Objective To investigate the effects of estrogen on P57 kip2 protein expression and cell morphology changes in EC Ishikawa(high differentiation)and JEC cells(medium differentiation).Methods In the experimental group,Ishikawa and JEC cells were disturbed with the same amount of medium and high concentration Estradiol(beta Estradiol,E 2)(10-6,10-8 and 10-10 mol/L)medium.Control group:cells were cultured in the same medium without drugs.After 24,48 and 72 h of culture,cell proliferation was detected by MTT method.After 24 and 72 h of culture,the protein expression of P57 kip2 was detected by Western blot assay and the morphological changes of cells were observed under light microscope and electron microscope.Results(1)MTT results showed that,in Ishikawa cells,compared with the control group,the proliferation of low and medium concentration E2 group was obvious(P<0.05),the cell proliferation of high-concentration E 2 group was not obvious(P>0.05).In JEC cells,all concentrations of E 2 promoted cell growth,and the promoting effect was more obvious with the increase of drug concentration(P<0.05).(2)The light and electron microscope results showed that,compared with the control group,the density of the two EC cells increased under the interference of low E 2 concentration.Under the interference of E 2 at medium concentration,the density of EC cells in the two strains increased.Under electron microscope,Ishikawa cell swelling was more severe than before,and irreversible damage,such as mitochondrial outer membrane rupture occurred.JEC cell reversible damage was aggravated and nuclear bag and nuclear pore appeared.After the interference of high concentration E 2,the cell density of Ishikawa was decreased,and the mitochondrial membrane was broken,the endoplasmic reticulum expanded into vesicles,and apoptotic corpuscles appeared.JEC cell density continued to increase,mitochondria were highly swollen and appeared flocculent sediments,chromatin in the nucleus was homogenized,and nuclear pores were increased.(3)Western blot assay showed that the expression of P57 kip2 protein in Ishikawa cells was increased gradually with the prolonged action time and increased concentration of E 2(P<0.05).P57 kip2 protein expression decreased gradually in JEC cells.Conclusion Female hormone and cell cycle regulation might be involved in the development and differentiation of EC cells.Low concentration of E 2 could cause morphological and structural changes in EC cells to enhance protein synthesis and promote growth and metabolism.It may also reduce the negative regulation of cell cycle and promote the growth of EC cells by down-regulating the expression of P57 kip2 protein.The higher concentration of E 2 could destroy the normal morphological structure of EC cells and cause irreversible cell damage,which may induce the development of EC cells toward low differentiation.