CAJ | 학술논문

目的观察蛋白激酶Cε(PKCε)在再生障碍性贫血(AA)患者血清体外诱导小鼠髓系前体细胞(32D细胞)凋亡中的作用.方法重型AA(SAA)患者血清、非重型AA(NSAA)患者血清及正常对照者血清分别与小鼠32D细胞孵育,用流式细胞术检测细胞凋亡指数,用蛋白免疫印迹法检测磷酸化PKCε(pPKCε)的表达.结果血清孵育32D细胞0、12、24、36及48 h,饥饿4 h,SAA组和NSAA组pPKCε表达在24 h上升到最高水平,与正常对照组相比较,差异有统计学意义(P＜0.05),血清孵育32D细胞24 h饥饿4 h后,SAA组pPKCε表达水平(0.90±0.10)较NSAA组(0.64±0.08)和正常对照组(0.54±0.08)明显上调(P值均＜0.05),NSAA组较正常对照组pPKCε表达水平也明显增加(P＜0.05),SAA组细胞凋亡率[(4.05±1.05)%]较正常对照组[(2.45±0.51)%]明显增加(P＜0.05),NSAA组[(3.24±0.56)%]较正常对照组无明显增加(P＞0.05).SAA组经同一份血清孵育的细胞的凋亡率与pPKCε表达水平之间有明显相关性(r=0.869,P＜0.05).结论SAA和NSAA患者血清均可诱导32D细胞pPKCε的表达,前者可导致32D细胞凋亡,后者对32D细胞凋亡无明显影响.
목적 관찰단백격매Cε(PKCε)재재생장애성빈혈(AA)환자혈청체외유도소서수계전체세포(32D세포)조망중적작용.방법 중형AA(SAA)환자혈청、비중형AA(NSAA)환자혈청급정상대조자혈청분별여소서32D세포부육,용류식세포술검측세포조망지수,용단백면역인적법검측린산화PKCε(pPKCε)적표체.결과 혈청부육32D세포0、12、24、36급48 h,기아4 h,SAA조화NSAA조pPKCε표체재24 h상승도최고수평,여정상대조조상비교,차이유통계학의의(P＜0.05),혈청부육32D세포24 h기아4 h후,SAA조pPKCε표체수평(0.90±0.10)교NSAA조(0.64±0.08)화정상대조조(0.54±0.08)명현상조(P치균＜0.05),NSAA조교정상대조조pPKCε표체수평야명현증가(P＜0.05),SAA조세포조망솔[(4.05±1.05)%]교정상대조조[(2.45±0.51)%]명현증가(P＜0.05),NSAA조[(3.24±0.56)%]교정상대조조무명현증가(P＞0.05).SAA조경동일빈혈청부육적세포적조망솔여pPKCε표체수평지간유명현상관성(r=0.869,P＜0.05).결론SAA화NSAA환자혈청균가유도32D세포pPKCε적표체,전자가도치32D세포조망,후자대32D세포조망무명현영향.
Objective To investigate the effect of phosphorylated protein kinase Cε(pPKCε) on apoptosis of 32D cells induced by sera from patients with aplastic anemia (AA). Methods The expression of pPKCε and apoptosis in 32D cells were measured by Western blotting and flow cytometry after incubation with sera from healthy individuals( controls, n=8 ), patients with severe AA(SAA, n=8) and non severe AA ( NSAA, n = 6). Results After incubation for 0, 12, 24, 36 and 48 hours in the presence of serum and for another 4 hours in medium deprived of serum, the levels of pPKCε in cells in SAA and NSAA group increased gradually, peaked at 24 hours, and then declined (P＜0.05 ). Compared with that in control group(0.54±0.08) , pPKCε was overexpressed in both SAA group (0.90±0.10) and NSAA group (0.64±0.08 ) ( P＜0.05 ) after 24 hours incubation with serum and subsequent 4 hours without serum, pPKCε level was higher in SAA group than in NSAA group ( P＜0.05 ). A greater proportion of 32D cells showed apoptosis after 24 hours incubation with sera from SAA patients [ (4.05±1.05 )%] and subsequent 4 hours incubation without serum than that in controls [ (2.45±0.51)%, P＜0.05 ], which was correlated with the same serum-induced expression of pPKCε( r = 0. 869, P＜0.05 ). Although the mean level of pPKCε expression was higher in NSAA group than in control group, no significant difference of apoptosis was found between the two groups[ (2.45±0.51 )% vs ( 3.24±0.56)%, P＞0.05 ]. Conclusion Sera from both SAA and NSAA patients could upregulate the expression of pPKCε in 32D cells. The SAA sera induce apoptosis in 32D cells significantly, but the latter do not.