CAJ | 학술논문

目的：探讨冬凌草甲素对急性单核细胞白血病细胞株THP-1的抗白血病效应及其作用机制。方法以急性单核细胞白血病细胞株THP-1为研究对象,应用改良四甲基偶氮唑盐法测定4、6、8μmol/L浓度冬凌草甲素处理72 h后THP-1细胞的增殖抑制率,以及计算2、4、6、8、10μmol/L冬凌草甲素处理24、72、120 h后的细胞生存率并绘制生长曲线；显微镜下观察4、6、8μmol/L冬凌草甲素处理24 h后THP-1细胞的形态学变化；采用流式细胞术检测0、4、6、8μmol/L冬凌草甲素处理24 h后THP-1细胞凋亡率；采用Western blot法检测6μmol/L冬凌草甲素处理THP-1细胞0、12、24 h后Akt/mTOR、Raf/MEK/ERK细胞信号转导通路的变化,以及凋亡调节蛋白Bcl-2和Bax的变化。结果①4、6、8μmol/L冬凌草甲素处理72 h后细胞增殖抑制率分别为(20.02±11.15)%、(45.99±12.76)%、(86．39±9.18)%,与4μmol/L冬凌草甲素比较,6、8μmol/L冬凌草甲素处理后细胞增殖抑制率均升高,差异均有统计学意义(P<0．05或P<0．01)；冬凌草甲素处理72 h后细胞半抑制浓度为(6.12±1.48)μmol/L；细胞生长曲线显示,冬凌草甲素对THP-1细胞的生长抑制作用呈时间和浓度依赖性。②冬凌草甲素处理24 h后,THP-1细胞出现凋亡,形成凋亡小体,浓度越高细胞凋亡小体形成越多。③0、4、6、8μmol/L 冬凌草甲素(分别设为对照组及4、6、8μmol/L组)处理24 h后,THP-1细胞凋亡率分别为(3.7±1.1)%、(16.2±3.3)%、(30.1±4.3)%、(49.5±6.7)%,对照组与各浓度组凋亡率比较差异均有统计学意义( P<0．05或P<0．01)；与4μmol/L组比较,6、8μmol/L组凋亡率均升高,差异均有统计学意义(P<0．05)。④经Western blot法检测6μmol/L冬凌草甲素处理THP-1细胞24 h后可抑制细胞内Akt/mTOR、Raf/MEK/ERK信号转导通路的活化,并下调Bcl-2、上调Bax的表达。结论冬凌草甲素通过抑制Akt/mTOR、Raf/MEK/ERK信号转导通路的活化,以及调节凋亡调节蛋白Bax和Bcl-2的表达而发挥抗白血病效应。目的：探討鼕凌草甲素對急性單覈細胞白血病細胞株THP-1的抗白血病效應及其作用機製。方法以急性單覈細胞白血病細胞株THP-1為研究對象,應用改良四甲基偶氮唑鹽法測定4、6、8μmol/L濃度鼕凌草甲素處理72 h後THP-1細胞的增殖抑製率,以及計算2、4、6、8、10μmol/L鼕凌草甲素處理24、72、120 h後的細胞生存率併繪製生長麯線；顯微鏡下觀察4、6、8μmol/L鼕凌草甲素處理24 h後THP-1細胞的形態學變化；採用流式細胞術檢測0、4、6、8μmol/L鼕凌草甲素處理24 h後THP-1細胞凋亡率；採用Western blot法檢測6μmol/L鼕凌草甲素處理THP-1細胞0、12、24 h後Akt/mTOR、Raf/MEK/ERK細胞信號轉導通路的變化,以及凋亡調節蛋白Bcl-2和Bax的變化。結果①4、6、8μmol/L鼕凌草甲素處理72 h後細胞增殖抑製率分彆為(20.02±11.15)%、(45.99±12.76)%、(86．39±9.18)%,與4μmol/L鼕凌草甲素比較,6、8μmol/L鼕凌草甲素處理後細胞增殖抑製率均升高,差異均有統計學意義(P<0．05或P<0．01)；鼕凌草甲素處理72 h後細胞半抑製濃度為(6.12±1.48)μmol/L；細胞生長麯線顯示,鼕凌草甲素對THP-1細胞的生長抑製作用呈時間和濃度依賴性。②鼕凌草甲素處理24 h後,THP-1細胞齣現凋亡,形成凋亡小體,濃度越高細胞凋亡小體形成越多。③0、4、6、8μmol/L 鼕凌草甲素(分彆設為對照組及4、6、8μmol/L組)處理24 h後,THP-1細胞凋亡率分彆為(3.7±1.1)%、(16.2±3.3)%、(30.1±4.3)%、(49.5±6.7)%,對照組與各濃度組凋亡率比較差異均有統計學意義( P<0．05或P<0．01)；與4μmol/L組比較,6、8μmol/L組凋亡率均升高,差異均有統計學意義(P<0．05)。④經Western blot法檢測6μmol/L鼕凌草甲素處理THP-1細胞24 h後可抑製細胞內Akt/mTOR、Raf/MEK/ERK信號轉導通路的活化,併下調Bcl-2、上調Bax的錶達。結論鼕凌草甲素通過抑製Akt/mTOR、Raf/MEK/ERK信號轉導通路的活化,以及調節凋亡調節蛋白Bax和Bcl-2的錶達而髮揮抗白血病效應。
목적：탐토동릉초갑소대급성단핵세포백혈병세포주THP-1적항백혈병효응급기작용궤제。방법이급성단핵세포백혈병세포주THP-1위연구대상,응용개량사갑기우담서염법측정4、6、8μmol/L농도동릉초갑소처리72 h후THP-1세포적증식억제솔,이급계산2、4、6、8、10μmol/L동릉초갑소처리24、72、120 h후적세포생존솔병회제생장곡선；현미경하관찰4、6、8μmol/L동릉초갑소처리24 h후THP-1세포적형태학변화；채용류식세포술검측0、4、6、8μmol/L동릉초갑소처리24 h후THP-1세포조망솔；채용Western blot법검측6μmol/L동릉초갑소처리THP-1세포0、12、24 h후Akt/mTOR、Raf/MEK/ERK세포신호전도통로적변화,이급조망조절단백Bcl-2화Bax적변화。결과①4、6、8μmol/L동릉초갑소처리72 h후세포증식억제솔분별위(20.02±11.15)%、(45.99±12.76)%、(86．39±9.18)%,여4μmol/L동릉초갑소비교,6、8μmol/L동릉초갑소처리후세포증식억제솔균승고,차이균유통계학의의(P<0．05혹P<0．01)；동릉초갑소처리72 h후세포반억제농도위(6.12±1.48)μmol/L；세포생장곡선현시,동릉초갑소대THP-1세포적생장억제작용정시간화농도의뢰성。②동릉초갑소처리24 h후,THP-1세포출현조망,형성조망소체,농도월고세포조망소체형성월다。③0、4、6、8μmol/L 동릉초갑소(분별설위대조조급4、6、8μmol/L조)처리24 h후,THP-1세포조망솔분별위(3.7±1.1)%、(16.2±3.3)%、(30.1±4.3)%、(49.5±6.7)%,대조조여각농도조조망솔비교차이균유통계학의의( P<0．05혹P<0．01)；여4μmol/L조비교,6、8μmol/L조조망솔균승고,차이균유통계학의의(P<0．05)。④경Western blot법검측6μmol/L동릉초갑소처리THP-1세포24 h후가억제세포내Akt/mTOR、Raf/MEK/ERK신호전도통로적활화,병하조Bcl-2、상조Bax적표체。결론동릉초갑소통과억제Akt/mTOR、Raf/MEK/ERK신호전도통로적활화,이급조절조망조절단백Bax화Bcl-2적표체이발휘항백혈병효응。
Objective To investigate the anti-leukemia effect of oridonin on acute monoblastic leukemia cell line THP-1. Methods Acute monoblastic leukemia cell line THP-1 was cultured in vitro. Cell proliferation Inhibition rate after 72 h of treatment of 4, 6, 8 μmol/L oridonin was examined using modified MTT assay, cell survival rate after 24, 72, 120 h of treatment of 2, 4, 6, 8,10 μmol/L oridonin was calculated and the growth curve was drawn. The cellular morphologic changes after 24 h of treatment of 4, 6, 8μmol/L oridonin were observed under a light microscope. The percentage of apopto-sis of THP-1 cells after 24 h of treatment of 0, 4, 6, 8μmol/L oridonin was evaluated using flow cytometric analysis. The ac-tivation levels of Akt/mTOR, Raf/MEK/ERK signaling pathways and the expression levels of Bcl-2 and Bax after 0, 12, 24 h of treatment of 6 μmol/L oridonin were examined by Western blot. Results ① Cell proliferation inhibition rate after 72 h of treatment of 4, 6, 8 μmol/L oridonin was (20. 02 ± 11. 15)%, (45. 99 ± 12. 76)%, (86. 39 ± 9. 18)% respectively, com-pared with 4 μmol/L oridonin treatment group, cell proliferation inhibition rate after 6, 8 μmol/L oridonin treatment were higher, with statistically significant differences (P<0. 05 or P<0. 01). Cell growth curve showed that oridonin inhibited the growth of THP-1 cells in time-and dose-dependent manner and the IC50 of oridonin was (6. 12 ± 1. 48)μmol/L after 72 h of treatment. ② After 24 h of treatment of oridonin, THP-1 cells apoptosis occurred and apoptotic bodies were formed, the high-er the concentration of oridonin, the more apoptotic bodies were. ③The percentage of apoptosis rate after 24 h of treatment of 0, 4, 6, 8 μmol/L oridonin were (3. 7 ± 1. 1)%, (16. 2 ± 3. 3)%, (30. 1 ± 4. 3)% and (49. 5 ± 6. 7)% respectively. The differences of apoptosis rate between various oridonin concentration groups and control group were statistically significant (P<0. 05 or P<0. 01). The differences between various oridonin concentration groups were also statistically significant (P<0. 05). ④ 6 μmol/L oridonin treatment inhibited the activation of Akt/mTOR and Raf/MEK/ERK signaling pathways in THP-1 cells, and down-regulated the level of anti-apoptotic protein Bcl-2 and up-regulated the expressions of pro-apoptotic protein Bax. Conclusion Oridonin exerts anti-leukemia effect in THP-1 cells by inhibiting the activation of Akt/mTOR and Raf/MEK/ERK signaling pathways, regulating the expression of Bcl-2 and Bax apoptosis-modulating proteins.