目的 探讨澳洲茄胺盐酸盐(SBHL)在三氧化二砷(As2O3)诱导的HeLa细胞凋亡中的作用及对细胞端粒酶活性的影响.方法 采用细胞培养的方法体外培养宫颈癌HeLa细胞.用四甲基偶氮唑盐(MTT)法在SBHL(0,10,20,40,80,460,320 μmol/L)中筛选出最佳浓度.将HeLa细胞按1640培养液含As2O3和最佳SBHL浓度分为3组:对照组(0 μmol/L As2O3)、As2O3组(5 μmol/L As2O3)、As2O3+SBHL组(5μmoL/L As2O3+40μmol/L SBHL),培养时间分别为24、48、72 h.倒置显微镜下观察的HeLa细胞生长状况;透射电镜下观察HeLa细胞凋亡状况;MTT法检测HeLa细胞存活率;流式细胞术检测HeLa细胞周期和凋亡率;抗酒石酸酸性磷酸酶-酶联免疫(TRAP-ELISA)法检测HeLa细胞端粒酶活性的变化.结果 倒置相差显微镜下,对照组HeLa细胞排列密集,细胞呈圆形;As2O3组细胞数量减少,细胞间距逐渐增大;As2O3+SBHL组细胞收缩明显,细胞核碎裂为花瓣状,细胞间隙变得更大.电镜下,对照组Hela细胞表面有丰富的微绒毛,细胞间隔清晰,可见幼稚连接,连接不紧密,细胞核内多为常染色体,核仁清晰,核浆比约1∶1;As2O3组细胞超微结构呈现典型的凋亡特征,核内异染色质增多,染色质浓缩成块状,边集于核膜;As2O3+SBHL组细胞表面的微绒毛断裂、减少,核致密,呈块状分布,可见到凋亡小体.细胞存活率在24、48、72 h,组间比较差异均有统计学意义(X2分别为10.39、13.88、17.21,P均<0.05);在72 h,As2O3+SBHL组细胞存活率(52.80%)明显低于As2O3组(77.51%,X2=9.29,P<0.05).细胞周期G1期、S期组间比较差异有统计学意义(F值分别为7.46、22.14,P均<0.05);与对照组[(41.57±1.56)%、(50.45±2.37)%]比较,As2O3组[(27.10±5.32)%]和As2O3+SBHL组[(20.06±4.98)%]S期细胞减少(P<0.05或<0.01),G1期细胞增加[(58.70±5.18)%、(69.67±4.17)%,P<0.05或<0.01].细胞凋亡率组间比较差异有统计学意义(F=4.01,P<0.05);与对照组[(1.18±1.40)%]比较,As2O3组[(6.04±2.53)%]和As2O3+SBHL组[(21.08±1.22)%]细胞凋亡率明显升高(P<0.05或<0.01),其中As2O3+SBHL组高于As2O3组(P<0.01).端粒酶活性组间比较差异有统计学意义(F=21.28,P<0.05);与对照组(2.107±0.057)比较,As2O3组(1.214±0.621)和As2O3+SBHL组(0.865±0.284)细胞端粒酶活性降低(P均<0.05),其中As2O3+SBHL组低于As2O3组(P<0.05).结论 SBHL能促进As2O3诱导HeLa细胞凋亡,并能通过抑制端粒酶活性促进As2O3诱导HeLa细胞凋亡.
目的 探討澳洲茄胺鹽痠鹽(SBHL)在三氧化二砷(As2O3)誘導的HeLa細胞凋亡中的作用及對細胞耑粒酶活性的影響.方法 採用細胞培養的方法體外培養宮頸癌HeLa細胞.用四甲基偶氮唑鹽(MTT)法在SBHL(0,10,20,40,80,460,320 μmol/L)中篩選齣最佳濃度.將HeLa細胞按1640培養液含As2O3和最佳SBHL濃度分為3組:對照組(0 μmol/L As2O3)、As2O3組(5 μmol/L As2O3)、As2O3+SBHL組(5μmoL/L As2O3+40μmol/L SBHL),培養時間分彆為24、48、72 h.倒置顯微鏡下觀察的HeLa細胞生長狀況;透射電鏡下觀察HeLa細胞凋亡狀況;MTT法檢測HeLa細胞存活率;流式細胞術檢測HeLa細胞週期和凋亡率;抗酒石痠痠性燐痠酶-酶聯免疫(TRAP-ELISA)法檢測HeLa細胞耑粒酶活性的變化.結果 倒置相差顯微鏡下,對照組HeLa細胞排列密集,細胞呈圓形;As2O3組細胞數量減少,細胞間距逐漸增大;As2O3+SBHL組細胞收縮明顯,細胞覈碎裂為花瓣狀,細胞間隙變得更大.電鏡下,對照組Hela細胞錶麵有豐富的微絨毛,細胞間隔清晰,可見幼稚連接,連接不緊密,細胞覈內多為常染色體,覈仁清晰,覈漿比約1∶1;As2O3組細胞超微結構呈現典型的凋亡特徵,覈內異染色質增多,染色質濃縮成塊狀,邊集于覈膜;As2O3+SBHL組細胞錶麵的微絨毛斷裂、減少,覈緻密,呈塊狀分佈,可見到凋亡小體.細胞存活率在24、48、72 h,組間比較差異均有統計學意義(X2分彆為10.39、13.88、17.21,P均<0.05);在72 h,As2O3+SBHL組細胞存活率(52.80%)明顯低于As2O3組(77.51%,X2=9.29,P<0.05).細胞週期G1期、S期組間比較差異有統計學意義(F值分彆為7.46、22.14,P均<0.05);與對照組[(41.57±1.56)%、(50.45±2.37)%]比較,As2O3組[(27.10±5.32)%]和As2O3+SBHL組[(20.06±4.98)%]S期細胞減少(P<0.05或<0.01),G1期細胞增加[(58.70±5.18)%、(69.67±4.17)%,P<0.05或<0.01].細胞凋亡率組間比較差異有統計學意義(F=4.01,P<0.05);與對照組[(1.18±1.40)%]比較,As2O3組[(6.04±2.53)%]和As2O3+SBHL組[(21.08±1.22)%]細胞凋亡率明顯升高(P<0.05或<0.01),其中As2O3+SBHL組高于As2O3組(P<0.01).耑粒酶活性組間比較差異有統計學意義(F=21.28,P<0.05);與對照組(2.107±0.057)比較,As2O3組(1.214±0.621)和As2O3+SBHL組(0.865±0.284)細胞耑粒酶活性降低(P均<0.05),其中As2O3+SBHL組低于As2O3組(P<0.05).結論 SBHL能促進As2O3誘導HeLa細胞凋亡,併能通過抑製耑粒酶活性促進As2O3誘導HeLa細胞凋亡.
목적 탐토오주가알염산염(SBHL)재삼양화이신(As2O3)유도적HeLa세포조망중적작용급대세포단립매활성적영향.방법 채용세포배양적방법체외배양궁경암HeLa세포.용사갑기우담서염(MTT)법재SBHL(0,10,20,40,80,460,320 μmol/L)중사선출최가농도.장HeLa세포안1640배양액함As2O3화최가SBHL농도분위3조:대조조(0 μmol/L As2O3)、As2O3조(5 μmol/L As2O3)、As2O3+SBHL조(5μmoL/L As2O3+40μmol/L SBHL),배양시간분별위24、48、72 h.도치현미경하관찰적HeLa세포생장상황;투사전경하관찰HeLa세포조망상황;MTT법검측HeLa세포존활솔;류식세포술검측HeLa세포주기화조망솔;항주석산산성린산매-매련면역(TRAP-ELISA)법검측HeLa세포단립매활성적변화.결과 도치상차현미경하,대조조HeLa세포배렬밀집,세포정원형;As2O3조세포수량감소,세포간거축점증대;As2O3+SBHL조세포수축명현,세포핵쇄렬위화판상,세포간극변득경대.전경하,대조조Hela세포표면유봉부적미융모,세포간격청석,가견유치련접,련접불긴밀,세포핵내다위상염색체,핵인청석,핵장비약1∶1;As2O3조세포초미결구정현전형적조망특정,핵내이염색질증다,염색질농축성괴상,변집우핵막;As2O3+SBHL조세포표면적미융모단렬、감소,핵치밀,정괴상분포,가견도조망소체.세포존활솔재24、48、72 h,조간비교차이균유통계학의의(X2분별위10.39、13.88、17.21,P균<0.05);재72 h,As2O3+SBHL조세포존활솔(52.80%)명현저우As2O3조(77.51%,X2=9.29,P<0.05).세포주기G1기、S기조간비교차이유통계학의의(F치분별위7.46、22.14,P균<0.05);여대조조[(41.57±1.56)%、(50.45±2.37)%]비교,As2O3조[(27.10±5.32)%]화As2O3+SBHL조[(20.06±4.98)%]S기세포감소(P<0.05혹<0.01),G1기세포증가[(58.70±5.18)%、(69.67±4.17)%,P<0.05혹<0.01].세포조망솔조간비교차이유통계학의의(F=4.01,P<0.05);여대조조[(1.18±1.40)%]비교,As2O3조[(6.04±2.53)%]화As2O3+SBHL조[(21.08±1.22)%]세포조망솔명현승고(P<0.05혹<0.01),기중As2O3+SBHL조고우As2O3조(P<0.01).단립매활성조간비교차이유통계학의의(F=21.28,P<0.05);여대조조(2.107±0.057)비교,As2O3조(1.214±0.621)화As2O3+SBHL조(0.865±0.284)세포단립매활성강저(P균<0.05),기중As2O3+SBHL조저우As2O3조(P<0.05).결론 SBHL능촉진As2O3유도HeLa세포조망,병능통과억제단립매활성촉진As2O3유도HeLa세포조망.
Objective To study whether solasodine hydrochloride (SBHL) could enhance the effect of arsenic trioxide in inducing apoptosis and affecting telomerase activity in cervical cancer HeLa cells. Methods Using cell culture methods, cervical cancer HeLa cells were cultured in vitro. The optimal concentration of SBHL was determined by MTT method from 0, 10, 20, 40, 80, 160, to 320 μmol/L. HeLa cells were grown in improved RPMI1640 supplemented respectively with arsenic trioxide(5 μmol/L As2O3), As2O3(5 μmol/L)+ SBHL( 40 μmol/L) and none (control group). The growth morphology of HeLa cells was observed under phase contrast microscopy after culture for 24, 48, and 72 h. Apoptosis of HeLa cells was determined under transmission electronic microscopy. The method of MTT was used to study the cell survival percentage. The technique of flow cytometry was used to measure cell cycle and cell apoptosis percentage. The method of tartrate-resistant acid phosphatase-enzyme linked immunosorbent assay (TRAP-ELISA) was used to determine telomerase activity of HeLa cells. Results Under phase contrast microscopy, in control group HeLa cells were round, densely packed; in As2O3 group the numbers of the cells were less, cell spacing increased; in As2O3 + SBHL group the cells shrinked significantly, nuclear fragmented as a petal-like, gap became larger. Under transmission electronic microscopy, there were rich microvillus on the cell surface in control group, cell intervals clear, immature connections, and the intervals did not close. The structure of the mitochondria in the cytoplasm was integrated. Most of the chromatin in the nucleus were, euchromatin and characteristics of apoptosis with heterochromatin increased and the chromatin condensed into masses, on the boundary of nuclear membrane. The microvillud on the cell surface were ruptured and decreased in As2O3 + SBHL group. The chromatin condensed into masses. The formation of apoptotic bodies was observed. The difference was statistically significant between groups in cell survival percentage at 24, 48, 72h(x2 = 10.39 , 13.88 , 17.21,respectively, all P < 0.05). Cell survival percentage in SBHL + As2O3 group (52.80%) was significantly less than that of As2O3 group(77.51%, x2 = 9.29, P < 0.05) at 72 h. In cell cycles, the difference was statistically significant between groups in C1 phase and S phase(F = 7.46,22.14, all P < 0.05), respectively. Compared with , control group[ (41.57 ± 1.56)%, (50.45 ± 2.37)%], cell percentages in S phase in As2O3 + SBHL group[(20.06 ± 4.98)%] and As2O3 group[(27.10 ± 5.32)%] were decreased(P< 0.05 or < 0.01), while cell percentage in C1 phase was increased[(58.70 ± 5.18)%, (69.67 ± 4.17)%, P< 0.05 or < 0.01]. The difference was statistically significant between groups in apoptotic percentage of HeLa cells (F = 4.01, P < 0.05). Compared with control group[ (1.18 ± 1.40)%], apoptosis percentage was significantly increased in As2O3 + SBHL group and As2O3 group [(21.08± 1.22)%, (6.04±2.53)%, P< 0.05 or < 0.01], respectively, and As2O3 + SBHL group was higher than As2O3 group(P < 0.01). The difference was statistically significant between groups in telomerase activity (F = 21.28, P< 0.05). Telomerase activity was inhibited in As2O3 group(1.214 ± 0.621) and As2O3A + SBHL group(0.865 ± 0.284) compared to control group (2.107 ± 0.057, all P < 0.05), and telomerase activity in As2O3 + SBHL group was lower than that of As2O3 group (P < 0.05). Conclusions SBHL enhances the effect of As2O3 in inducing apoptosis in HeLa cells, which is related to its inhibiting telomerase activity in HeLa cells.
