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目的研究人端粒酶逆转录酶(hTERT)基因反义寡核苷酸(ASODN)对 HL-60细胞致瘤性的影响及其诱导凋亡作用。方法用流式细胞术检测 hTERT ASODN 转染 HL-60细胞72 h 后细胞凋亡,琼脂糖凝胶电泳检测凋亡细胞 DNA 梯带;将转染及未转染寡核苷酸的 HL-60细胞分别接种BALB/c 裸鼠,观察成瘤情况,切片观察移植瘤组织形态;另取10只 BALB/c 裸鼠,改进成瘤方法使其均匀成瘤后分成两组,在瘤体局部分别注射 ASODN、磷酸盐缓冲液(PBS),经7d 治疗后测量瘤体并切片观察其细胞形态变化,采用 TUNEL 方法检测细胞凋亡。结果 hTERT ASODN 作用于 HL-60细胞72 h 后,流式细胞术检测出早期凋亡峰,琼脂糖凝胶电泳显示 DNA 梯形条带,SODN 组及空白对照组未检测出凋亡峰及 DNA 梯形条带 ASODN 转染的 HL-60细胞组 BALB/c 裸鼠接种后第16～17天成瘤,5只中2只成瘤,未转染组在第12～13天成瘤,5只中4只成瘤,成瘤后 ASODN 治疗组瘤组织间质及血管较少,PBS 治疗对照组瘤组织间质发达,血管丰富。成瘤后 ASODN 治疗前肿瘤体积为(100.9±24.6)mm~3,治疗后肿瘤与 PBS 对照组比较,生长明显减缓[瘤体积分别为(422.7±326.4)mm~3和(786.4±357.6)mm~3](P<0.05) 组织切片显示,ASODN 治疗组瘤组织中可见大量细胞呈现凋亡形态,PBS 治疗组则少见凋亡细胞 TUNEL 检测显示,ASODN 治疗组瘤组织中阳性细胞数较 PBS 治疗组明显增多(P<0.05)。结论 hTERT 基因 ASODN 能够诱导 HL-60细胞凋亡并降低其在 BALB/c 裸鼠体内的致瘤性,抑制移植瘤增长速度。
Objective To study the effect of human telomerase reverse transcriptase (hTERT) gene antisense oligonucleotide (ASODN) on the tumorigenicity of HL-60 cells and its induction of apoptosis. Methods The apoptosis of HL-60 cells was detected by flow cytometry 72 h after hTERT ASODN transfection. The DNA ladder of apoptotic cells was detected by agarose gel electrophoresis. The HL-60 The cells were inoculated BALB / c nude mice respectively to observe the tumorigenesis. The morphological changes of the transplanted tumor were observed by sectioning. Another 10 BALB / c nude mice were used to improve the tumorigenesis method to divide the tumor into two groups. ASODN and phosphate buffered saline (PBS) were injected. After 7 days of treatment, the tumors were observed and sliced to observe the changes of cell morphology. TUNEL method was used to detect apoptosis. Results The apoptotic peak was detected by flow cytometry after hTERT ASODN was applied to HL-60 cells for 72 h. The results of agarose gel electrophoresis showed no apoptotic peak and DNA ladder in DNA ladder, SODN group and blank control group BALB / c nude mice were infected with ASODN-transfected HL-60 cells on day 16-17 after inoculation, and 2 of 5 tumors formed tumors. On day 12-13, untransfected tumors formed tumors and 4 of 5 Tumor formation, tumor formation after ASODN treatment group interstitial and blood vessels less, PBS control group tumor tissue developed, rich in blood vessels. The tumor volume of ASODN before treatment was (100.9 ± 24.6) mm ~ 3 after treatment, and the growth of the tumor was slowed down significantly compared with PBS control group [tumor volume was (422.7 ± 326.4) mm 3 and (786.4 ± 357.6) mm ~ 3] (P <0.05) Tissue sections showed that a large number of cells showed apoptotic morphology in the ASODN treated group, but rare apoptotic cells in the PBS treated group showed that the number of positive cells in the ASODN treated group was higher than that in the PBS treated group Group increased significantly (P <0.05). Conclusion hTERT gene ASODN can induce HL-60 cell apoptosis and reduce its tumorigenicity in BALB / c nude mice and inhibit the growth of xenografts.