c-myc反义核苷酸对淋巴瘤细胞系CA46凋亡的作用

Apoptosis of Lymphoma CA46 Cells Induced by Inhibition of c-myc with Antisense Oligoxynucleotides

目的 研究 c- m yc反义核苷酸对体外培养的淋巴瘤细胞系 CA4 6基因表达及诱导凋亡的作用。 方法 c- myc反义核苷酸 (ASPO)为人工合成的针对 c- myc基因的一段外源性基因片段 ,以无义的寡核苷酸作为对照 ,导入 CA4 6细胞后观察其基因表达及诱导凋亡的作用。流式细胞仪检测 c- m yc基因产物表达 ,荧光染色观察 CA4 6细胞的凋亡形态 ,流式细胞仪定量检测凋亡百分率 ,TU NEL 检测加用 VP1 6后的 CA4 6原位凋亡。 结果 c- m yc反义核苷酸 (ASPO)作用 CA4 6细胞后 ,RT- PCR示 c- myc m RNA表达降低。荧光染色 ASPO组细胞胞体缩小 ,胞膜完整 ,核质浓缩 ,出现黄绿色不规则或局部致密荧光 ,凋亡细胞约占 2 3.3% ,而 SPO组及空白组未见明显凋亡细胞 ,细胞呈均匀绿色荧光。流式细胞仪检测仅用 VP1 6 1 0 m g/ L 3d,ASPO作用 2 d后加 VP1 6 1 0 m g/ L 1 d及 AS-PO作用 3d均出现凋亡峰 ,凋亡率分别为 1 6 % ,72 %及 5 2 % ,而 SPO组及空白但均未见凋亡峰。流式细胞仪检测CA4 6细胞 c- myc蛋白为 95 .3% ,经 ASPO作用后降为 2 3.7% ,同时 SPO组 c- m yc表达率为 90 .6 % ,TU NEL 检测ASPO组凋亡细胞为 2 5 .5 % ,加用 VP1 6后的 CA4 6凋亡率为 36 .7%。 结论 体外实验中 CA4 6特异地被 c- m yc反义核酸抑制?

Objective\ To investigate the effect of c\|myc antisense oligodeoxynucleotide(ASPO) on apoptosis, and the expression of c\|myc gene in lymphoma CA46 cells.\ Methods\ The c\|myc ASPO were artificial synthetic gene segment transducted into tumor cells to bind to specific sites of target genes.\ The expression of c\|myc mRNA was determined by reverse transcription and polymerase chain reaction(RT\|PCR); the morphologic signs of apoptotic cells was observed by transmission electron microscope, the proportion of apoptotic cells was detected by flow cytometry and the DNA fragment was analysed by electrophoresis.\ Results\ The expression of the c\|myc mRNA in ASPO treated group was markedly decreased.\ After treatment ASPO, cells showed cell body decreased with compele embran, high\|density nucleus.\ Rate of apoptotic cells ASPO were 23 3% investigated by fluorscent staining.\ SPO group cells showed even yellow\|green, without apoptotic cells obviously.\ The levels of c\|myc proteins after treatment decreased from 95 3% to 23 7%, while apoptotic rates of only treatment with etoposide(VP16) 10 mg/L 72 hours, after ASPO for 48 hours then with VP16 10 mg/L 24 hours groups, and after ASPO for 72 hours were 16%, 72% and 52% respectively.\ The positive rates of c\|myc protein in CA46 cells treatment with SPO are 90 6% rate of apoptotic cells after treatment ASPO for 48 hours were 25 5% investigated by terminal deoxynucleotidyl transferase mediatted nick end labeling(TUNEL), while apoptotic rates of with VP16 10 mg/L 24 hours were 36 7%.\ Conclusion\ \ C\|myc antisense phosphorothioate oligodeoxynucleotide can inhibit the cellular proliferation specificlly, decrease the levels of c-myc mRNA, and induce apoptosis of CA46 cells.