中华临床医师杂志（电子版）
中華臨床醫師雜誌（電子版）
중화림상의사잡지（전자판）
CHINESE JOURNAL OF CLINICIANS(ELECTRONIC VERSION)
2014年
24期
4422-4427
,共6页
刘飞%亓春玲%李健%李宝江
劉飛%亓春玲%李健%李寶江
류비%기춘령%리건%리보강
寡核苷酸类,反义%乳腺肿瘤%肿瘤干细胞%端粒酶反转录酶%生物学行为
寡覈苷痠類,反義%乳腺腫瘤%腫瘤榦細胞%耑粒酶反轉錄酶%生物學行為
과핵감산류,반의%유선종류%종류간세포%단립매반전록매%생물학행위
Oligonucleotides,antisense%Breast neoplasms%Neoplastic stem cells%Telomerase reverse transcriptase%Biological behavior
目的：观察人端粒酶反转录酶反义寡核苷酸（hTERT ASODN）对乳腺癌干细胞生物学行为的影响。方法合成针对人端粒酶反转录酶（hTERT）的反义寡核苷酸，以脂质体转染的方法将其转染入乳腺癌干细胞，设为 ASODN 组，以正义序列转染作为阴性对照组，并设未处理对照组。RT-PCR和Western blot分别检测各组hTERT基因和蛋白表达。应用CCK8法检测转染对乳腺癌干细胞增殖的影响，Transwell小室法检测干细胞侵袭、迁移能力。结果 RT-PCR和Western blot检测结果显示，ASODN组hTERT基因相对表达量为0.40±0.03，低于阴性对照组（0.81±0.05）和未处理对照组（0.79±0.04），F＝137.33，P＜0.001；hTERT基因蛋白相对表达量为0.39±0.06，低于阴性对照组（0.87±0.04）和未处理对照组（0.90±0.02），F＝59.29，P＜0.001。CCK8结果显示，乳腺癌干细胞培养12、24、36、48和60 h，ASODN组细胞增殖能力明显低于阴性对照组和未处理对照组，差异有统计学意义（P＜0.05）。Transwell侵袭实验，ASODN组穿越Transwell小室的细胞数为8.40±0.36，低于阴性对照组（15.64±0.13）和未处理对照组（14.31±1.12），F＝94.56，P＜0.001。Transwell迁移实验，ASODN组穿越Transwell小室的细胞数为30.6±8.03，低于阴性对照组（51.40±5.66）和未处理对照组（53.11±6.34），F＝20.11，P＝0.002。结论 hTERT ASODN可显著抑制乳腺癌干细胞体外增殖、侵袭和迁移能力。
目的：觀察人耑粒酶反轉錄酶反義寡覈苷痠（hTERT ASODN）對乳腺癌榦細胞生物學行為的影響。方法閤成針對人耑粒酶反轉錄酶（hTERT）的反義寡覈苷痠，以脂質體轉染的方法將其轉染入乳腺癌榦細胞，設為 ASODN 組，以正義序列轉染作為陰性對照組，併設未處理對照組。RT-PCR和Western blot分彆檢測各組hTERT基因和蛋白錶達。應用CCK8法檢測轉染對乳腺癌榦細胞增殖的影響，Transwell小室法檢測榦細胞侵襲、遷移能力。結果 RT-PCR和Western blot檢測結果顯示，ASODN組hTERT基因相對錶達量為0.40±0.03，低于陰性對照組（0.81±0.05）和未處理對照組（0.79±0.04），F＝137.33，P＜0.001；hTERT基因蛋白相對錶達量為0.39±0.06，低于陰性對照組（0.87±0.04）和未處理對照組（0.90±0.02），F＝59.29，P＜0.001。CCK8結果顯示，乳腺癌榦細胞培養12、24、36、48和60 h，ASODN組細胞增殖能力明顯低于陰性對照組和未處理對照組，差異有統計學意義（P＜0.05）。Transwell侵襲實驗，ASODN組穿越Transwell小室的細胞數為8.40±0.36，低于陰性對照組（15.64±0.13）和未處理對照組（14.31±1.12），F＝94.56，P＜0.001。Transwell遷移實驗，ASODN組穿越Transwell小室的細胞數為30.6±8.03，低于陰性對照組（51.40±5.66）和未處理對照組（53.11±6.34），F＝20.11，P＝0.002。結論 hTERT ASODN可顯著抑製乳腺癌榦細胞體外增殖、侵襲和遷移能力。
목적：관찰인단립매반전록매반의과핵감산（hTERT ASODN）대유선암간세포생물학행위적영향。방법합성침대인단립매반전록매（hTERT）적반의과핵감산，이지질체전염적방법장기전염입유선암간세포，설위 ASODN 조，이정의서렬전염작위음성대조조，병설미처리대조조。RT-PCR화Western blot분별검측각조hTERT기인화단백표체。응용CCK8법검측전염대유선암간세포증식적영향，Transwell소실법검측간세포침습、천이능력。결과 RT-PCR화Western blot검측결과현시，ASODN조hTERT기인상대표체량위0.40±0.03，저우음성대조조（0.81±0.05）화미처리대조조（0.79±0.04），F＝137.33，P＜0.001；hTERT기인단백상대표체량위0.39±0.06，저우음성대조조（0.87±0.04）화미처리대조조（0.90±0.02），F＝59.29，P＜0.001。CCK8결과현시，유선암간세포배양12、24、36、48화60 h，ASODN조세포증식능력명현저우음성대조조화미처리대조조，차이유통계학의의（P＜0.05）。Transwell침습실험，ASODN조천월Transwell소실적세포수위8.40±0.36，저우음성대조조（15.64±0.13）화미처리대조조（14.31±1.12），F＝94.56，P＜0.001。Transwell천이실험，ASODN조천월Transwell소실적세포수위30.6±8.03，저우음성대조조（51.40±5.66）화미처리대조조（53.11±6.34），F＝20.11，P＝0.002。결론 hTERT ASODN가현저억제유선암간세포체외증식、침습화천이능력。
Objective To investigate the effects of telomerase reversetranscriptase antisense oligonucleotide (hTERT-ASODN) on biological behavior of breast cancer stem cells. Methods Antisense oligonucleotides according to hTERT target gene was synthesized and transfected into breast cancer stem cells using lipofectamine. Set up ASODN transfection group. Sense oligodeoxy nucleotide transfection was treated as negative control and untreated cells as blank control group. The mRNA and protein expression levels of hTERT were tested by real-time PCR and Western blot. The cell proliferation was tested by CCK-8. Transwell systems were used to examine the invasion and migration abilities of the breast cancer stem cells. Results Real-time PCR analysis and Western blot assay showed that hTERT gene relative expression in ASODN group (0.40±0.03) was lower than that in negative control group (0.81±0.05), as compared with untreated control group (0.79±0.04), F=137.33, P<0.001;hTERT protein expression level (0.39±0.06) was lower than that in negative control group (0.87±0.04), and the untreated control group (0.90±0.02), F=59.29, P<0.001. CCK-8 assay showed that the breast stem cells proliferation at 12 h, 24 h, 36 h, 48 h and 60 h was obviously lower than that in negative control group and in untreated control group (P<0.05). The invasion and migration assay in Transwell systems showed that the number of cells that passed the Transwell membrane was 8.40±0.36, 30.6±8.03, respectively, compared with (15.64±0.13, 51.40±5.66) in negative control group and (14.31±1.12, 53.11±6.34) in untreated control group, F=94.56,P<0.001 and F=20.11, P=0.002. Conclusion Telomerase reverse transcriptase antisense oligonucleotides can significantly inhibit the proliferation, invasion and migration ability of breast cancer stem cells in vitro.
