中华妇产科杂志
中華婦產科雜誌
중화부산과잡지
CHINESE JOUNAL OF OBSTETRICS AND GYNECOLOGY
2009年
11期
851-855
,共5页
韩冰%向阳%沙桂华%张浩%刘欣
韓冰%嚮暘%沙桂華%張浩%劉訢
한빙%향양%사계화%장호%류흔
绒毛膜癌%细胞系%肿瘤%氟尿苷%抗药性%肿瘤%胸苷酸合酶
絨毛膜癌%細胞繫%腫瘤%氟尿苷%抗藥性%腫瘤%胸苷痠閤酶
융모막암%세포계%종류%불뇨감%항약성%종류%흉감산합매
Choriocarcinoma%Cell line%tumor%Floxuridine%Drug resistance%neoplasm%Thymidylate synthetase
目的 建立对氟尿苷(FUDR)耐药的绒毛膜癌(绒癌)细胞系,鉴定其生物学特性,并探讨胸苷合成酶(TS)在绒癌FUDR耐药细胞中的表达.方法 采用间断诱导法诱导绒癌细胞系JeG-3,建立绒癌FUDR耐药细胞系JeG-3/FUDRA(诱导浓度为2.3×10~(-3) mol/L).倒置显微镜观察细胞形态;活细胞计数(CCK-8)法观察敏感细胞和耐药细胞的生长和耐药指数;化学发光法测定培养液中的激素水平;流式细胞仪检测细胞周期比例和染色体倍性;荧光定量PCR技术检测不同浓度FUDR(2.3×10~(-3)、5.1×10~(-5)、5.1×10~(-7) mol/L)诱导的JeG-3细胞(JeG-3/FUDRA、JeG-3/FUDRC、JeG-3/FUDRE)中TS mRNA的表达.结果 (1)绒癌耐药细胞系JeG-3/FUDRA的建立及生物学鉴定:历时10个月成功建立了绒癌耐药细胞系JeG-3/FUDRA,其耐药指数为31.62.与JeG-3亲本细胞相比,JeG-3/FUDRA耐药细胞的形态发生明显改变,细胞生长明显减慢(P＜0.05),群体倍增时间明显延长[分别为(27.4±0.8)和(47.3±2.1)h,P＜0.01];G_2/M期细胞比例明显下降[分别为(27±6)%和(9±3)%,P＜0.05],G_0/G_1,期细胞比例明显增加[分别为(29±3)%和(63±7)%,P＜0.01];染色体数目明显增加[分别为(76±5)和(143±5)条,P＜0.01];其培养液中人绒毛膜促性腺激素(hCG)水平明显下降[分别为(29.1±9.9)和(8.9±0.9)mU/ml,P＜0.05],孕酮水平明显下降[分别为(31±8)和(16±3)ng/ml,P＜0.05].(2)耐药细胞中TS mRNA的表达:经低浓度FUDR诱导后,JeG-3/FUDRE细胞中TS mRNA的表达水平下调;随着FUDR诱导浓度的增加和作用时间的延长,细胞中TS mRNA的表达水平逐渐恢复,其中JeG-3/FUDRC细胞中TS mRNA表达水平和JeG-3亲本细胞比较,差异无统计学意义(P＞0.05);而JeG-3/FUDRA细胞中TS mRNA表达水平则明显高于JeG-3亲本细胞(P＜0.05).结论 本研究成功建立了绒癌FUDR耐药细胞系JeG-3/FUDRA,随着FUDR浓度的增加和作用时间的延长,其TS mRNA的表达有阶段性的差异.就本研究结果而言,TS mRNA表达水平的高低不适用于作为肿瘤是否对FUDR耐药的指标.
目的 建立對氟尿苷(FUDR)耐藥的絨毛膜癌(絨癌)細胞繫,鑒定其生物學特性,併探討胸苷閤成酶(TS)在絨癌FUDR耐藥細胞中的錶達.方法 採用間斷誘導法誘導絨癌細胞繫JeG-3,建立絨癌FUDR耐藥細胞繫JeG-3/FUDRA(誘導濃度為2.3×10~(-3) mol/L).倒置顯微鏡觀察細胞形態;活細胞計數(CCK-8)法觀察敏感細胞和耐藥細胞的生長和耐藥指數;化學髮光法測定培養液中的激素水平;流式細胞儀檢測細胞週期比例和染色體倍性;熒光定量PCR技術檢測不同濃度FUDR(2.3×10~(-3)、5.1×10~(-5)、5.1×10~(-7) mol/L)誘導的JeG-3細胞(JeG-3/FUDRA、JeG-3/FUDRC、JeG-3/FUDRE)中TS mRNA的錶達.結果 (1)絨癌耐藥細胞繫JeG-3/FUDRA的建立及生物學鑒定:歷時10箇月成功建立瞭絨癌耐藥細胞繫JeG-3/FUDRA,其耐藥指數為31.62.與JeG-3親本細胞相比,JeG-3/FUDRA耐藥細胞的形態髮生明顯改變,細胞生長明顯減慢(P＜0.05),群體倍增時間明顯延長[分彆為(27.4±0.8)和(47.3±2.1)h,P＜0.01];G_2/M期細胞比例明顯下降[分彆為(27±6)%和(9±3)%,P＜0.05],G_0/G_1,期細胞比例明顯增加[分彆為(29±3)%和(63±7)%,P＜0.01];染色體數目明顯增加[分彆為(76±5)和(143±5)條,P＜0.01];其培養液中人絨毛膜促性腺激素(hCG)水平明顯下降[分彆為(29.1±9.9)和(8.9±0.9)mU/ml,P＜0.05],孕酮水平明顯下降[分彆為(31±8)和(16±3)ng/ml,P＜0.05].(2)耐藥細胞中TS mRNA的錶達:經低濃度FUDR誘導後,JeG-3/FUDRE細胞中TS mRNA的錶達水平下調;隨著FUDR誘導濃度的增加和作用時間的延長,細胞中TS mRNA的錶達水平逐漸恢複,其中JeG-3/FUDRC細胞中TS mRNA錶達水平和JeG-3親本細胞比較,差異無統計學意義(P＞0.05);而JeG-3/FUDRA細胞中TS mRNA錶達水平則明顯高于JeG-3親本細胞(P＜0.05).結論 本研究成功建立瞭絨癌FUDR耐藥細胞繫JeG-3/FUDRA,隨著FUDR濃度的增加和作用時間的延長,其TS mRNA的錶達有階段性的差異.就本研究結果而言,TS mRNA錶達水平的高低不適用于作為腫瘤是否對FUDR耐藥的指標.
목적 건립대불뇨감(FUDR)내약적융모막암(융암)세포계,감정기생물학특성,병탐토흉감합성매(TS)재융암FUDR내약세포중적표체.방법 채용간단유도법유도융암세포계JeG-3,건립융암FUDR내약세포계JeG-3/FUDRA(유도농도위2.3×10~(-3) mol/L).도치현미경관찰세포형태;활세포계수(CCK-8)법관찰민감세포화내약세포적생장화내약지수;화학발광법측정배양액중적격소수평;류식세포의검측세포주기비례화염색체배성;형광정량PCR기술검측불동농도FUDR(2.3×10~(-3)、5.1×10~(-5)、5.1×10~(-7) mol/L)유도적JeG-3세포(JeG-3/FUDRA、JeG-3/FUDRC、JeG-3/FUDRE)중TS mRNA적표체.결과 (1)융암내약세포계JeG-3/FUDRA적건립급생물학감정:력시10개월성공건립료융암내약세포계JeG-3/FUDRA,기내약지수위31.62.여JeG-3친본세포상비,JeG-3/FUDRA내약세포적형태발생명현개변,세포생장명현감만(P＜0.05),군체배증시간명현연장[분별위(27.4±0.8)화(47.3±2.1)h,P＜0.01];G_2/M기세포비례명현하강[분별위(27±6)%화(9±3)%,P＜0.05],G_0/G_1,기세포비례명현증가[분별위(29±3)%화(63±7)%,P＜0.01];염색체수목명현증가[분별위(76±5)화(143±5)조,P＜0.01];기배양액중인융모막촉성선격소(hCG)수평명현하강[분별위(29.1±9.9)화(8.9±0.9)mU/ml,P＜0.05],잉동수평명현하강[분별위(31±8)화(16±3)ng/ml,P＜0.05].(2)내약세포중TS mRNA적표체:경저농도FUDR유도후,JeG-3/FUDRE세포중TS mRNA적표체수평하조;수착FUDR유도농도적증가화작용시간적연장,세포중TS mRNA적표체수평축점회복,기중JeG-3/FUDRC세포중TS mRNA표체수평화JeG-3친본세포비교,차이무통계학의의(P＞0.05);이JeG-3/FUDRA세포중TS mRNA표체수평칙명현고우JeG-3친본세포(P＜0.05).결론 본연구성공건립료융암FUDR내약세포계JeG-3/FUDRA,수착FUDR농도적증가화작용시간적연장,기TS mRNA적표체유계단성적차이.취본연구결과이언,TS mRNA표체수평적고저불괄용우작위종류시부대FUDR내약적지표.
Objective To establish human choriocarcinoma JeG-3 cell line resistant to floxuridine (FUDR)and describe the characteristics of this FUDR-resistant subline.The thymidylate synthase (TS) expression level in FUDR-resistant subline was also discussed.Methods The FUDR-resistant sub-line JeG-3/FUDRA was established by intermitted exposure to grads increased FUDR.Reversed microscope was used to observe the morphological changes in FUDR-resistant sub-line.Population doubling time was calculated and compared based on the growth curve of these two cell lines,cell cycles and chromosomal ploidy were assayed with flow cytometry methods.The chemo-luminescence assay was used to detect the hormone secretion by two kinds of cell lines.The resistant index (RI) was measured by cell counting kit-8 (CCK-8)assay.Quantitative RT-PCR was used to detect the mRNA expression level of TS and we also detected the TS mRNA expression level in different doses exposed subline.Results The RI of JeG-3/FUDRA was 31.62.Compared with the JeG-3 cell,the FUDR-resistant cell line had gross changes in morphological,cell growth,cell cycles and chromosomal numbers.The ability of human chorionic gonadotrop(hCG) and progesterone secretion was lower in JeG-3/FUDRA subline.The trend of TS mRNA expression was:while exposed to low concentration of FUDR,the TS mRNA expression level was downregulated,then followed the increasing dose of the drug,the expression level of TS mRNA ascended gradually.When the terminal concentration was reached,the expression level of TS mRNA in JeG-3/FUDRA subline was higher than that of JeG-3 cell line (P＜0.05).Conclusions We established the FUDR-resistant subline of JeG-3 successfully.The TS mRNA expression level is stage-related to the different concentration and different phase in FUDR exposure.Our data suggested that TS mRNA expression level may not be used as a biomarker to predict the chemosensitivity in FUDR-based chemotherapy.
