FN在人绒毛膜癌细胞的表达及FUDR对绒癌细胞抑制作用研究
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摘要
[目的]
(1)检测纤维粘连蛋白(fibronectin,FN)在人绒毛膜癌不同侵袭力细胞JEG-3和JAR内的表达,探讨FN高表达与绒毛膜癌侵袭转移的关系；
(2)研究氟尿嘧啶脱氧核苷(floxuridine,FUDR)对绒毛膜癌高侵袭力JEG-3细胞生长能力的抑制作用,并与5-氟尿嘧啶进行比较,观察两种药物影响JEG-3细胞生长能力的差异并比较药物作用后JEG-3细胞中FN的表达改变。
【方法】
(1)运用Matrigel体外侵袭实验方法,检测两种绒毛膜癌细胞JEG-3和JAR侵袭能力,并比较差异；
(2)运用荧光定量—聚合酶链反应(real time-polymerase chain reaction,RT-PCR)及蛋白免疫印迹(Western blotting)方法,检测绒毛膜癌细胞JEG-3和JAR中FN mRNA和蛋白表达情况,并比较两者间的差异；
(3)用不同浓度(0.05、0.25、0.5、2.5和5 ug/m1)氟尿嘧啶脱氧核苷(FUDR)和5-氟尿嘧啶(5-FU),分别处理体外培养的高侵袭性绒毛膜癌细胞株JEG-3,在处理后48小时,以MTT比色法检测两种药物对JEG-3细胞生长增殖的影响,计算相对生存率,比较两种药物抑制JEG-3细胞生长能力的差异并检测FN蛋白表达。
【结果】
(1)Matrigel体外侵袭实验证实,JEG-3细胞较JAR细胞的侵袭能力明显增强(P<0.01)；
(2)在两种细胞中均检测到FN mRNA表达,相对低侵袭性JAR细胞,高侵袭性JEG-3细胞中FN mRNA表达明显增加(P<0.001),JEG-3细胞中FN蛋白表达水平较JAR细胞亦明显增加(P<0.05)；
(3)MTT抑制实验证实,FUDR和5-FU均对JEG-3细胞的生长能力具有抑制作用,且呈剂量依赖性。在5、2.5和0.5ug/ml药物浓度处理下,FUDR对JEG-3细胞生长抑制能力明显强于5-FU的作用(P<0.05)：最低有效浓度0.5ug/ml处理后,JEG-3细胞FN蛋白表达水平以FUDR处理的较5-FU处理要明显降低。
【结论】
(1)FN参与绒毛膜癌的发生和发展,与绒毛膜癌的侵袭和转移相关。
(2)FUDR能抑制高侵袭力绒毛膜癌细胞JEG-3生长能力,在一定剂量作用下,抑制能力强于5-FU。JEG-3细胞以FUDR处理的较5-FU处理FN蛋白表达水平降低。
Objective
(1)To detect the expression of FN (fibronectin) in different aggressive cells (JEG-3 and JAR) of choriocarcinoma, and investigate the relationships between the high expression of FN and the invasion and metastasis of choriocarcinoma.
(2)To explore the inhibitory effect of FUDR (floxuridine) on the high metastasis-ability of human choriocarcinoma JEG-3 cell line in vitro, and to compare the efficacy and the expression of FN that of floxuridine with 5-FU.
Methods
(1)Transwell Chamber assay was performed to compare the effect of the JEG-3 and JAR cell lines in invasion capacity;
(2) Expression of FN mRNA and protein level in the human choriocarcinoma cell lines(JEG-3 cells and JAR cells)were detected by real time-PCR and western blotting, and then compare their differences;
(3)The human choriocarcinoma cells(JEG-3 cells and JAR cells)cultured in vitro were treated by FUDR and 5-FU with concentration of 0.05, 0.25,0.5,2.5,5ug/ml for period of 48h. MTT assay was used to determine the growth inhibition by FUDR and 5-FU on the JEG-3 cells,and calculate the relative survival rate.Western Blotting assay was used to detect the expression of FN by FUDR and 5-FU with concentration as 0.5ug/ml.
Results
(1)This exprement investigate that the invasion ability of JEG-3 cell line was stronger than that of JAR cell line(P<0.01);
(2) In both of the two kinds of cells can detect the expression of FNmRNA,The expression of FN mRNA and protein level in JEG-3 cells were significantly stronger than that in JAR cells(P<0.001 and P<0.05);
(3)FUDR and 5-FU can inhibit growth of JEG-3 cell and manner of the concentration-dependent, the inhibition ability of FUDR was significantly stronger than that of 5-FU with concentration as 5,2.5, 0.5ug/ml(P<0.05).The expression of FN protein level in JEG-3 cells were treated by FUDR with concentration as 0.5ug/ml significantly lower than that by 5-FU.
Conclusion
(1)The existence of FN gene in JEG-3 and JAR cell lines may play an important role in the metastasis and invasion of human
choriocarcinoma.
(2)FUDR can inhibit growth of the high-metastasis JEG-3 cell line,the inhibition ability of FUDR was significantly stronger than that of 5-FU in the certain concentration.FN protein level in JEG-3 cells were treated by FUDR with concentration as 0.5ug/ml significantly lower than that by 5-FU.
(1)检测纤维粘连蛋白(fibronectin,FN)在人绒毛膜癌不同侵袭力细胞JEG-3和JAR内的表达,探讨FN高表达与绒毛膜癌侵袭转移的关系；
(2)研究氟尿嘧啶脱氧核苷(floxuridine,FUDR)对绒毛膜癌高侵袭力JEG-3细胞生长能力的抑制作用,并与5-氟尿嘧啶进行比较,观察两种药物影响JEG-3细胞生长能力的差异并比较药物作用后JEG-3细胞中FN的表达改变。
【方法】
(1)运用Matrigel体外侵袭实验方法,检测两种绒毛膜癌细胞JEG-3和JAR侵袭能力,并比较差异；
(2)运用荧光定量—聚合酶链反应(real time-polymerase chain reaction,RT-PCR)及蛋白免疫印迹(Western blotting)方法,检测绒毛膜癌细胞JEG-3和JAR中FN mRNA和蛋白表达情况,并比较两者间的差异；
(3)用不同浓度(0.05、0.25、0.5、2.5和5 ug/m1)氟尿嘧啶脱氧核苷(FUDR)和5-氟尿嘧啶(5-FU),分别处理体外培养的高侵袭性绒毛膜癌细胞株JEG-3,在处理后48小时,以MTT比色法检测两种药物对JEG-3细胞生长增殖的影响,计算相对生存率,比较两种药物抑制JEG-3细胞生长能力的差异并检测FN蛋白表达。
【结果】
(1)Matrigel体外侵袭实验证实,JEG-3细胞较JAR细胞的侵袭能力明显增强(P<0.01)；
(2)在两种细胞中均检测到FN mRNA表达,相对低侵袭性JAR细胞,高侵袭性JEG-3细胞中FN mRNA表达明显增加(P<0.001),JEG-3细胞中FN蛋白表达水平较JAR细胞亦明显增加(P<0.05)；
(3)MTT抑制实验证实,FUDR和5-FU均对JEG-3细胞的生长能力具有抑制作用,且呈剂量依赖性。在5、2.5和0.5ug/ml药物浓度处理下,FUDR对JEG-3细胞生长抑制能力明显强于5-FU的作用(P<0.05)：最低有效浓度0.5ug/ml处理后,JEG-3细胞FN蛋白表达水平以FUDR处理的较5-FU处理要明显降低。
【结论】
(1)FN参与绒毛膜癌的发生和发展,与绒毛膜癌的侵袭和转移相关。
(2)FUDR能抑制高侵袭力绒毛膜癌细胞JEG-3生长能力,在一定剂量作用下,抑制能力强于5-FU。JEG-3细胞以FUDR处理的较5-FU处理FN蛋白表达水平降低。
Objective
(1)To detect the expression of FN (fibronectin) in different aggressive cells (JEG-3 and JAR) of choriocarcinoma, and investigate the relationships between the high expression of FN and the invasion and metastasis of choriocarcinoma.
(2)To explore the inhibitory effect of FUDR (floxuridine) on the high metastasis-ability of human choriocarcinoma JEG-3 cell line in vitro, and to compare the efficacy and the expression of FN that of floxuridine with 5-FU.
Methods
(1)Transwell Chamber assay was performed to compare the effect of the JEG-3 and JAR cell lines in invasion capacity;
(2) Expression of FN mRNA and protein level in the human choriocarcinoma cell lines(JEG-3 cells and JAR cells)were detected by real time-PCR and western blotting, and then compare their differences;
(3)The human choriocarcinoma cells(JEG-3 cells and JAR cells)cultured in vitro were treated by FUDR and 5-FU with concentration of 0.05, 0.25,0.5,2.5,5ug/ml for period of 48h. MTT assay was used to determine the growth inhibition by FUDR and 5-FU on the JEG-3 cells,and calculate the relative survival rate.Western Blotting assay was used to detect the expression of FN by FUDR and 5-FU with concentration as 0.5ug/ml.
Results
(1)This exprement investigate that the invasion ability of JEG-3 cell line was stronger than that of JAR cell line(P<0.01);
(2) In both of the two kinds of cells can detect the expression of FNmRNA,The expression of FN mRNA and protein level in JEG-3 cells were significantly stronger than that in JAR cells(P<0.001 and P<0.05);
(3)FUDR and 5-FU can inhibit growth of JEG-3 cell and manner of the concentration-dependent, the inhibition ability of FUDR was significantly stronger than that of 5-FU with concentration as 5,2.5, 0.5ug/ml(P<0.05).The expression of FN protein level in JEG-3 cells were treated by FUDR with concentration as 0.5ug/ml significantly lower than that by 5-FU.
Conclusion
(1)The existence of FN gene in JEG-3 and JAR cell lines may play an important role in the metastasis and invasion of human
choriocarcinoma.
(2)FUDR can inhibit growth of the high-metastasis JEG-3 cell line,the inhibition ability of FUDR was significantly stronger than that of 5-FU in the certain concentration.FN protein level in JEG-3 cells were treated by FUDR with concentration as 0.5ug/ml significantly lower than that by 5-FU.
引文
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