中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
CHINESE JOURNAL OF EXPERIMENTAL SURGERY
2014年
6期
1309-1311
,共3页
黑素瘤%畸胎瘤衍生生长因子-1%黏附%侵袭%尿激酶纤溶酶原激活物
黑素瘤%畸胎瘤衍生生長因子-1%黏附%侵襲%尿激酶纖溶酶原激活物
흑소류%기태류연생생장인자-1%점부%침습%뇨격매섬용매원격활물
Melanoma%Teratocarcinoma-derived growth factor-1%Adhesion%Invasion%Urokinase-type plasminogen activator
目的 观察畸胎瘤衍生生长因子-1(TDGF-1)基因小干扰RNA对黑素瘤细胞黏附和侵袭的影响.方法 培养人黑素瘤A-375、C-918及M14细胞株,以实时荧光定量聚合酶链反应(FQ-PCR)方法检测TDGF-1基因mRNA水平,筛选出TDGF-1表达最高者.采用TDGF-1基因小干扰RNA(siRNA)转染黑素瘤细胞株,分别以FQ-PCR和免疫荧光方法观察TDGF-1基因mRNA和蛋白水平,然后以噻唑蓝(MMT)法检测细胞黏附,以Boyden方法检测癌细胞侵袭力.结果 FQ-PCR方法显示,A-375、C-918及M14细胞株TDGF-1 mRNA水平分别为0.589±0.081、0.712±0.065、1.517±0.085;以TDGF-1siRNA转染黑素瘤M14细胞后,癌细胞TDGF-1基因mRNA和蛋白表达明显下降,且呈浓度依赖性(P＜0.01);细胞黏附结果显示,Con-A、Con-B、3.125nmol/L、6.250 nmol/L和12.500 nmol/L组吸光度A值分别为0.89±0.15、0.85 ±0.12、0.62±0.09、0.51±0.08、0.33±0.06.Boyden小室试验结果显示,Con-A、Con-B、3.125 nmol/L、6.250 nmol/L和12.500 nmol/L组穿膜细胞数分别为(37.8±1.6)、(36.9±1.5)、(21.8±1.2)、(11.6±0.8)和(5.6±0.5)个.酶联免疫吸附试验(ELISA)结果显示,Con-A、Con-B、3.125 nmol/L、6.250 nmol/L和12.500 nmol/L组尿激酶型纤溶酶原激活物(uPA)含量分别为(85.2±1.8)、(84.8±1.5)、(51.6±1.2)、(35.6±0.8)、(17.8±0.6)ng/L.结论 TDGF-1基因在黑素瘤细胞黏附和侵袭中发挥着重要作用;以siRNA转染黑素瘤细胞,可抑制黑素瘤细胞黏附和侵袭能力,抑制uPA是其重要机制之一.
目的 觀察畸胎瘤衍生生長因子-1(TDGF-1)基因小榦擾RNA對黑素瘤細胞黏附和侵襲的影響.方法 培養人黑素瘤A-375、C-918及M14細胞株,以實時熒光定量聚閤酶鏈反應(FQ-PCR)方法檢測TDGF-1基因mRNA水平,篩選齣TDGF-1錶達最高者.採用TDGF-1基因小榦擾RNA(siRNA)轉染黑素瘤細胞株,分彆以FQ-PCR和免疫熒光方法觀察TDGF-1基因mRNA和蛋白水平,然後以噻唑藍(MMT)法檢測細胞黏附,以Boyden方法檢測癌細胞侵襲力.結果 FQ-PCR方法顯示,A-375、C-918及M14細胞株TDGF-1 mRNA水平分彆為0.589±0.081、0.712±0.065、1.517±0.085;以TDGF-1siRNA轉染黑素瘤M14細胞後,癌細胞TDGF-1基因mRNA和蛋白錶達明顯下降,且呈濃度依賴性(P＜0.01);細胞黏附結果顯示,Con-A、Con-B、3.125nmol/L、6.250 nmol/L和12.500 nmol/L組吸光度A值分彆為0.89±0.15、0.85 ±0.12、0.62±0.09、0.51±0.08、0.33±0.06.Boyden小室試驗結果顯示,Con-A、Con-B、3.125 nmol/L、6.250 nmol/L和12.500 nmol/L組穿膜細胞數分彆為(37.8±1.6)、(36.9±1.5)、(21.8±1.2)、(11.6±0.8)和(5.6±0.5)箇.酶聯免疫吸附試驗(ELISA)結果顯示,Con-A、Con-B、3.125 nmol/L、6.250 nmol/L和12.500 nmol/L組尿激酶型纖溶酶原激活物(uPA)含量分彆為(85.2±1.8)、(84.8±1.5)、(51.6±1.2)、(35.6±0.8)、(17.8±0.6)ng/L.結論 TDGF-1基因在黑素瘤細胞黏附和侵襲中髮揮著重要作用;以siRNA轉染黑素瘤細胞,可抑製黑素瘤細胞黏附和侵襲能力,抑製uPA是其重要機製之一.
목적 관찰기태류연생생장인자-1(TDGF-1)기인소간우RNA대흑소류세포점부화침습적영향.방법 배양인흑소류A-375、C-918급M14세포주,이실시형광정량취합매련반응(FQ-PCR)방법검측TDGF-1기인mRNA수평,사선출TDGF-1표체최고자.채용TDGF-1기인소간우RNA(siRNA)전염흑소류세포주,분별이FQ-PCR화면역형광방법관찰TDGF-1기인mRNA화단백수평,연후이새서람(MMT)법검측세포점부,이Boyden방법검측암세포침습력.결과 FQ-PCR방법현시,A-375、C-918급M14세포주TDGF-1 mRNA수평분별위0.589±0.081、0.712±0.065、1.517±0.085;이TDGF-1siRNA전염흑소류M14세포후,암세포TDGF-1기인mRNA화단백표체명현하강,차정농도의뢰성(P＜0.01);세포점부결과현시,Con-A、Con-B、3.125nmol/L、6.250 nmol/L화12.500 nmol/L조흡광도A치분별위0.89±0.15、0.85 ±0.12、0.62±0.09、0.51±0.08、0.33±0.06.Boyden소실시험결과현시,Con-A、Con-B、3.125 nmol/L、6.250 nmol/L화12.500 nmol/L조천막세포수분별위(37.8±1.6)、(36.9±1.5)、(21.8±1.2)、(11.6±0.8)화(5.6±0.5)개.매련면역흡부시험(ELISA)결과현시,Con-A、Con-B、3.125 nmol/L、6.250 nmol/L화12.500 nmol/L조뇨격매형섬용매원격활물(uPA)함량분별위(85.2±1.8)、(84.8±1.5)、(51.6±1.2)、(35.6±0.8)、(17.8±0.6)ng/L.결론 TDGF-1기인재흑소류세포점부화침습중발휘착중요작용;이siRNA전염흑소류세포,가억제흑소류세포점부화침습능력,억제uPA시기중요궤제지일.
Objective To study the effects of teratocarcinoma-derived growth factor-1 (TDGF-1) gene small interfering RNA (siRNA) on adhesion and invasion of human melanoma cell.Methods Realtime fluorescent quantitative polymerase chain reaction(FQ-PCR) was used to evaluate the TDGF-1 mRNA expression of human melanoma cell lines A-375,C-918 and M14.The highest expression of TDGF-1 was transfected with different dose of TDGF-1 siRNA.The expression of TDGF-1 mRNA and protein were were determined by Real-time quantitative PCR and Western blotting,respectively.Cell adhesion was exmined by methyl thiazol tetrazolium (MTT) assay,and cell invasion was evaluated by boyden chamber,respectiv ely.The matrix metalloproteinase-13 (MMP-13) were examined by enzyme linked immunosorbent assay (ELISA) assay.Results The results from FQ-PCR showed that the TDGF-1 mRNA of A-375,C-918 and M14 cell line is 0.589 ±0.081,0.712 ±0.065,and 1.517 ±0.085.After M14 cell line was transfected by TDGF-1 siRNA,the results of the MTT assay showed that the A Values of Con-A,Con-B,3.125 nmol/L,6.250 nmol/L,and 12.500 nmol/L siRNA were 0.89 ±0.15,0.85 ±0.12,0.62 ±0.09,0.51 ±0.08,0.33 ± 0.06,respectively (P ＜ 0.05) ; the results of Boyden assay showed that the membrane cell number of Con-A,Con-B,3.125 nmol/L siRNA,6.250 nmol/L siRNA,and 12.500 nmol/L siRNA were 37.8 ± 1.6,36.9±1.5,21.8±1.2,11.6±0.8,and 5.6±0.5,respectively (P＜0.05).The results from ELISA showed that urokinase-type plasminogen activator (uPA) content of Con-A,Con-B,3.125 nmol/L siRNA,6.250 nmol/L siRNA,and 12.500 nmol/L siRNA were (85.2 ± 1.8),(84.8 ± 1.5),(51.6 ± 1.2),(35.6 ± 0.8),(17.8 ± 0.6) ng/L.Conclusion TDGF-1 gene might play an important role in adhesion and invasion of human melanoma cancer cell.The siRNA targeted TDGF-1 could effectively inhibit adhesion and invasion of human melanoma cancer cell through downregulation uPA.
