肿瘤研究与临床
腫瘤研究與臨床
종류연구여림상
CANCER RESEARCH AND CLINIC
2015年
5期
320-327,331
,共9页
游锦梅%许雅鑫%吴彬%Yang Yong%李钰%陈显久
遊錦梅%許雅鑫%吳彬%Yang Yong%李鈺%陳顯久
유금매%허아흠%오빈%Yang Yong%리옥%진현구
干扰素%口腔鳞状细胞癌%酪氨酸蛋白激酶%信号转导通路
榦擾素%口腔鱗狀細胞癌%酪氨痠蛋白激酶%信號轉導通路
간우소%구강린상세포암%락안산단백격매%신호전도통로
Interferon%Oral squamous cell carcinoma%Protein tyrosine kinases%Signal transduction pathway
目的 以3种不同癌变程度的口腔细胞为工具,观察在干扰素α(IFN-α)作用下,JAK-信号转导子和转录激活子(STAT)-细胞因子信号抑制因子(SOCS)信号分子的表达变化,为深入理解口腔鳞状细胞癌(OSCC)细胞免疫逃逸的机制提供研究基础.方法 常规培养NOK、DOK、KB细胞.空白组加入完全培养液,二甲基亚砜(DMSO)对照组加入含有0.1％ DMSO的完全培养液,实验组设10、100、500U/ml三个不同浓度组,每孔分别加入含不同浓度IFN-α的完全培养液,JAK抑制剂干预组在加入IFN-α前1h加入JAK抑制剂CP-690550 100 μmol/L.细胞培养24 h后检测.进行反转录聚合酶链反应(RT-PCR)和Western blot实验.结果 采用RT-PCR法检测显示:JAK1和JAK2在NOK细胞均有少量表达,IFN-α和CP-690550对NOK细胞组的JAK1和JAK2表达无影响,差异无统计学意义(P＞0.05).100和500 U/ml的IFN-α能刺激DOK和KB细胞的JAK1和JAK2表达增加,差异均具有统计学意义(P＜0.05).CP-690550能有效减低DOK和KB细胞的JAK1表达,差异均具有统计学意义(P＜0.05),而对JAK2表达无作用.采用Western blot法检测显示:STAT1、STAT3和pSTAT3(Tyr705)在对照组均有表达,pSTAT1(Tyr701)在对照组无表达;IFN-α和CP-690550对NOK细胞组的STAT1、STAT3和pSTAT3(Tyr705)表达无影响,差异无统计学意义(P＞0.05).100、500 U/ml的IFN-α能刺激DOK和KB细胞的pSTAT3(Tyr705)表达增加,差异均具有统计学意义(P＜0.05);而对pSTAT1(Tyr701)的表达无影响.CP-690550能有效减低DOK和KB细胞的pSTAT3(Tyr705)表达,差异均具有统计学意义(P＜0.05).采用Western blot法检测显示,SOCS1和SOCS3在对照组均有表达;IFN-α和CP-690550对NOK细胞组的SOCS1和SOCS3表达无影响,差异无统计学意义(P＞0.05).100 U/ml和500 U/ml的IFN-α能刺激DOK和KB细胞的SOCS1表达增加,差异均具有统计学意义(P＜0.05);而对SOCS3的表达无影响.CP-690550能有效减低DOK和KB细胞的SOCS1表达,差异均具有统计学意义(P＜0.05).结论 IFN-α激活DOK和KB细胞JAK1和JAK2的表达,以JAK1激活为主;IFN-α通过激活DOK和KB细胞JAK1和JAK2的表达促进STAT3在Tyr705位点磷酸化;IFN-α通过促进STAT3磷酸化进一步促进SOCS1的表达,发挥对IFN-α作用的抑制作用,减少细胞凋亡.
目的 以3種不同癌變程度的口腔細胞為工具,觀察在榦擾素α(IFN-α)作用下,JAK-信號轉導子和轉錄激活子(STAT)-細胞因子信號抑製因子(SOCS)信號分子的錶達變化,為深入理解口腔鱗狀細胞癌(OSCC)細胞免疫逃逸的機製提供研究基礎.方法 常規培養NOK、DOK、KB細胞.空白組加入完全培養液,二甲基亞砜(DMSO)對照組加入含有0.1％ DMSO的完全培養液,實驗組設10、100、500U/ml三箇不同濃度組,每孔分彆加入含不同濃度IFN-α的完全培養液,JAK抑製劑榦預組在加入IFN-α前1h加入JAK抑製劑CP-690550 100 μmol/L.細胞培養24 h後檢測.進行反轉錄聚閤酶鏈反應(RT-PCR)和Western blot實驗.結果 採用RT-PCR法檢測顯示:JAK1和JAK2在NOK細胞均有少量錶達,IFN-α和CP-690550對NOK細胞組的JAK1和JAK2錶達無影響,差異無統計學意義(P＞0.05).100和500 U/ml的IFN-α能刺激DOK和KB細胞的JAK1和JAK2錶達增加,差異均具有統計學意義(P＜0.05).CP-690550能有效減低DOK和KB細胞的JAK1錶達,差異均具有統計學意義(P＜0.05),而對JAK2錶達無作用.採用Western blot法檢測顯示:STAT1、STAT3和pSTAT3(Tyr705)在對照組均有錶達,pSTAT1(Tyr701)在對照組無錶達;IFN-α和CP-690550對NOK細胞組的STAT1、STAT3和pSTAT3(Tyr705)錶達無影響,差異無統計學意義(P＞0.05).100、500 U/ml的IFN-α能刺激DOK和KB細胞的pSTAT3(Tyr705)錶達增加,差異均具有統計學意義(P＜0.05);而對pSTAT1(Tyr701)的錶達無影響.CP-690550能有效減低DOK和KB細胞的pSTAT3(Tyr705)錶達,差異均具有統計學意義(P＜0.05).採用Western blot法檢測顯示,SOCS1和SOCS3在對照組均有錶達;IFN-α和CP-690550對NOK細胞組的SOCS1和SOCS3錶達無影響,差異無統計學意義(P＞0.05).100 U/ml和500 U/ml的IFN-α能刺激DOK和KB細胞的SOCS1錶達增加,差異均具有統計學意義(P＜0.05);而對SOCS3的錶達無影響.CP-690550能有效減低DOK和KB細胞的SOCS1錶達,差異均具有統計學意義(P＜0.05).結論 IFN-α激活DOK和KB細胞JAK1和JAK2的錶達,以JAK1激活為主;IFN-α通過激活DOK和KB細胞JAK1和JAK2的錶達促進STAT3在Tyr705位點燐痠化;IFN-α通過促進STAT3燐痠化進一步促進SOCS1的錶達,髮揮對IFN-α作用的抑製作用,減少細胞凋亡.
목적 이3충불동암변정도적구강세포위공구,관찰재간우소α(IFN-α)작용하,JAK-신호전도자화전록격활자(STAT)-세포인자신호억제인자(SOCS)신호분자적표체변화,위심입리해구강린상세포암(OSCC)세포면역도일적궤제제공연구기출.방법 상규배양NOK、DOK、KB세포.공백조가입완전배양액,이갑기아풍(DMSO)대조조가입함유0.1％ DMSO적완전배양액,실험조설10、100、500U/ml삼개불동농도조,매공분별가입함불동농도IFN-α적완전배양액,JAK억제제간예조재가입IFN-α전1h가입JAK억제제CP-690550 100 μmol/L.세포배양24 h후검측.진행반전록취합매련반응(RT-PCR)화Western blot실험.결과 채용RT-PCR법검측현시:JAK1화JAK2재NOK세포균유소량표체,IFN-α화CP-690550대NOK세포조적JAK1화JAK2표체무영향,차이무통계학의의(P＞0.05).100화500 U/ml적IFN-α능자격DOK화KB세포적JAK1화JAK2표체증가,차이균구유통계학의의(P＜0.05).CP-690550능유효감저DOK화KB세포적JAK1표체,차이균구유통계학의의(P＜0.05),이대JAK2표체무작용.채용Western blot법검측현시:STAT1、STAT3화pSTAT3(Tyr705)재대조조균유표체,pSTAT1(Tyr701)재대조조무표체;IFN-α화CP-690550대NOK세포조적STAT1、STAT3화pSTAT3(Tyr705)표체무영향,차이무통계학의의(P＞0.05).100、500 U/ml적IFN-α능자격DOK화KB세포적pSTAT3(Tyr705)표체증가,차이균구유통계학의의(P＜0.05);이대pSTAT1(Tyr701)적표체무영향.CP-690550능유효감저DOK화KB세포적pSTAT3(Tyr705)표체,차이균구유통계학의의(P＜0.05).채용Western blot법검측현시,SOCS1화SOCS3재대조조균유표체;IFN-α화CP-690550대NOK세포조적SOCS1화SOCS3표체무영향,차이무통계학의의(P＞0.05).100 U/ml화500 U/ml적IFN-α능자격DOK화KB세포적SOCS1표체증가,차이균구유통계학의의(P＜0.05);이대SOCS3적표체무영향.CP-690550능유효감저DOK화KB세포적SOCS1표체,차이균구유통계학의의(P＜0.05).결론 IFN-α격활DOK화KB세포JAK1화JAK2적표체,이JAK1격활위주;IFN-α통과격활DOK화KB세포JAK1화JAK2적표체촉진STAT3재Tyr705위점린산화;IFN-α통과촉진STAT3린산화진일보촉진SOCS1적표체,발휘대IFN-α작용적억제작용,감소세포조망.
Objective To observe the change of JAK-STAT-SOCS signal molecules after interferons alpha acting on the cancerous oral cells in 3 different degrees,namely NOK,DOK and KB cells,and to provide research foundation for the deep understanding of OSCC (oral squamous cell cancer) tumor ceils immune escape mechanism.Methods NOK,DOK,and KB cells were all cultured respectively,and then the third passage cells in the logarithmic growth phase were inoculated in cell culture plate.Blank control group of each hole was added 2 ml complete medium containing 10 ％ FPS.DMSO control group of each hole was added 2 ml complete medium containing 0.1％ DMSO.And in experimental groups containing 10 U/ml,100 U/ml,and 500 U/ml interferons,complete culture medium were added to each hole containing different concentrations of interferons alpha.CP-690550 (100 μmol/L) was added before interferons alpha was added 1 h.All were detected by RT-PCR test and Western blot test after conventional cultured for 24 h.Results RT-PCR detection showed that JAK1 and JAK2 in NOK cells had a small amount of expression,interferons alpha and CP-690550 cells could not influence the expression of JAK1 and JAK2 of NOK group,and there was no statistically significant difference (P ＞ 0.05).Interferons alpha in 100 and 500 U/ml could stimulate the increase of JAK1 and JAK2 expression in DOK and KB cells,and the differences were statistically significant (P ＜ 0.05).CP-690550 could effectively reduce the JAK1 expression of DOK and KB cells,while had no effect on the expression of JAK2,and the differences were statistically significant (P ＜ 0.05).Western blot showed that STAT1,STAT3 and pSTAT3 (Tyr705) all expressed in the control group,while pSTAT1 (Tyr701) didn't express in the control group.Interferons alpha and CP-690550 cells had no effect on STAT1,STAT3 and pSTAT3 (Tyr705) expression of NOK group,and there was no statistically significant difference (P ＞ 0.05).100 U/ml and 500 U/ml of interferons alpha could stimulate the increases of pSTAT3 (Tyr705) expression of DOK and KB cells,and the differences were statistically significant (P ＜ 0.05).While they had no effect on pSTAT1 (Tyr701) expression.CP-690550 could effectively reduce the pSTAT3 DOK and KB cells (Tyr705) expression,and the differences were statistically significant (P ＜ 0.05).Western blot showed that there were expression of SOCS1 and SOCS3 in control group.Interferons alpha and CP-690550 had no effect on SOCS1 and SOCS3 expression of NOK cell group,and there was no statistically significant difference (P ＞ 0.05).100 U/ml and 500 U/ml of interferons alpha could stimulate the increase of SOCS 1 expression of DOK and KB cells,and differences were statistically significant (P ＜ 0.05).For the expression of SOCS3,no influence.CP-690550 could effectively reduce the expression of SOCS1 of DOK and KB cells,and the differences were statistically significant (P ＜ 0.05).Conclusions Interferons alpha activate DOK JAK1 and KB cells and the expression of JAK2,mainly JAK1 activation.Interferons alpha,by activating DOK JAK1 and KB cells and the expression of JAK2,promote STAT3 phosphorylation in Tyr705 locus.Interferons alpha,by promoting STAT3 phosphorylation,further promote the expression of SOCS1,which plays the role in inhibiting interferons alpha and reducing the apoptosis.
