【作者】 林敏；

【导师】 纪超；

【作者基本信息】 福建医科大学 ， 皮肤病与性病学， 2020， 硕士

【摘要】 目的:1.研究SDF-1与皮肤鳞状细胞癌之间的关系。2.研究SDF-1与皮肤鳞状细胞癌细胞迁移之间的关系。3.研究塞来昔布通过SDF-1在皮肤鳞状细胞癌细胞迁移的作用机制。方法:1.检测SDF-1在人皮肤鳞状细胞癌皮损组织及正常皮肤组织中的表达水平:收集2017年9月起我院门诊和住院部确诊为皮肤鳞状细胞癌患者的皮损组织及正常皮肤组织,通过免疫组织化学技术检测SDF-1表达水平。2.检测人鳞状细胞癌细胞系A431和SCL1受到UV照射后SDF-1的表达水平:常规培养人鳞状细胞癌细胞系细胞(A431和SCL1),经不同强度UV照射后(0 m J/cm~2,3 m J/cm~2,5 m J/cm~2,10 m J/cm~2,20 m J/cm~2及30m J/cm~2),通过免疫印迹(Western Blot)、酶联免疫吸附实验(Elisa)、实时定量荧光PCR(RT-PCR)技术,从蛋白质、RNA水平检测SDF-1蛋白表达水平及转录水平。3.检测SDF-1/CXCR4通路在人鳞状细胞癌细胞迁移作用:以内吞抑制剂MDC/CXCR4拮抗剂AMD3100/CXCR7拮抗剂CCX771预处理人鳞状细胞癌细胞系1小时,再以不同剂量的SDF-1(0ng/m L、10 ng/m L、50 ng/m L、100 ng/m L)处理12小时,通过Transwell实验评估其细胞侵袭迁移能力。再用不同剂量SDF-1(0ng/m L、30 ng/m L、100 ng/m L、300 ng/m L)处理人鳞状细胞癌细胞系,通过流式细胞术观察细胞膜表面CXCR4+细胞数,确定SDF-1是通过与CXCR4受体作用继而诱导人鳞状细胞癌细胞发生迁移。4.检测塞来昔布与SDF-1/CXCR4通路在人鳞状细胞癌细胞迁移关系:以不同剂量塞来昔布(0μM、10μM、25μM、50μM、100μM)处理人鳞状细胞癌细胞系12小时,并用CCK8检测细胞活力。塞来昔布(100μM)预处理人鳞状细胞癌细胞系1小时,再以不同剂量SDF-1(0ng/m L、10 ng/m L、50 ng/m L、100 ng/m L)处理12小时,通过Transwell实验评估皮肤鳞状细胞癌细胞迁移能力。最后流式细胞术观察空白对照组、SDF-1(100 ng/m L)组、塞来昔布(100μM)组、SDF-1(100ng/m L)联合塞来昔布(100μM)组细胞膜表面CXCR4+细胞数,确定塞来昔布通过SDF-1/CXCR4通路抑制人鳞状细胞癌细胞迁移。5.检测塞来昔布与SDF-1/CXCR4介导人鳞状细胞癌细胞迁移的下游信号通路:人鳞状细胞癌细胞系分别经内吞抑制剂MDC、CXCR4抑制剂AMD3100、塞来昔布处理后加入SDF-1,通过免疫印迹(Western Blot)检测细胞中p ERK1/2、p Akt、Akt表达水平。结果:1.皮肤鳞状细胞癌组织中SDF-1表达水平明显上调:与正常皮肤组织对比,皮肤鳞状细胞癌患者皮损组织行SDF-1免疫组织化学染色呈强阳性表达。2.紫外线照射可呈剂量依赖性地上调SDF-1在皮肤鳞状细胞癌细胞中的表达:皮肤鳞状细胞癌细胞系(A431和SCL1)予不同强度UV照射后,Western Blot检测SDF-1蛋白表达水平明显高于空白对照组;ELISA检测SDF-1蛋白表达水平明显高于空白对照组;实时定量荧光PCR检测SDF-1的m RNA转录水平明显高于空白对照组。3.SDF-1通过与CXCR4受体作用促进皮肤鳞状细胞癌细胞迁移:内吞抑制剂MDC、CXCR4拮抗剂AMD3100、CXCR7拮抗剂CCX771预处理人鳞状细胞癌细胞系1小时,再用不同剂量的SDF-1(0ng/m L、10ng/m L、50ng/m L、100ng/m L)处理12小时后,通过Transwell实验评估其细胞迁移能力,发现SDF-1诱导细胞迁移呈剂量依赖性。内吞抑制剂MDC和CXCR4拮抗剂AMD3100可抑制SDF-1诱导的皮肤鳞状细胞癌细胞迁移。另外,用不同剂量SDF-1(0ng/m L、30ng/m L、100ng/m L、300ng/m L)刺激A431细胞,通过流式细胞术观察细胞膜表面CXCR4+细胞数,提示SDF-1是通过与CXCR4受体作用继而诱导人鳞状细胞癌细胞发生迁移,且呈剂量依赖性。4.塞来昔布通过SDF-1/CXCR4通路抑制皮肤鳞状细胞癌细胞迁移:塞来昔布(100μM)预处理人鳞状细胞癌细胞系1小时后,再用不同剂量的SDF-1(0ng/m L、10ng/m L、50ng/m L、100ng/m L)处理12小时,Transwell侵袭实验结果显示塞来昔布可以抑制SDF-1诱导的CSCC细胞迁移。流式细胞仪观察SDF-1(100ng/m L)组和SDF-1(100ng/m L)联合塞来昔布(100μM)组细胞膜上CXCR4的表达情况,结果表明塞来昔布通过SDF-1/CXCR4通路抑制人鳞状细胞癌细胞迁移。5.塞来昔布通过抑制SDF-1/CXCR4通路介导的ERK/Akt细胞信号通路抑制皮肤鳞状细胞癌细胞迁:与移SDF-1组相比,抑制剂组ERK–Akt信号通路中p ERK1/2、p Akt、Akt蛋白表达水平降低。另外,塞来昔布(100μM)联合SDF-1(100ng/m L)共处理24小时,同样可观察ERK/Akt细胞信号通路激活程度下降。结论:1.在皮肤鳞状细胞癌中,紫外线照射可呈剂量依赖性地上调皮肤鳞状细胞癌细胞中的SDF-1表达。2.SDF-1通过与CXCR4受体作用促进CSCC细胞迁移。3.塞来昔布通过SDF-1/CXCR4通路抑制CSCC细胞迁移。ERK-Akt细胞信号通路在塞来昔布抑制SDF-1/CXCR4通路诱导CSCC细胞迁移起关键作用。更多还原

【Abstract】 Objective:1.To investigate the relationship between SDF-1 and cutaneous squamous cell carcinoma.2.To investigate the relationship between SDF-1 and the migration of cutaneous squamous cell carcinoma cells.3.To investigate the mechanism of celecoxib in cutaneous squamous-cell carcinoma cell migration.Method:1.Study the expression of SDF-1 in human cutaneous squamous cell carcinoma:we performed immunohistochemistry on normal human skin and cutaneous squamous cell carcinoma samples that from outpatient and inpatient departments of our hospital since September 2017.2.The UV radiation stimulated SDF-1 release and increased SDF-1 expression in cutaneous squamous cell carcinoma cells(A431 and SCL1):to determine whether UV radiation altered SDF-1 release and expression in cutaneous squamous cell carcinoma cells,SCL1 and A431 cells were challenged with UVB at different intensities(0 m J/cm~2,3 m J/cm~2,5 m J/cm~2,10 m J/cm~2,20 m J/cm~2,and 30m J/cm~2)and subjected to quantitative analyses by Western Blot,Elisa and RT-PCR.We intend to explore the SDF-1 in cutaneous squamous cell carcinoma cells(A431 and SCL1)both in protein and in RNA levels.3.Study the role of SDF-1/CXCR4 pathway in the migration of human squamous cell carcinoma cells:cutaneous squamous cell carcinoma cells(A431)were pretreated with MDC,AMD3100 or CCX771 for 1 hour prior to SDF-1(0ng/m L、10 ng/m L、50 ng/m L、100 ng/m L)challenge at indicated doses for 12 hours.Cell migration was examined by chemotaxis assay.To verify whether SDF-1 affected endocytosis of CXCR4 in cutaneous squamous cell carcinoma cells further,we challenged cutaneous squamous cell carcinoma cells with SDF-1 at different doses(0ng/m L、10 ng/m L、50 ng/m L、100 ng/m L)and assessed CXCR4 expression on the plasma membrane.4.Study the relationship between celecoxib and SDF-1/CXCR4 pathway in human squamous cell carcinoma cell migration:cutaneous squamous cell carcinoma cells were treated with celecoxib in different concentrations(0μM、10μM、25μM、50μM、100μM)as indicated for 12 hours,and cell viability was measured with CCK8assays.Cutaneous squamous cell carcinoma cells(A431)were pretreated with celecoxib for 1 hour prior to SDF-1(0ng/m L、10 ng/m L、50 ng/m L、100 ng/m L)challenge at indicated doses for 12 hours.Cell migration was examined by chemotaxis assay.Finally the experiment was divided into 4 groups:control group,SDF-1 treatment group,celecoxib treatment group,celecoxib in combination with SDF-1 treatment group,and then assessed CXCR4 expression on the plasma membrane.5.Study the downstream signaling pathways of celecoxib and SDF-1/CXCR4mediated migration of human squamous cell carcinoma cells:A431 and SCL1 cells were pretreated with MDC,a CXCR4-neutralizing antibody,or AMD3100 prior to SDF-1 challenge.After treatment,cells were collected and subjected to Western Blot analysis.Result:1.SDF1 expression was significantly upregulated in cutaneous squamous cell carcinoma samples:we performed immunohistochemistry on normal human skin and cutaneous squamous cell carcinoma samples.We found that SDF1 expression was significantly upregulated in CSCC samples.2.UV radiation stimulated SDF-1 release and increased SDF-1 expression in cutaneous squamous cell carcinoma cells,and SDF-1 were robustly increased in a dose-dependent manner:cutaneous squamous cell carcinoma cells,SCL1 and A431cells were challenged with UVB at different intensities(0,3,5,10,20,and 30m J/cm2)and subjected to quantitative analyses.Western blot analysis revealed that protein levels of SDF1 were robustly increased by UVB radiation in cutaneous squamous cell carcinoma cells.The expression level of SDF-1 detected by ELISA was significantly higher than that of the blank control group.The m RNA level of SDF-1 detected by real-time quantitative fluorescence PCR was significantly higher than that of the blank control group,.3.SDF-1 promotes the migration of cutaneous squamous cell carcinoma cells by interacting with CXCR4:cutaneous squamous cell carcinoma cells were pretreated with MDC,AMD3100 or CCX771 for 1 hour prior to SDF1(0ng/m L、10ng/m L、50ng/m L、100ng/m L)challenge at indicated doses for 12 hours.Cell migration was examined with Boyden assays.Quantification of migrated cells showed that SDF1induced cell migration in a dose-dependent manner.Compared with the blank control group.MDC and AMD3100 abrogated the effect of SDF1 in CSCC-cell migration.Compared with the blank control group.We challenged A431 cells with SDF1 at different doses(0ng/m L、30ng/m L、100ng/m L、300ng/m L)and assessed CXCR4expression on the plasma membrane.SDF1 notably induced CXCR4 endocytosis in CSCC cells in a dose-dependent manner.These results suggest that CXCR4endocytosis is the key regulator in CSCC-cell migration induced by SDF1.Compared with the blank control group.4.CXCR4 endocytosis is the key regulator in cutaneous squamous cell carcinoma cell migration induced by SDF-1:cutaneous squamous cell carcinoma cells were also challenged with SDF1 at different doses(0ng/m L、10ng/m L、50ng/m L、100ng/m L)with celecoxib(100μM).Celecoxib robustly abrogated the cutaneous squamous cell carcinoma cell migration induced by SDF1.Compared with the blank control group.Flow cytometry was used to observe the expression of CXCR4 on the cell membrane of SDF-1(100ng/m L)group and SDF-1(100ng/m L)group combined with celecoxib(100μM)group.CXCR4 endocytosis was suppressed by cotreatment with celecoxib,indicating the inhibitory influence of celecoxib on SDF1-induced endocytosis of CXCR4.5.Celecoxib inhibits the migration of cutaneous squamous cell carcinoma cells via inhibition of SDF-1/CXCR4-mediated ERK/Akt signal pathway:compared with the SDF-1 group,the expression levels of p ERK1/2,p Akt and Akt proteins in the ERK-Akt signaling pathway were decreased in the inhibitor group.In addition,celecoxib(100μM)combined with SDF-1(100ng/m L)for 24 hours could also observe the decrease in the activation degree of ERK/Akt cell signaling pathway.Conclusion:1.UV radiation stimulated SDF-1 release and increased SDF-1 expression in cutaneous squamous cell carcinoma cells,and SDF-1 were robustly increased in a dose-dependent manner.2.SDF-1 can induce cell migration in cutaneous squamous cell carcinoma cells mediated by CXCR4 endocytosis.3.Celecoxib suppresses cutaneous squamous cell carcinoma cell migration via inhibition of SDF-1-induced endocytosis of CXCR4.ERK-Akt pathways played a critical role in the inhibitory effect of celecoxib in SDF-1-induced cutaneous squamous cell carcinoma cell migration and CXCR4 endocytosis更多还原