【作者】 郑科；

【导师】 任国胜；

【作者基本信息】 重庆医科大学 ， 外科学， 2010， 博士

【摘要】 第一部分趋化因子受体CXCR7在人肝细胞肝癌及肝癌细胞株中的表达及意义目的探讨趋化因子受体CXCR7在人肝细胞肝癌组织、癌旁组织、正常肝脏组织及肝癌细胞中的表达,从而初步推测CXCR7在肝癌发生发展中的作用。方法应用免疫组织化学检测CXCR7在人肝癌组织、癌旁组织、正常肝脏组织中的表达。Western-blot和RT-PCR在蛋白和mRNA水平上检测CXCR7,CXCR4在肝癌细胞株(HepG2, Hep3B, SMMC-7721, MHCC97L, MHCC97H和HCCLM6)及人脐静脉内皮细胞(HUVECs)中的表达情况。结果(1)CXCR7在人肝细胞肝癌组织中的表达高于癌旁组织、正常肝脏组织。(2)与HUVECs相比,CXCR7在人肝癌细胞中的表达水平较高。(3)侵袭能力强的肝癌细胞中CXCR7表达水平高于侵袭能力弱的肝癌细胞。(4)在6株肝癌细胞及HUVECs中,均可见CXCR4的表达。结论①在人肝细胞肝癌组织中CXCR7表达较高,而癌旁组织、正常肝脏组织中表达较低。②CXCR7在高侵袭力肝癌细胞中表达较高,从而初步推测CXCR7在肝癌细胞的侵袭能力中可能具有一定作用。第二部分CXCR7shRNA真核表达载体的构建及鉴定目的构建CXCR7shRNA真核表达载体,将CXCR7shRNA重组质粒转染人肝癌细胞SMMC-7721,特异性沉默肝癌细胞中CXCR7的表达。经过G418稳定筛选,获得稳定表达CXCR7shRNA重组质粒的SMMC-7721细胞。方法设计合成针对CXCR7的转录模板序列,构建针对CXCR7的shRNA重组质粒,并通过酶切和测序鉴定。将CXCR7shRNA重组质粒转染人肝癌细胞SMMC-7721,经过G418筛选获得长效表达CXCR7shRNA重组质粒的细胞,荧光显微镜下观察转染效率。采用Western-blot检测稳定筛选细胞中CXCR7蛋白表达水平,RT-PCR检测CXCR7mRNA表达水平。结果(1)重组质粒经酶切鉴定,电泳结果显示,阳性重组质粒被BamH I切开,而不能被Pst I切开。(2)荧光显微镜下观察,CXCR7shRNA重组质粒转染人肝癌细胞SMMC-7721,经G418稳定筛选后,大量细胞均可见绿色荧光。(3)经G418稳定筛选后获得的细胞,用RT-PCR检测CXCR7mRNA表达,转染了CXCR7shRNA的细胞中CXCR7mRNA水平明显低于对照组(P<0. 05)。(4)经G418稳定筛选后获得的细胞,用Western-blot检测CXCR7蛋白表达,转染了CXCR7shRNA的细胞中CXCR7蛋白表达明显下调,和对照组比较具有明显差异(P<0. 05)。结论①经酶切和测序鉴定,成功构建了针对CXCR7的shRNA重组质粒。②荧光显微镜下观察,稳定转染了CXCR7shRNA重组质粒的SMMC-7721可见大量荧光。③运用RT-PCR和Western-blot检测发现,稳定转染了CXCR7shRNA重组质粒的SMMC-7721中CXCR7的表达被有效沉默。第三部分CXCL12/CXCR7对人肝癌细胞生物学行为的影响目的特异性沉默CXCR7的表达后,研究其对SMMC-7721细胞的侵袭、粘附、VEGF分泌和诱导血管生成能力的影响。VEGF刺激能否调节CXCR7的表达,以及对SMMC-7721细胞侵袭能力的影响。方法①运用Transwell小室侵袭实验,检测CXCL12诱导对SMMC-7721细胞侵袭能力的影响。②运用Transwell小室侵袭实验,检测特异性沉默CXCR7对SMMC-7721细胞侵袭能力的影响。③运用肿瘤细胞与细胞外基质粘附实验,检测特异性沉默CXCR7对SMMC-7721细胞粘附能力的影响。④运用ELISA法,检测特异性沉默CXCR7对SMMC-7721细胞VEGF分泌的影响。⑤运用肿瘤细胞与HUVECs共培养模型,检测特异性沉默CXCR7对SMMC-7721细胞诱导体外血管生成能力的影响。⑥运用RT-PCR和Western-blot,检测VEGF刺激对SMMC-7721细胞和HUVECs中CXCR7表达的影响。⑥运用Transwell小室侵袭实验,检测VEGF刺激对SMMC-7721细胞侵袭能力的影响。结果(1)不同浓度CXCL12(0, 10,100 ng/ml)诱导下,SMMC-7721细胞侵袭能力随CXCL12浓度的增加而增强,表现为浓度依赖性。特异性沉默CXCR7的表达后,CXCL12(100 ng/ml)诱导的SMMC-7721细胞侵袭能力减弱。(2) CXCL12(100 ng/ml)能够诱导增强SMMC-7721细胞与细胞外基质(FN和LN)的粘附能力。SMMC-7721细胞与LN的粘附能力比与FN的粘附能力强。特异性沉默CXCR7的表达后,CXCL12(100 ng/ml)诱导的SMMC-7721细胞与FN和LN的粘附能力减弱。(3) CXCL12(100 ng/ml)能够诱导SMMC-7721细胞分泌VEGF。特异性沉默CXCR7的表达后,CXCL12(100 ng/ml)诱导的SMMC-7721细胞分泌VEGF的能力减弱。(4)特异性沉默CXCR7的表达后,SMMC-7721细胞诱导体外血管生成的能力减弱。(5) VEGF(50 ng/ml )刺激下,SMMC-7721细胞和HUVECs中CXCR7mRNA表达上调,表现为时间依赖性。VEGF(50 ng/ml )刺激下,SMMC-7721细胞和HUVECs中CXCR7蛋白表达水平上调,表现为时间依赖性。(6) VEGF(50 ng/ml )预刺激后,SMMC-7721细胞的趋化侵袭能力增强。结论①CXCL12能够诱导增强SMMC-7721细胞的侵袭、粘附和VEGF分泌能力。②CXCR7表达被有效沉默后:CXCL12诱导的细胞侵袭、粘附和VEGF分泌的能力减弱;SMMC-7721细胞诱导体外血管生成的能力减弱。这些结果说明CXCL12/ CXCR7轴能够调节SMMC-7721细胞的侵袭、粘附、体外血管生成的能力。CXCL12/ CXCR7轴可能在肝癌细胞的远处转移中起重要作用。③VEGF刺激能够上调CXCR7的表达,且上调的CXCR7是具有功能的。第四部分CXCR7沉默对人肝癌细胞裸鼠皮下种植瘤生长的影响目的研究特异性沉默SMMC-7721细胞中CXCR7表达后,对SMMC-7721细胞裸鼠皮下种植瘤生长的影响。方法将未转染的SMMC-7721细胞、转染了阴性shRNA的SMMC-7721细胞和稳定转染了CXCR7shRNA的SMMC-7721细胞,分别接种到裸鼠背部皮下,定时测量肿瘤体积,绘制肿瘤生长曲线。裸鼠处死后,完整剥离肿瘤,称取瘤重,并将肿瘤固定于4%多聚甲醛,石蜡包埋,切片。切片用于免疫组织化学检测CD31,计算MVD。取出裸鼠肺和肝脏,观察有无转移灶形成。结果(1) CXCR7的表达被有效沉默后,稳定转染CXCR7shRNA组肿瘤体积明显较小,生长速度明显低于对照组,差异具有统计学意义(P<0.05)。而转染了阴性shRNA组与对照组之间无明显差异(P>0.05)。(2) CXCR7的表达被有效沉默后,稳定转染CXCR7shRNA组肿瘤的重量明显低于对照组,差异具有统计学意义(P<0.05)。而转染了阴性shRNA组与对照组之间肿瘤重量无明显差异(P>0.05)。(3) CXCR7的表达被有效沉默后,稳定转染CXCR7shRNA组肿瘤血管CD31的表达明显较少,其MVD明显小于对照组MVD,差异具有统计学意义(P<0.05)。而转染了阴性shRNA组与对照组之间肿瘤血管生成均较多,两组之间无明显差异(P>0.05)。结论①CXCR7的表达被有效沉默后,能够调节SMMC-7721细胞裸鼠皮下种植瘤的生长。②CXCR7对裸鼠皮下种植瘤生长的调节,可能是通过抑制肿瘤血管生成而实现的。③CXCR7的表达被有效沉默后,未影响裸鼠皮下种植瘤的转移。然而,我们并不能因此而推论CXCR7的表达不能调节裸鼠移植瘤的转移,因为原位移植瘤模型才是研究CXCR7调节肿瘤转移更合适的模型。更多还原

【Abstract】 PART ONE EXPRESSION AND SIGNIFICANCE OF CHEMOKINE RECEPTOR CXCR7 IN HCC AND LIVER CANER CELL LINESObjectiveTo study the expression of CXCR7 in hepatocellular carcinoma tissues, matched adjacent non-neoplastic tissues , normal liver tissues and liver cancer cells. To evaluate the role of CXCR7 in HCC development.MethodsImmunohistochemistry was used to detect the expression of CXCR7 in human hepatocellular carcinoma tissues, matched adjacent non-neoplastic tissues and normal liver tissues. RT-PCR and Western-blot was used to evaluate CXCR7mRNA and CXCR4mRNA and protein levels in liver cancer cell lines(HepG2, Hep3B, SMMC-7721, MHCC97L, MHCC97H ,HCCLM6)and HUVECs.Results(1)HCC tissues displayed higher CXCR7 expression than matched adjacent non-neoplastic tissues and normal liver tissues.(2)Human liver cancer cells displayed higher CXCR7 expression than HUVECs.( 3 ) High aggressive liver cancer cells displayed higher CXCR7 expression than low aggressive liver cancer cells.(4)The expression of CXCR4 was detected in six liver cancer cells and HUVECs.Conclusion①The expression of CXCR7 was high elevated in HCC. However, CXCR7 expression was low in matched adjacent non-neoplastic tissues and normal liver tissues.②CXCR7 was overexpressed in high aggressive liver cancer cells. CXCR7 might play a role in invasion of liver cancer cells.PART TWO CONSTRUCTION AND IDENTIFICATION OF CXCR7shRNA EUKARYOTIC EXPRESSION VECTORObjectiveTo construct siRNA eukaryotic expression vectors targeting to CXCR7 gene.To specifically silence CXCR7 expression through transfecting recombinant plasmid of CXCR7shRNA into SMMC-7721 cells. To obtain SMMC-7721 cells stably expressing recombinant plasmid of CXCR7shRNA through G418 selection. MethodsTranscription template sequence targeting to CXCR7 was designed and synthesized. The recombinant plasmid of CXCR7shRNA was constructed and further identified by enzyme digestion and sequencing. The recombinant plasmid of CXCR7shRNA was transfected into SMMC-7721 cells and further selected by G418. Transfection efficiency was evaluated by fluorescent microscope. CXCR7mRNA levels in SMMC-7721 cells transfected with CXCR7shRNA were detected by RT-PCR. The protein levels of CXCR7 in SMMC-7721 cells transfected with CXCR7shRNA were detected by Western-blot.Results(1)The positive recombinant plasmid was cut by BamH I but not Pst I after recombinant plasmid was digested.(2) The recombinant plasmid of CXCR7shRNA was successfully transfected into SMMC-7721 cells. After G418 selection, a great quantity of cells transfected with recombinant plasmid displayed green fluorescence.(3)After G418 selection, CXCR7mRNA levels in SMMC-7721 cells were detected by RT-PCR. The cells transfected with CXCR7shRNA exhibited lower CXCR7mRNA levels compared with the control cells(P<0. 05).(4)After G418 selection, protein levels of CXCR7 in SMMC-7721 cells were detected by Western-blot. Expression of CXCR7 in cells transfected with CXCR7shRNA was significantly downregulated compared with the control cells(P<0. 05).Conclusion①The recombinant plasmid of CXCR7shRNA was successfully constructed and identified by enzyme digestion and sequencing.②Under fluorescent microscope, SMMC-7721 cells stably transfected with recombinant plasmid of CXCR7shRNA displayed a great quantity of green fluorescence. Transfection efficiency was approximate 80%-90%。③Expression of CXCR7 in cells stably transfected with recombinant plasmid of CXCR7shRNA was efficiently silenced through RT-PC R and Western-blot assay.PART THREE EFFECT OF CXCL12/CXCR7 ON BIOLOGICAL BEHAVIOR OF HUMAN LIVER CANCER CELLSObjectiveTo investigate the effect of CXCR7 silencing on invasion, adhesion, VEGF secretion and angiogenesis of SMMC-7721 cells. To evaluate the effect of VEGF stimulation on CXCR7 expression and invasive ability of SMMC-7721 cells.Methods①Invasive ability of SMMC-7721 cells induced by CXCL12 was evaluated by Transwell chamber assay.②Effect of CXCR7 silencing on invasive ability of SMMC-7721 cells was evaluated by Transwell chamber assay.③Effect of CXCR7 silencing on adhesive ability of SMMC-7721 cells was evaluated by tumor cell- Extra Cellular Matrix adhesion assay.④Effect of CXCR7 silencing on VEGF secretion in SMMC-7721 cells was investigated by ELISA assay.⑤Effect of CXCR7 silencing on SMMC-7721 cells induced-angiogenesis was detected by tumor cell- HUVECs coculture system.⑥Effect of VEGF stimulation on CXCR7 expression in SMMC-7721 cells and HUVECs was evaluated by RT-PCR and Western-blot assay.⑥Effect of VEGF stimulation on invasive ability of SMMC-7721 was evaluated by Transwell chamber assay.Results(1) We found that CXCL12(0, 10,100 ng/ml) induced a significant and dose-dependent increase of invasion in SMMC-7721 cells.(2) CXCL12(100 ng/ml) induced invasion in SMMC-7721 cells was decreased after silencing of CXCR7.(3)CXCL12(100 ng/ml) induced a significant increase of cell-Extra Cellular Matrix (FN and LN) adhesion. Adhesion of SMMC-7721 cells to LN was greater than adhesion to FN.(4)CXCL12(100 ng/ml)induced adhesion of SMMC-7721 cells to LN and FN was decreased after silencing of CXCR7.(5)CXCL12(100 ng/ml)could induce VEGF secretion in SMMC-7721 cells.(6)VEGF secretion induced by CXCL12(100 ng/ml)was decreased after silencing of CXCR7.(7)In vitro tube formation induced by SMMC-7721 cells was decreased after silencing of CXCR7.(8)VEGF(50 ng/ml )stimulation induced a time-dependent increase of CXCR7mRNA levels in SMMC-7721 cells and HUVECs.(9)VEGF(50 ng/ml )stimulation induced a time-dependent increase of protein levels of CXCR7 in SMMC-7721 cells and HUVECs.(10)Invasive ability of SMMC-7721 cells was enhanced after VEGF(50 ng/ml )stimulation.Conclusion①CXCL12 could induce invasion, adhesion, VEGF secretion in SMMC-7721 cells.②Invasion, adhesion and VEGF secretion was decreased after silencing of CXCR7. Also, in vitro tube formation induced by SMMC-7721 cells was decreased after silencing of CXCR7. These results demonstrated that CXCL12/ CXCR7 could regulate invasion and adhesion of SMMC-7721 cells. CXCL12/ CXCR7 axis might play an important role in metastasis of SMMC-7721 cells.③Up-regulation of CXCR7 expression by VEGF stimulation was functional. PART FOUR EFFECT OF CXCR7 SILENCING ON TUMOR GROWTH OF HUMAN LIVER CANCER CELL XENOGRAFT IN NUDE MOUSEObjectiveTo study the effect of CXCR7 silencing on tumor growth of SMMC-7721 cells xenograft in nude mice.MethodsThe control, negative shRNA and stable CXCR7shRNA transfected SMMC-7721 cells were inoculated subcutaneously into the back of nude mice. The tumor size was measured and the growth curve of tumor was drawed. The tumors were completely separated and weighed after the mice were sacrificed. Subsequently, the tumors were fixed in 4% polyformaldehyde and embedded with paraffin. The tumor sections were excised for immunohistochemical analysis of CD31 and calculation of MVD. Metastasis was evaluated in lung and liver of nude mice.Results(1) The cells stably transfected with CXCR7shRNA showed smaller tumor size compared with control cells(P<0.05). However, no significant difference was observed between control and negative shRNA group(P>0.05).(2) The cells stably transfected with CXCR7shRNA showed significantly reduced tumor weight compared with control cells(P<0.05). However, no significant difference of tumor weight was observed between control and negative shRNA group(P>0.05). (3) The cells stably transfected with CXCR7shRNA showed significantly reduced expression of CD31 and decreased MVD compared with control cells. Statistic differences were obtained between control and CXCR7shRNA group(P<0.05). However, no significant difference of tumor angiogenesis was observed between control and negative shRNA group(P>0.05).Conclusion①Tumor growth of SMMC-7721 cells xenograft in nude mice could be regulated by silencing of CXCR7.②Silencing of CXCR7 substantially suppressed angiogenesis and subsequently regulated the tumor growth..③We did not find metastasis of xenograft in nude mice after silencing of CXCR7.However, we could not conclude that CXCR7 was unable to regulate metastasis of transplanted tumor in nude mice. Orthotopic transplanted tumor model is a better way to further evaluate the role of CXCR7 in regulating tumor metastasis.更多还原