【作者】 孙仁虎；

【导师】 王国斌；

【作者基本信息】 华中科技大学 ， 外科学， 2009， 博士

【摘要】 第一部分结肠癌中CCR7、MMP-9的表达及其临床意义目的：检测趋化因子受体CCR7(C-C chemokine receptor 7)、金属基质蛋白酶-9(matrix metalloproteinases，MMP-9)在结肠癌组织中的表达，并探讨其与临床病理特征的关系；分析4株结肠癌细胞中CCR7的表达，为相关体外实验提供基础。方法：免疫组织化学链霉亲和素-生物素-过氧化物酶复合物法(streptavidin-biotinperoxidase complex，SABC)检测65例结肠癌根治术后组织标本中CCR7、MMP-9的表达，结合临床病理资料进行回顾分析；RT-PCR和Western blot检测结肠癌细胞株SW480、LoVo、HT-29、Caco-2中CCR7 mRNA及蛋白的表达水平。结果：结肠癌组织中CCR7的表达率为63．1％(41／65)，MMP-9的表达率为75．4％(49／65)；CCR7和MMP-9的表达与结肠癌的浸润深度、淋巴结转移相关(P＜0．05)，而与患者年龄、性别、肿瘤部位及大小无关(P＞0．05)。CCR7(+)和CCR7(-)结肠癌组织中MMP-9的表达率分别为90．2％(37／41)和50．0％(12／24)；MMP-9(+)和MMP-9(-)结肠癌组织中CCR7的表达率分别为85．5％(37／49)和25．0％(4／16)，两者的表达呈相关性(P＜0．05)。RT-PCR与Western blot结果显示，CCR7 mRNA和蛋白在4株结肠癌细胞中均有表达，以SW480中表达最强，LoVo、HT-29次之，Caco-2表达量最弱。结论：结肠癌中CCR7、MMP-9蛋白表达与结肠癌淋巴结转移密切有关。结肠癌细胞株SW480可作为CCR7相关体外实验的研究对象。第二部分CCR7-shRNA表达载体的构建及对SW480细胞的沉默效应目的：设计并构建靶向CCR7基因短发夹RNA(short hairpin RNA，shRNA)真核表达质粒，并检测其对人结肠癌SW480细胞中CCR7表达的干扰效果。方法：以CCR7为靶基因设计具有短发夹结构的模板寡核苷酸，退火形成互补双链结构，再克隆至pGC-silencer-CRM30载体，构建的3条重组shRNA真核表达质粒分别命名为pGC-silencer-CRM30_CCR7A、_CCR7B和_CCR7C。采用测序法鉴定寡核苷酸序列。将构建好的3条shRNA真核表达质粒转染人结肠癌SW480细胞，荧光显微镜观察转染效果，RT-PCR法检测CCR7 mRNA干扰效果，优化转染条件，以实现最佳干扰效果；选取干扰效果最佳的1条shRNA真核表达质粒转染SW480细胞，并通过G418筛选稳定表达CCR7-shRNA的细胞株。结果：重组质粒测序结果与Genebank中的CCR7 cDNA序列相符，转染SW480细胞后，荧光显微镜下可观察到绿色荧光蛋白；RT-PCR结果显示，pGC-silencer-CRM30_CCR7 C干扰效果最强。将pGC-silencer-CRM30_CCR7C转染SW480细胞，并通过G418筛选稳定表达CCR7-shRNA的细胞株，命名为CCR7-shRNA SW480细胞；RT-PCR及Westen blot结果显示，CCR7表达被沉默。结论：靶向CCR7基因shRNA表达载体构建无误，并获得稳定表达CCR7-shRNA的细胞株；为进一步探讨趋化因子受体CCR7在胃肠道恶性肿瘤生物学行为中的作用奠定了基础。第三部分CCL21／CCR7在人结肠癌SW480细胞体外侵袭中的作用研究目的：研究趋化因子CCL21及其受体CCR7对人结肠癌SW480细胞体外侵袭能力的影响，并探讨其作用机制。方法：不同浓度的CCL21刺激亲本SW480细胞、shRNA SW480细胞及CCR7-shRNA SW480细胞，划痕实验和Transwell侵袭小室实验检测SW480细胞侵袭能力变化，逆转录-聚合酶链反应(RT-PCR)检测SW480细胞中Snail mRNA的表达，免疫印迹(Western Blot)法检测金属基质蛋白酶-9(MMP-9)和E-钙黏蛋白(E-cadherin)的表达。结果：CCL21促进亲本SW480细胞向划痕处爬行的迁移；10 ng／ml组(76±6)和100ng／ml组(113±7)中穿膜细胞数显著多于空白对照组(48±4)(P＜0．05)。CCL21刺激亲本SW480细胞，Snail mRNA的表达增强，E-钙黏蛋白表达减弱，MMP-9的表达增强，与空白对照组相比，均有差异有统计学意义(P＜0．05)。CCL21对shRNA SW480细胞的影响与对亲本SW480细胞的影响基本相似。CCR7-shRNA可以阻断CCL21对SW480细胞的各项刺激效应。结论：CCL21／CCR7相互作用，可通过促进Snail mRNA和MMP-9蛋白表达，降低E-钙黏蛋白水平，以增强SW480细胞的侵袭能力。第四部分CCL21／CCR7在结肠癌SW480细胞增殖及凋亡中的作用研究目的：探讨趋化因子CCL21及其受体CCR7对人结肠癌SW480增殖及凋亡的影响，并探讨及其作用机制。方法：绘制亲本SW480和CCR7-shRNA SW480的生长曲线；流式细胞仪检测亲本SW480和CCR7-shRNA SW480的细胞周期。CCL21预处理的SW480细胞在含足叶乙甙(VP-16)的体系中培养，MTT法检测细胞增殖活力，流式细胞术和Hoechst 33258染色检测细胞凋亡；免疫印迹检测凋亡相关基因Bcl-2／Bax的表达。结果：细胞生长曲线图显示，CCR7-shRNASW480细胞的生长特性无明显变化；与亲本SW480细胞相比，CCR7--shRNASW480细胞周期未发生变化。CCL21单独作用不能促进SW480细胞增殖；VP-16组中SW480细胞的增殖抑制率为68．3％，CCL21预处理增强SW480细胞活力，CCL21(100ng／mL)中抑制率降至47．4％；VP-16组与CCL21(100ng／mL)组中凋亡率分别为(65．2±5．2)％和(48．7±3．1)％，差异有统计学意义(P＜0．05)。CCL21作用后，SW480细胞中Bcl-2表达升高，Bax表达降低，与空白对照组相比，差异有统计学意义(P＜0．05)，CCR7-shRNA可以阻断此效应。结论：CCL21／CCR7增强人结肠癌SW480细胞抗凋亡能力，提高其在非最适微环境中的存活能力。更多还原

【Abstract】 PartⅠ:Expression of CCR7 and MMP-9 in Colorectal Carcinomaand its Clinical SignificanceObjective:To investigate the expressions of C-C chemokine receptor(CCR7)andmatrix metalloproteinases-9(MMP-9),to explore their correlation to clinical pathologicalfeatures of colorectal carcinoma,and the relationship between expressions of CCR7 andMMP-9.Methods:The expressions of CCR7 and MMP-9 proteins in 65 cases of colorectalcarcinoma were detected by SABC immunohistochemistry,the expressions of CCR7 in 4strains of colorectal carcinoma cell line examined by RT-PCR and Western blot.Results:41 of the 65 colorectal carcinoma specimens(63.1%)were positive in CCR7,the expression of CCR7 was correlated with invasion depth and lymph node metastasis ofcolorectal carcinoma(P＜0.05).MMP-9 expression was detected in 75.4%(49/65)ofcolorectal carcinoma specimens,the expression of MMP-9 was assiociated with invasiondepth and lymph node metastasis of colorectal carcinoma(P＜0.05).Significant relevancewas found between the expression of MMP-9 and CCR7(P＜0.05).Conclusion:The expression of the chemokine receptor CCR7 and MMP-9 incolorectal carcinoma tissues may be associated with its progression and metastasis.Molecular targets of CCR7 and MMP-9 may also be important factors in the metastasis ofcolorectal carcinoma. PartⅡ:Construction and Identification of pGC-silencer-CRM30CCR7 short hairpin RNA Expression VectorsObjective:To construct CCR7 shRNA eukaryotic expression vectors to transfect intoSW480 cells in order to further study the silencing effects of the vector on the targetinggene CCR7.Methods:The shRNA oligonucleotides targeting for CCR7 gene were synthesized andcloned into pGC-silencer-CRM30 to generate shRNA eukaryotic expression vectors.Therecombinants were named pGC-silencer-CRM30_CCR7A,_CCR7B and _CCR7C.shRNAexpression plasmids were identificated by sequencing.The eukaryotic expression vectorswere transfected into SW480 cells.The green fluorescent protein(GFP)was detected byfluorescence microscope,and the silencing effects of the recombinant vectors weredetermined by RT-PCR.Procedures were optimized to maximize the silencing effect.Two-weeks after addition of G-418,clones stablly expressing pGC-silencer-CRM30_CCR7C were individually selected and expanded.Results:The recombinant sequence identified by sequencing was the same as thetargeting one.In SW480 cells transfected with the recombinant vectors,the expression ofgreen fluorescent protein(GFP)was detected,the silencing effect of pGC-silencer-CRM30_CCR7C was more evident than other recombinant vectors.After screening withG418,CCR7-silencing stable SW480 cells were obtained,and named CCR7-shRNASW480.Conclusion:shRNA recombinant was established successfully by RNAi techniqueand transfected into SW480 cells.The expression of CCR7 was completely knocked downin SW480 cells. PartⅢ:Effect of CCL21/CCR7 on Invasion ofColorectal Carcinoma cell line SW480Objective:To investigate the role of CCL21/CCR7 in invasion of colorectalcarcinoma cell line SW480.Methods:parental SW480 and CCR7-shRNA cell SW480 were exposed to varyingconcentrations of CCL21(10,100 ng/ml)for 48 h.The invasive ability was detected byWound healing assay and Transwell assay,the expression of Snail mRNA was evaluated byRT-PCR,the expressions of MMP-9 and E-cadherin were determined by Western Blot.Results:CCL21 drove more parental SW480 cell migrated into the gap than controlgroup at same time-points after inducing the lesion.The counts of parental SW480penetrating through membrane in 100ng/ml group were 113±7,significantly more than48±4 in control group(P＜0.05).The expression of MMP-9 in 100ng/ml group was0.83±0.02,significantly higher than 0.38±0.01 in control group(P＜0.05),the expression ofE-cadherin was significantly lowered after exposure to CCL21(P＜0.05),the expression ofSnail mRNA was significantly up-regulated(P＜0.05);these functions could be blocked bytransfecting the CCR-shRNA.Conclusion:CCL21 increased the invasive ability in SW480 cells,induced theMMP-9 expression and activity,and enhanced the survival capacity of SW480 cells. PartⅣ:Effects of CCL21/CCR7 on the Survival andApoptosis of Colorectal Carcinoma line SW480Objective:To investigate the role of CCL21/CCR7 in survival of colon cancer cellline SW480 at suboptimal circumstance and explore the mechanism.Methods:The growth curve of CCR7-shRNA SW480 was drwaed by using MTTmethod,the cell cycle was detected by flow cytometry.Parental SW480 cells werepre-incubated with CCL21 for 2h before exposure to VP-16(20 ng/mL),the cellproliferation was detected by MTT assay,cell apoptosis by flow cytometry and Hoechst33258 staining;the expression of Bcl-2/Bax was examined by Western blot.Results:CCL21 alone did not promote the proliferation,but pre-incubation withCCL21 confer SW480 resistance to death induced by VP-16,inhibition rate reduced from68.3% to 47.4% with treatment of 100ng/mL CCL21;consistently,apoptosis decreasedfrom 65.2% to 48.7% after exposure to 100ng/ml CCL21.The expression of Bcl-2 wassignificantly elevated,and the expression of Bax was significantly decreased with treatmentwith CCL21,the effect was blocked by Transfecting SW480 cell with CCR-shRNA.Conclusion:CCL21 enhanced the invasive ability in SW480 cells,induced MMP-9expression,and promoted the survival of SW480 cells under the suboptimal circumstance.更多还原