【作者】 王红鲜；

【导师】 陈道瑾；

【作者基本信息】 中南大学 ， 普通外科学， 2009， 博士

【摘要】 恶性肿瘤的发生发展过程涉及多个趋化因子和受体的异常表达,CXCL12和受体CXCR4是肿瘤生长和转移的关键因子。最近在前列腺癌的研究中发现仅抑制CXCR4的表达,只能部分地阻断恶性肿瘤的生长、转移。进一步研究发现,CXCR7(孤儿受体RDC1)与CXCL12具有高亲和力,表达于许多肿瘤细胞系,直接参与调节肿瘤的发生发展。CXCR7信号传导机制还不确切,推测可能是通过结构性激活机制来活化细胞,此结构性激活机制通过可逆性地与配体结合的形式活化。体外实验证实CXCR7能提高肿瘤细胞的生长、粘附和侵袭能力。导入CXCR7序列的乳腺癌细胞MDA-MB435s,体内成瘤性显著提高;而CXCR7siRNA可使乳腺癌细胞4T1体内成瘤性显著降低。用CXCR7的小分子拮抗剂也可使肿瘤生长显著减小。目前研究CXCR7对肿瘤生物学行为的影响仅限于乳腺癌、肺癌、前列腺癌、膀胱癌等。本文拟展开“CXCR7与结肠癌”的实验研究,首先假定CXCR7是人结肠癌发生发展的促进因子,在结肠癌组织中高表达,并与结肠癌细胞的增殖、迁移相关,靶向沉默CXCR7的表达能有效抑制结肠癌的恶性进展,由此实验研究设计如下:1.研究CXCR7在人结肠癌组织、正常结肠组织以及结肠癌细胞系中的表达,探讨其表达水平与淋巴结转移以及肿瘤分化程度的关系。2.设计、筛选有效抑制人结肠癌细胞CXCR7表达的siRNA序列,并构建重组慢病毒CXCR7shRNA(lentiviraus-CXCR7shRNA,LV-CXCR7shRNA)表达载体。3.研究靶向CXCR7的慢病毒shRNA表达载体对人结肠癌细胞增殖、迁移活性的调节作用,从细胞水平探讨CXCR7对结肠癌的生物学行为的影响。4.研究靶向CXCR7的慢病毒shRNA表达载体对人结肠癌荷瘤裸鼠瘤体的治疗作用,在动物体内进一步证实CXCR7对结肠癌的生物学行为的影响。第一章CXCR7在人结肠癌组织和结肠癌细胞系中的表达及其意义目的研究CXCR7在人结肠癌组织、正常结肠组织以及结肠癌细胞系中的表达水平。方法1.CXCR7在人结肠癌组织中的表达水平:分别采用实时荧光定量PCR(Real-time fluorescent quantitative PCR,FQ-PCR)和Westernblotting(WB)方法测定人结肠癌组织和正常结肠组织中CXCR7mRNA和蛋白的表达水平,结合临床资料分析CXCR7的表达水平与淋巴结转移和肿瘤分化程度的关系。2.CXCR7在人结肠癌细胞系中的表达水平:FQ-PCR方法测定CXCR7mRNA在2种人结肠癌细胞系HT-29和SW480中的表达水平,选择其中CXCR7表达较高的一株结肠癌细胞进入后续部分的实验。结果1.人结肠癌组织和正常结肠组织中CXCR7mRNA相对表达的中位数分别为1.14和0.22,差异有统计学意义(P＜0.05)。结肠癌组织中CXCR7蛋白的相对表达量为0.245±0.059,与正常结肠组织中的0.17±0.064相比差异有统计学意义(P＜0.05)。2.在伴有和不伴有淋巴结转移的结肠癌组织中,CXCR7mRNA相对表达的中位数分别为1.455和0.74,差异有统计学意义(P＜0.05);CXCR7蛋白的相对表达量分别为0.276±0.055和0.2±0.026,差异有统计学意义(P＜0.05)。3.在不同分化程度的肿瘤组织中,CXCR7mRNA和蛋白的表达无显著性差异(P＞0.05)。4.CXCR7mRNA在人结肠癌细胞系HT-29中的表达水平高于在SW-480中的表达水平。结论1.CXCR7在人结肠癌组织中的表达显著增高,并且在伴有区域淋巴结转移的结肠癌组织中表达上调。2.在不同分化程度的结肠癌组织中,CXCR7的表达无差异。3.HT-29细胞中CXCR7mRNA的表达较高。第二章构建CXCR7shRNA重组慢病毒表达载体目的筛选有效的CXCR7siRNA序列,构建重组慢病毒CXCR7shRNA表达载体。方法1.筛选有效抑制人结肠癌细胞CXCR7表达的siRNA序列:设计3个针对CXCR7靶基因序列的siRNA序列,分别构建重组shRNA质粒,小量包装shRNA慢病毒载体,并转染人结肠癌细胞HT-29,FQ-PCR和WB方法分别测定转染后HT-29细胞中CXCR7mRNA和蛋白的表达水平,证实沉默效果,筛选出有效靶点。2.构建LV-CXCR7shRNA:用所筛选出的siRNA序列构建重组shRNA质粒,大量包装LV-CXCR7shRNA,并检测病毒滴度。结果1.FQ-PCR检测3个siRNA序列对CXCR7基因的沉默效应,其中以LV-CXCR7shRNA-2下调最为明显(72%),WB检测结果与其一致。2.选择沉默效应为72%的siRNA序列包装shRNA慢病毒载体,测定病毒滴度为4.0×10~8TU/mL。结论成功构建慢病毒CXCR7shRNA表达载体,可有效下调人结肠癌HT-29细胞中CXCR7mRNA和蛋白的表达。第三章靶向抑制CXCR7的表达对人结肠癌细胞生物学特性的影响目的研究靶向抑制CXCR7的表达对人结肠癌细胞增殖、迁移活性的影响。方法1.将人结肠癌细胞HT-29分为3组:实验组、阴性对照组和空白对照组,实验组加入LV-CXCR7shRNA;阴性对照组加入LV-shRNA阴性对照;空白对照组不予任何处理。2.FQ-PCR和WB方法分别测定各组细胞CXCR7mRNA和蛋白的表达水平3.细胞活性实验(MTT)、Transwell细胞迁移实验检测各组细胞的增殖和迁移活力。结果1.CXCR7mRNA和蛋白的表达水平:FQ-PCR结果显示实验组CXCR7mRNA的相对表达水平低于阴性对照组和空白对照组(P＜0.05),而两组对照组之间相比较无显著性差异(P＞0.05)。WB结果与FQ-PCR一致,提示LV-CXCR7shRNA对HT-29细胞的CXCR7基因沉默有效。2.细胞活性实验(MTT):结果显示实验组细胞生长曲线较平缓,细胞生长速度较阴性对照组和空白对照组显著降低(P＜0.05);阴性对照组细胞生长曲线较空白对照组低平,提示靶向沉默CXCR7的表达明显抑制结肠癌细胞的增殖活性,同时慢病毒载体本身具有的潜在细胞毒性。3.Transwell细胞迁移实验:结果显示实验组穿透滤过膜细胞数量较阴性对照组和空白对照组明显减少(P＜0.05),两对照组比较无显著性差异(P＞0.05)。结果提示靶向抑制CXCR7的表达明显抑制人结肠癌细胞HT-29的迁移活性。结论靶向沉默CXCR7的表达抑制结肠癌细胞增殖和迁移活性第四章CXCR7shRNA重组慢病毒表达载体对人结肠癌荷瘤裸鼠皮下种植瘤的治疗作用目的探讨慢病毒载体介导的CXCR7shRNA对人结肠癌裸鼠皮下种植瘤生长的抑制作用,为进一步研究以CXCR7为靶点的结肠癌基因治疗奠定基础。方法1.建立人结肠癌荷瘤鼠模型,计算成瘤率。2.动物分组:将人结肠癌荷瘤裸鼠随机分为3组,每组8只:治疗组:瘤体内注射LV-CXCR7shRNA 50μL;阴性对照组:瘤体内注射LV-shRNA negative control 50μL;空白对照组:瘤体内注射PBS 50μL。3.给药后各组裸鼠每3天测量荷瘤裸鼠肿瘤体积,绘制瘤体增长曲线。4.24天后处死动物,取出种植瘤,测量其体积、重量,计算抑瘤率。5.FQ-PCR和WB方法分别测定肿瘤组织的CXCR7mRNA和CXCR7蛋白表达水平。结果1.人结肠癌荷瘤鼠模型构建成功,成瘤率为100%。2.与阴性对照组和空白对照组比较,治疗组瘤体的增长速度慢、体积小(P＜0.05),重量减轻(P＜0.05),抑瘤率分别为38%和34.04%。3.治疗组的瘤体组织中CXCR7mRNA和蛋白的表达水平低于阴性对照组和空白对照组(P＜0.05),阴性对照组和空白对照组比较无显著性差异(P＞0.05),提示瘤体内注射靶向CXCR7shRNA慢病毒表达载体能有效下调荷瘤鼠瘤体组织中CXCR7的表达。结论CXCR7-shRNA慢病毒表达载体能显著抑制人结肠癌荷瘤裸鼠的瘤体生长。更多还原

【Abstract】 The occurrence and deterioration of malignant tumor involve abnormal expression of a number of chemokine receptors.CXCL12 and its receptor CXCR4 are the key factors in tumor growth and metastasis. Recent study of prostate cancer showed that the expression of CXCR4 inhibited could only partially block the malignant growth and metastasis. Further study found that CXCR7(orphan receptor RDC1),with high affinity to CXCL12,is expressed in many tumor cell lines and directly involved in regulating the occurrence and deterioration of tumor.The signal pathway of CXCR7 is unclear and presumed to activate cells by a structural activate mechanism of reversible binding with its ligand.CXCR7 can enhance growth,adhesion and invasion of tumor cell in vitro.Tumorigenicity was significantly increased by transfecting CXCR7 sequence into breast cancer cells MDA-MB435s,but suppressed by transfecting CXCR7siRNA into other breast cancer cells 4T1 in vivo.The small molecule antagonist of CXCR7 could also significantly suppress the tumor growth.Present investigation of the effects of CXCR7 on biological behavior of tumor only involve in breast cancer,lung cancer,prostate cancer, bladder cancer and so on.This article intended to study "CXCR7 and colon cancer".First of all,CXCR7 was envisaged as a promotive factor of colon cancer,high-expression in colon cancer tissue and to associate with proliferation and migration of colon cancer cell.Furthermore,it was also assumed that gene silence of CXCR7-targeting could effectively inhibit malignant progression of colon cancer.Accordingly,the design of this experiment was as follows:1.To investigate the expression levels of CXCR7 in human colon cancer,normal colon tissue and colon cancer cell lines and to explore the relationships between CXCR7 expression level and colon cancer,lymph node metastasis and tumor differentiation.2.Designing and screening the siRNA sequence which can effectively inhibit the expression of CXCR7 of human colon cancer cell for constructing the recombinant lentiviral vector of CXCR7shRNA (lentivirus-CXCR7shRNA,LV-CXCR7shRNA).3.Research on the regulatory roles of LV-CXCR7shRNA on proliferative and migratory activities of human colon cancer cell for exploring the effect of CXCR7 on biological behaviors of colon cancer from the cellular level.4.Research on the therapeutic effect of LV-CXCR7shRNA on bearing-tumor nude mice of human colon cancer,which further confirm the role of CXCR7 on biological behaviors of colon cancer from in vivo of animals.Part one To investigate the expressive significance of CXCR7 in human colon cancer tissues and cell linesObjective Study the expression level and significance of CXCR7 in human colon cancer tissues,normal colon tissues and cell lines.Methods1.Expressive level of CXCR7 in human colon cancer tissues:the expression levels of CXCR7mRNA and protein were determined by Real-time fluorescent quantitative PCR(FQ-PCR) and Western blotting (WB) in human colon cancer tissues and normal colon tissue, respectively.Data were analyzed together with lymph node metastasis and tumor differentiation.2.Expression level of CXCR7 in human colon cancer cell lines:the expression levels of CXCR7mRNA were determined by FQ-PCR in HT-29 and SW480 of human colon cancer cell lines and the higher was selected for further experiment.Results1.The medians of CXCR7mRNA relative expression were 1.14 and 0.22,with statistically significant(P＜0.05),in human colon cancer and normal colon tissue,respectively.The relative expression levels of CXCR7 protein were 0.245±0.0591 and 0.17±0.064 in colon cancer and normal colon tissue,the difference was statistically significant(P＜0.05).2.The medians of CXCR7mRNA relative expression were 1.455 and 0.74 in colon cancer tissues with and without lymph node metastasis, respectively.The difference was statistically significant(P＜0.05).The relative expression levels of CXCR7 protein were 0.276±0.055 and 0.2±0.026,with significance difference(P＜0.05),in above two tissues, respectively. 3.The expression levels of CXCR7mRNA and protein were no significant difference(P＞0.05) in tumor tissues with different differentiations.4.The expression level of CXCR7mRNA of human colon cancer cell line HT-29 was higher than that of SW-480.Conclusions1.The expression of CXCR7 is significantly up-regulated in the colon cancer tissue with higher expressions as lymph node metastasis.2.The expressions of CXCR7 are not difference in differentiations.3.HT-29 cell was selected for higher expression of CXCR7mRNA for further experiment.Part two To construction the recombinant lentiviral expression vector of CXCR7shRNAObjectiveScreening effective sequence of CXCR7siRNA and constructing the LV-CXCR7shRNA.Methods1.Screening effective sequence of CXCR7siRNA in human colon cancer cell:3 siRNA interference sequences of CXCR7-targeting gene were designed for constructing recombinant plasmid-shRNA,respectively. A small amount of shRNA lentiviral vectors of 3 kinds of plasmid-shRNA was constructed and then transfected into human colon cancer cells HT-29,respectively.CXCR7 expressions of mRNA and protein were detected by FQ-PCR and WB,respectively.Then the effects of gene silence were determined for screening the effective target.2.Constructing LV-CXCR7shRNA:to construct the recombinant plasmid using the selected target sequence of siRNA for constructing a large number of LV-CXCR7shRNA,and then detecting the titer of virus.Results1.FQ-PCR detected the effects of 3 siRNA sequences on CXCR7 gene silencing.The decline of CXCR7mRNA was most marked(72%) by LV-CXCR7shRNA-2.This result was consistent with that of WB.2.To construct the lentvirus-shRNA vector using the siRNA sequence of 72%silence effect.The titer of virus detected was 4.0×10~8 TU/mL.ConclusionsLV-CXCR7shRNA vector was constructed successfully which effectively reduced the expressions of CXCR7mRNA and protein in human colon cancer cell HT-29. Part three Effects of gene silence of CXCR7-targeting on biological characteristics of human colon cancer cellObjectiveTo study the proliferative and migratory activities of human colon cancer cell after CXCR7-targeting inhibition.Methods1.Human colon cancer cells HT-29 were divided into 3 groups: experimental group,negative control group and blank control group.The experimental group was administrated with LV-CXCR7shRNA,negative control group with LV-shRNA negative control and blank control group without the any.2.CXCR7mRNA and protein of every group were detected using FQ-PCR and WB methods,respectively.3.The proliferative and migratory activities of every group were detected using cell active assay(MTT) and Transwell cell migratory assay.Results1.Expression levels of CXCR7mRNA and protein:CXCR7mRNA was lower expression in experimental group than the negative control group and blank control group(P＜0.05),but no significant difference (P＞0.05) between the two control groups.These results were consistent with protein expression.It is prompted that LV-CXCR7shRNA can effectively silence the gene expression of CXCR7 in HT-29 cell.2.Cell activity assay(MTT):the cell growth curve of experimental group was flatter and the level of cell growth was significantly lower(P ＜0.05)than the negative control group and blank control group.The cell growth curve of negative control group was lower than blank control group.These results suggest that the gene silencing of CXCR7-targetting significantly inhibit the proliferative activity of colon cancer cell.In addition,lentiviral vector has potential cytotoxicity.3.Transwell cell migratory assay:the cell populations of penetrating the filtrative membrane in experimental group were significantly lower(P＜0.05)than negative control group and blank control group,with no significant difference(P＞0.05) between the two control groups, suggesting that inhibition of CXCR7 expression suppress migratory activity of colon cancer cell.ConclusionsGene silence of CXCR7-targeting suppresses the proliferative and migratory activities of colon cancer cell.Part four The therapeutic effect of recombinant lentiviral-CXCR7shRNA vector on planted subcutaneous tumors of human colon tumor-bearing nude miceObjectiveTo explore the growth inhibition of planted subcutaneous tumors in human colon tumor-bearing nude mice using lentivirus-CXCR7shRNA, for enlightening further study of CXCR7-targeting gene therapy toward human.Methods1.To establish the animal model of human colon tumor-bearing mice and calculate the tumor-formation rate.2.Tumor-bearing nude mice were randomized into 3 groups and each group contained 8.Treatment groups:injecting 50μL of LV-CXCR7shRNA into tumor;Negative control group:injecting 50μL of LV-shRNA negative control into tumor;Blank control group:injecting 50μL PBS into tumor.3.Tumor volumes were measured per 3 days after administration for mapping tumor growth curves.4.To sacrifice animals after 24 days,remove tumors,measure tumor volumes and weights and calculate inhibition rates,respectively.5.CXCR7mRNA and protein of tumors were measured by FQ-PCR and WB.Results1.The model animals of human colon tumor-bearing mice were established successfully by 100%.2.As compared with negative control and blank control group,the tumor growth of treatment group was slow and the tumor volume and weight of treatment group decreased significantly(P＜0.05) with 38% and 34.04%tumor-growth-inhibition rates,respectively.3.As compared with negative control and blank control group,the expression of CXCR7mRNA and protein in tumors of treatment group were down-regulated significantly(P＜0.05),with no significant difference(P＞0.05) between the negative control group and blank control group.It was prompted that lentiviral-CXCR7shRNA effectively inhibited the expression of CXCR7 in tumors of human colon cancer-bearing nude mice.ConclusionsThe LV-CXCR7shRNA can effectively inhibit the growth of tumor of human colon tumor-bearing nude mouse.更多还原