【作者】 王磊；

【导师】 陈卫昌；

【作者基本信息】 苏州大学 ， 内科学， 2008， 博士

【摘要】 第一部分人COX-2小分子干扰RNA的设计、合成及转染条件的优化目的设计并化学合成人COX-2基因的小分子干扰RNA,并进一步优化转染人结肠癌HT-29细胞的条件。方法遵循RNA干扰目标序列的选取原则,利用互联网资源对人COX-2基因mRNA序列设计4条小干扰RNA(siRNA),经退火合成。以阴性荧光素特异性siRNA为报告基因,通过脂质体lipofectamineTM2000转染人结肠癌HT-29细胞,利用荧光显微镜计数观察转染效率,MTT法检测转染后对HT-29细胞的毒性。结果成功设计并合成4对人COX-2基因的小分子干扰RNA,在24孔板中筛选出最优化的转染条件为1.5ul lipofectamineTM2000和40pmol siRNA。结论成功设计并合成了人COX-2基因的小分子干扰RNA,同时优化的最佳转染条件为进一步研究人COX-2小干扰RNA功能提供了条件。第二部分COX-2小干扰RNA对人结肠癌HT-29细胞COX-2表达及细胞生长影响的研究目的研究COX-2 siRNA对环氧合酶-2(COX-2)高表达的人结肠癌HT-29细胞增殖和凋亡的影响。方法从化学合成的4对小干扰RNA中筛选出最佳的靶向COX-2 siRNA转染人结肠癌HT-29细胞,采用逆转录-聚合酶链反应(RT-PCR)和Western印迹分别从mRNA和蛋白水平检测COX-2表达,四甲基偶氮唑蓝(MTT)和Hoechst染色检测COX-2表达被抑制后细胞增殖和凋亡的情况。结果COX-2 siRNA能在mRNA和蛋白水平上下调人结肠癌HT-29细胞中COX-2基因的表达(P<0.05),以转染72h时显著。COX-2表达被抑制后,HT-29细胞生长受到明显抑制(P<0.05),凋亡细胞显著增加。结论COX-2与结肠癌细胞生长和凋亡密切相关,抑制结肠癌细胞中COX-2表达可以抑制结肠癌细胞生长,促进结肠癌细胞凋亡。第三部分人COX-2shRNA真核表达载体的构建、鉴定和转染条件的优化目的构建环氧合酶-2(COX-2)基因靶向性的shRNA真核表达载体,并进一步优化在转染人结肠癌HT-29的转染条件,以观察转染结肠癌细胞株HT-29细胞后,其对COX-2活性的抑制效率。方法设计短发夹结构的COX-2 siRNA对应模版DNA序列,退火处理后克隆至pGPH1-GFP-Neo质粒,构建重组质粒pshCOX-2。酶切并测序鉴定。优化转染条件后,将重组质粒转染人结肠癌HT-29细胞,采用逆转录-聚合酶链反应(RT-PCR)、Western印迹分别从mRNA和蛋白水平检测COX-2表达。结果pshCOX-2经鉴定与设计目的序列完全一致。转染重组质粒72h后可以显著抑制HT-29细胞COX-2 mRNA表达与对照组比较有显著性差异(F=349.58,P=0.0001),对照组之间则无差异(P>0.05),蛋白水平的抑制效率与mRNA水平一致。结论成功构建无内毒素活性的COX-2 shRNA真核表达质粒pshCOX-2,可稳定抑制人结肠癌HT-29细胞COX-2基因表达,为利用RNAi技术在体内和体外水平研究COX-2在结肠癌中的作用机制打下了基础。第四部分抑制COX-2蛋白表达对人结肠癌HT-29细胞生物学活性的影响目的体外观察pshCOX-2抑制COX-2活性对HT-29细胞生物学活性的影响,并探讨可能的作用机制。方法应用pshCOX-2瞬时转染HT-29细胞,MTT法检测细胞的生长曲线,放射免疫法(RIA)和ELISA法分别检测转染后细胞培养上清中PGE2和VEGF的表达;优化G418浓度、筛选和鉴定稳定表达pshCOX-2的HT-29细胞株,应用过河实验观察细胞迁徙能力的改变,集落形成实验观察细胞增殖能力的改变。免疫组化法观察稳定细胞株中COX-2和EGFR的表达,采用RT-PCR检测稳定细胞株中VEGF、MMP-2、EGFR mRNA的表达。结果pshCOX-2转染HT-29后72h,细胞增长明显受抑,细胞培养上清中PGE2和VEGF的表达明显下降。稳定表达pshCOX-2细胞株内COX-2蛋白和mRNA均下调,细胞越过划痕的时间为(7.67±1.53)d,与对照组有显著性差异(F=9.50,P=0.0138);细胞集落密度为(257.67±39.53)个,明显低于其平行对照组(F=37.07,P=0.0004)。免疫组化提示pshCOX-2细胞株内COX-2和EGFR表达均有下调,RT-PCR显示pshCOX-2组VEGF、MMP-2、EGFR mRNA表达下调。结论抑制COX-2活性能抑制HT-29细胞增殖和迁徙能力,同时可以抑制VEGF、EGFR、MMP-2表达和PGE2产生,有潜在的抑制肿瘤血管形成作用。第五部分抑制COX-2蛋白对结肠癌细胞凋亡影响的实验研究目的观察pshCOX-2抑制COX-2活性对HT-29细胞凋亡和对于化疗药物敏感性的影响,并探讨可能的作用机制。方法应用pshCOX-2瞬时转染HT-29细胞,联合使用5-Fu处理HT-29细胞,MTT法检测细胞的生长曲线,流式细胞仪分析凋亡率。应用TUNEL法观察稳定表达pshCOX-2的HT-29细胞凋亡形态,应用RT-PCR和Western blot法从转录水平和蛋白水平检测Bcl-2/Bax和Fas/FasL表达的变化。结果pshCOX-2瞬时转染3d后可以明显抑制HT-29细胞生长,与空白和阴性对照相比(P<0.05),联合使用5-Fu后在7d时细胞生长受抑作用,且呈协同作用,差异有显著性(F=231.94,P=0.0001)。流式细胞仪检测细胞DNA含量,发现转染组凋亡指数(7.07%)明显高于对照组的0.30%和0.27%(F=24.71,P=0.0013),低于联合处理组13.57%,差异有显著性(P<0.05)。TUNEL法原位凋亡指数结果与流式细胞仪结果类似,RT-PCR和Western blot结果提示稳定表达pshCOX-2细胞株Bax和Fas表达上调,Bcl-2表达呈下调趋势,而FasL表达与对照组相无显著性差异(P<0.05)。结论COX-2shRNA可以抑制细胞增殖,促进凋亡,增强肿瘤细胞对5-Fu的敏感性,COX-2shRNA促进凋亡的机制可能与上调Bax和Fas表达,下调Bcl-2表达有关。第六部分荷瘤鼠模型建立及COX-2shRNA对结肠癌细胞致瘤性的影响目的通过裸鼠体内实验,进一步验证抑制COX-2表达对结肠癌致瘤的影响并探讨可能的作用机制。方法选用BALB/C裸鼠进行体内实验。裸鼠25只。随机分为5组:A.空白对照组5只;B.阴性质粒组5只;C.转染pshCOX-2组5只;D.5-Fu处理组5只;E.5-Fu+转染pshCOX-2联合组5只。于每只裸鼠右胁腹前肢皮下分别接种等量的不同转染细胞(5×106个细胞/100ul/只),接种42d后处死老鼠。观察肿瘤生长状况、成瘤潜伏期,计算抑瘤率。免疫组化法检测瘤组织内COX-2和EGFR蛋白表达情况,测定瘤块中MVD;TUNEL法检测肿瘤的凋亡指数,透视电镜观察瘤块中肿瘤细胞凋亡情况。结果转染pshRNA组成瘤潜伏期延长、生长曲线、瘤体积和瘤重与对照组比较(P均<0.05),联合治疗组抑瘤作用更强,与转染pshRNA组相比较(P均<0.05);pshRNA组原位凋亡明显增多,透视电镜观察pshRNA组出现较多明显凋亡形态的细胞。免疫组化提示pshRNA组COX-2和EGFR表达均较对照组下调(P<0.05),瘤组织中MVD,对照组明显高于pshRNA组(P<0.05)。结论体内实验表明抑制肿瘤细胞COX-2表达可以有效抑制肿瘤的生长,促进凋亡,抑制肿瘤血管形成,同时可以增加肿瘤细胞对化疗药物敏感性,以此基因为靶点进行的小干扰RNA基因治疗,将有可能成为人类结肠癌治疗提供有效的手段。更多还原

【Abstract】 The First Section: Study on designing and synthesis of COX-2 siRNA and optimizing transfection conditionsObjective To design and chemically synthesize siRNA targeted on human COX-2 and optimize transfection conditions. Methods To follow the principle of selecting RNA interference target sequence, to utilize the web resource,design four potential small interference RNA (siRNA) of COX-2,and then were annealed. The FAM- negative-siRNA was transfected to HT-29 cells by lipofectamineTM2000. To optimize transfecting conditions, the fluorescence microscope was used for deciding the efficiency of transfection and the activity of cells were measured by MTT after transfecting. Results The four siRNA targeted on COX-2 were successfully constructed. The best transfection conditions was the combination with 1.5ul lipofectamineTM2000 and 40pmol siRNA in the 24 wells board. Conclusions The four siRNA targeted on COX-2 were successfully designed and constructed. The study on effects of COX-2 siRNA on HT-29 cells will be performed in the most optimal transfection conditions.The Second Section: Study on effects of COX-2 siRNA-mediated gene silencing on the expression of COX-2 and proliferation of human colon cancer cellsObjective To investigate the effect of synthesized COX-2 specific siRNA on the cell proliferation and apoptosis of COX-2 overexpressing human colon cancer cells. Methods Human colon cancer cells of the line HT-29 were cultured and transfected with the most optimal COX-2 siRNA by screening from four siRNA. RT-PCR and Western blot were used to detect the expression of COX-2 mRNA and protein. The method of MTT was used to examine the proliferation of the cells. The apoptosis of the cells was detected by Hoechest staining. Results The expression of COX-2 mRNA and protein in the COX-2 siRNA group decreased remarkably(P<0.05),especially 72 hours after transfecting. The proliferation of HT-29 cells transfected with COX-2 siRNA did not changed significantly 24 hours after transfecting, however, decreased 48 hours after and especially 72 hours and 1 week after(P<0.05), Meanwhile, the apoptosis of the HT-29 cells transfected with COX-2 siRNA was significantly higher than that of the cells transfected with the negative COX-2 siRNA and that of the untransfected cells. Conclusions COX-2 is closely related to the proliferation and apoptosis of tumor cells, transfection of the specific siRNA targeting on COX-2 helps inhibiting the expression of COX-2, thus inhibiting the growth and enhancing the apoptosis of the tumor cells.The Third Section: Study on construction and identification of the eukaryotic expression plasmid for cyclooxygenase-2 specific shRNA and optimizing transfection conditionsObjective To clone the recombinant eukaryotic expression plasmid of specific small interfering RNA (siRNA)against COX-2 gene and optimize transfection conditions. Methods The COX-2 siRNA template DNA sequence for short hairpin RNA (shRNA)was designed and synthesized. The annealed siRNA template was inserted into pGPH1-GFP-Neo plasmid. The recombinant plasmid (pshCOX-2) was transformed into DH5αstrain and identified by restrictive enzyme digestion and sequence analysis. The effect of the recombinant plasmid on the COX-2 expression of human colon cancer HT-29 cells was detected by RT-PCR and Western blot after optimizing transfection conditions. Results It was confirmed by restrictive enzyme digestion and sequence analysis that the recombinant plasmid was cloned and the aim sequence was obtained. The COX-2 expression of HT-29 cells was inhibited at mRNA and protein levels 72 hours after transfected with the recombinant plasmid. The difference is significant between the group transfected with the recombinant plasmid and the control groups (F=349.58,P=0.0001), but the difference between the control groups is not significant(P>0.05). Conclusion COX-2 shRNA expression plasmid pshCOX-2 successfully constructed can lastingly inhibit the expression of COX-2 gene in human colon cancer HT-29 cells. It may be a tool to study the relations between COX-2 and colon carcinoma in vitro and in vivo.The Fourth Section: Study on effects of gene silencing of COX-2 on the biological activity of HT-29 cellsObjective To observe the effect on proliferation of HT-29 cells with short hairpin RNA targeted on COX-2 gene in vitro, and to explore the potential mechanisms. Methods After HT-29 cells were transient transfected with pshCOX-2 plasmid, growth curves were detected by MTT methods, and the methods of RIA and ELISA were respectively used to estimate the content of PGE2 and VEGF in supernatant. Cell clones which stably expressed pshCOX-2 were obtained in the medium which contained G418 after 3-4 weeks. The time of healing the wound was utilized to observe the effect on cell migration, invasion and locomotion of HT-29-pshCOX-2 cells. The numbers of clone formation were used to observe the ability of proliferation of HT-29-pshCOX-2 cells.The expressions of COX-2 and EGFR of HT-29- pshCOX-2 cells was judged by immunohistochemistry and the expression of VEGF, MMP-2 and EGFR mRNA was detected by RT-PCR. Results The growth of HT-29 cells was inhibited at 72h after transient transfected with pshCOX-2 and the content of PGE2 and VEGF in supernatant decreased. The expression of COX-2 of HT-29 cells which stably expressed pshCOX-2 down-regulated. Time when cells healed the wound was (7.67±1.53) d, which is longer than control groups (F=9.50 , P=0.0138). The number of clone formation was 257.67±39.53, which is few than control groups(F=37.07,P=0.0004)The expression of COX-2 and EGFR of HT-29-pshCOX-2 decreased and VEGF, MMP-2 and EGFR mRNA down-regulated significantly. Conclusions Inhibiting COX-2 expression can decrease the proliferation and invasion in HT-29 cells, downregulate the expression of VEGF,MMP-2 and EGFR mRNA and decrease the synthesis of PGE2; It may potentially inhibit angiopoiesis of tumor, which is foundation to the next in vivo experiment.The Fifth Section: Study on blocking COX-2 protein expression to promote HT-29 apoptosisObjective To observe the effect on chemotherapy- sensibility and apoptosis of HT-29 cells with short hair-pin RNA targeted on COX-2 gene in vitro, and to explore the potential mechanisms. Methods After HT-29 cells were transient transfected with pshCOX-2 plasmid, 5-Fu was sequently added or singly used, growth curves were detected by MTT methods, apoptosis rate were analyzed by flow cytometry. Morphocytology of apoptotic HT-29-pshCOX-2 cells were observed by TUNEL methods, and the expression of Bcl-2/Bax and Fas/FasL of these cells was measured by RT-PCR and Western blot. Results The growth of HT-29 cells was inhibited at 72h after transient transfected with pshCOX-2 in vitro, and it is significant compared with control groups(P>0.05). The synergistic inhibition can be found in the HT-29-pshCOX-2 cells 7d after 5-Fu used(F=231.94,P=0.0001). FCM show the apoptosis ratio is (7.07%) in pshCOX-2 group, it is statistically significance between pshCOX-2 group and control groups(0.3 % and 0.27%)(F=24.71,P=0.0013), but lower than combination group(13.57%). The results of TUNEL were the same as the FCM. The results of RT-PCR and Western blot showed that the expression of Bax and Fas up-regulated, Bcl-2 down-regulated and FasL had no changes in pshCOX-2 group. Conclusions: The stably expression of COX-2shRNA in HT-29 cells can inhibit the proliferation of HT-29 cells and promote the apoptosis of HT-29 cells. Down-regulating the expression of COX-2 by pshCOX can increase the chemotherapy- sensibility of 5-Fu.The Sixth Section: The therapeutic effect of COX-2 shRNA on colon carcinoma HT-29 cells in vivoObjective To investigate the effect of COXsiRNA gene therepy in vivo, and to explore the potential mechanisms. Methods We carried out vivo study with BALB/C/nu/nu nude mice. 25 nude mice were randomly divided into five groups, and 5×106 cells per mouse was subcutaneously injected on the right HS abdomen of mice(A)control group: 5 mice injected with HT-29 cells;(B)negative control group: 5 mice injected with HT-29pshNC cells;(C) pshCOX-2 group: 5 mice injected with HT-29pshCOX-2cells;(D)5-Fu group:5 mice injected in the abdomen with 5-Fu 50ug/kg/d×5d;(E)5-Fu+HT-29pshCOX-2 group(combination group):C+D. All mice were sacrificed 42 days later and the changes of weight of all nude mice were observed. The volume and weight of tumor were measured and the rates of tumor inhibition were evaluated. The growth curves and stage of latency of tumor were observed, and morphocytology of apoptotic cells were detected by TUNEL and TEM. The expression of COX-2, EGFR and CD34 were stained by immunohistochemistry. The microvessel density (MVD) of tumor tissue was evaluated. Results Stage of latency lengthen and growth of tumor in group C was inhibited. The volume and weight of tumors were significantly different between group C and control groups(P<0.05). The effect of inhibition of tumor in group C was weaker than combination group (P<0.05).The apoptosis in situ of tumors in group C increased, it is statistically significance compared with the control groups (P<0.05). The expression of COX-2 and EGFR decreased in group C,and the MVD of group C were scarce and punctiform. But in control group, MVD are intensive and annuliform, it is statistically significance between group C and control groups (P<0.05). Under TEM,more apoptosic cells in groupC can be found. Conclusions: By inhibiting the expression of COX-2 of mice, the growth of tumor can be inhibited; more apoptosic cells can be found in tumor, Inhibiting COX-2 expression can decrease vascularization of tumor and improve the sensibility of chemotherapy. The therapeutic effect of COXsiRNA in vivo suggested that COX-2 gene be a good target for siRNA gene therapy of colon carcinoma. We obtain a useful theoretical basis and measure for gene therapy of colon carcinoma.更多还原