【作者】 邢志军；

【导师】 高忠礼；

【作者基本信息】 吉林大学 ， 外科学， 2009， 博士

【摘要】 本研究以体外培养的人成骨肉瘤细胞系-MG63等细胞为对象,利用RNAi沉默等分子生物学技术,观察Cofilin表达、激活以及Cofilin介导的信号通路对人成骨肉瘤细胞迁移的影响并详细探讨了其作用机制。通过检测人成骨肉瘤细胞中CXCR4基因蛋白表达发现,CXCR4在人成骨肉瘤细胞系MG63及U2OS细胞中高表达。提示SDF-1/ CXCR4生物学轴可能对调控骨肉瘤的生物学行为具有重要作用。进一步研究发现经SDF-1预处理的骨肉瘤细胞其迁移能力明显提高,同时SDF-1诱导了AKT磷酸化/激活以及Cofilin去磷酸化/激活。Wortmannin有效抑制了SDF-1诱导的骨肉瘤细胞迁移以及SDF-1/PI3’K/cofilin信号通路的激活;另外、抑癌基因PTEN瞬时转染骨肉瘤细胞后可导致AKT去磷酸化/失活,以及Cofilin磷酸化/失活。上述结果提示SDF-1/PI3’K/Cofilin信号通路对骨肉瘤细胞迁移起着关键的调控作用。本研究成功构建了靶向Cofilin基因的siRNA逆转录病毒表达载体,并将其稳定转染至MG63细胞系。细胞趋化实验结果显示沉默MG63细胞中Cofilin后,细胞运动、迁移能力明显降低。说明Cofilin是调控细胞的运动能力的重要因素。本研究为以Cofilin为靶点的肿瘤治疗学研究提供了新的理论基础。更多还原

【Abstract】 Osteosarcoma is a kind of common primary malignant neoplasms of bone. Most Osteosarcoma attacks the adolescent, and causes great harm and high mortality. At the outset the disease is latent and in high-degree malignancy. Its early clinical symptoms may be confused with trauma of bone and pulmonary metastasis may occur in the early stage of the disease.At present, the main treatment of osteosarcoma is still surgical treatment. Yet some patients encounter treatment failure because of tumor recurrence and metastasis. Along with the development of experimentations on the molecular level and clinical research into tumor growth and invasion, gene therapy of osteosarcoma proves a broad prospect for research.Chemokines is a micromolecule cytokine which can generate cell movement,they can orientation generate cell to high concentration. Chemokines and it’s recipient developed important and complicated role in the development of tumor.some chemokines can promote tumor cell multiplication,cause movement of tumor cell and endothelial cell ,accelerate tumor cell diffusion and corestriction and the form of tumor blood vessels;otherwise some chemokines inhibit the growth of tumor cell and blood vessels,stimulate body specificity and non- specificityImmunological response inhibit the corestriction of tumor.Cofilin is an important member of ADF∕Cofilin family,It’s function Exist in accelerate the depolymerization of actin filaments and futher develop the recycle of actin filaments after its binding to F-actin.The fidelity vacuity regulation of actin deploy-merization and cutting activity which cofilin depended.Recent research shows cofilin’s function inducing act as regulatory factor of cell anterior border migration,regulate the interaction of lamelliform pseudopodia through answering upstream signal.Cofilin and it’s passway signal plays an important role not only in normal body ,but also in the development and progress of disease.It is close concerned with their function,however,the correct actin site,conduction path of signal and complicated function needs further research.RNAi is a kind of sequence-specific and post-transcriptional gene silencing mechanism which is dsRNA triggered and which results in the degradation of sequence homology mRNA . At present, RNAi has been widely used in the study of genome function, anti-virus and anti-tumor.1 The expression and meaning of CXCR4 in human osteosarcoma .CXCR4 is the rceptor of SDF-1,Although CXCR4 is widely spread,the expression is different in various kinds of tissues and cells. SDF-1 can activate MAPK and NF-KB after binding with CXCR4,then cause the form of pseudopodia in tumor cells,induce the invasion and migration of tumor cells.This research check the expression of CXCR4 through western blot.The result shows,the expression of CXCR4 in human osteosarcoma Mg63 and U2OS is higher 2-3 times than in normal HFOB cells,the difference shows statistics meaning (P﹤0.05);but the expression of CXCR4 shows not much difference.Which means the biological axis of SDF-1∕ CXCR4 plays an important role in the enhancement and migration of oseteosarcoma .2 The function of SDF-1∕PI3’K∕Cofilin signal passway regulate the migration of osteosarcoma cells.The migration of tumor is a procedure which means malignant tumor cells spread from primary position to other position through lymph,blood and body cavity etc.This is one of biological character difference from normal cells.The point reason of endanger host life and affect the effectiveness of therapy is the migration of tumor.Recent research shows SDF-1∕CXCR4 biological axis plays an important role in the regulation of osteosarcoma orientation migration,but it’s detail function is not clear.This research act SDF-1 as the inducing factor of MG63 cell epimatrix set migration,check the ability of set migration of cell by using Borden room invasion experiment.3 hours after inoculate to cells,operate the distance of cell invasion.Through microscope we found that cells invase at the back of membranes spread in much round fenestra irregularly.Contract with control group,the migration ability of cells induced by SDF-1 was increased about 2 times,otherwise,developing with the density of Wortmannin,the ability of cell invasion was apparently inhibited.The result checked by Western blot shows,the expression of P-AKT in human osteosarcoma MG63 was apparently inhibited by Wortmannin,depend on concentration,which means Wortmannin was the effective inhibitor of PI3’K∕AKT passway.Using 200nm Wortmannin act on human osteosarcoma MG63 0hour ,1hour ,2hours,4hour3,the result checked by Western blot shows Wortmannin apparently increased the P-cofilin of human osteosarcoma. Using 5nm SDF-1 act on human osteosarcoma MG63 cells 0min,5min,20min,40min, the result checked by Western blot shows SDF-1 decreased the P-cofilin of human osteosarcoma,and depend on time,which shows SDF-1 signal passway activate cofilin;1hour after treated with 200nm Wortmannin,inhibit the activation of cofilin induced by SDF-1,which means PI3’K∕AKT play a decisive role in SDF-1∕confilin signal passway.Further check the phosphory lation of P- AKT,shows incrase phosphorylation of AKT by stimulation by SDF-1,otherwise, the function of Wortmannin is on the contrary.On the other hand,after transient transfect wild PTEN(WT)plasmid in MG63 cells, the result checked by Western blot shows the expression of P- AKT decrease ,at the same time the expression of P-confilin step-up;Otherwise the expressing of P-confilin apparently decreas in MG63 cells which non-active mutant PTEN(cs) overexpressed,shows PTEN inhibit the activation of AKT,so did cofilin.All the result show,confilin plays an important role in osteosarcoma cell migration induced by SDF-1∕PI3’K signal passway.3 The function of cofilin in human osteosarcoma cell invasionRNAi technology is using little Double-Strandfd RNA,high performance and exceptionally block the expression of some special gene,induce the degradation of mRNA,make cells express lack special gene,it is an important conservancy mechanism which prevent gene from damage and infection viral,which can act as a simple and effective tool to replace gene knockout.According to the difference mechanism of tumor formation and development, RNAi technology is widly used in the field of phymatology.This research we transfect thereverse transcription viral epression∕carrier carrying target cofilin special siRNA in human osteosarcoma cells with PRev-H1-cofilin,the transcript create hairpin-like cofilin-siRNA under the effect of Dicer,then lead cofilin mRNA degradation specially.Effectively silence the expression of cofilin in human osteosarcoma MG63 cells,and set up steady transfection cell line successfully.The result of tendency experiments shows,after silence cofilin gene,the ability of cell movement and cell tendency movement was apparently dcreased,which means cofilin is an important factor in regulate the invasion and metastasis of tumor cell.All of these provide act cofilin as targets for further gene therapy of osteosarcoma.更多还原