【作者】 周立宏；

【导师】 李桂英；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2006， 硕士

【摘要】 细胞周期蛋白D1(Cyclin D1)是一类重要的细胞周期正性调控因子,它通过激活CDK4/6来推进细胞周期G1期到S期的演进。Cyclin D1在很多恶性肿瘤中呈过度表达,并与肿瘤的发生、恶性程度与患者的预后密切相关。Cyclin D1成为人们进行肿瘤基因治疗的靶点之一。 研究者们曾尝试向肿瘤细胞中注射抗Cyclin D1抗体、转录导入反义Cyclin D1,两种方法均可改变转化细胞形态,抑制肿瘤细胞增殖,但是由于存在免疫原性较强,后者又存在体内半衰期短、不稳定等缺点,限制了该法在肿瘤治疗中的应用。近年来兴起的以抗体作为效应分子的能够在细胞内高效、特异灭活靶蛋白的胞内抗体技术为肿瘤的基因治疗开辟了新的思路。 为此,本研究克隆了内质网滞留型抗人Cyclin D1胞内scFv基因(ER-ADx),并构建其重组真核表达载体pER-ADx,通过细胞转染实验、RT-PCR、免疫组化、Dot-blot、MTT法生长增殖特性分析、流式细胞术等方法和技术分别对ER-ADx的表达及胞内定位情况、体外生物活性等进行了研究。结果表明,ER-ADx基因能够在肿瘤细胞中有效表达并实现胞内抗体的目标定位。ER-ADx基因的稳定表达能够显著抑制细胞生长和增殖,引起细胞周期阻滞,并明显诱导细胞凋亡。综上所述,以Cyclin D1为靶点的胞内抗体治疗作为一种新型肿瘤基因治疗方法,有潜在的应用价值。更多还原

【Abstract】 Cyclin D1, a putative mammalian G1 cyclin, plays an important role in controlling the transition from G1 to S in cell cycle. Amplification and over-expressi- on of Cyclin D1 has been found in tumorigenesis of several types of human cancer. It becomes a potential target for the treatment of human diseases in which the control of cell proliferation is deregulated.At present, there are several other methods to inhibit biological activity of molecules intracellularly. RNA based therapies, such as ribozymes or antisense RNA or RNAi are less stable and can only be used against RNA. Other protein-based therapies, such as dominant negative inhibitors, rely on competition with a native binding species and therefore require excessive concentrations In addition, these targets generally are proteins. Whereas intrabodies, by linking appropriate intracellular trafficking signal sequences to their coding genes, can be directed to all intracellular compartments including protein, nucleic acid, carbohydrate, lipid, or other cellular components. Recently intrabodies have been studied in terms of treatments for tumor growth and even clinical trials for cancer. As potential direct therapeutics, intrabodies are distinguished from small-molecule and peptide drugs in their enhanced targetbinding specificity and stability. Thus intrabody technique may pioneer a new approach for cancer therapy.In present study, an expression plasmid pER-ADic habouring an endoplasmic reticulum(ER)-retained ER-ADk scFv gene against human Cyclin Dl was constructed. In order to test the expression and activity of pER-ADic, MCF-7 cells and HeLa cells were transfected with pER-ADK and pcDNA3.1, respectively. Then expression and subcellular distribution of ER-ADk was detected by RT-PCR, Dot blot and fluorescence staining analysis. MTT analysis was used for cell proliferation analysis, the cell cycle and apoptosis changes of the transfection cells were detected by Flow Cytometry.Confocal microscope analysis demonstrated that both MCF-7/pER-ADK and HeLa/pER-ADK cells showed positive staining in cytoplasm, but the MCF-7, HeLa, MCF-7/pcDNA3.1 and HeLa/pcDNA3.1 cells did not show any fluorescence staining. RT-PCR analysis demonstrated that the ER-ADk mRNA was expressed only in MCF-7/pER-ADK and HeLa/pER-ADK cells, not in MCF-7, HeLa, MCF-7/pcDNA3.1 and HeLa/pcDNA3.1 cells. Dot blotting analysis showed that ER-ADk was detected only in MCF-7/pER-ADK and HeLa/pER-ADK cells, but was not detected in MCF-7, MCF-7/pcDNA3.1, HeLa, and HeLa/pcDNA3.1 cells. These results suggested that ER-ADk gene was already transferred into MCF-7 and HeLa cells and was successfully expressed.The growth curve showed that the expression of ER-ADranhibited the growth of MCF-7 cells and HeLa cells significantly. Cell cycle analysis by FCM showed that the expression of ER-ADk arrested the cell cycle of MCF-7/pER-ADK and HeLa/pER-ADK cells at the Gl phase, inhibiting the cells to enter the S phase of MCF-7/pER-ADK and HeLa/pER-ADK cells. Cell apoptosis analysis indicated that expression of ER-ADk inducedMCF-7/pER-ADK and HeLa/pER-ADK cells apoptosis distinctly.In conclusion, stable expression of ER-ADk regulate cell cycle and cell proliferation of MCF-7/pER-ADK and HeLa/pER-ADK cells. These data strongly suggested that intracellular scFv may become an effective approach for tumor gene therapy.更多还原