抗Cyclin D1人源胞内单链抗体在肿瘤细胞中的表达及生物活性分析

发布时间：2018-05-20 08:17

本文选题：Cyclin + D1　；参考：《吉林大学》2006年硕士论文

【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。
[Abstract]:Cyclin D1 (cyclin D1) is an important positive regulator of cell cycle, which activates CDK4/6 to promote the progression from G1 to S phase of cell cycle. Cyclin D1 is overexpressed in many malignant tumors and is associated with tumorigenesis. The degree of malignancy is closely related to the prognosis of patients. Cyclin D1 has become one of the targets of gene therapy for cancer. The researchers have tried to inject anti-Cyclin D1 antibodies into tumor cells and transcribe them into antisense Cyclin D1. Both methods can change the morphology of transformed cells and inhibit the proliferation of tumor cells, but because of their strong immunogenicity, The latter has the shortcomings of short half-life and instability in vivo, which limits the application of this method in tumor therapy. In recent years, the technology of intracellular antibody with antibody as effector molecule can be highly efficient and specific to kill target protein in cells, which opens a new way for gene therapy of tumor. The recombinant eukaryotic expression vector pER-ADX was constructed by cloning endoplasmic reticulum (ER) retained anti-human Cyclin D1 scFv gene (ER-ADxX), and the proliferation characteristics of the recombinant plasmid pER-ADX were analyzed by cell transfection assay with RT-PCR and immunohistochemistry with Dot-blot MTT assay. The expression and intracellular localization of ER-ADx and its bioactivity in vitro were studied by flow cytometry. The results showed that ER-ADX gene could effectively express in tumor cells and realize the target localization of intracellular antibody. The stable expression of ER-ADX gene could significantly inhibit cell growth and proliferation, induce cell cycle arrest and induce apoptosis. In conclusion, intracellular antibody therapy targeting Cyclin D1 as a new tumor gene therapy has potential application value.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R730.5;R392

【引证文献】