【作者】 高歌；

【导师】 牛朝诗； 傅先明；

【作者基本信息】 安徽医科大学 ， 外科学， 2009， 硕士

【摘要】 背景胶质瘤是最常见的脑恶性肿瘤,具有恶性增殖、浸润性生长等生物学特性,临床治疗效果差。抑制肿瘤细胞增殖、抗浸润性和抗血管生成是治愈胶质瘤的焦点。PEX是新近发现的一种人基质金属蛋白酶-2(MMP-2)自身催化降解的片段,它对胶质瘤的增殖、血管形成以及侵袭性都有强大抑制作用,能够干预恶性胶质瘤的侵袭行为。MSCs是近年来干细胞研究领域中引人注目的一种成体干细胞,具有多向分化潜能,具有较弱的免疫原性和免疫调节功能、强大的迁移力和趋瘤性等优点,作为一种靶向肿瘤治疗的新型载体,其在胶质瘤基因治疗中具有很好的应用前景。本实验在成功分离、培养、鉴定大鼠MSCs和构建pEGFP-C1-PEX真核表达载体的基础上,将重组质粒转染MSCs,并检测其瞬时转染效率,为进一步的体内外实验研究PEX修饰的MSCs对胶质瘤的治疗作用奠定基础。目的(1)探讨成体MSCs的体外培养方法并研究培养细胞的生物学行为特征,建立体外分离培养MSCs程序方法,为进一步实验和临床应用提供基础;(2)构建PEX基因真核表达载体pEGFP-C1-PEX,并用其转染MSCs,检测其瞬时转染效率,探讨外源基因转染MSCs的可行性:(3)确定获取稳定转染细胞所需的G418筛选浓度,为进一步实验需要做准备。方法(1)采用全贴壁细胞分离法分离大鼠骨髓间质干细胞,体外培养扩增,倒置相差显微镜下观察细胞形态特点,传代培养后流式细胞术鉴定细胞表面分子标记物的表达;(2)采用Trizol试剂提取C6胶质瘤细胞株(高表达PEX)总RNA,RT-PCR扩增目的基因,扩增产物经核酸纯化、酶切、连接等步骤克隆到pEGFP-C1载体中,经DH5α感受态细胞转化,小提质粒,酶切鉴定,测序验证;(3)采用高转染效率的Lipofectmine2000将质粒pEGFP-C1-PEX转染于增殖中的MSCs细胞。通过荧光显微镜观察绿色荧光蛋白(EGFP)的表达,并用半定量RT-PCR法检测其瞬时转染效率;(4)通过预实验确定获得稳定转染细胞所需的G418筛选浓度。结果(1)大鼠MSCs的生物学特征及鉴定:细胞主要呈“成纤维样”。CD90、CD105呈阳性表达,CD45阴性,提示MSCs分离培养成功;(2)pEGFP-C1-PEX真核表达载体构建成功;(3)重组质粒转染大鼠MSCs24后小时可见EGFP表达,提示载体基因转染入靶细胞内;(4)转染前/后PEX基因在大鼠MSCs中表达情况的检测:瞬时转染pEGFP-C1-PEX质粒的MSCs的PEX mRNA表达增高;(5)经预实验确定G418筛选浓度:300ng/ml为第9天细胞全部死亡的最低浓度,以400ng/ml定为进一步实验获取稳定转染细胞的筛选浓度。结论(1)建立了稳定的大鼠MSCs体外培养体系,按照此培养体系进行培养可以成功获得大鼠MSCs;(2)成功构建了携带有PEX基因的pEGFP-C1-PEX真核表达载体,可以用于转染大鼠MSCs;(3)用所构建的pEGFP-C1-PEX真核表达载体转染大鼠骨髓间充质干细胞,转染效率约为20%;(4)本实验将pEGFP-C1-PEX真核表达载体成功转染大鼠MSCs,而且确定获得稳定转然细胞的G418筛选浓度,为后续的实验奠定了坚实的基础。更多还原

【Abstract】 Background Gliomas are the most common malignant tumor of the brain with malignant proliferation, invasive growth and other biological characteristics,the effects of clinical treatment are poor. The keys to cure it was inhibition of cancer cell proliferation,invasion and angiogenesis. PEX is a newly discovered fragment degradated from matrix metalloproteinase-2 (MMP-2), which has a strong inhibitory effect on glioma proliferation, angiogenesis and invasiveness,and is able to interfere with malignant gliomas invasive behavior. MSCs is one kind of adult stem cells with multilineage differentiation potential,excellent migratory ability, tropism, low immunogenicity and immunological regulation and showed great potential in gene therapy of glioma.In this experiment , based on the success of isolation, cultivation and identification of rat MSCs, the recombinant plasmid of pEGFP-Cl-PEX eukaryotic expression vector was to transfected to MSCs and the transient transfection efficiency was detected. Our data lay the foundation to further studies in vitro and in vivo of MSCs modified PEX on glioma for the therapeutic effect.Objective (1) To built the adult MSCs culture system and to study the biological characteristics of MSCs in vitro; (2)To construct PEX eukaryotic expression vector pEGFP-C1-PEX and to transfect MSCs,then teste the efficiency of transient transfection and discuss the feasibility of examine exogenous gene transfect the MSCs; (3)To determine the concentration of G418 on selection stability of transfected cells. Method (1) Isolation of rat MSCs using the whole adherent cells method and culturing cells in vitro.Inverted phase contrast microscope was used to observe cell morphology characteristic and flow cytometry to detect the molecular markers of cell surface; (2 ) Extraction total RNA of C6 glioma cell line (high expression of PEX) using Trizol reagent, gene amplification with reverse transcription PCR kit, nucleic acid amplification product was purified, digested, and cloned into the vector pEGFP-C1 by the transformation of DH5αcompetent cells, small reference plasmid, restriction enzyme digestion, sequencing verification; (3) Use the high transfection efficiency of plasmid Lipofectmine2000 to transfect the pEGFP-C1-PEX on cell proliferation of MSCs. Through fluorescence microscopy of green fluorescent protein (EGFP) expression, and by semi-quantitative RT-PCR method to detect the transient transfection efficiency; (4) Through the pre-test to determine the likelihood stable cell concentration required for G418 selection.Results (1) The biological characteristics of rat MSCs and identification : cells were "fibroblast-like". CD90, CD105 positive expression and CD45 negetive expression, indicating successful isolation and culture of MSCs; (2) The pEGFP-C1-PEX eukaryotic expression vector was successfully constructed; (3) Observation of fluorescence after 24 hours of recombinant plasmid transfection of rat MSCs: EGFP expression can be seen, suggesting that recombinant plasmid was transfered into the target cells; (4) The detection of gene expression before / after the transfection of PEX gene in rat MSCs: Our data showed that PEX mRNA expression increased in MSCs which was transfected by pEGFP-C1-PEX plasmid ; (5) By the pre-test to determine the concentration of G418 selection: 300ng/ml is the minimum concentration of all cells death within nine days ,so with 400ng/mlas a stable selection of transfected cells concentration for further experiments .Conclusion (1)A stable cultur system of rat MSCs were established in vitro, in accordance with this training system can be successfully cultured rat MSCs ; (2) we have successfully constructed pEGFP-C1-PEX eukaryotic expression vector carrying PEX gene, which can be transfected into rat MSCs efficently; (3) use the constructed pEGFP-C1-PEX eukaryotic expression vector transfected into rat MSCs, and the transfection efficiency is about 20%; (4)This experiment successfully transfected the pEGFP-C1-PEX eukaryotic expression vector into rat MSCs and also determined the stability of the G418 selection concentration,which laid a solid foundation for the further experiments.更多还原