脐带间质干细胞的功能及转基因抗肿瘤的研究

发布时间：2018-03-02 07:41

  本文关键词： 间质干细胞 脐带 分化 基因治疗　出处：《江苏大学》2007年硕士论文　论文类型：学位论文

【摘要】： 目的探讨人脐带间质干细胞(human umbilical cord mesenchymal stem cells，hUCMSC)的分离培养及其生物学特性。开展hUCMSC慢病毒转基因抗肿瘤的初步研究。 方法采用组织块贴壁培养方法获得hUCMSC，并对其进行体外培养，应用PKH26和二咪基苯基吲哚(DAPI)染色方法进行形态学观察；应用流式细胞术测定细胞周期及表面抗原特征FITC-CD34，CD71，HLA-DR，PE-CD29，CD38，CD44，CD105和HLA-Ⅰ；染色体遗传分析及绘制生长曲线；利用地塞米松、维生素C、β-磷酸甘油、bFGF和地塞米松、3-异丁基-1-甲基黄嘌呤、胰岛素、消炎痛诱导第3代hUCMSC分别向成骨细胞和脂肪细胞分化，并通过碱性磷酸酶(ALP)、Von Kossa和油红O染色进行检测；通过PCR，，RT-PCR等方法检测相关基因的表达。构建融合基因TNFα-Tumstatin的慢病毒载体，采用磷酸钙转染方法转染293T细胞和hUCMSC，通过PCR，RT-PCR，ELISA，~3H-TdR检测融合基因及其蛋白的表达，观察抑制肿瘤生长的效果。 结果hUCMSC在培养了两周后，大多呈纤维状生长，细胞传至第2代时，则为均一的长梭形细胞。在经PKH26和DAPI染色后，可见细胞有一个蓝色的平滑的核，胞浆呈红色，并可维持20天左右。在传代4个月后(约26代)，细胞仍然保持了其生物学特性。流式分析显示：CD29，CD44，CD95，CD105，HLA-Ⅰ表达阳性，CD34，CD38，CD71，HLA-DR不表达。染色体核型正常。第3代细胞周期分析显示：G_0／G_1，G_2／M和S期分别为88.86％，5.69％和5.45％。经过体外诱导，ALP、Von Kossa和油红O染色均呈不同程度的阳性，RT-PCR结果显示，诱导后的细胞分别表达了骨形成蛋白-3(BMP-3)和过氧化物酶体增殖体激活受体γ2(PPAR_γ2)等基因。hUCMSC还表达了骨髓MSC所表达的BMI-1和nucleostemin基因。成功构建融合基因TNFα-Tumstatin的慢病毒载体，包装293T细胞后可见绿色荧光，转染效率可达50％以上，在转化293T和hUCMSC后，基因组中可检测到融合基因，表明转染成功。慢病毒上清中可以检测到融合蛋白的表达并且对肿瘤细胞U937(人髓系白血病细胞株)具有抑制作用。 结论HUCMSC具有与骨髓MSC相似的生物学特性，尤其具有向成骨细胞和成脂肪细胞分化的多潜能性特点。hUCMSC的慢病毒转基因抗肿瘤的研究已取得初步进展，在转基因治疗肿瘤中具有重要的前景，将成为基因治疗和组织工程中一种新的间质干细胞来源。
[Abstract]:Objective to investigate the isolation, culture and biological characteristics of human umbilical cord mesenchymal stem cells (hUCMSC).
Methods hUCMSC by tissue adherent culture method, and the application of PKH26 in vitro, and two with phenyl indole (DAPI) staining method was used to observe the morphology; Determination of cell cycle and surface antigen FITC-CD34 by flow cytometry. CD71, HLA-DR, PE-CD29, CD38, CD44, CD105 and HLA- 1; genetic analysis and growth curve; the use of dexamethasone, vitamin C, beta glycerophosphate and dexamethasone, bFGF 3- -1-, isobutyl methylxanthine, insulin, indomethacin induced third generation of hUCMSC to osteoblast and adipocyte differentiation, and the alkaline phosphatase (ALP), Von Kossa and oil red O staining; through the PCR, to detect the expression of related gene RT-PCR and other methods. To construct a lentiviral vector of TNF fusion gene transfection method using alpha -Tumstatin, calcium phosphate transfection of 293T cells and hUCMSC, RT-PCR, ELISA, by PCR, ~ 3H-TdR was used to detect the expression of the fusion gene and its protein, and to observe the effect of inhibiting the growth of the tumor.
The results of hUCMSC in culture after two weeks, mostly showed fibrous growth when cells spread to the second generation, but long spindle cell uniform. After PKH26 and DAPI staining, the cells have a blue smooth nucleus, cytoplasm is red, and can be maintained for about 20 days. At the 4 passage a month later (26 generation), the cells still retained their biological characteristics. Flow cytometry analysis showed that CD29, CD44, CD95, CD105, HLA- of CD34, CD38 positive expression, CD71, HLA-DR respectively. Normal karyotype. The third generation of cell cycle analysis showed that G_0 / G_1, G_2 / M and S were 88.86%, 5.69% and 5.45%. after induction in vitro, ALP, Von Kossa and oil red O staining were positive, RT-PCR results showed that the induced cells were expressed in bone morphogenetic protein -3 (BMP-3) and peroxisome proliferator activated receptor gamma 2 (PPAR_ gamma 2 gene.HUCMSC) etc. also the expression of MSC in bone marrow 鎵

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