發(fā)布時(shí)間：2018-03-02 07:41

  本文關(guān)鍵詞： 間質(zhì)干細(xì)胞 臍帶 分化 基因治療　出處：《江蘇大學(xué)》2007年碩士論文　論文類型：學(xué)位論文

【摘要】： 目的探討人臍帶間質(zhì)干細(xì)胞(human umbilical cord mesenchymal stem cells，hUCMSC)的分離培養(yǎng)及其生物學(xué)特性。開展hUCMSC慢病毒轉(zhuǎn)基因抗腫瘤的初步研究。 方法采用組織塊貼壁培養(yǎng)方法獲得hUCMSC，并對(duì)其進(jìn)行體外培養(yǎng)，應(yīng)用PKH26和二咪基苯基吲哚(DAPI)染色方法進(jìn)行形態(tài)學(xué)觀察；應(yīng)用流式細(xì)胞術(shù)測(cè)定細(xì)胞周期及表面抗原特征FITC-CD34，CD71，HLA-DR，PE-CD29，CD38，CD44，CD105和HLA-Ⅰ；染色體遺傳分析及繪制生長(zhǎng)曲線；利用地塞米松、維生素C、β-磷酸甘油、bFGF和地塞米松、3-異丁基-1-甲基黃嘌呤、胰島素、消炎痛誘導(dǎo)第3代hUCMSC分別向成骨細(xì)胞和脂肪細(xì)胞分化，并通過堿性磷酸酶(ALP)、Von Kossa和油紅O染色進(jìn)行檢測(cè)；通過PCR，，RT-PCR等方法檢測(cè)相關(guān)基因的表達(dá)。構(gòu)建融合基因TNFα-Tumstatin的慢病毒載體，采用磷酸鈣轉(zhuǎn)染方法轉(zhuǎn)染293T細(xì)胞和hUCMSC，通過PCR，RT-PCR，ELISA，~3H-TdR檢測(cè)融合基因及其蛋白的表達(dá)，觀察抑制腫瘤生長(zhǎng)的效果。 結(jié)果hUCMSC在培養(yǎng)了兩周后，大多呈纖維狀生長(zhǎng)，細(xì)胞傳至第2代時(shí)，則為均一的長(zhǎng)梭形細(xì)胞。在經(jīng)PKH26和DAPI染色后，可見細(xì)胞有一個(gè)藍(lán)色的平滑的核，胞漿呈紅色，并可維持20天左右。在傳代4個(gè)月后(約26代)，細(xì)胞仍然保持了其生物學(xué)特性。流式分析顯示：CD29，CD44，CD95，CD105，HLA-Ⅰ表達(dá)陽(yáng)性，CD34，CD38，CD71，HLA-DR不表達(dá)。染色體核型正常。第3代細(xì)胞周期分析顯示：G_0／G_1，G_2／M和S期分別為88.86％，5.69％和5.45％。經(jīng)過體外誘導(dǎo)，ALP、Von Kossa和油紅O染色均呈不同程度的陽(yáng)性，RT-PCR結(jié)果顯示，誘導(dǎo)后的細(xì)胞分別表達(dá)了骨形成蛋白-3(BMP-3)和過氧化物酶體增殖體激活受體γ2(PPAR_γ2)等基因。hUCMSC還表達(dá)了骨髓MSC所表達(dá)的BMI-1和nucleostemin基因。成功構(gòu)建融合基因TNFα-Tumstatin的慢病毒載體，包裝293T細(xì)胞后可見綠色熒光，轉(zhuǎn)染效率可達(dá)50％以上，在轉(zhuǎn)化293T和hUCMSC后，基因組中可檢測(cè)到融合基因，表明轉(zhuǎn)染成功。慢病毒上清中可以檢測(cè)到融合蛋白的表達(dá)并且對(duì)腫瘤細(xì)胞U937(人髓系白血病細(xì)胞株)具有抑制作用。 結(jié)論HUCMSC具有與骨髓MSC相似的生物學(xué)特性，尤其具有向成骨細(xì)胞和成脂肪細(xì)胞分化的多潛能性特點(diǎn)。hUCMSC的慢病毒轉(zhuǎn)基因抗腫瘤的研究已取得初步進(jìn)展，在轉(zhuǎn)基因治療腫瘤中具有重要的前景，將成為基因治療和組織工程中一種新的間質(zhì)干細(xì)胞來源。
[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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