本文選題：高頻脈沖電磁場　切入點(diǎn)：骨髓間充質(zhì)干細(xì)胞　出處：《廣西醫(yī)科大學(xué)》2012年碩士論文

【摘要】：骨髓間充質(zhì)干細(xì)胞(MSCs)因具有多向分化潛能、免疫耐受、取材方便、自我更新等特點(diǎn)而成為組織工程重要的種子細(xì)胞及近年來研究的一個熱點(diǎn)。本課題的前期研究表明高頻脈沖電磁場促進(jìn)髓間充質(zhì)干細(xì)胞的凋亡,但其機(jī)理尚不清楚,蛋白質(zhì)組學(xué)是指研究和鑒定一個細(xì)胞、器官、組織在一特定時間所表達(dá)的總蛋白質(zhì)的過程,蛋白質(zhì)組學(xué)研究的興起,為我們從蛋白質(zhì)水平研究高頻脈沖電磁場促進(jìn)髓間充質(zhì)干細(xì)胞的凋亡的機(jī)理奠定了基礎(chǔ)。 目的：1.利用雙向電泳技術(shù)(2-DE)創(chuàng)建體外正常培養(yǎng)的和高頻脈沖電磁場干預(yù)培養(yǎng)的骨髓間充質(zhì)干細(xì)胞蛋白表達(dá)圖譜。2.利用圖像分析軟件對蛋白表達(dá)圖譜進(jìn)行對比分析查找差異蛋白點(diǎn),經(jīng)質(zhì)譜鑒定后找出高頻脈沖電磁場促進(jìn)骨髓間充質(zhì)干細(xì)胞的凋亡有關(guān)的功能蛋白,探討功能蛋白的作用機(jī)制。 方法：1.用全骨髓貼壁法進(jìn)行體外分離,培養(yǎng),傳代,純化骨髓間充質(zhì)干細(xì)胞,取P3代作為研究的靶細(xì)胞。2.以1×106/cm2密度接種至底面積100cm2培養(yǎng)瓶,分為A、B兩組進(jìn)行培養(yǎng),A組為高頻脈沖電磁場干預(yù)組,B組為對照組。3.分別提取A、B兩組細(xì)胞總蛋白,純化蛋白樣本,Bradford法進(jìn)行蛋白定量,進(jìn)行雙向電泳,獲取A、B兩組雙向電泳圖譜并進(jìn)行對比分析,查找差異蛋白點(diǎn)。切取蛋白差異點(diǎn)、酶解,進(jìn)行質(zhì)譜鑒定,根據(jù)質(zhì)譜鑒定結(jié)果進(jìn)行數(shù)據(jù)庫檢索查找功能蛋白。 結(jié)果：1.獲得分辨率和重復(fù)性較好的正常培養(yǎng)的和高頻脈沖電磁場干預(yù)培養(yǎng)的骨髓間充質(zhì)干細(xì)胞蛋白表達(dá)圖譜。2.用圖像分析軟件對蛋白表達(dá)圖譜進(jìn)行對比分析出差異蛋白點(diǎn)19個,其中17個蛋白點(diǎn)在A組表達(dá)量上升,2個表達(dá)量下降,經(jīng)質(zhì)譜成功鑒定11個,其中EF-hand domain-containing protein D2(又稱Swiprosin-1),有促進(jìn)細(xì)胞凋亡作用。 結(jié)論：利用雙向電泳技術(shù)(2-DE)創(chuàng)建了正常培養(yǎng)的和高頻脈沖電磁場干預(yù)培養(yǎng)的骨髓間充質(zhì)干細(xì)胞比較理想的蛋白表達(dá)圖譜；EF-hand domain-containing protein D2可能是高頻脈沖電磁場促進(jìn)BMSCs凋亡的一個功能蛋白。
[Abstract]:Bone marrow mesenchymal stem cell (MSCs) has become an important seed cell in tissue engineering and has been a hot research topic in recent years because of its multidirectional differentiation potential, immune tolerance, convenient material collection, self-renewal and so on.Previous studies in this study have shown that high frequency pulsed electromagnetic fields promote apoptosis of marrow mesenchymal stem cells, but its mechanism is not clear. Proteomics refers to the study and identification of a cell or organ.The process of tissue expression of total protein at a specific time and the rise of proteomics provide a basis for the study of the mechanism of high frequency pulsed electromagnetic field (HFPEM) promoting the apoptosis of mesenchymal stem cells (MMSCs) from the protein level.Purpose 1.Two-dimensional electrophoretic technique (2-DE) was used to create protein expression map of bone marrow mesenchymal stem cells (BMSCs) in vitro and in high frequency pulsed electromagnetic field (HFPEM) intervention.The protein expression patterns were analyzed by image analysis software to find the differential protein spots. The functional proteins related to the apoptosis of bone marrow mesenchymal stem cells (BMSCs) were identified by high frequency pulsed electromagnetic field (HPEM) and the mechanism of functional proteins was discussed.Method 1: 1.Bone marrow mesenchymal stem cells (BMSCs) were isolated, cultured, subcultured and purified by whole-bone marrow adherent method. P3 generation was used as target cells.1 脳 106/cm2 density inoculation to the bottom area of 100cm2 culture bottle was divided into two groups: group A: high frequency pulsed electromagnetic field intervention group, group B: control group. 3.The total proteins were extracted from two groups of Agna B cells, and the protein samples were purified by Bradford method for protein quantification. The two dimensional electrophoresis patterns of two groups of Agna B were obtained and compared and analyzed to find the differential protein spots.The difference point of protein was digested, hydrolyzed by enzyme, identified by mass spectrometry, and the functional protein was searched in database according to the result of mass spectrometry.The result is 1: 1.The protein expression patterns of bone marrow mesenchymal stem cells in normal culture and in high frequency pulsed electromagnetic field were obtained with good resolution and reproducibility.19 differentially expressed protein spots were analyzed by image analysis software. Among them, 17 protein spots increased in group A and 2 decreased. 11 spots were successfully identified by mass spectrometry.Among them, EF-hand domain-containing protein D 2 (also known as Swiprosin-1) can promote apoptosis.Conclusion: the ideal protein expression profile of bone marrow mesenchymal stem cells (BMSCs) in normal culture and high frequency pulsed electromagnetic field intervention has been created by using two-dimensional electrophoresis technique. EF-hand domain-containing protein D2 may be the high frequency pulsed electromagnetic field to promote BMSCs regulation.A functional protein that kills.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R329

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