【摘要】： 骨髓間充質(zhì)干細(xì)胞(MSCs)具有多向分化潛能,可在體外大量擴(kuò)增,并且具有調(diào)節(jié)細(xì)胞免疫反應(yīng)的功能,其來源不存在倫理爭議問題。因此是細(xì)胞治療和組織工程中理想的種子細(xì)胞來源。胚胎干細(xì)胞(ESCs)可作為藥物篩選的理想資源。而現(xiàn)今面臨的主要問題是如何獲得大量優(yōu)質(zhì)的種子干細(xì)胞來滿足其在醫(yī)學(xué)上的應(yīng)用,現(xiàn)今的技術(shù)手段仍無法解決干細(xì)胞的大規(guī)模擴(kuò)增問題,因此需要研究和發(fā)展干細(xì)胞的三維培養(yǎng)平臺技術(shù)。針刺無紡聚對苯二甲酸乙二酯(Polyethylene Terephthalate,PET)纖維材料可作為三維的支架材料來支持MSCs和ESCs的增殖,它具備高孔隙率、比表面積大、滲透性好、機(jī)械強(qiáng)度高的優(yōu)點(diǎn),其獨(dú)特的三維結(jié)構(gòu)可提供給細(xì)胞一個(gè)仿生的環(huán)境。 研究目的:本實(shí)驗(yàn)采用PET纖維材料對hMSCs和mESCs進(jìn)行三維擴(kuò)增培養(yǎng),探討并優(yōu)化hMSCs的培養(yǎng)條件及評定三維擴(kuò)增的效果。為將來研發(fā)大規(guī)模生產(chǎn)干細(xì)胞的系統(tǒng),進(jìn)而建立一個(gè)相對成熟高效的培養(yǎng)系統(tǒng)平臺做基礎(chǔ) 實(shí)驗(yàn)方法:為了優(yōu)化hMSCs在PET上的條件培養(yǎng),CCK-8法檢測了bFGF對hMSCs的增殖影響,分析了在PET纖維材料上的接種密度對細(xì)胞增殖的影響,檢測了乳酸對細(xì)胞增殖的抑制作用;進(jìn)一步探討了hMSCs在PET上的增殖和代謝的特點(diǎn);利用Cell tracker Green熒光染色,激光共聚焦顯微鏡檢測細(xì)胞在材料上的活性;掃描電鏡檢測細(xì)胞在PET上的分布、形態(tài)及粘附情況;檢測了低濃度的bFGF對PET上細(xì)胞的影響;流式細(xì)胞儀鑒定三維擴(kuò)增后的hMSCs的表面標(biāo)記物CD29、CD44、CD34和CD45。對hMSCs進(jìn)行向成骨、成脂方向的誘導(dǎo)培養(yǎng);最后通過倒置顯微鏡觀察、接種量的優(yōu)化和激光共聚焦顯微鏡、掃描電鏡的觀察檢測PET對mESs的擴(kuò)增培養(yǎng)情況。 結(jié)果:實(shí)驗(yàn)研究表明,培養(yǎng)基中5 ng/mL的bFGF,每孔接種1×104個(gè)hMSCs及保持培養(yǎng)基中的乳酸濃度少于0.9 g/L有利于促進(jìn)PET上hMSCs的增殖。PET能夠支持更長期的細(xì)胞的培養(yǎng),而且PET上培養(yǎng)的hMSCs的對營養(yǎng)物的利用率更高。觀察顯示hMSCs及mESCs在PET纖維材料上成立體,多層生長,有較大的生長空間。三維擴(kuò)增后的細(xì)胞保持了原有未分化狀態(tài)和分化潛能;bFGF對三維材料上hMSCs的增殖有更明顯的促進(jìn)作用。1~2×104個(gè)細(xì)胞/孔為mESCs在PET纖維材料上接種的最適密度。
[Abstract]:Bone marrow mesenchymal stem cells (BMSCs) have the potential of multidirectional differentiation, can be expanded in vitro, and have the function of regulating cellular immune response. Therefore, it is an ideal seed cell source for cell therapy and tissue engineering. Embryonic stem cell (ESCs) can be used as an ideal resource for drug screening. But the main problem now is how to obtain a large number of high-quality seed stem cells to meet its medical application, but the current technology can not solve the problem of stem cell expansion on a large scale. Therefore, it is necessary to research and develop the three-dimensional culture platform technology of stem cells. The needle-free poly (ethylene terephthalate) (Polyethylene Terephthalate,PET) fiber can be used as a three-dimensional scaffold material to support the proliferation of MSCs and ESCs. It has the advantages of high porosity, large specific surface area, good permeability and high mechanical strength. Its unique three-dimensional structure provides a bionic environment for cells. Objective: to study and optimize the culture conditions of hMSCs and mESCs and evaluate the effect of three dimensional amplification by using PET fiber material. In order to develop a large-scale stem cell production system in the future, and then establish a relatively mature and efficient culture system platform for basic experimental methods: in order to optimize the condition culture of hMSCs on PET, The effect of bFGF on the proliferation of hMSCs, the effect of inoculation density on the proliferation of hMSCs and the inhibition of lactic acid on cell proliferation were analyzed by CCK-8 method. The characteristics of proliferation and metabolism of hMSCs on PET were further discussed. The activity of cells on the material was detected by Cell tracker Green fluorescence staining and confocal microscopy. The distribution, morphology and adhesion of cells on PET were detected by scanning electron microscope (SEM). The effect of low concentration of bFGF on the cells on PET was detected. The surface markers CD29,CD44,CD34 and CD45. of hMSCs were identified by flow cytometry HMSCs was cultured in the direction of osteogenesis and fat-forming. Finally, the amplification and culture of mESs by PET were detected by inverted microscope, optimization of inoculation amount and laser confocal microscope, scanning electron microscope (SEM). Results: the experimental study showed that 5 ng/mL bFGF, inoculated with 1 脳 104 hMSCs per well and kept lactic acid concentration less than 0.9 g / L in the culture medium could promote the proliferation of hMSCs on PET. PET could support the long-term cell culture. Moreover, the nutrient utilization of hMSCs cultured on PET was higher. The results showed that hMSCs and mESCs were three dimensional and multilayer growth on PET fiber material. The undifferentiated state and differentiation potential of the expanded cells were maintained, the proliferation of hMSCs on the three dimensional material was promoted more obviously by bFGF, and 1 2 脳 10 4 cells / well was the best density of mESCs inoculation on the PET fiber material. 2 脳 10 4 cells / well was the most suitable density of mESCs inoculation on PET fiber material.
【學(xué)位授予單位】：華南理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R329

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