本文關鍵詞：羊膜間充質干細胞克隆的分離和生物學特性的研究　出處：《華東理工大學》2010年碩士論文　論文類型：學位論文

  更多相關文章： 羊膜間充質干細胞 克隆 擴增 動態(tài)培養(yǎng)

【摘要】： 羊膜因來源充足、含有豐富的間充質干細胞以及沒有倫理爭議等優(yōu)點,而成為再生治療新的細胞來源。人羊膜間充質干細胞(Human amnion derived mesenchymal stem cells, hAMCs)具多向分化潛能和低免疫原性等特點,日漸成為細胞移植治療的熱點。本文以人類羊膜為研究對象,通過酶解-貼壁方法分離到羊膜間充質干細胞,考察其自我更新能力和多向分化潛能；通過有限稀釋法獲得形態(tài)各異的3支細胞克隆,對比克隆間以及克隆和雜合細胞間形態(tài)、自我更新能力、多向分化潛能、表面標記和干細胞基因表達等生物學特性；最后,對比靜態(tài)和動態(tài)培養(yǎng)前后克隆與雜合細胞在數(shù)量及質量上的差異,探索規(guī)�；瘮U增克隆細胞的可行方法,為臨床和基礎研究提供基礎。 胰酶和膠原酶作用羊膜組織,通過貼壁培養(yǎng)可以獲得以細長梭形為主兼有其它形態(tài)的單核細胞,該細胞具有形成成纖維樣的細胞集落和多向分化的能力,在體外可向骨、脂肪和軟骨細胞分化,鑒定其為羊膜間充質干細胞,并命名為雜合細胞。通過有限稀釋法獲得的3支克隆細胞分別命名為8B、11D和11F,在體外傳代過程中保持其各自特有的細胞形態(tài)。第6代克隆和雜合細胞表現(xiàn)出不同的體外擴增特性,細長型克隆(8B和11F)具有更強的集落形成能力�？寺『碗s合細胞均表達CD29、CD44和CD105,不表達CD14、CD34和CD45,其中CD105在克隆細胞系的表達明顯高于雜合細胞�？寺『碗s合細胞在干細胞基因Oct-4和Nanog-3表達上有差異�？寺『碗s合細胞表現(xiàn)出不同的三向分化能力：8B和11F細胞體外成骨和成脂肪能力明顯優(yōu)于雜合細胞,而11D細胞略低于雜合細胞；除11D外,其它細胞系均能向軟骨分化。轉瓶動態(tài)培養(yǎng)能夠實現(xiàn)克隆細胞的擴增。與靜態(tài)培養(yǎng)系統(tǒng)中收獲的細胞相比,在轉瓶動態(tài)培養(yǎng)中收獲的克隆和雜合細胞,能維持較好的集落形成能力和成骨成脂肪分化潛能。 研究表明,可以從羊膜中分離獲得間充質干細胞,但該細胞群為多種細胞形態(tài)構成的雜合細胞。通過克隆分析發(fā)現(xiàn),Nanog-3和CD105高表達的克隆具有更強的三向分化潛能,并且克隆細胞可以通過轉瓶動態(tài)培養(yǎng)實現(xiàn)規(guī)�；瘮U增。
[Abstract]:Amniotic membrane due to adequate sources, rich in mesenchymal stem cells and the lack of ethical controversy has become a new cell source for regeneration. Human amniotic mesenchymal stem cells (Human amnion derived mesenchymal stem cells, hAMCs) with the characteristics of multilineage differentiation and low immunogenicity, has gradually become a hot spot in cell transplantation. The human amniotic membrane as the research object, through enzymatic hydrolysis and adherent separation method to amniotic mesenchymal stem cells, investigate its self-renewal and multilineage differentiation potential; 3 cell clones of different forms are obtained by the method of limited dilution cloning and comparison between cloned and heterozygous cell morphology, self-renewal capacity, differentiation potential, surface markers and gene expression in stem cells and other biological characteristics; finally, comparison of static and dynamic culture before and after cloning and heterozygous cells in the quality and quantity of the difference, explore The feasible method of scale amplification of cloned cells provides a basis for clinical and basic research.
Trypsin and collagenase effects of amniotic tissue by adherent culture can be mononuclears with other forms with a slender spindle, the formation of cells with fibroblast like cell colony and differentiation ability in vitro into bone, fat and cartilage cell differentiation, identification of the amniotic mesenchymal stem cells, and named heterozygous cells. 3 clones obtained by limited dilution method respectively named 8B, 11D and 11F, it maintains its own unique form in the process of body cell generation. The sixth generation of cloned and heterozygous cells showed different growth characteristics in vitro expansion, slender clones (8B and 11F) has a stronger ability of colony formation. Cloning and heterozygous cells were positive for CD29, CD44 and CD105, the expression of CD14, CD34 and CD45, the expression of CD105 in clonal cell lines was significantly higher than that in heterozygous cells. Cloning and heterozygous cells in stem cell gene Oct-4 and Na Nog-3. Cloning and differential expression of heterozygous cells showed three different differentiation capacity: 8B and 11F cells in vitro osteogenic and adipogenic ability is obviously better than that of heterozygous cells, while 11D cells were slightly lower than heterozygous cells; in addition to 11D, other cells can differentiation into cartilage. Dynamic culture bottle to achieve the amplification of cloned cells. Compared with the static culture system of harvested cells, in turn bottle dynamic culture harvested and heterozygous clone cells can maintain good colony forming ability and osteogenic and adipogenic differentiation potential.
Research shows that can isolate mesenchymal stem cells from amniotic membrane, but the cells were heterozygous cells composed of various cell morphology. By cloning analysis, cloning and high expression of Nanog-3 and CD105 three have stronger differentiation potential, and you can turn the bottle cell dynamic culture to achieve the scale of expansion.

【學位授予單位】：華東理工大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R329

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本文編號：1368905