本文選題：羊水干細胞 + 細胞培養(yǎng)�。� 參考：《第四軍醫(yī)大學(xué)》2011年碩士論文

【摘要】：再生醫(yī)學(xué)的飛速發(fā)展使干細胞在現(xiàn)代醫(yī)學(xué)的基礎(chǔ)研究與臨床應(yīng)用中發(fā)揮越來越重要的作用。因倫理道德問題,胚胎干細胞的研究和臨床應(yīng)用受到了極大的制約;成體干細胞自身分化潛能和增殖能力均有限,只能分化出部分細胞和組織。羊水干細胞的發(fā)現(xiàn)為干細胞的研究開辟了新的領(lǐng)域。羊水來源干細胞有望作為一種新的種子細胞,應(yīng)用于組織工程領(lǐng)域的研究與細胞治療。雖然羊水干細胞的培養(yǎng)方法不斷改進,但利用現(xiàn)有技術(shù)獲得的干細胞并不適合應(yīng)用于人體器官再生醫(yī)學(xué)的研究。通過免疫磁珠法分選獲得的干細胞雖然純度高,但細胞易受動物抗體的污染;連續(xù)傳代培養(yǎng)法雖可避免干細胞被其他成分污染,但體外培養(yǎng)周期長。我們在實驗中通過改良培養(yǎng)方法,建立了一種高速有效的獲得羊水干細胞的新方法。 目的: 1.通過改良培養(yǎng)方法,建立一種高速有效的人羊水來源多潛能干細胞的體外培養(yǎng)體系,并對其生物學(xué)特性進行探討。 2.對培養(yǎng)的羊水干細胞進行鑒定。 3.在適宜的培養(yǎng)條件下誘導(dǎo)羊水干細胞向成骨細胞、脂肪細胞、神經(jīng)細胞進行分化,檢測其體外分化能力。 方法: 1.取人孕中期羊水標(biāo)本進行原代培養(yǎng),細胞貼壁后挑選出長梭形、呈纖維樣細胞,將其稱之為起始細胞,利用起始細胞產(chǎn)生干細胞集落。 2.流式細胞儀和RT-PCR技術(shù)檢測胚胎干細胞和間充質(zhì)干細胞部分標(biāo)志基因Oct-4、SSEA-4、Nanog、CD29、CD44、CD105和CD117的表達情況,鑒定分離培養(yǎng)的羊水干細胞。 3.使用特定培養(yǎng)基,在適宜條件下誘導(dǎo)羊水干細胞向成骨細胞、脂肪細胞、神經(jīng)細胞進行分化。分別用茜素紅S染色、油紅O染色以及免疫細胞化學(xué)方法對分化出的細胞進行鑒定。 結(jié)果: 1.應(yīng)用改良后的培養(yǎng)方法,獲得的羊水干細胞集落均一性好。體外增殖能力強,倍增時間僅為24h,明顯快于常規(guī)使用的培養(yǎng)方法。 2.流式細胞儀檢測細胞表達Oct-4、SSEA-4、CD29、CD44、CD105、CD117等胚胎干細胞和間充質(zhì)干細胞標(biāo)志基因,不表達造血干細胞表面標(biāo)志基因CD45、CD133。RT-PCR結(jié)果顯示細胞中Oct-4基因mRNA和Nanog基因mRNA均明顯表達。 3.成骨誘導(dǎo)3周后茜素紅S染色見細胞內(nèi)有鈣結(jié)節(jié)的形成;成脂誘導(dǎo)3周后油紅O染色見細胞內(nèi)有大量脂滴形成;向神經(jīng)細胞誘導(dǎo)后免疫細胞化學(xué)方法檢測細胞巢蛋白(Nestin)的表達呈陽性。 結(jié)論: 1.將改良的培養(yǎng)方法與常規(guī)方法良好的結(jié)合,使細胞的培養(yǎng)周期明顯縮短,獲得的羊水干細胞集落均一性好,純度高。此方法培養(yǎng)得到的干細胞可以作為一種理想的再生醫(yī)學(xué)的種子細胞。 2. AFS表達胚胎干細胞和間充質(zhì)干細胞部分標(biāo)志基因,體外增值能力強,符合多潛能干細胞的特點。 3.在適宜的培養(yǎng)條件下,羊水干細胞可以向成骨細胞、脂肪細胞、神經(jīng)細胞分化。
[Abstract]:With the rapid development of regenerative medicine, stem cells play a more and more important role in the basic research and clinical application of modern medicine. The research and clinical application of embryonic stem cells have been greatly restricted due to ethical and moral problems. The ability of differentiation and proliferation of adult stem cells is limited, and only some cells and tissues can be differentiated. The discovery of amniotic fluid stem cells opens up a new field for stem cell research. Amniotic fluid derived stem cells are expected to be used as a new seed cell in tissue engineering research and cell therapy. Although the culture methods of amniotic fluid stem cells have been improved continuously, the stem cells obtained by using the existing techniques are not suitable for the research of human organ regeneration medicine. Although the purity of stem cells obtained by immunomagnetic bead method was high, but the cells were easily contaminated by animal antibodies, the continuous passage culture method could avoid the contamination of stem cells by other components, but the culture cycle in vitro was long. We established a high-speed and effective method to obtain amniotic fluid stem cells. Objective: 1. A high speed and effective culture system of human amniotic fluid derived pluripotent stem cells was established and its biological characteristics were discussed. 2. The cultured amniotic fluid stem cells were identified. 3. Amniotic fluid stem cells were induced to differentiate into osteoblasts, adipocytes and nerve cells under suitable culture conditions. Methods: 1. Human amniotic fluid specimens were collected for primary culture. Long fusiform fibroid cells were selected after adherent to the wall of human amniotic fluid. The cells were called initial cells and stem cell colonies were produced by the initial cells. 2. Flow cytometry (FCM) and RT-PCR technique were used to detect the expression of the partial marker gene Oct-4SEA-4 of embryonic stem cells (ESCs) and mesenchymal stem cells (MSCs), and to identify the isolated and cultured amniotic fluid stem cells (amniotic fluid stem cells). 3. Under suitable conditions, amniotic fluid stem cells were induced to differentiate into osteoblasts, adipocytes and nerve cells. The differentiated cells were identified by alizarin red S staining, oil red O staining and immunocytochemistry. Results: 1. Using the improved culture method, the colony uniformity of amniotic fluid stem cells was good. The multiplication time was only 24 h, which was faster than the conventional culture method. 2. Flow cytometry was used to detect the expression of embryonic stem cell and mesenchymal stem cell marker genes such as Oct-4SSEA-4 + CD49 + CD105- CD117. The results of RT-PCR showed that Oct-4 gene mRNA and Nanog gene mRNA were significantly expressed in the cells without expression of hematopoietic stem cell surface marker gene CD45 + CD133. 3. 3 weeks after osteogenesis induction, alizarin red S staining showed the formation of calcium nodules in the cells, oil red O staining showed a large number of lipid droplets in the cells after 3 weeks of adipogenic induction. The positive expression of nestin was detected by immunocytochemistry after induction of nerve cells. Conclusion: 1. By combining the improved culture method with the conventional method, the cell culture cycle was shortened obviously, and the colony homogeneity and purity of amniotic fluid stem cells were obtained. The stem cells obtained by this method can be used as an ideal seed cell for regenerative medicine. 2. AFS expressed some marker genes of embryonic stem cells and mesenchymal stem cells. 3. Under suitable culture conditions, amniotic fluid stem cells can differentiate into osteoblasts, adipocytes and nerve cells.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R329

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