本文關鍵詞：人源羊水干細胞分離培養(yǎng)、生物學特性檢測及誘導分化研究　出處：《西北農林科技大學》2008年博士論文　論文類型：學位論文

  更多相關文章： 人 羊水干細胞 類胚體 誘導分化 心肌細胞 神經細胞

【摘要】： 由于胚胎干細胞的研究受到倫理道德問題的束縛與限制,很多研究者開始嘗試尋找新的干細胞來源。2003年,Prusa等在羊水中發(fā)現Oct-4陽性細胞,提示羊水中可能存在多能干細胞。2007年,Paolo等報道,他們在人的孕中期羊水中發(fā)現少量具有ES細胞特性的干細胞,將其命名為人羊水源干細胞(human Amniotic Fluid Stem Cell,hAFS cell)。這種細胞表達ES細胞和成體干細胞標志基因,體外誘導可分化為包括三個胚層的細胞,且通過了功能測試。hAFS細胞的特點是容易獲取,不會損害母體及胚胎,可為細胞和組織工程治療提供新的種子細胞來源。本實驗自人孕中期、孕晚期羊水中分離培養(yǎng)hAFS細胞,檢測了羊水標本性狀對細胞原代培養(yǎng)的影響,篩選并優(yōu)化了細胞培養(yǎng)體系。同時,通過RT-PCR、免疫細胞化學和流式細胞儀技術分選等技術,對hAFS細胞的生物學特性進行了檢測,并誘導hAFS細胞向心肌細胞和神經細胞分化。 (1)羊水標本性狀對hAFS細胞原代培養(yǎng)的影響 從醫(yī)院婦產科收集孕中期及孕晚期羊水標本,離心收集細胞,加入羊水干細胞培養(yǎng)液(ACM)進行培養(yǎng),記錄原代細胞貼壁時間及貼壁細胞數量,探討人羊水標本性狀等因素對原代分離培養(yǎng)的影響。實驗結果表明,孕中期(4.7±0.6 d)與孕晚期(6±0.5 d)羊水標本中細胞的貼壁時間有明顯差異(P0.05),孕晚期貼壁時間較長。羊水中血細胞污染程度對貼壁時間有明顯影響,重度污染組(10.8±0.3 d)與無污染組(6±0.5 d)、輕度污染組(6.3±0.6 d)貼壁時間差異顯著(P0.05)。羊水體積對細胞貼壁時間沒有明顯影響,但對貼壁細胞數量有顯著影響(P0.05)。整個實驗系統(tǒng)地檢測了羊水干細胞原代培養(yǎng)的影響因素,為羊水干細胞研究提供了可以借鑒的資料。 (2)hAFS細胞分離培養(yǎng)及生物學特性檢測 培養(yǎng)人孕中期及孕晚期羊水標本,通過機械方法分離純化hAFS細胞,采用RT-PCR、免疫細胞化學和流式細胞儀分選等技術對其生物學特性和分化潛能進行了檢測。結果表明,在原代細胞培養(yǎng)的基礎上,通過機械分離方法,可得到成纖維樣hAFS細胞。這種細胞表達胚胎干細胞的特異性基因標志Oct-4、hTERT、Nanog、SSEA-1、SSEA-4和CD117,不表達SSEA-3;表達間充質干細胞的特異性基因標志CD29、CD44、CD73、CD90和CD105;不表達造血干細胞和造血細胞的特異性基因標志CD34、CD133和CD45;另外,這種細胞還表達HLA-ABC,弱表達HLA-DR。將hAFS細胞在體外多次傳代后(已傳至45代),仍具有較強的增殖能力,細胞核型正常。hAFS細胞在懸浮培養(yǎng)條件下可聚集形成類胚體,堿性磷酸酶(AP)檢測呈陽性,表達三胚層特異性基因標志,如,外胚層:fgf5;中胚層:ζ-globin;內胚層:α-fetoprotein,證明其具有向三個胚層分化的潛能。同時,本實驗篩選含不同濃度FBS及KSR的ACM培養(yǎng)液,表明hAFS細胞可在含KSR的無血清培養(yǎng)體系中擴增。 (3)hAFS細胞向心肌細胞誘導分化 采用人孕晚期羊水中分離得到hAFS細胞,通過形成類胚體(EB)誘導和單層誘導兩種方法,結合不同的誘導液,誘導hAFS細胞向心肌細胞分化,比較誘導效果,篩選最適的誘導體系。結果表明,在不同誘導條件下,均得到α-actin染色陽性細胞,表達心肌細胞特異標志基因Tbx5、Nkx2.5、GATA4和α-MHC。比較不同誘導液對細胞誘導效果的影響發(fā)現,條件誘導液組的誘導效果顯著優(yōu)于RA和DMSO組(P0.05)。在相同誘導液誘導條件下,通過形成類胚體誘導hAFS細胞向心肌細胞分化的效果顯著優(yōu)于單層誘導組(DMSO和條件培養(yǎng)液誘導,P0.05)。以上結果表明,誘導hAFS細胞向心肌細胞分化時,通過形成類胚體并采用條件培養(yǎng)液的誘導效果最好。 (4)hAFS細胞向神經細胞誘導分化 由人孕晚期羊水中分離得到hAFS細胞,采用形成類胚體(EB)誘導和單層誘導兩種方法,結合不同的誘導液,誘導hAFS細胞向神經細胞分化,通過比較誘導效果,篩選最適的誘導體系。結果表明,在不同誘導條件下,均得到Nestin、NSE陽性細胞,分化的細胞體積變小,胞質收縮,細胞逐漸呈錐形或三角形,表達神經細胞特異性基因標志fgf-5和CD56。在采用RA進行誘導時,單層誘導組誘導結果與類胚體誘導組類似,但相應階段Nestin、NSE的相對表達量及陽性細胞百分比高于類胚體誘導組,且差異顯著(P0.05)。比較不同濃度β-Me的單層誘導效果,表明5mmol/Lβ-Me組誘導效果較好,且誘導時間應控制在3~4h之內,但在β-Me誘導條件下,未檢測到NSE陽性細胞。以上結果表明,誘導hAFS細胞向神經細胞分化時,采用RA誘導液并進行單層誘導的效果較好。
[Abstract]:Due to the limitation and restriction of embryonic stem cell research by the ethical problem, many researchers try to find out a new source of stem cells in.2003, Prusa found that Oct-4 positive cells in amniotic fluid, amniotic fluid that may exist in pluripotent stem cells.2007, Paolo reported that they found a small amount of ES cells with stem cell characteristics in the second trimester of human amniotic fluid, named human stem cells (human Amniotic sheep water Fluid Stem Cell, hAFS cell). The expression of ES cells and adult stem cell markers in vitro can differentiate into three germ layers including cells, and through the characteristic function test of.HAFS cells is easy to obtain, maternal and embryo will not damage, can provide a new source of seed cells for cell therapy and tissue engineering. This experiment from the second trimester, hAFS cells were isolated in late pregnancy amniotic fluid, amniotic fluid detection standard Influence of nature on primary cell culture, screening and optimization of the cell culture system. At the same time, by RT-PCR, immunocytochemistry and flow cytometer, the biological characteristics of hAFS cells were detected, and induce hAFS cells to differentiate to cardiomyocytes and neural cells.
(1) the effect of amniotic fluid specimen on primary culture of hAFS cells
From the hospital of Obstetrics and gynecology from second trimester and third trimester amniotic fluid samples were collected by centrifugation, cells with amniotic fluid stem cell culture medium (ACM) were cultured in primary record cell attachment time and the number of cells, to explore the factors of human amniotic fluid specimens traits influence on primary culture. The experimental results show that the second trimester (4.7 + 0.6 d) and late pregnancy (6 + 0.5 d) cells in amniotic fluid samples were adherent time significantly (P0.05), late pregnancy adherent time longer. The pollution degree of blood cells in amniotic fluid has significant effect on the adherence time, severe pollution group (10.8 + 0.3 D) and group (no pollution 6 + 0.5 d), light pollution group (6.3 + 0.6 d) adherent time difference (P0.05). Amniotic fluid volume had no obvious effect on cell attachment time, but has a significant impact on the number of adherent cells (P0.05). The whole experimental system to examine factors affecting cell cultured amniotic fluid stem, amniotic fluid Stem cell research provides information that can be used for reference.
(2) isolation and culture of hAFS cells and detection of biological characteristics
Cultured human second trimester and third trimester amniotic fluid samples by mechanical method for separation and purification of hAFS cells by RT-PCR, immunocytochemistry and flow cytometry sorting technology on the biological characteristics and differentiation potential were detected. The results showed that in primary cell culture, can be obtained by mechanical separation method, fiber like hAFS cells. The expression of embryonic stem cell specific gene cell markers Oct-4, hTERT, Nanog, SSEA-1, SSEA-4 and CD117, the expression of SSEA-3; expression of mesenchymal stem cell specific gene markers CD29, CD44, CD73, CD90 and CD105; the expression of specific genes in hematopoietic stem cells and hematopoietic cells mark CD34, CD133 and CD45; in addition, the cells also expressed HLA-ABC, weak expression of HLA-DR. hAFS cells in vitro after several passages (has been passaged for 45 generations), still have strong proliferation ability and cell normal karyotype of.HAFS cells in suspension Cultured together to form embryoid bodies can be under the condition of alkaline phosphatase (AP) positive expression, three embryo specific gene markers, such as: FGF5; ectoderm mesoderm endoderm: alpha zeta: -globin; -fetoprotein, three to prove its differentiation ability. At the same time, the screening experiments with different concentrations of FBS KSR and ACM medium, suggesting that hAFS cells can be amplified in non serum culture system containing KSR.
(3) induction of differentiation from hAFS cells to cardiomyocytes
The hAFS cells isolated from human amniotic fluid in late pregnancy, through the formation of embryoid bodies (EB) and induced monolayer induced two kinds of methods, combined with different induced liquid, induce hAFS cells to differentiate into cardiomyocytes, induce effect, screening the best induction system. The results show that under different induction conditions were obtained alpha -actin staining positive cells, the expression of myocardial cell specific marker genes Tbx5, Nkx2.5, GATA4 and -MHC. to compare the different effects of alpha induced liquid induced effects on cells that induced conditions induced group was significantly better than that of RA and DMSO group (P0.05). In the same induction medium under the induction of embryoid body differentiation induced by the effect of myocardial cells was significantly higher than that of monolayer induced formation of hAFS cells (DMSO group and conditioned medium induced P0.05). These results indicate that hAFS cells induced differentiation into cardiomyocytes, medium through the formation of embryoid bodies under the condition The effect of induction is the best.
(4) induction of differentiation from hAFS cells to neural cells
Isolated hAFS cells from human amniotic fluid in late pregnancy, the formation of embryoid bodies (EB) and induced monolayer induced two kinds of methods, combined with different induction medium, inducing hAFS cells differentiation into neural cells, by inducing effect, screening the best induction system. The results show that under different induction conditions were obtained. Nestin, NSE positive cells, differentiation of cell size smaller, cytoplasmic contraction, the cells gradually tapered or triangular, the expression of neuron specific gene markers FGF-5 and CD56. were induced by RA in monolayer, induced group induced results and embryoid induction group is similar, but the corresponding stage of Nestin, the relative expression percentage of NSE the amount of positive cells and higher embryoid induction group, and the difference was significant (P0.05). The monolayer of different concentrations of beta -Me induced effects, suggest that 5mmol/L beta -Me group were better, and the induction time should be controlled within 3~4h, but in the beta -M Under the condition of e induction, no NSE positive cells were detected. The above results showed that when inducing hAFS cells to differentiate into neurons, RA induced solution and monolayer induction were effective.

【學位授予單位】：西北農林科技大學
【學位級別】：博士
【學位授予年份】：2008
【分類號】：R329

【引證文獻】

相關期刊論文 前2條

1 胡斯樂;達賴;吳江鴻;周歡敏;張家新;榮威恒;朱憲光;王鳳武;;蒙古羊羊水源干細胞分離培養(yǎng)及其成骨分化研究[J];中國畜牧獸醫(yī);2011年11期

2 李歡;高W，

本文編號：1377289