本文關(guān)鍵詞：人源羊水干細(xì)胞分離培養(yǎng)、生物學(xué)特性檢測及誘導(dǎo)分化研究　出處：《西北農(nóng)林科技大學(xué)》2008年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 人 羊水干細(xì)胞 類胚體 誘導(dǎo)分化 心肌細(xì)胞 神經(jīng)細(xì)胞

【摘要】： 由于胚胎干細(xì)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(xì)胞來源。2003年,Prusa等在羊水中發(fā)現(xiàn)Oct-4陽性細(xì)胞,提示羊水中可能存在多能干細(xì)胞。2007年,Paolo等報道,他們在人的孕中期羊水中發(fā)現(xiàn)少量具有ES細(xì)胞特性的干細(xì)胞,將其命名為人羊水源干細(xì)胞(human Amniotic Fluid Stem Cell,hAFS cell)。這種細(xì)胞表達(dá)ES細(xì)胞和成體干細(xì)胞標(biāo)志基因,體外誘導(dǎo)可分化為包括三個胚層的細(xì)胞,且通過了功能測試。hAFS細(xì)胞的特點(diǎn)是容易獲取,不會損害母體及胚胎,可為細(xì)胞和組織工程治療提供新的種子細(xì)胞來源。本實(shí)驗(yàn)自人孕中期、孕晚期羊水中分離培養(yǎng)hAFS細(xì)胞,檢測了羊水標(biāo)本性狀對細(xì)胞原代培養(yǎng)的影響,篩選并優(yōu)化了細(xì)胞培養(yǎng)體系。同時,通過RT-PCR、免疫細(xì)胞化學(xué)和流式細(xì)胞儀技術(shù)分選等技術(shù),對hAFS細(xì)胞的生物學(xué)特性進(jìn)行了檢測,并誘導(dǎo)hAFS細(xì)胞向心肌細(xì)胞和神經(jīng)細(xì)胞分化。 (1)羊水標(biāo)本性狀對hAFS細(xì)胞原代培養(yǎng)的影響 從醫(yī)院婦產(chǎn)科收集孕中期及孕晚期羊水標(biāo)本,離心收集細(xì)胞,加入羊水干細(xì)胞培養(yǎng)液(ACM)進(jìn)行培養(yǎng),記錄原代細(xì)胞貼壁時間及貼壁細(xì)胞數(shù)量,探討人羊水標(biāo)本性狀等因素對原代分離培養(yǎng)的影響。實(shí)驗(yàn)結(jié)果表明,孕中期(4.7±0.6 d)與孕晚期(6±0.5 d)羊水標(biāo)本中細(xì)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(xì)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(xì)胞貼壁時間沒有明顯影響,但對貼壁細(xì)胞數(shù)量有顯著影響(P0.05)。整個實(shí)驗(yàn)系統(tǒng)地檢測了羊水干細(xì)胞原代培養(yǎng)的影響因素,為羊水干細(xì)胞研究提供了可以借鑒的資料。 (2)hAFS細(xì)胞分離培養(yǎng)及生物學(xué)特性檢測 培養(yǎng)人孕中期及孕晚期羊水標(biāo)本,通過機(jī)械方法分離純化hAFS細(xì)胞,采用RT-PCR、免疫細(xì)胞化學(xué)和流式細(xì)胞儀分選等技術(shù)對其生物學(xué)特性和分化潛能進(jìn)行了檢測。結(jié)果表明,在原代細(xì)胞培養(yǎng)的基礎(chǔ)上,通過機(jī)械分離方法,可得到成纖維樣hAFS細(xì)胞。這種細(xì)胞表達(dá)胚胎干細(xì)胞的特異性基因標(biāo)志Oct-4、hTERT、Nanog、SSEA-1、SSEA-4和CD117,不表達(dá)SSEA-3;表達(dá)間充質(zhì)干細(xì)胞的特異性基因標(biāo)志CD29、CD44、CD73、CD90和CD105;不表達(dá)造血干細(xì)胞和造血細(xì)胞的特異性基因標(biāo)志CD34、CD133和CD45;另外,這種細(xì)胞還表達(dá)HLA-ABC,弱表達(dá)HLA-DR。將hAFS細(xì)胞在體外多次傳代后(已傳至45代),仍具有較強(qiáng)的增殖能力,細(xì)胞核型正常。hAFS細(xì)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(dá)三胚層特異性基因標(biāo)志,如,外胚層:fgf5;中胚層:ζ-globin;內(nèi)胚層:α-fetoprotein,證明其具有向三個胚層分化的潛能。同時,本實(shí)驗(yàn)篩選含不同濃度FBS及KSR的ACM培養(yǎng)液,表明hAFS細(xì)胞可在含KSR的無血清培養(yǎng)體系中擴(kuò)增。 (3)hAFS細(xì)胞向心肌細(xì)胞誘導(dǎo)分化 采用人孕晚期羊水中分離得到hAFS細(xì)胞,通過形成類胚體(EB)誘導(dǎo)和單層誘導(dǎo)兩種方法,結(jié)合不同的誘導(dǎo)液,誘導(dǎo)hAFS細(xì)胞向心肌細(xì)胞分化,比較誘導(dǎo)效果,篩選最適的誘導(dǎo)體系。結(jié)果表明,在不同誘導(dǎo)條件下,均得到α-actin染色陽性細(xì)胞,表達(dá)心肌細(xì)胞特異標(biāo)志基因Tbx5、Nkx2.5、GATA4和α-MHC。比較不同誘導(dǎo)液對細(xì)胞誘導(dǎo)效果的影響發(fā)現(xiàn),條件誘導(dǎo)液組的誘導(dǎo)效果顯著優(yōu)于RA和DMSO組(P0.05)。在相同誘導(dǎo)液誘導(dǎo)條件下,通過形成類胚體誘導(dǎo)hAFS細(xì)胞向心肌細(xì)胞分化的效果顯著優(yōu)于單層誘導(dǎo)組(DMSO和條件培養(yǎng)液誘導(dǎo),P0.05)。以上結(jié)果表明,誘導(dǎo)hAFS細(xì)胞向心肌細(xì)胞分化時,通過形成類胚體并采用條件培養(yǎng)液的誘導(dǎo)效果最好。 (4)hAFS細(xì)胞向神經(jīng)細(xì)胞誘導(dǎo)分化 由人孕晚期羊水中分離得到hAFS細(xì)胞,采用形成類胚體(EB)誘導(dǎo)和單層誘導(dǎo)兩種方法,結(jié)合不同的誘導(dǎo)液,誘導(dǎo)hAFS細(xì)胞向神經(jīng)細(xì)胞分化,通過比較誘導(dǎo)效果,篩選最適的誘導(dǎo)體系。結(jié)果表明,在不同誘導(dǎo)條件下,均得到Nestin、NSE陽性細(xì)胞,分化的細(xì)胞體積變小,胞質(zhì)收縮,細(xì)胞逐漸呈錐形或三角形,表達(dá)神經(jīng)細(xì)胞特異性基因標(biāo)志fgf-5和CD56。在采用RA進(jìn)行誘導(dǎo)時,單層誘導(dǎo)組誘導(dǎo)結(jié)果與類胚體誘導(dǎo)組類似,但相應(yīng)階段Nestin、NSE的相對表達(dá)量及陽性細(xì)胞百分比高于類胚體誘導(dǎo)組,且差異顯著(P0.05)。比較不同濃度β-Me的單層誘導(dǎo)效果,表明5mmol/Lβ-Me組誘導(dǎo)效果較好,且誘導(dǎo)時間應(yīng)控制在3~4h之內(nèi),但在β-Me誘導(dǎo)條件下,未檢測到NSE陽性細(xì)胞。以上結(jié)果表明,誘導(dǎo)hAFS細(xì)胞向神經(jīng)細(xì)胞分化時,采用RA誘導(dǎo)液并進(jìn)行單層誘導(dǎo)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(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2008
【分類號】：R329

【引證文獻(xiàn)】

相關(guān)期刊論文 前2條

1 胡斯樂;達(dá)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(xì)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