人源羊水干细胞分离培养、生物学特性检测及诱导分化研究

发布时间：2018-01-04 05:33

本文关键词：人源羊水干细胞分离培养、生物学特性检测及诱导分化研究　出处：《西北农林科技大学》2008年博士论文　论文类型：学位论文

【摘要】： 由于胚胎干细胞的研究受到伦理道德问题的束缚与限制,很多研究者开始尝试寻找新的干细胞来源。2003年,Prusa等在羊水中发现Oct-4阳性细胞,提示羊水中可能存在多能干细胞。2007年,Paolo等报道,他们在人的孕中期羊水中发现少量具有ES细胞特性的干细胞,将其命名为人羊水源干细胞(human Amniotic Fluid Stem Cell,hAFS cell)。这种细胞表达ES细胞和成体干细胞标志基因,体外诱导可分化为包括三个胚层的细胞,且通过了功能测试。hAFS细胞的特点是容易获取,不会损害母体及胚胎,可为细胞和组织工程治疗提供新的种子细胞来源。本实验自人孕中期、孕晚期羊水中分离培养hAFS细胞,检测了羊水标本性状对细胞原代培养的影响,筛选并优化了细胞培养体系。同时,通过RT-PCR、免疫细胞化学和流式细胞仪技术分选等技术,对hAFS细胞的生物学特性进行了检测,并诱导hAFS细胞向心肌细胞和神经细胞分化。 (1)羊水标本性状对hAFS细胞原代培养的影响从医院妇产科收集孕中期及孕晚期羊水标本,离心收集细胞,加入羊水干细胞培养液(ACM)进行培养,记录原代细胞贴壁时间及贴壁细胞数量,探讨人羊水标本性状等因素对原代分离培养的影响。实验结果表明,孕中期(4.7±0.6 d)与孕晚期(6±0.5 d)羊水标本中细胞的贴壁时间有明显差异(P0.05),孕晚期贴壁时间较长。羊水中血细胞污染程度对贴壁时间有明显影响,重度污染组(10.8±0.3 d)与无污染组(6±0.5 d)、轻度污染组(6.3±0.6 d)贴壁时间差异显著(P0.05)。羊水体积对细胞贴壁时间没有明显影响,但对贴壁细胞数量有显著影响(P0.05)。整个实验系统地检测了羊水干细胞原代培养的影响因素,为羊水干细胞研究提供了可以借鉴的资料。 (2)hAFS细胞分离培养及生物学特性检测培养人孕中期及孕晚期羊水标本,通过机械方法分离纯化hAFS细胞,采用RT-PCR、免疫细胞化学和流式细胞仪分选等技术对其生物学特性和分化潜能进行了检测。结果表明,在原代细胞培养的基础上,通过机械分离方法,可得到成纤维样hAFS细胞。这种细胞表达胚胎干细胞的特异性基因标志Oct-4、hTERT、Nanog、SSEA-1、SSEA-4和CD117,不表达SSEA-3;表达间充质干细胞的特异性基因标志CD29、CD44、CD73、CD90和CD105;不表达造血干细胞和造血细胞的特异性基因标志CD34、CD133和CD45;另外,这种细胞还表达HLA-ABC,弱表达HLA-DR。将hAFS细胞在体外多次传代后(已传至45代),仍具有较强的增殖能力,细胞核型正常。hAFS细胞在悬浮培养条件下可聚集形成类胚体,碱性磷酸酶(AP)检测呈阳性,表达三胚层特异性基因标志,如,外胚层:fgf5;中胚层:ζ-globin;内胚层:α-fetoprotein,证明其具有向三个胚层分化的潜能。同时,本实验筛选含不同浓度FBS及KSR的ACM培养液,表明hAFS细胞可在含KSR的无血清培养体系中扩增。 (3)hAFS细胞向心肌细胞诱导分化采用人孕晚期羊水中分离得到hAFS细胞,通过形成类胚体(EB)诱导和单层诱导两种方法,结合不同的诱导液,诱导hAFS细胞向心肌细胞分化,比较诱导效果,筛选最适的诱导体系。结果表明,在不同诱导条件下,均得到α-actin染色阳性细胞,表达心肌细胞特异标志基因Tbx5、Nkx2.5、GATA4和α-MHC。比较不同诱导液对细胞诱导效果的影响发现,条件诱导液组的诱导效果显著优于RA和DMSO组(P0.05)。在相同诱导液诱导条件下,通过形成类胚体诱导hAFS细胞向心肌细胞分化的效果显著优于单层诱导组(DMSO和条件培养液诱导,P0.05)。以上结果表明,诱导hAFS细胞向心肌细胞分化时,通过形成类胚体并采用条件培养液的诱导效果最好。 (4)hAFS细胞向神经细胞诱导分化由人孕晚期羊水中分离得到hAFS细胞,采用形成类胚体(EB)诱导和单层诱导两种方法,结合不同的诱导液,诱导hAFS细胞向神经细胞分化,通过比较诱导效果,筛选最适的诱导体系。结果表明,在不同诱导条件下,均得到Nestin、NSE阳性细胞,分化的细胞体积变小,胞质收缩,细胞逐渐呈锥形或三角形,表达神经细胞特异性基因标志fgf-5和CD56。在采用RA进行诱导时,单层诱导组诱导结果与类胚体诱导组类似,但相应阶段Nestin、NSE的相对表达量及阳性细胞百分比高于类胚体诱导组,且差异显著(P0.05)。比较不同浓度β-Me的单层诱导效果,表明5mmol/Lβ-Me组诱导效果较好,且诱导时间应控制在3~4h之内,但在β-Me诱导条件下,未检测到NSE阳性细胞。以上结果表明,诱导hAFS细胞向神经细胞分化时,采用RA诱导液并进行单层诱导的效果较好。
[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.

【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R329

【引证文献】