【作者】 王晗；

【导师】 王华岩； 窦忠英；

【作者基本信息】 西北农林科技大学 ， 临床兽医学， 2008， 博士

【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(P<0.05),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(P<0.05)。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(P<0.05)。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(P<0.05)。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,P<0.05)。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(P<0.05)。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。更多还原

【Abstract】 Researchers try to find out new resource of stem cell because of the ethical argument of embryonic stem cells. In 2003, Oct-4 positive cells were isolated in amniotic fluid, which showed that there might be multipotent stem cell in amniotic fluid. In 2007, Paolo demonstrated that they found a few stem cells in amniotic fluid,which was named human amniotic fluid stem cell (hAFS cell). hAFS cells that express embryonic and adult stem cell markers can differentiate into cell types of three embryonic germ layer and display specialized functions. hAFS cells can be obtained easily and do not damage the mother body and the embryo, which may offer an alternative approach for the therapy of cell and tissue engineering. In this study, hAFS cells were isolated from human amniotic fluid at the 2nd or 3rd trimester of gestation. Effects of the characteristics of amniotic fluid on the culture of hAFS cells were evaluated and culture system were established. At the same time, the biological characteristics of hAFS cells were detected by immunocytochemistry, RT-PCR and flow cytometer. hAFS cells were induced to differentiate to cardiomyocytes and neural cells.(1) Effects of the characteristics of amniotic fluid on the culture of hAFS cells Human amniotic fluid samples were obtained at the 2nd or 3rd trimester of gestation. Cells were isolated after centrifugation and were cultured with amniotic fluid stem cell culture medium (ACM). The cell attachment time and numbers were recorded to investigate the factors that affect the growth of hAFS cell primary culture. The results showed that the cell attachment time in the 2nd trimester of gestation (4.7±0.6 d) was significantly different with that in the 3rd trimester of gestation (6±0.5 d) (P<0.05), suggesting that cells collected from the fluid of 3rd trimester of gestation need longer time to attachment. We also find that blood contamination can significantly affect the time of cell attachment. The attachment time of maroon group (10.8±0.3 d) was significantly different with the clear group (6±0.5 d) and yellow group (6.3±0.6 d) (P<0.05). The effects of volume of amniotic fluid were also investigated on numbers and time of cell attachment. The volume of amniotic fluid did not affect cell attachment time, but affected the cell attachment numbers to a certain extent (P<0.05). The present studies systematically examine factors affecting on the primary culture of human AFS cells and provide useful data for AFS cell research.(2) The isolation, culture and detection of hAFS cells Human amniotic fluid samples at the 2nd or 3rd trimester of gestation were cultured and hAFS cells were isolated by the manual dissociation. The biological characteristics and the differentiation potential of hAFS cells were studied by the methods of RT-PCR, immunocytochemistry and flow cytometer. hAFS cells showing fibroblastoid-type were obtained and were positively express specific makers of embryonic stem cells, such as Oct-4, hTERT, Nanog, SSEA-1, SSEA-4 and CD117, but not SSEA-3. hAFS cells were stained positively for mesenchymal stem cells markers, including CD29, CD44, CD73, CD90 and CD105, but negatively for hematopoietic stem cells and hematopoietic lineage markers, including CD34, CD133 and CD45. hAFS cells were also positive for HLA-ABC and weakly positive for HLA-DR. hAFS cells maintain the proliferative potential and normal karyotypes even after expansion to several populations. In the suspension culture, hAFS cells could form embryoid bodies which were alkaline phosphatase (AP) positive and expressed fgf5,ζ-globin andα-fetoprotein which were the specific makers of three germs. hAFS cells proliferation rates were compared under two different medium, containing either FBS or KSR. The results showed that hAFS cells can be expanded in the absence of animal serum.(3) Differentiation of hAFS cells into cardiomyocyteshAFS cells were isolated from human amniotic fluid samples at the 3rd trimester of gestation. The induce effect were compared with different induce conditions. The results showed that the differentiated cells were positive forα-actin, Tbx5, Nkx2.5, GATA4 andα-MHC. The average optical density ofα-actin positive cells in conditional medium was higher than RA and DMSO treatments (P<0.05). In the same medium, the induce effect in EB group was well (P<0.05). The conclusion showed that the induce effect of hAFS cells differentiation into cardiomyocytes through the formation of embryoid bodies under the conditional medium was advantageous over other induce conditions.(4) Differentiation of hAFS cells into neural cellshAFS cells were isolated from human amniotic fluid samples at the 3rd trimester of gestation. The induce effect were compared with different induction conditions. The results showed that the differentiated cells were smaller,cytoplasmic contraction,cone-shaped or triangular,which were positive for Nestin, NSE, fgf-5 and CD56. Under RA medium, the average optical density of Nestin and NSE positive cells in monolayer group was higher than in EB group (P<0.05). Underβ-Me medium, no NSE positive cells were observed and the induce time should be changed in 3~4 hours. The conclusion showed that the induce effect of hAFS cells differentiation into neural cells under RA medium in monolayer group was advantageous over other induce conditions.更多还原