羊水来源干细胞的分离培养及其生物学特征的研究

发布时间：2018-01-05 21:20

本文关键词：羊水来源干细胞的分离培养及其生物学特征的研究　出处：《泰山医学院》2011年硕士论文　论文类型：学位论文

【摘要】：目的研究羊水来源干细胞(amniotic fluid-drived stem cells,AFS)的确切来源。目前,鉴定羊水来源干细胞没有什么好方法,只是通过细胞表面干细胞因子的表达情况和生物学特性来确定其是否为AFS。本实验在分离培养干细胞的基础上,研究其生物学特性;通过细胞形态及细胞原位免疫组化来明确羊水干细胞的确切来源,为下一步的临床治疗提供实验基础和理论依据。研究方法在知情同意的情况下,通过B超引导下腹腔穿刺抽取人孕16-24周的羊水18-20ml,原位培养2周后,待其达到50%融合后,将一部分P1培养瓶中的细胞分为两部分,倒置显微镜下,将培养瓶一分为二,用进口刮刀将其分别刮下,倒入新的培养瓶中,进行传代,记为P21、P22。由于刮刀对细胞的损伤较大,故继续传代用胰酶消化的方法,一瓶用来倒置显微镜下观察各种细胞的形态,绘制生长曲线、构建类胚胎及分析染色体核型;另一瓶,通过细胞免疫组化研究OCT-4、CD117的表达情况;另一部分P1,倒置显微镜下观察其形态并通过细胞原位免疫组化来研究其表面分子的表达情况,检测各种标志物如波形蛋白Vimen、广谱角蛋白CK、CD44、CD29、CD34、CD45的表达情况。结果 1、通过细胞的原位培养,根据倒置显微镜下细胞形态的不同,将羊水中的细胞分为四类,为小岛样细胞、E样细胞、羊水特异细胞及成纤维样细胞。 2、多次传代后,细胞不再呈集落样生长,而呈重叠样生长,以梭形为主,排列紧密,相互间界限不清。形态比原代更均匀,生长旺盛。可以连续传10代,没有发现细胞老化现象。 3、羊水细胞生长曲线呈S形,P10比P3、P5生长缓慢。生长曲线的特征为:接种后2天左右为潜伏适应期,大约从第3天起为对数生长期,第8天达到高峰期,之后就进入了平台期,说明其在体外具有很强的增殖能力。 4、将P7羊水细胞倒置悬浮培养3天,显微镜下可见类胚体样结构。证明了其具有向三个胚层分化的潜能,有待进一步的研究。 5、取每代羊水细胞做染色体核型分析,都为正常二倍体核型,说明了其具有稳定遗传的特性。 6、取P10做细胞免疫组化,结果显示其表达OCT-4、CD117,提示羊水中含有干细胞。 7、小岛样细胞表达CK、Vimen、CD44、OCT-4,不表达CD117、CD34、CD45;E样细胞表达CK、Vimen、CD44、Oct-4、CD117,不表达CD34、CD45;羊水特异细胞表CK、Vimen、CD44、OCT-4,不表达CD117、CD34、CD45;成纤维样细胞表达Vimen、CD44、Oct-4、CD117,不表达CD34、CD45。结论及意义 1、本实验证明上皮样细胞和成纤维样细胞都为羊水干细胞的确切来源。 2、本实验根据其形态的不同,将羊水细胞分为小岛样细胞、E样细胞、羊水特异细胞及成纤维样细胞。 3、本实验证明羊水中含有表达CD44、Vimen的间充质干细胞、表达干细胞因子CD117的干细胞、表达OCT-4的多能干细胞。 4、本实验证明了羊水来源的干细胞在体外具有很强的增殖能力、分化潜能及稳定遗传的特性。
[Abstract]:Purpose To study the exact origin of amniotic fluid-drived stem cells (AFSs) derived from amniotic fluid. Identification of amniotic fluid derived stem cells is not a good method, only through the cell surface stem cell factor expression and biological characteristics to determine whether it is AFS. this experiment is based on the isolation and culture of stem cells. To study its biological characteristics; The exact origin of amniotic fluid stem cells was determined by cell morphology and in situ immunohistochemistry to provide experimental and theoretical basis for the next clinical treatment. Research method In the case of informed consent, the amniotic fluid of 16-24 weeks gestation was extracted by abdominal puncture guided by B-ultrasound. After in situ culture for 2 weeks, it reached 50% fusion. The cells in the P1 culture bottle were divided into two parts. Under the inverted microscope, the culture bottle was divided into two parts. The culture bottle was scraped off separately with an imported scraper, then poured into the new culture bottle and subcultured as P21. P22. because of the large damage to cells caused by scraper, the method of trypsin digestion was continued. A bottle was used to observe the morphology of various cells under inverted microscope and to draw the growth curve. Construction of embryoid and karyotype analysis; In the other bottle, the expression of OCT-4 CD117 was studied by immunohistochemistry. In the other part, the morphology of P1 was observed under inverted microscope, and the expression of surface molecules was studied by in situ immunohistochemistry, and various markers such as vimentin Vimenin and broad-spectrum keratin CK were detected. Expression of CD44, CD29, CD34, CD45. Results 1. The cells in amniotic fluid were divided into four groups by in situ culture. According to the different morphology of the cells under inverted microscope, the cells in amniotic fluid were divided into small island like cells, amniotic fluid specific cells and fibroblast cells. (2) after repeated passages, the cells were no longer colony like growth, but overlapped like growth, mainly fusiform, closely arranged, with unclear boundaries between each other. The morphology was more uniform than the original generation, and the growth was exuberant. The cells could be passed on for 10 generations in succession. No cell aging was found. 3. The growth curve of amniotic fluid cells showed S-shaped P10 was slower than P3P5. The characteristic of the growth curve was that the incubation period was about 2 days after inoculation and the logarithmic growth period was about 3 days after inoculation. On the 8th day, it reached its peak and then entered the platform stage, which indicated that it had strong proliferative ability in vitro. (4) when the P7 amniotic fluid cells were cultured in reverse suspension for 3 days, the embryoid structure could be seen under microscope. It was proved that P7 amniotic fluid cells had the potential to differentiate into three endosperms, which should be further studied. 5. The chromosome karyotype analysis of every generation of amniotic fluid cells is normal diploid karyotype, which shows that it has stable genetic characteristics. 6. P10 was taken as cell immunohistochemistry. The results showed that OCT-4 and CD117 were expressed in amniotic fluid, suggesting that there were stem cells in amniotic fluid. (7) the small island like cells expressed CK-Vimenus CD44-OCT-4, and did not express CD117- CD34- CD45; E-like cells expressed CK-Vimenn ~ (4) -Oct-4 + CD117, but not CD34 ~ (4) ~ (+) CD45; In amniotic fluid specific cell surface, Vimenus CD4and OCT-4 did not express CD117, CD34 and CD45; Fibroblasts expressed Vimenin CD44, Oct-4, CD117, but not CD34, CD45. Conclusion and significance 1. It was proved that both epithelioid cells and fibroblasts were the exact sources of amniotic fluid stem cells. 2. According to its morphology, amniotic fluid cells were divided into small island like cells, amniotic fluid specific cells and fibroblasts. 3. The results showed that the amniotic fluid contained mesenchymal stem cells expressing CD44-Vimen, stem cells expressing stem cell factor CD117 and pluripotent stem cells expressing OCT-4. 4. The results showed that the stem cells derived from amniotic fluid had strong proliferative ability, differentiation potential and stable heredity in vitro.
【学位授予单位】：泰山医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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