基于原位光学成像的卵母细胞纺锤体和染色体形态的数值化表征研究

发布时间：2018-08-16 13:10

【摘要】：光学成像与图像处理结合是认识生物结构空间构象的重要技术手段。而生物结构的空间构象对认识其生理功能,发育机制有主要意义。基于免疫荧光方法,本文采用双光子荧光显微成像技术,原位且立体地获取生物样品图像。进一步,本文通过图像三维分割、三维重构及数字化建模,量化表征生物结构的构象信息。采用该方法,本文研究了卵母细胞纺锤体和染色体的空间构象。染色体的空间构象与染色体的密度,基因转录活跃程度,以及细胞核的功能有重要关联。纺锤体的空间构象与染色体的排列与分离以及细胞分裂有关,其完整性对细胞能否正常繁殖有重要作用。卵母细胞中纺锤体和染色体的构象研究对评估卵母细胞质量,分析生殖发育问题有重要意义(如出生缺陷问题)。基于体外培养技术,本文首先研究了氨甲喋呤对卵母细胞纺锤体和染色体的影响。氨甲喋呤是一种抗叶酸代谢类药物,具有抑制细胞增殖的功能,广泛用于临床医治恶性肿瘤、类风湿性关节炎及异位妊娠等疾病。许多研究证明,氨甲喋呤对快速分裂的体细胞很强的副作用。然而,其对女性生殖细胞的影响尚不明确。本文研究发现与对照组(84%)相比,生发泡破裂比率在氨甲喋呤组下降至73%(p0.05)。氨甲喋呤组的第一极体排出率(53%)也明显低于对照组(63%;p0.05)。氨甲喋呤组染色体异常排布比率和纺锤体形态异常比率分别为60%和49%,均明显高于对照组情况(24%,17%;p0.05)。以上结果提示氨甲喋呤可影响卵母细胞的成熟质量。随后,本文又研究了激素超排对卵母细胞质量的影响。激素超数排卵作为一种非自然排卵方式,在临床辅助生殖及基础科学研究中应用广泛。但此种排卵方式对卵母细胞成熟质量的影响仍不明确。我们研究发现,激素超排造成染色体空间排布异常,但不影响染色体的体积、表面积等几何构型。此结果可能提示激素超排干扰了染色体的运动与迁移功能。本论文建立了一套数字化表征纺锤体和染色体空间构象的方法,研究结果对认识生殖发育问题有重要意义。
[Abstract]:The combination of optical imaging and image processing is an important technique for understanding the spatial conformation of biological structures. The spatial conformation of biological structure is of great significance in understanding its physiological function and developmental mechanism. Based on the immunofluorescence method, two-photon fluorescence microscopic imaging technique is used to obtain in situ and stereoscopic images of biological samples. Furthermore, the conformational information of biological structure is quantitatively represented by 3D segmentation, 3D reconstruction and digital modeling. By using this method, the spatial conformation of oocyte spindles and chromosomes was studied. The spatial conformation of chromosomes is closely related to the density of chromosomes, the degree of transcriptional activity of genes, and the function of nuclei. The spatial conformation of the spindle is related to the arrangement and separation of chromosomes and cell division, and its integrity plays an important role in the normal reproduction of cells. The study of the conformation of spindle and chromosome in oocytes is of great significance in evaluating the quality of oocytes and analyzing reproductive development problems (such as birth defects). Based on in vitro culture, the effects of methotrexate on oocyte spindle and chromosome were studied. Methotrexate is a kind of anti-folic acid metabolite with the function of inhibiting cell proliferation and is widely used in clinical treatment of malignant tumor rheumatoid arthritis and ectopic pregnancy and other diseases. Many studies have shown that methotrexate has a strong side effect on rapidly dividing somatic cells. However, its effect on female germ cells is unclear. Compared with the control group (84%), the rupture rate of foaming decreased to 73% (p0.05) in methotrexate group. The first polar body excretion rate (53%) in methotrexate group was significantly lower than that in control group (63% p0.05). The chromosomal abnormal arrangement ratio and spindle morphology abnormality rate of methotrexate group were 60% and 49%, respectively, which were significantly higher than those in the control group (24%, 1717%, p0.05). These results suggest that methotrexate may affect the maturation quality of oocytes. Then, the effect of hormone superovulation on oocyte quality was studied. Hormone superovulation, as an unnatural ovulation, is widely used in clinical assisted reproduction and basic scientific research. However, the effect of ovulation on the maturation quality of oocytes is still unclear. We found that hormone superovulation causes abnormal spatial distribution of chromosomes, but does not affect the geometric configuration of chromosomes, such as volume and surface area. These results may suggest that hormone superovulation interferes with the movement and migration of chromosomes. In this paper, a method of digitally characterizing the spatial conformation of spindles and chromosomes is established, and the results are of great significance to the understanding of reproductive development.
【学位授予单位】：沈阳师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q343;TP391.41

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