胚胎干细胞体外培养体系的初步探讨

发布时间：2018-01-11 22:17

本文关键词：胚胎干细胞体外培养体系的初步探讨　出处：《广西医科大学》2009年硕士论文　论文类型：学位论文

【摘要】： 目的:在前期课题组研究的基础之上,优化饲养层体系,稳定培养条件,探索适合自身实验室条件的胚胎干细胞体外培养体系,对实验细胞的描述记录实行标准化和规范化。方法:(1).使用胰酶消化法体外分离培养MEF,MTT法测定P3、P8和复苏P3 MEF生长活力,优化原代取材、传代、冻存和复苏细胞的条件,免疫组化检测MEF特异性标志——波形蛋白的表达,使用丝裂霉素C处理MEF,建立饲养层培养体系。(2).自然受孕法和超排卵法获取昆明小鼠3.5dpc胚胎,体外培养发育至囊胚,全胚培养法获取ICM,在饲养层上增殖,然后离散ICM种于新鲜的饲养层上,观察ESC集落的形成,探索ESC体外培养体系。(3).收集生殖中心IVF-ET后剩余无冷冻价值的胚胎,医院伦理委员会讨论通过,签署患者知情同意书后用于体外培养,探索其体外培养条件。(4).依据《实验细胞资源的描述标准与管理规范》,整理实验数据,对实验细胞的描述记录实行标准化和规范化。结果:(1).使用胰酶消化法可从10~18dpc小鼠胚胎中获取MEF,倒置显微镜下观察,13~15dpc小鼠胚胎获得的MEF质量较佳;第五代之后MEF逐渐呈衰老相;MEF生长曲线显示:在第3天时三组细胞均进入对数增长期,P8组细胞的增殖速度明显低于其他两组(P0.05)。SABC免疫组化染色鉴定MEF,90%以上细胞中间丝波形蛋白呈棕色颗粒或棕色着染,即为阳性。丝裂霉素C处理MEF,终浓度为10μg/ml,作用时间2h,实验显示在抑制细胞有丝分裂的同时又不引起大量细胞死亡。(2).自然受孕法每只昆明小鼠可获取8~12个3.5dpc胚胎,质量较好的胚胎在饲养层体系中可逐渐发育至囊胚扩张期,自然从透明带孵出,ICM附着于饲养层上快速增殖。(3).整理了近三年课题组培养和冻存实验细胞数据,制定了规范化的实验细胞记录表格,标准化了实验细胞的描述。结论:(1).优化稳定了饲养层培养体系并鉴定饲养层细胞。(2).初步探索了胚胎培养及ICM集落形成的条件。
[Abstract]:Objective: to optimize the feeding layer system, to stabilize the culture conditions, and to explore an in vitro culture system of embryonic stem cells suitable for their laboratory conditions. The description of experimental cells is standardized and standardized. Methods the growth activities of P3P8 and P3 MEF were determined by trypsin digestion in vitro, and the primary material was optimized and subcultured. Under the condition of cryopreservation and resuscitation, the expression of vimentin, a specific marker of MEF, was detected by immunohistochemistry. Mitomycin C was used to treat MEF. The 3.5dpc embryos of Kunming mice were obtained by natural fertilization and superovulation. The embryos were cultured to blastocysts in vitro, and ICMs were obtained from the whole embryo culture method and proliferated on the feeder layer. Then discrete ICM was seeded on fresh feeder layer to observe the formation of ESC colony and to explore the culture system of ESC in vitro. The remaining embryos without freezing value after IVF-ET in reproductive center were collected. The hospital ethics committee discussed and approved, signed the informed consent of patients and used it for in vitro culture, and explored its culture condition in vitro. According to the description Standard and Management Standard of Experimental Cell Resources. Collate the experimental data and standardize the description and record of experimental cells. Results using trypsin digestion method, MEF could be obtained from 10 ~ 18dpc mouse embryos. The MEF obtained from 13 ~ (13) D ~ (PC) mouse embryos was better under inverted microscope. After the fifth generation, MEF gradually showed senescence phase. The growth curve of MEF showed that the proliferation rate of P8 cells in the three groups was significantly lower than that in the other two groups by immunohistochemical staining on the third day. More than 90% cells showed brown or brown staining of vimentin, which was positive. The final concentration of mitomycin C was 10 渭 g / ml for 2 h. The experiment showed that while inhibiting cell mitosis without causing a large number of cell death, 8 ~ 12 3.5dpc embryos could be obtained from each Kunming mouse by natural conception. The embryos with good quality can gradually develop into blastocyst dilation in the feeder layer system, and naturally hatched from the pellucida. ICM was attached to the feeder layer to proliferate rapidly. In the last three years, the experimental cell data were collected and frozen by our group, and a standardized record table of experimental cells was established, and the description of experimental cells was standardized. Conclusion the culture system of the feeder layer was optimized and stable, and the cells of the feeder layer were identified. The conditions of embryo culture and colony formation of ICM were preliminarily explored.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329

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