论文标题:表达绿色荧光蛋白的小鼠胚胎干细胞的分离与鉴定
Isolation and Characterization of Mouse Embryonic Stem Cells Expressing Green Fluorescence Protein
论文作者
论文导师 扈廷茂,论文学位 硕士,论文专业 微生物学
论文单位 内蒙古大学,点击次数 99,论文页数 62页File Size5334K
2007-05-01论文网 http://www.lw23.com/lunwen_624294552/
EGFP-transgenic mouse; embryonic stem cells; green fluorescence protein; blastocyst; inner cell mass (ICM)
目的：本实验旨在研究绿色荧光蛋白在EGFP转基因小鼠各组织、器官中的表达情况，使用国产丝裂霉素在胎儿成纤维细胞饲养层制备中的应用以及建立来源EGFP转基因小鼠受精囊胚的表达绿色荧光蛋白的多能性小鼠胚胎干细胞系。并分析其部分生物学特性。表达绿色荧光蛋白的胚胎干细胞在研究细胞分化方面具有巨大优势。 方法：采用0.05％胰蛋白酶—0.02％EDTA混合消化液消化EGFP转基因小鼠各组织、器官制备单细胞悬液，通过流式细胞仪分析绿色荧光蛋白在EGFP转基因小鼠不同组织、器官中的表达情况。使用标准化试剂，采用国际上小鼠胚胎干细胞建系的通用路线，直接从强表达绿色荧光蛋白的转基因小鼠的受精囊胚的内胚团分离胚胎干细胞。通过将超排卵后受孕3.5天的EGFP转基因小鼠囊胚与由受孕13.5天的昆明种小鼠胚胎制备的胚胎成纤维细胞饲养层共培养，以高糖DMEM添加10％胎牛血清、2mmol／1L-谷氨酰胺、0.1mmol／1β—巯基乙醇，以及1000u／ml的鼠白血病抑制因子(mLIF)等为培养基，利用自制的玻璃微针在解剖显微镜下机械剥离增殖4-6天的内细胞群(ICM)，在0.05％胰蛋白酶—0.02％EDTA混合消化液的液滴中消化3～5min，其间辅助机械吹打然后再接种到新的饲养层上培养。观察记录消化后细胞集落的生长情况，4-6天对ES细胞样集落继续消化传代培养，在第五代时，在传代同时冻存处理，在传至第十代时进行。碱性磷酸酶染色(AKP)、免疫组化(Oct-4，SSEA-1)、畸胎瘤实验、体外分化实验以及流式细胞仪分析。 结果显示：绿色荧光蛋白在EGFP转基因鼠不同组织、器官中的表达能力存在较大差异，在脾、脑、心、胸腺、骨髓组织中表达绿色荧光蛋白的细胞所占比率较高，分别达到99.75％、91.76％、87.95％、83.32％、83.16％；而在肠上皮、皮肤表皮、肝脏、肌肉组织中表达绿色荧光蛋白的细胞所占比率较低，分别为26.67％、36.21％、46.51％、52.88％。浙江海正药业生产的丝裂霉素浓度为30μg／ml作用2h，可有效抑制小鼠胎儿成纤维细胞的分裂而不影响细胞活力。 获得的小鼠胚胎干细胞能够形成畸胎瘤、类胚体，表达碱性磷酸酶活性，Oct-4阳性，SSEA-1表面抗原呈阳性。经流式细胞仪分析显示该胚胎干细胞能够表达绿色荧光蛋白。 结论： (1)绿色荧光蛋白在EGFP转基因鼠的不同组织、器官中表达水平存在差异。 (2)来自EGFP转基因小鼠的胚胎干细胞适合用于脾、脑、心、胸腺、骨髓组织的分化研究。 (3)丝裂霉素浓度为20μg／mL处理4h或30μg／mL处理2h、4h3，能够抑制小鼠胎儿成纤维细胞的分裂而不影响细胞活力。 (4)可以从EGFP转基因小鼠囊胚的内细胞群中分离胚胎干细胞，该胚胎干细胞表达碱性磷酸酶活性，SSEA-1和Oct-4阳性。分化实验表明ES细胞能够形成类胚体和具有三个胚层的分化能力的畸胎瘤。且带有强绿色荧光蛋白标记。
Objective: The target of this study is to investigate GFP-expressing cell rate in organ tissues derived from EGFP mouse, to observe the effects of the homemade mytomycin on growth of the MEFs in vitro, to isolate pluripotent mouse embryonic stem cells (ES cells) from blastocysts of EGFP mouse and to analysis some biological characteristics of ES cells. GFP-expressing ES cells have great advantage in researching cell differentiation. Methods : The cell suspensions derived from organ tissues of EGFP mouse using 0.05%Trypsin -0.02%EDTA solution was used to analysis GFP-expressing cells with a flow cytometry. In our experiment, the MTT methods was adopted. The mouse embryonic fibroblasts (MEF) were treated by different concentrations of the homemade mytomycin for different duration to observe the effects of division and vitality in vitro. The GFP-expressing embryonic stem cells were isolated from inner cell mass (ICM) which were separated from EGFP-transgenic mouse blastocysts of 4 dpc. The blastocysts from EGFP mouse were collected and cultured on mouse embryonic fibroblast (MEF) feeder. The culture medium is DMEM with 4500mg/1L glucose , supplemented with 10% fetal bovine serum (FBS), 2mmol/1 L-glutamine, 0.1mmol/1L B-mercaptoethanol, 1000IU/ml mouse leukemia inhibitory factor (mLIF) and penicillin and streptomycin. Inner cell mass (ICM) was transferred into liquid drops of 0.05%Trypsin-0.02%EDTA. After incubation for 3～5minutes, the disaggregated clumps of ICM were implanted into newly-prepared feeder layer and were cultured continuously. After 4-6 days, ES cells-like colonies were continually disaggregated and were cultured ES cells were freezed in passage 5. To testify those cell colonies, AKP staining and immunocytochemistry of Oct-4、SSEA-1 was carried out. These ES cells in passage 10 was also to analysis the GFP-expressing cell rate with a flow cytometry. Resu Its : The rate of GFP-expressing cells in organ tissues of EGFP mouse is different. The rate of GFP-expressing cells in the spleen ,brain, heart, thymus and marrow is 99.75%, 91.76%, 87.95%, 83.32%, 83.16%, respectively. The rate GFP-expressing cells in the intestinal epithelium, epidermis, liver and muscle is 26.67%, 36.21%, 46.51%, 52.88%, respectively. The results show that the mytomycin produced by Zhejiang Hisun Pharmaceutical Co.,Ltd (20μg/mL for 4h, 30μg/mL for 2 or 4 h) can inhibit effectively the division of the MEFs, but not affect vitality of the MEFs and that resultant ES cells express high levels of green fluorescent protein(GFP) with a flow cytometry analysis and maintain the developmental potential to form teratoma with all three embryonic germ layers. AKP staining and immunocytochemistry of Oct-4、SSEA-1 is positive. Conclusions: (1) The rate of GFP-expressing cells in organ of EGFP mouse is different. (2)The ES cell derived from EGFP mouse can be used to study differentiatation from ES cells to the spleen, brain, heart, thymus. (3) The homemade mytomycin produced by Zhejiang Hisun Pharmaceutical Co.,Ltd (30μg/mL for 2) can inhibit effectively the division of the MEFs, but not affect vitality of the MEFs. (4) ES cells colonies which express AKP, SSEA-land Oct-4 positive can be isolated from ICM of blastocyst of EGFP mouse. Differentiation experiment showed that ES cells have ability to form EBs and teratomas with three embryonic germ layers which express strong green fluorescent protein.
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