胚胎心脏祖细胞的生物学特性及Islet1基因的初步研究
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摘要
本试验对北京油鸡胚胎心脏祖细胞的分离培养、鉴定、诱导分化等生物学特性以及自分化现象进行了探索,对鸡Islet1基因进行了初步研究,得出以下结果:
1.试验对两种不同做样方法进行了比较,最终选择酶消化法进行鸡胚心脏祖细胞的常规分离方法。酶消化法分离培养得到的鸡胚心脏祖细胞,经过纯化扩增,在体外培养条件下能传到第20代。
2.试验对分离培养时所用鸡胚的孵化时间做了研究,结果发现在孵化6d-11d之间的鸡胚心脏大小适合试验操作,而且能够得到大量相对纯净的目的细胞。
3.鸡胚心脏祖细胞对培养基有很强的选择性,对培养基酸碱度非常敏感,PH变化较大时会导致细胞大量凋亡,适合条件的培养基能使ECPCs快速增殖,最终我们选用的培养基是ECPCs DMEM/F12增殖培养基。
4.分离培养的鸡胚心脏祖细胞呈克隆性增殖,增殖速度较快,形态上以椭圆形、多角形居多,细胞核质较大,镜下观察能明显看到其立体状结构。
5.对第3代和第11代的鸡胚心脏祖细胞进行免疫化学及RT-PCR鉴定,结果显示鸡胚心脏祖细胞均能表达Isl1、GATA4、Flk1和Nkx2.5四个标志物。
6.鸡胚心脏祖细胞在诱导条件下能够分化成为成熟心肌细胞和平滑肌细胞,诱导后免疫组化及分子生物学鉴定结果显示:诱导后的心肌细胞能发生节律性收缩,cTnT表达呈阳性;诱导后的平滑肌细胞α-SMA表达呈阳性。
7.成功克隆了Islet1基因CDS区和LIM同源结构域片段,并完成真核表达载体的构建。
8.通过生物信息学手段分析Islet1蛋白序列发现,Islet1蛋白在不同物种间有较高的同源性,蛋白分为三个明显的结构域。
To clarify the biological characteristics of the chicken embryonic cardiac progenitor cells, we made a study on isolation, isolated culture, identification, differentiation, biological characteristics, Self-differentiation of the ECPCs and we also made a preliminary exploration of the chicken gene Islet1. We got the following results:
1. The test compared two isolation methods: tissue culture and enzyme digestion culture method. Finally, we made a selection for enzyme digestion culture as a conventional isolation method for chicken cardiac progenitor cells. The chicken cardiac progenitor cells obtained by Enzyme digestion can be expanded stably in vitro, and sub-cultured to 18th passage.
2. We explored the chick hatching time for experiment and found that the chick embryos amoung 6d-11d were suited for operation, and could got a large number of relatively pure target cells.
3. Different culture conditions on proliferation of chick embryo cardiac progenitor cells have greater impact: chicken cardiac progenitor cells are very sensitive to pH in culture medium, the dramatic changes of PH will lead to cell apoptosis; chicken embryonic cardiac progenitor cells have a serious selection for the conditions of the culture medium, they could proliferated rapidly while we used the ECPCs DMEM/F12 proliferation medium.
4. In morphology, the chick embro cardiac progenitor cells showed a faster, clonal proliferation, morphology with oval, polygonal, a high ratio on nucleus and cytoplasm. We can observe a three-dimensional-like structure of cells clearly by Phase contrast microscope.
5. We made immunohistochemical and RT-PCR identification on the 3rd and 11th passsge of chicken cardiac progenitor cells and the results showed that chicken cardiac progenitor cells can express Isl1, GATA4, Flk1, and Nkx2.5.
6. Chicken cardiac progenitor cells can differentiate into mature cardiac cells and smooth muscle cells in appropriate inducing conditions, the immunohistochemical and molecular identification results of the induction showed that: The rhythmic beat contraction of myocardial cells occurred after induced, cTnT expression was positive; theα-SMA expression of smooth muscle cells after induced was positive.
7. The CDS region and LIM homeodomain fragment of Islet1 gene was successfully cloned, and the eukaryotic expression vectors were completed.
8. The bioinformatics analysis of Islet1 protein sequence showed that Islet1 protein has three structure domains and high homology in different species.
1.试验对两种不同做样方法进行了比较,最终选择酶消化法进行鸡胚心脏祖细胞的常规分离方法。酶消化法分离培养得到的鸡胚心脏祖细胞,经过纯化扩增,在体外培养条件下能传到第20代。
2.试验对分离培养时所用鸡胚的孵化时间做了研究,结果发现在孵化6d-11d之间的鸡胚心脏大小适合试验操作,而且能够得到大量相对纯净的目的细胞。
3.鸡胚心脏祖细胞对培养基有很强的选择性,对培养基酸碱度非常敏感,PH变化较大时会导致细胞大量凋亡,适合条件的培养基能使ECPCs快速增殖,最终我们选用的培养基是ECPCs DMEM/F12增殖培养基。
4.分离培养的鸡胚心脏祖细胞呈克隆性增殖,增殖速度较快,形态上以椭圆形、多角形居多,细胞核质较大,镜下观察能明显看到其立体状结构。
5.对第3代和第11代的鸡胚心脏祖细胞进行免疫化学及RT-PCR鉴定,结果显示鸡胚心脏祖细胞均能表达Isl1、GATA4、Flk1和Nkx2.5四个标志物。
6.鸡胚心脏祖细胞在诱导条件下能够分化成为成熟心肌细胞和平滑肌细胞,诱导后免疫组化及分子生物学鉴定结果显示:诱导后的心肌细胞能发生节律性收缩,cTnT表达呈阳性;诱导后的平滑肌细胞α-SMA表达呈阳性。
7.成功克隆了Islet1基因CDS区和LIM同源结构域片段,并完成真核表达载体的构建。
8.通过生物信息学手段分析Islet1蛋白序列发现,Islet1蛋白在不同物种间有较高的同源性,蛋白分为三个明显的结构域。
To clarify the biological characteristics of the chicken embryonic cardiac progenitor cells, we made a study on isolation, isolated culture, identification, differentiation, biological characteristics, Self-differentiation of the ECPCs and we also made a preliminary exploration of the chicken gene Islet1. We got the following results:
1. The test compared two isolation methods: tissue culture and enzyme digestion culture method. Finally, we made a selection for enzyme digestion culture as a conventional isolation method for chicken cardiac progenitor cells. The chicken cardiac progenitor cells obtained by Enzyme digestion can be expanded stably in vitro, and sub-cultured to 18th passage.
2. We explored the chick hatching time for experiment and found that the chick embryos amoung 6d-11d were suited for operation, and could got a large number of relatively pure target cells.
3. Different culture conditions on proliferation of chick embryo cardiac progenitor cells have greater impact: chicken cardiac progenitor cells are very sensitive to pH in culture medium, the dramatic changes of PH will lead to cell apoptosis; chicken embryonic cardiac progenitor cells have a serious selection for the conditions of the culture medium, they could proliferated rapidly while we used the ECPCs DMEM/F12 proliferation medium.
4. In morphology, the chick embro cardiac progenitor cells showed a faster, clonal proliferation, morphology with oval, polygonal, a high ratio on nucleus and cytoplasm. We can observe a three-dimensional-like structure of cells clearly by Phase contrast microscope.
5. We made immunohistochemical and RT-PCR identification on the 3rd and 11th passsge of chicken cardiac progenitor cells and the results showed that chicken cardiac progenitor cells can express Isl1, GATA4, Flk1, and Nkx2.5.
6. Chicken cardiac progenitor cells can differentiate into mature cardiac cells and smooth muscle cells in appropriate inducing conditions, the immunohistochemical and molecular identification results of the induction showed that: The rhythmic beat contraction of myocardial cells occurred after induced, cTnT expression was positive; theα-SMA expression of smooth muscle cells after induced was positive.
7. The CDS region and LIM homeodomain fragment of Islet1 gene was successfully cloned, and the eukaryotic expression vectors were completed.
8. The bioinformatics analysis of Islet1 protein sequence showed that Islet1 protein has three structure domains and high homology in different species.
引文
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