蒙古羊羊水源干细胞的分离鉴定及其定向成骨分化研究
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摘要
本研究旨在分离鉴定蒙古羊羊水源干细胞(Amniotic fluid-derived stem cells,AFSC),并对其生物学特征及分化潜能进行探究,为以AFSC为种质资源生产克隆羊、转基因羊奠定理论基础。
经过培养不同孕期蒙古羊AFSC,发现羊水采集时间对AFSC的贴壁率及细胞活性影响较大,孕中期AFSC贴壁率比孕早期的高,细胞活性比孕晚期的强,采集AFSC的最佳时期为孕中期。
15% FBS可维持孕中期蒙古羊AFSC增殖50代以上,其形态及生物特性保持不变,细胞生长速度及增殖能力无明显差异。
细胞培养初期,通过机械分离法,并有效配合胰蛋白酶消化时间,去除成纤维状细胞中混杂的上皮样细胞,可在5代之内纯化。纯化的AFSC形态上为梭型,细胞汇合有方向性,集落呈典型涡旋状排列。
利用RT-PCR检测不同代数AFSC,结果表明蒙古羊AFSC表达干细胞标志基因Oct-4, Nanog, SH2, SSEA-1, CD117和HLA-A等。
蒙古羊AFSC悬浮培养时,可形成典型的类胚体,并表达三胚层标志基因fgf5 (外胚层)、α-fetoprotein (内胚层)和ζ-globin (中胚层)。
利用地塞米松、抗坏血酸、β-甘油磷酸钠对蒙古羊AFSC诱导后细胞生长相对变缓,边缘变模糊,局部出现复层生长,并形成颗粒状结节。诱导三周后细胞碱性磷酸酶活性显著增加,茜素红染色呈红色,表明诱导成功。
研究结果表明本试验从蒙古羊羊水中分离得到的细胞具有一定的干细胞特性,在体外有很强的增殖和分化能力,并在特定条件下可形成成骨细胞。
The aim of this study was to isolate and identify Mongolian sheep amniotic fluid-derived stem cells, and to detect the biological characteristics and differentiation potential of AFSC. This study lay a theoretical foundation for generating the cloned sheep and the transgenic sheep which derived from AFSC.
The Mongolian sheep AFSC from different gestation stage were cultured, the result showed that the adhere-rate and activity of cells was greatly affected by the time of amniotic fluid acquisition. The adhere-rate of cells in the mid-gestation was higher than that of early gestation. The activity of cells in the mid-gestation was higher than that of late-gestation. The best time of AFSC acquisition was the mid-gestation.
Biological characteristics and configuration of AFSC after a long-term cultivation with 50th or more cell passage sustained stable when 15% FBS added in cell medium.
AFSC could be purified within 5th passage by mechanical separation combinated with defined digesting time with trypsin to remove the epithelioid cells from fibrous cells. These cells were shuttle-type, cell confluence directional, and the colony was vortex-like arrangement.
The result showed that Oct-4, Nanog, SH2, SSEA-1, CD117 and HLA-A were expressed in different passage AFSC by RT-PCR. AFSC were able to form the embryoid body in vitro, which express marker genes of three embryonic germ layers including fgf5 (ectoderm)、α-fetoprotein (endoderm) andζ-globin (mesoderm)。
AFSC were induced by dexamethasone,β-glycerophosphate and ascorbic acid. As a result, cells growth was reduced, the edges blurred and multilayer growth and nodules occurred over time. Three weeks after induction, the activity of alkaline phosphatase increased remarkably and Alizarin red staining showed red. These results showed that induction into osteoblasts have been successfully.
In summary, these results show that the AFSC obtained in this study have stem cell characteristic, strong proliferation and differentiation potency, and could be induced to differentiate into osteoblasts under certainly condition.
经过培养不同孕期蒙古羊AFSC,发现羊水采集时间对AFSC的贴壁率及细胞活性影响较大,孕中期AFSC贴壁率比孕早期的高,细胞活性比孕晚期的强,采集AFSC的最佳时期为孕中期。
15% FBS可维持孕中期蒙古羊AFSC增殖50代以上,其形态及生物特性保持不变,细胞生长速度及增殖能力无明显差异。
细胞培养初期,通过机械分离法,并有效配合胰蛋白酶消化时间,去除成纤维状细胞中混杂的上皮样细胞,可在5代之内纯化。纯化的AFSC形态上为梭型,细胞汇合有方向性,集落呈典型涡旋状排列。
利用RT-PCR检测不同代数AFSC,结果表明蒙古羊AFSC表达干细胞标志基因Oct-4, Nanog, SH2, SSEA-1, CD117和HLA-A等。
蒙古羊AFSC悬浮培养时,可形成典型的类胚体,并表达三胚层标志基因fgf5 (外胚层)、α-fetoprotein (内胚层)和ζ-globin (中胚层)。
利用地塞米松、抗坏血酸、β-甘油磷酸钠对蒙古羊AFSC诱导后细胞生长相对变缓,边缘变模糊,局部出现复层生长,并形成颗粒状结节。诱导三周后细胞碱性磷酸酶活性显著增加,茜素红染色呈红色,表明诱导成功。
研究结果表明本试验从蒙古羊羊水中分离得到的细胞具有一定的干细胞特性,在体外有很强的增殖和分化能力,并在特定条件下可形成成骨细胞。
The aim of this study was to isolate and identify Mongolian sheep amniotic fluid-derived stem cells, and to detect the biological characteristics and differentiation potential of AFSC. This study lay a theoretical foundation for generating the cloned sheep and the transgenic sheep which derived from AFSC.
The Mongolian sheep AFSC from different gestation stage were cultured, the result showed that the adhere-rate and activity of cells was greatly affected by the time of amniotic fluid acquisition. The adhere-rate of cells in the mid-gestation was higher than that of early gestation. The activity of cells in the mid-gestation was higher than that of late-gestation. The best time of AFSC acquisition was the mid-gestation.
Biological characteristics and configuration of AFSC after a long-term cultivation with 50th or more cell passage sustained stable when 15% FBS added in cell medium.
AFSC could be purified within 5th passage by mechanical separation combinated with defined digesting time with trypsin to remove the epithelioid cells from fibrous cells. These cells were shuttle-type, cell confluence directional, and the colony was vortex-like arrangement.
The result showed that Oct-4, Nanog, SH2, SSEA-1, CD117 and HLA-A were expressed in different passage AFSC by RT-PCR. AFSC were able to form the embryoid body in vitro, which express marker genes of three embryonic germ layers including fgf5 (ectoderm)、α-fetoprotein (endoderm) andζ-globin (mesoderm)。
AFSC were induced by dexamethasone,β-glycerophosphate and ascorbic acid. As a result, cells growth was reduced, the edges blurred and multilayer growth and nodules occurred over time. Three weeks after induction, the activity of alkaline phosphatase increased remarkably and Alizarin red staining showed red. These results showed that induction into osteoblasts have been successfully.
In summary, these results show that the AFSC obtained in this study have stem cell characteristic, strong proliferation and differentiation potency, and could be induced to differentiate into osteoblasts under certainly condition.
引文
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3 Sarkar S, Chang HC, Porreco RP, Jones OW. Neural origin of cells in amniotic fluid. [J]Am J Obstet Gynecol 1980;136 (1):67-72
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5 Gosden CM. Amniotic fluid cell types and culture.[J]Br Med Bull 1983;39 (4):348-354
6 Brock DJ. Amniotic fluid tests for fetal neural tube defects. [J]Br Med Bull 1983;39 (4):373-377
7 Smith AD. Amniotic-fluid acetylcholinesterase and neural-tube defects: plea for standardisation. [J]Lancet 1979;2 (8137):307-308
8 Chubb IW, Pilowsky PM, Springell HJ, Pollard AC. Acetylcholinesterase in human amniotic fluid: An index of fetal neural development? [J]Lancet 1979;1 (8118):688-690
9 Buamah PK, Taylor P, Ward AM. Concanavalin A binding of alpha-fetoprotein in amniotic fluid as an aid in the diagnosis of neural tube defects. [J]Clin Chem 1981;27 (10):1658-1660
10 Milunsky A. Clinical Genetics.[M] New York,Plenum Press,1979.215-220
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