| 摘要: |
| 【目的】建立新生山羊肾小管上皮细胞(Renal tubular epithelial cell，RTECs)的分离培养方法。【方法】采集刚出生未哺乳健康山羊的肾脏，分别采用胶原酶Ⅰ和胶原酶Ⅳ消化法分离培养RTECs，选择孔径0.15 mm的筛网对RTECs进行纯化，观察各组RTECs的生长情况；对分离的RTECs进行免疫组化和碱性磷酸酶染色鉴定，并进行透射电镜观察；用流式细胞仪检测RTECs的增殖周期和凋亡情况。【结果】胶原酶Ⅳ对RTECs的消化效果优于胶原酶Ⅰ，所得的细胞团块多，纯度高，细胞生长快，经孔径0.15 mm的筛网过滤后，RTECs的纯度达95%以上，并可连续传至第12代。分离获得的细胞经免疫组化法和碱性磷酸酶染色法鉴定呈阳性；透射电镜观察显示，细胞表面有微绒毛和桥粒连接。用流式细胞仪检测的结果显示，第4代新生山羊RTECs中G1期和S期细胞分别占细胞总数的25.01%和74.99%，G2/G1为1.97%，第10代细胞中G1期细胞和S期细胞均占50.00%，G2/G1为1.88%；第4代细胞的早期凋亡率在0.32%左右，而第10代可达3.7%，远高于第4代细胞。【结论】建立了增殖快、细胞活力高的山羊RTECs分离培养方法。 |
| 关键词: 新生山羊肾小管上皮细胞 原代培养 酶消化 纯化 |
| DOI： |
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| 基金项目:陕西省重大科技攻关项目(2006kz07-G2) |
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| Culture and identification of renal tubular epithelial cells of newborn goat |
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| Abstract: |
| 【Objective】The aim of study was to establish the culture method of goat renal tubular epithelial cells.【Method】Renal tubular epithelial cells was digested by collagenaseⅠ and collagenase Ⅳ.0.15 mm aperture sieve was used to purificate renal tubular epithelial cells,and growth curvature of RTECs was observed.The isolated RTECs were identificated by immunohistochemistry,alkaline phosphatase assay and observed with transmission electron microscopy.Cell program death and cell cycle were measured with flow cytometer.【Result】The results showed that,the collagenase Ⅳ digestion was better than collagenaseⅠdigestion and a number of pure and highly active cell aggregates were obtained.Filtered by 0.15 mm aperture sieve,the purity of renal tubular epithelial cells reached more than 95%,and cells could be successively propagated twelve times.The isolated cells were identified as cells of epithelial origin by immunohistochemistry and alkaline phosphatase assay.Electron microscopy analysis of the ultrastructural characteristics of primary cultures of renal tubular epithelial cells showed the presence of microvillus and bridge corpuscle.By flow cytometer,the results showed that the No.4 generation RTECs was 25.01% in G1 and 74.99% in S,G2/G1 was 1.97%.Meanwhile,the No.10 generation RTECs was 50.00% in G1 and S,G2/G1 was 1.88%;the apotosis rate of the No.10 generation RTECs reached 3.7%,which was much higher than the No.4 generation cells with 0.32%.【Conclusion】A method of culturing renal tubular ephithelia cells was developed in this research.The cultured cells showed a high activity and proliferation. |
| Key words: renal tubular epithelial cell of newborn goat primary culture enzymic digestion purification |
