Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China.
Mol Biol Rep. 2013 Jan;40(1):327-36. doi: 10.1007/s11033-012-2064-3. Epub 2012 Oct 14.
Establishment of fibroblast cell lines of endangered goat breeds and research on the gene or protein functions based on the cells made a significant contribution to the conservation and utilization of genetic resources. In this study, a fibroblast cell line of Liaoning cashmere goat, frozen in 174 cryovials with 5 × 10(6) cells each, was successfully established from 60 goats ear marginal tissues using explant culture and cryopreservation techniques. Biological analysis of in vitro cultured cell line showed that, the cells were morphologically consistent with fibroblasts; the average viability of the cells was 94.9 % before freezing and 90.1 % after thawing; the growth process of cells was consisted of a lag phase, a logarithmic phase and a plateau phase; cell population doubling time was 65.5 h; more than 90 % of cells were diploid prior to the 6th generation; Neither microbial contamination nor cross-contamination was detected. To determine cell permeability, intracellular path and stability of exogenous proteins during the transduction, a TAT protein transduction domain was fused to the C-terminus of enhanced green fluorescent protein, the established fibroblast cell line was treated with the purified exogenous proteins at various concentrations by adding them to the cell culture media for 1-24 h and assayed cell morphology and protein presence, it was found that the purified exogenous proteins readily entered cells at a concentration of 0.1 mg/ml within 1.5 h and some of them could translocate into nucleus, moreover, the exogenous proteins appeared to be stable inside cells for up to 24 h.
建立濒危山羊品种的成纤维细胞系，并基于这些细胞研究基因或蛋白质功能，为遗传资源的保护和利用做出了重要贡献。本研究采用组织块培养和冷冻保存技术，从 60 只辽宁绒山羊耳缘组织中成功建立了 174 支冻存管（每管 5×10(6)个细胞）的辽宁绒山羊成纤维细胞系。体外培养的细胞系生物学分析表明，细胞形态上与成纤维细胞一致；细胞冻存前的平均存活率为 94.9%，解冻后为 90.1%；细胞生长过程包括潜伏期、对数生长期和平台期；细胞群体倍增时间为 65.5 小时；在第 6 代之前，超过 90%的细胞为二倍体；未检测到微生物污染和交叉污染。为了确定转导过程中外源蛋白的细胞通透性、细胞内途径和稳定性，在增强型绿色荧光蛋白的 C 端融合了 TAT 蛋白转导结构域，用纯化的外源蛋白处理建立的成纤维细胞系，通过将其添加到细胞培养基中 1-24 小时，检测细胞形态和蛋白存在情况。结果发现，在 0.1mg/ml 的浓度下，纯化的外源蛋白在 1.5 小时内很容易进入细胞，其中一些可以转位到细胞核内，此外，外源蛋白在细胞内稳定存在长达 24 小时。
