豹猫体细胞建系及其生物学特性分析

Establishment of Fibroblast Cell Lines from Leopard Cat and Their Biological Characteristics

研究体外培养豹猫(Prionailurus bengalensis)细胞的形态、细胞贴壁率、冷冻存活率、生长曲线及细胞核型等生物学特性,为深入开展豹猫基因组学及濒危野生动物保护提供依据。本实验选择豹猫3种组织,即剑状软骨、心和肺,采用组织块贴壁培养法进行体细胞的原代培养;酶消化法完成细胞的传代培养;程序降温完成细胞的冷冻保存;通过细胞计数法计数细胞冷冻存活率;绘制细胞生长曲线;采用常规染色体标本制备技术,对豹猫的染色体核型及G带进行分析。细胞原代培养结果显示,剑状软骨组织在培养第3天出现纤维样细胞、培养6~7 d铺满培养瓶;心组织在培养第5天出现上皮样细胞、12 d铺满培养瓶;肺组织在培养4 d后出现成纤维细胞、8~9 d铺满培养瓶。3种来源体细胞均显示成纤维细胞特征,剑状软骨源细胞最易贴壁、肺源细胞次之、心源细胞最弱。对比3种不同来源体细胞从6代(P6)至12代(P12)冷冻存活率,剑状软骨源细胞冷冻存活率显著下降(冻存前91.0%~97.6%,冻存后76.8%~85.5%,P<0.05),心源细胞冷冻存活率亦显著下降(冻存前82.7%~88.1%,冻存后43.7%~80.5%,P<0.05),肺源细胞冷冻存活率有下降趋势,但无显著差异(冻存前83.4%~96.8%,冻存后73.9%~93.3%,P>0.05)。生长曲线分析显示,3种体细胞均呈"S"型,其中剑状软骨源细胞增殖最快、肺源细胞次之、心源细胞最慢。核型分析结果显示,3种不同来源的成纤维体细胞染色体数目均为2n=38,18对为常染色体,形态类型为6m+10sm+2st,1对为性染色体,X染色体形态类型为m。本研究建立了3种组织来源的豹猫成纤维细胞建系技术及体细胞系,揭示了该物种成纤维细胞的基本生物学特性,为动物遗传信息研究及豹猫保护提供了重要的实验材料和基础信息。

In order to study Leopard Cat（Prionailurus bengalensis） genomics and protect endangered Leopard Cat, the biological characteristics including the morphology, the adherence rate, the frozen survival rate, the growth curve and the cell karyotype of in vitro cultured fibroblast cells were investigated. Three types of Leopard Cat tissues of cartilage, heart and lung were collected. The adhesive culture with tissue block was carried out for the primary culture of somatic cells, and then subculture was conducted after digestion by the trypsin. The frozen cell viability was calculated by cell counting method and the growth curve was made. The chromosome karyotype and G band were analyzed by normal chromosome specimen preparation technology. Fibrod cells were observed from D3 of cartilage tissue block primary culture and they overspread all the bottom surface of cultured flask by Day 6﹣7, while these events took place by D5 and D12 of heart tissue culture, and by D4 and D8﹣9 of lung tissue culture（Fig. 1）. The cells derived from three organs or tissues all exhibited characteristics of fibroblast cells（Fig. 2, 3）. The adherence speed following culture showed lowest to fastest from the heart-derived cells, lung-derived cells and cartilage-derived cells, respectively（Fig. 4）. Comparing the frozen survival rates of three different sources of somatic cells, we showed significant reduction from P6 to P12 in both cartilage-derived cells（P6﹣P12： 91.0%﹣97.6% vs. 76.8%﹣85.5%, P〈0.05） and the heart-derived cells（P6﹣P12： 82.7%﹣88.1% vs. 43.7%﹣80.5%, P〈0.05）. Lung-derived cells also showed similar trend but no significant difference was observed from P6 to P12（P6﹣P12： 83.4%﹣96.8% vs. 73.9%﹣93.3%, P〉0.05）（Table 2）. The growth curve analysis of three types of fibroblast cells showed an ＂S-model＂, the cartilage-derived cells proliferated faster, next by the lung-derived cells, and the cardiac-derived cells proliferated slower（Fig. 5）. The results of karyotype analysis showed that the number of chromosomes of all three different sources of fibroblast cells were 2 n = 38（Fig. 6）, including 18 pairs of autosomes with 6 m ＋ 10 sm ＋ 2 st types, one pair of sex chromosomes, and X chromosome was m type（Fig. 7, Table 3）. This study established three fibroblast cell lines derived from three tissues of leopard cat, and analyzed their biological characteristic and karyotype. The results provide important biological information for future genetic research and species protection of leopard cat.