不同条件下Percoll密度梯度离心分离组织细胞浮游生物的比较

A comparative research on planktons separated from liver by different density gradient centrifugation with Percoll in rabbits

目的实验评估不同条件下Percoll密度梯度离心分离组织中浮游生物的效果。方法大白兔肝组织匀浆后与3种藻类混合物混匀,分为混匀高速分离组、混匀低速分离组、叠加高速分离组、叠加低速分离组和未分离组(对照组)进行Percoll密度梯度离心分离浮游生物,镜检并提取其DNA,用PCR技术分别检测浮游生物16S rDNA和叶绿素基因特异性片段。结果混匀高速分离组有较多浮游生物呈分层条纹状分布于离心管中部,叠加高速分离组浮游生物紧邻组织细胞层下,低速分离组见较少浮游生物紧邻于组织细胞层之下,未分离组见浮游生物和组织细胞混杂沉于管底;经DNA检测,各分离组组织细胞层下液体均可检出浮游生物的1条447bp 16S rDNA片段及1条194bp叶绿体/叶绿素脱辅基蛋白基因片段,未分离组均未检出扩增产物且背景较深。结论混匀加样方式进行高速Percoll密度梯度离心,合并收集组织细胞层下所有液体,是分离浮游生物的最有效方法。

Objective To experimentally evaluate the effect of different density gradient centrifugation on plankton separation from tissues with Percoll. Methods Mixture of three algae species with the homogenate of rabbit liver were divided into 5 centrifugation groups： well-mixed homogenate at high-speed centrifugation, well-mixed homogenate at low-speed centrifugation, overlaying homogenate at high-speed centrifugation, overlaying homogenate at low-speed centrifugation, and mixted homogenate centrifugated at 12 000r/min as control group. The planktons were separated from the mixture by density gradient centrifugation with Percoll. After separation, the sediment of different layer was detected under microscope, and DNA was extracted. The specific fragment of algae 16S rDNA and chlorophyll-related gene were subsequently amplified by PCR. Results In well-mixed homogenate at high-speed centrifugation, the algae were localized in the middle of tube as stratified layers. In overlaying homogenate at high-speed centrifugation, algae were detected beneath the tissue layer. In homogenate at low-speed centrifugation, less algae were found closely under the layer of tissue, and for the control, algae was in the bottom of tube. With DNA analysis, a 447bp fragment of 16S rDNA and a 194bp fragment of chlorophyll-related gene were detected in sediments separated from all group with Percoll. However, in control homogenate mixed with algae, no amplified product was detected with darker background of electrophoresis gel because of the interference of tissue DNA. Conclusion It is recommended that tissues homogenate should be centrifugated coll, and sediments beneath the tissue layer at high-speed density gradient centrifugation with Pershould be collected for plankton isolation.