CAJ | 학술논문

利用改良后的Percoll连续密度梯度分离方法，分离不同酵母菌株稳定期的同步细胞，并在单细胞水平上，应用激光光镊拉曼光谱对分离的同步细胞进行鉴别。采用高速离心机角式转头和2 mL聚丙烯离心管，20℃，19320 g离心15 min ，将大约1.75 mL Percoll溶液形成1.00～1.31 g · mL －1连续密度梯度；将样品均匀铺加在离心管连续密度梯度液顶层，20℃，400 g离心60 min ，酵母分成明显的2层细胞带；微分干涉差显微镜（DIC ）、相差显微镜以及同步生长曲线分析显示，下层酵母细胞以单个细胞为主，大小均匀、致密，折光性强，生长呈现阶梯式增长，具有同步细胞生长的特性，确定其为同步的G0细胞。利用单细胞光镊拉曼光谱系统对所分离的G0同步细胞与非G0细胞进行分析，结果显示G0同步细胞和非G0细胞的特征峰位置基本一致，但G0细胞对应的蛋白质、糖类、核酸等生物大分子特征峰强度大于非G0细胞，说明G0细胞大分子物质含量要比非G0细胞的高；对G0细胞和非G0细胞进行主成分分析，结果显示，分离的同步化G0细胞之间成分含量差异少，比较均一，而非同步细胞之间内含物质差异大，呈不均一性，说明单细胞光镊拉曼光谱系统可以鉴别同步和非同步细胞。改良后的Percoll连续密度梯度分离方法，能够分离多数酵母菌株同步化细胞，是一种易操作、成本低、效率高的细胞分离方法。
이용개량후적Percoll련속밀도제도분리방법，분리불동효모균주은정기적동보세포，병재단세포수평상，응용격광광섭랍만광보대분리적동보세포진행감별。채용고속리심궤각식전두화2 mL취병희리심관，20℃，19320 g리심15 min ，장대약1.75 mL Percoll용액형성1.00～1.31 g · mL －1련속밀도제도；장양품균균포가재리심관련속밀도제도액정층，20℃，400 g리심60 min ，효모분성명현적2층세포대；미분간섭차현미경（DIC ）、상차현미경이급동보생장곡선분석현시，하층효모세포이단개세포위주，대소균균、치밀，절광성강，생장정현계제식증장，구유동보세포생장적특성，학정기위동보적G0세포。이용단세포광섭랍만광보계통대소분리적G0동보세포여비G0세포진행분석，결과현시G0동보세포화비G0세포적특정봉위치기본일치，단G0세포대응적단백질、당류、핵산등생물대분자특정봉강도대우비G0세포，설명G0세포대분자물질함량요비비G0세포적고；대G0세포화비G0세포진행주성분분석，결과현시，분리적동보화G0세포지간성분함량차이소，비교균일，이비동보세포지간내함물질차이대，정불균일성，설명단세포광섭랍만광보계통가이감별동보화비동보세포。개량후적Percoll련속밀도제도분리방법，능구분리다수효모균주동보화세포，시일충역조작、성본저、효솔고적세포분리방법。
A modified procedure of Percoll density gradient centrifugation was developed to isolate and fractionate synchronous cells from stationary phase (sp) cultures of different yeast strains ,as well as Raman spectra discrimination of single yeast cells was reported .About 1.75 mL Percoll solution in 2 mL polypropylene centrifugal tube was centrifuged at 19 320 g ,20 ℃ with an angle rotor for 15 min to form continuous densities gradient (1.00~1.31 g · mL -1 ) ,approximately 100μL sample was overlaid onto the preformed continuous density gradient carefully ,subsequently ,centrifuged at 400 g for 60 min in a tabletop centrifuge equipped with a angle rotor at 25 ℃ .Yeast samples could be observed that the suspensions were separated into two cell fractions obviously .Both fractions of different yeast strains were respectively determined by differential interference contrast (DIC ) , phase contrast microscope and synchronous culture to distinguish their morphological and growth trait .The results showed that the lower fraction cells were unbudded ,mostly unicellular ,highly refractive ,homogeneous and uniform in size ,and represented growth characteristic synchronously ;Their protoplasm had relatively high density ,and contained significant concentrations of glycogen ;all of which were accordant with description of quiescent yeast cells and G 0 cells in previously published paper .It was shown that lower fraction was quiescent cells ,synchronous G0 cells as well .A Raman tweezers setup was used to investigate the differences between two fractions ,G0 cells and non G0 cells ,at a single cell level .The result showed that both G0 cells and the non G0 cells had the same characteristic peaks corresponding biological macromolecules including proteins ,carbohydrates and nu‐cleic acids ,but all characteristic peak intensities of G0 cells were higher than that of non G0 cells ,implied that the macromolecu‐lar substance content of G0 cells was more higher .Principal component analysis (PCA) was performed between G0 cells and non G0 cells ,the results showed that the chemical composition content among the synchronization G 0 cells has less difference ,and G0 cells were homogeneous but non G0 cells were heterogeneous ,indicating single cell optical tweezers Raman spectroscopy could identify the synchronous and asynchronous cells .The modified method is feasible ,economical and efficient highly .G0 synchro‐nous cells of most yeast strains could be isolated by a modification of Percoll density gradient centrifugation .