PREPARATION OF RAT-LIVER CELLS .3. ENZYMATIC REQUIREMENTS FOR TISSUE DISPERSION
PREPARATION OF RAT-LIVER CELLS .3. ENZYMATIC REQUIREMENTS FOR TISSUE DISPERSION
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DOI：
10.1016/0014-4827(73)90357-1
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发表时间：
1973-01-01
影响因子：
3.7
通讯作者：
SEGLEN, PO
中科院分区：
文献类型：
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作者：
SEGLEN, PO
分离的灌流大鼠肝脏通过两步去除钙离子(可能包括去除依赖于钙的黏附因子)和随后的酶处理来分散。胶原酶，粗的或纯化的，将所有的实质组织转化为95%完好无损的细胞悬浮液。其他酶如透明质酸酶、溶菌酶和胰酶无效。由于酶处理过程中的低氧条件不会影响细胞的活性，因此可以省去氧合装置以方便无菌细胞的制备，通过差速离心法纯化实质细胞，将非实质细胞的污染从最初的10-20%减少到最终的1-2%，实质细胞恢复了40%，活力没有损失。纯化的细胞有20%的双核，其生化组成与完整的肝组织相同。细胞活跃于合成RNA、蛋白质、糖原和各种代谢物，对类固醇和多肽激素敏感。许多新分离的细胞有明显的收缩皱纹，孵育后这些皱纹消失。
Isolated perfused rat livers were dispersed by a two-step procedure of Ca2+removal (probably including the removal of a Ca2+-dependent adhesion factor) followed by enzymatic treatment. Collagenase, crude or purified, converted all of the parenchymal tissue to a suspension of cells which were 95% intact. Other enzymes such as hyaluronidase, lysozyme and trypsin were ineffective. Since hypoxic conditions during enzymatic treatment did not affect cellular viability, the oxygenation apparatus can be omitted to facilitate sterile preparation of cells.Purification of the parenchymal cells by differential centrifugation reduced contamination by non-parenchymal cells from 10–20% initially to 1–2% finally, with 40% of the parenchymal cells recovered and no loss in viability. The purified cells were 20% binucleated and had the same biochemical composition as intact liver tissue. The cells were active in the synthesis of RNA, protein, glycogen and various metabolites, and showed sensitivity towards steroid and polypeptide hormones. Many of the freshly isolated cells had marked constriction furrows which disappeared upon incubation.