Wright W E, Hayflick L
Fed Proc. 1975 Jan;34(1):76-9.
Mass populations of normal human lung fibroblasts were enucleated by centrifugation at greater than or equal to 25,000 g in 4 mug/ml cytochalasin B. The 1 per cent of cells that did not enucleate where rendered nonviable by treatment with mitomycin C. Whole cells were poisoned with a 99 per cent lethal dose of the sulfhydryl reagent iodoacetate. The washed cells were then mixed with the anucleate cytoplasms, fused with inactivated Sendai virus, and planted in rotenone for 20 hours. Whereas normal cells are able to survive this rotenone treatment, the 1 per cent surviving iodoacetate-treated cells cannot withstand this additional stress. However, iodoacetate treated cells that fuse to untreated cytoplasms receive sufficient amounts of active enzymes to allow them to survive. Since this selective system does not rely on using enzymatic mutants, it should permit the selection of hybrids between anucleate cytoplasms and any type of whole cell. Cytoplasmic hybrids were cultured in order to determine their proliferative capacity. The life-spans of cytoplasmic hybrids between young and old cells were compared to those of young/young and old/old controls. Cytoplasmic factors do not appear to control in vitro cellular senescence.
将大量正常人肺成纤维细胞在含有4μg/ml细胞松弛素B的条件下，以大于或等于25,000g的离心力去核。未去核的1%细胞用丝裂霉素C处理使其失去活力。用99%致死剂量的巯基试剂碘乙酸对完整细胞进行毒害。然后将洗涤后的细胞与无核细胞质混合，用灭活的仙台病毒进行融合，并在鱼藤酮中培养20小时。正常细胞能够在这种鱼藤酮处理下存活，而1%存活的经碘乙酸处理的细胞无法承受这种额外的压力。然而，与未处理的细胞质融合的经碘乙酸处理的细胞会获得足够量的活性酶，从而使其能够存活。由于这种选择系统不依赖于使用酶突变体，它应该允许选择无核细胞质与任何类型的完整细胞之间的杂种。培养细胞质杂种以确定其增殖能力。比较了年轻细胞与老年细胞之间的细胞质杂种以及年轻/年轻和老年/老年对照的寿命。细胞质因子似乎并不控制体外细胞衰老。
