Bioassays for parathyrois hormone using clutures chondrocytes

1. 用兔肋生长软骨（RGC）细胞测定甲状旁腺激素（PTH）。在添加甲状旁腺激素4小时后，利用鸟氨酸脱羧酶诱导进行甲状旁腺激素的生物测定。在该方法中，将原代培养的RGC细胞悬浮在低渗标准缓冲液中，并与^<14>Cornithine直接孵育。在0.01 ~ 1 IU/ml范围内，PTH制剂的相似对数剂量依赖曲线呈线性关系。该方法测定的甲状旁腺素各种合成类似物和片段的活性与体内测定和肾阿苯基酸环化酶测定结果基本一致。添加PTH后2-5 min细胞内环AMP （cAMP）水平的刺激也被发现作为PTH生物测定的新系统。其半对数剂量依赖曲线在10^<-9>M和10^<-7>M之间呈线性关系。该方法测定的甲状旁腺激素的各种合成类似物和片段的活性与其他方法测定的结果一致。添加甲状旁腺激素（PTH）后cAMP水平的变化太快，不能作为同时检测许多样品的方法，但使用异丁基甲基肟胺（isobutylmethylxamthine）可以解决这个问题，它可以保持PTH刺激的cAMP水平至少30分钟。小鼠生长软骨pth应答性克隆细胞系的建立。从小鼠生长软骨二次培养的细胞系中分离出对甲状旁腺激素有应答的克隆细胞系。添加PTH后，cAMP水平的变化与RGC细胞原代培养相似。cAm@p水平变化的剂量反应源也与RGC细胞相似。然而，克隆细胞的刺激程度比RGC细胞要大。克隆细胞系现在处于传代50，被认为是不朽的。因此，该细胞系将对PTH的简单、简便和时间稳定的生物测定非常有用。少

1. Bioassay for parathyrois hormone (PTH) using rabbit costal growth cartilage (RGC) celle in culture. A simple procedure of bioassay of PTH using ornithine decarboxylase induction obsarved 4 hr after PTH addition was developed. In this method, RGC cells in primary cultyres were suspended in hypotonic standard buffer and directely incubated wirh^<14>Cornithine. The simi-log dose-dependent curve using PTH preparation of MRC research Standard A was linear between 0.01 to 1 IU/ml. The actibeties of various synthetic analogs and framents of PTH determined by this method almost corresponded to those obtained by in vivo assay and renal afenylate cyclase assat.The stimulation of intracelluar cyclic AMP (cAMP) level obsearved 2-5 min after the addition of PTH was also found to be used as a new system fro the bioassay of PTH. Its semi-log dose-dependent curve was linear bvetween 10^<-9>M and 10^<-7>M. The activities of various synthetic analogs and fragments of PTH determined by this method als … More o corresponded to those obtained by other assay. The change in cAMP level after PTH addition was too fast to be used as a procedure by which many samples were assayed at the same time, byt this problem was solved using isobutylmethylxamthine which maintained the PTH-stimulated cAMP level at least for 30 min.2. Establishment of a PTH-responsive clonal cell line from mouse growth cartilage. A clonal cell line that respond to PTH was isolated from a cell line established from secondary cultures of mouse growth cartilage. The change in the level of cAMP after addition of PTH to this clonal line was similar to that in primary cultures of RGC cells. The dose response vurce of change in the cAm@p level was also similat to that in RGC cells. However, ther extent of stimulation in the clonal cells was graster than that in RGC cells. The clonal cell line is now in passage 50 and is thought to be immortalized. Therefore, this cell line will be very useful for a simple, easy and time-sdabvign bioassay of PTH. Less