酶释放法用于细胞介导的细胞毒试验问题分析

目的 :检验分析酶释放法用于细胞毒试验存在的问题。方法 :用51Cr释放法和MTT比色法、NAG比色法 ,单独或相互比较 ,检验分析细胞毒试验中效靶细胞相互作用规律 ,酶在细胞代谢中分布情况 ,细胞死伤前后酶释放特点。结果 :以MTT为底物的酶在细胞代谢中主要分布在细胞膜表面 ,而以NAG为底物的酶分布在细胞内。细胞有自主释放酶的功能 ,正常细胞与受损伤细胞释放酶速度不同。细胞毒作用过程中效应细胞也有损伤 ,也有酶释放 ,与靶细胞死伤后释放的酶属于同一类 ,而且混合在一起 ,目前尚无可行的定量分离和鉴别方法。酶释放法用于细胞毒试验 ,对于不同状态的标本检验结果没有规律性 ,不能反映机体免疫功能状态。结论 :酶释放法不宜用于细胞介导的细胞毒试验。

The analysis of the problems of enzyme release way used for cell mediated cytotoxicity assay

Objective:?To examine and analysis the problems revealed in the enzyme release way used in cytotoxicity assay, and to pose better some improvement. Methods: 51 Cr release way and the colorimetric assay of MTT and NAG used in the experiment of cell metabolism and cytotoxicity assay. Results:?It is found that a some of enzymes are distributed on surface of cell membrane, such as the mitochondrial enzyme succinate dehydrogenase, and an others are distributed inside cell membrane, such as N acely β D amino caproic carbohydrase, and all of them can be secreted out of cells through cellular metabolism. The effective cell can be killed yet to release enzyme, and speed of the enzyme released from injured cells is faster than healthy cells. The enzymes released from effective cells and target cells are mixed together ,and to identify and quantify them are very difficult. It is not relativity that the activities of NK cell tested by enzyme release way compared with by 51 Cr release way. Conclusion:?The enzyme release way used for cytotoxicity assay is not appropriate.