应用MTT法观察人皮肤培养细胞的形态学特征

The investigation on the way to obtain the histological information of the cells during performing the MTT method

目的：在创面愈合细胞生物学研究时，应用ＭＴＴ比色法测定细胞活性和生长增殖；探讨及时观察细胞生长状态下的形态变化的可行性．方法：人角朊细胞（ＨＫ）和人真皮成纤维细胞（ＨＤＦ）不同生长密度时加入ＭＴＴ溶液后，观察不同时间点细胞的形态，测定细胞内形成镜下可见蓝色甲月赞及胞外形成针状结晶的时间；比较镜下观察时间长短对ＭＴＴ比色法最终测定值的影响．结果：在不同生长密度条件下，加入ＭＴＴ溶液后，ＨＫ和ＨＤＦ置于３７℃孵育，分别于１０ｍｉｎ及５ｍｉｎ即可形成细胞内镜下可见的蓝色甲月赞结晶物，并随孵育时间延长而增加．该活细胞染色法，可将处在培养生长状态的活细胞清晰地显现出来；在９０ｍｉｎ～６０ｍｉｎ及１２０ｍｉｎ～９０ｍｉｎ时，两种细胞分别形成胞外针状结晶，干扰细胞形态的进一步观察；不论细胞密度高低，标准实验室环境内进行短时间（≤１５ｍｉｎ）观察细胞形态及拍照记录均不对ＭＴＴ比色法测定细胞ＨＫ及ＨＤＦ的活性及生长增殖结果产生影响．结论：采用ＭＴＴ法测定培养的人皮肤附壁生长细胞活性及生长增殖量，可于适当时间内清楚地观察细胞生长状态；普通倒置显微镜下进行短时间的观察不会影响ＭＴＴ法的最终测定值．该方法简单、实用．

AIM: To investigate the available way, by which the morphological characteristics of living cells in culture could be observed easily during performance of MTT assay. METHODS: The normal human keratinocytes (HK) and dermal fibroblasts (HDF) seeded in different densities were observed under the ordinary inverted microscope in different time points after MTT solution was added to the cells. The times were measured when intracellular formazan and extracellular crystal appeared during the process in performing MTT assay. The possible influences upon the final colorimetric results caused by the observation time were also investigated further. RESULTS: The observable blue formazan appeared in HK and HDF at 10 min and 5 min respectively after MTT solution was added to the cells. With incubation time progressing, the living cells were showed very clearly by the formazan. The needle shape crystals were formed extracellularly from 60 min to 90 min in HK and from 90 to 120 min in HDF when the MTT reacted with the media out the cultivated cells, which interfered in the cells, appearances. A short period of observation time (less than 15 min) didn′t resulted in significant influence on the final colorimetric results measured by MTT assay used in the measurements of both activity and proliferation rate of HK or HDF. And during the observation, photographs could be taken under the ordinary inverted microscope in an environment of standard laboratory. CONCLUSION: According to the mechanism that the observable blue formazan was reduced by the mitochondrial dehydrogenase, so the formazan should be produced only in living cells. The morphologic characteristics of living cells could be seen clearly under an ordinary microscope during the performance of MTT assay. A suitable period of observation time doesn′t affect the measurement results by MTT assay. This method is easy to be adopted, which could add valuable morphologic information to analyze the whole experimental results.