摘要
目的:探索建立肿瘤坏死因子诱导蜕膜细胞凋亡模型,观察黄芩苷对早孕蜕膜细胞凋亡的影响,初步分析黄芩清热安胎作用机制。方法:实验于2006-04/09在湖南中医药大学国家重点二级实验室病理生理实验室完成。取湖南中医药大学第一附属医院门诊人流手术室正常妊娠40d健康孕妇人流组织,患者知情同意。体外常规进行蜕膜细胞培养,选取经鉴定的4、5代细胞蜕膜细胞,用不同浓度(0.5,2.0,10.0,50.0μg/L,共设4组,并设空白对照组)的肿瘤坏死因子α作用于蜕膜细胞,四甲基偶氮唑盐比色实验分析肿瘤坏死因子α对蜕膜细胞活力的影响,荧光细胞染色观察确定蜕膜细胞凋亡模型的建立。选取适宜浓度的黄芩苷作用于正常蜕膜细胞及肿瘤坏死因子α诱导的凋亡蜕膜细胞(设肿瘤坏死因子α模型组,黄芩苷组,肿瘤坏死因子α加黄芩苷组,并设空白对照组),培养24h后,四甲基偶氮唑盐比色实验分析黄芩苷对蜕膜细胞活力的影响,流式细胞术检测细胞凋亡率。结果:实验成功获取足够量的细胞,经泌乳素免疫组化鉴定为较高纯度的蜕膜细胞。①肿瘤坏死因子α作用后的蜕膜细胞活性均低于空白对照组,并且随着肿瘤坏死因子α浓度越大,活力越小。10.0,50.0μg/L肿瘤坏死因子α作用后蜕膜细胞活性与空白对照组比较,差异有显著性意义[(0.196±0.040),(0.106±0.020),(0.317±0.020),P<0.05]。但50.0μg/L时经形态学观察有部分细胞漂浮、死亡。②肿瘤坏死因子α模型组蜕膜细胞活性低于黄芩苷组和肿瘤坏死因子α加黄芩苷组,差异具有显著性意义[(0.27±0.03),(0.49±0.01),(0.38±0.02),P<0.05]。③流式细胞术示空白对照组蜕膜细胞凋亡率低于其他各组,差异均有显著性意义[(1.48±0.45)%,(16.40±0.82)%,(9.78±0.26)%,(10.96±0.92)%,P<0.05]。在荧光显微镜下,肿瘤坏死因子α作用后凋亡的蜕膜细胞细胞核变小皱缩,可见致密强荧光。结论:肿瘤坏死因子α能明显抑制人蜕膜细胞增殖分裂的过程,诱导该细胞凋亡,可用于人蜕膜细胞凋亡模型的建立。而黄芩苷对肿瘤坏死因子α诱导的蜕膜细胞凋亡具有一定对抗抑制作用。
AIM： To explore the establishment of decidual cells apoptotic model induced by tumor necrosis factorα （TNF-α）, and observe the effect of baicalin on decidual cells apeptosis, so as to preliminarily analyze the action mechanism of baicalin to clear heat and prevent miscarriage.
METHODS： The experiment was performed at the National Second Key Laboratory of Pathophysiology, Hunan University of Traditional Chinese Medicine from April to September 2006, The abortion tissues were harvested from the normal pregnant woman for 40 days of First Hospital of Hunan University of Traditional Chinese Medicine, and the informed consent was obtained. Human decidual cells were cultured in vitro, and the fourth or fifth generation cells were selected after immunohistochemistry identification, and induced by TNF-α with different concentrations （0,5, 2.0, 10.0, 50.0 μg/L and the control group was set up）, After cultured for 24 hours, the effect of TNF-α on the activity of decidual cells was examined by MTT assay, and fluorescent microscope was used to determine the establishment of apoptotic cells model. The baicalin with appropriate concentration was selected to induce normal decidual cells and apeptotic cells, in which there were TNF-α group, baicalin group, TNF-α plus baicalin group （integrated group） and blank control group, After cultured for 24 hours, the influence of baicalin on the activity of decidual cells was examined by MTT assay, and the apoptosis rate was examined by flow cytometry.
RESULTS： Enough decidual cells were cultured successfully, which were high purified decidual cells identified by prolactin immunohistochemistry, （1）The cell activity after induced by TNF-α of different concentrations was lower than the blank control group, and the activity was reduced with the increases in concentration of TNF-α, There were significant differences in the cell activity between the 10.0 and 50.0 μg/L TNF-α and black control group [（0.196±0.04）, （0.106± 0.02）, （0.317±0.02）, P 〈 0.05], but some floated and died cells were found in 50.0 μg/L TNF-α group in morphological observation, （2）The decidual cell activity of TNF-α model groups was lower than the baicalin group and the integrated group significantly [（0.27±0.03）, （0.49±0.01）, （0.38±0.02）, P 〈 0,05]. （3）Flow cytometry analysis showed that the apoptosis rate of the blank control group was lower than other groups, and the difference had markedly statistical significance [（1.48±0.45）%, （16.4±0.82）%, （9.78±0.26）%, （10.96±0.92）%, P 〈 0.05]. Under fluorescent microscope, the cell nucleus of apoptotic decidual cells induced by TNF-α were shrink and crenulation, and hyperfluorescence was observed.
CONCLUSION： TNF-α could induce human decidual cell apoptosis by restraining the course of multiplicative division obviously, so it could be used to establish the human decidual cells apoptotic model. Baicalin could restrain or resist decidual cell apoptosis induced by TNF-α.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2007年第19期3793-3796,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
国家自然科学基金项目(30472227)~~
