TNF-α对大鼠Leydig细胞的作用及机制研究

Studies on the effects of TNF-α on rats' Leydig cells and the mechanism of action

目的 观察TNF-α对睾丸细胞功能的影响并探讨其可能机制。方法 通过Percoll不连续密度梯度分离获取大鼠Leydig细胞进行原代培养。HE染色进行细胞形态学观察。Leydig细胞培养48h后在培养液中分别加入不同浓度的大鼠TNF-α，使其终浓度达到0．1、1、10、100ng／ml，分别在培养的第1、2．3、4天取上清，通过放免法测定上清液中睾酮的含量，应用MTT法测定细胞增殖情况，流式细胞术与丫啶橙（AO）染色检测及观察Leydig细胞凋亡情况。结果 原代细胞通过提纯后的细胞纯度可达70％～80%。HE染色后可见细胞胞质含量丰富，含有分泌颗粒，胞核圆。TNF-α在0．1～100ng／ml浓度范围内能呈剂量依赖的方式抑制Leydig细胞睾酮的基础分泌。0．1、1、10、100ng／ml TNF-α作用24h后，对Leydig细胞睾酮分泌量的抑制率分别为22．03％、34．98％、52．95％、74．83％。各组Leydig细胞睾酮的分泌量随时间延长呈减少的趋势，但除100ng／ml组外，其他各组各时间点比较差异无统计学意义。高浓度TNF-α（10、100ng／m1）组具有抑制Leydig细胞增殖和促进其凋亡作用，其增殖抑制率分别达到38．35％±4．17％、76．35％±8．65％，而凋亡率分别为13．23％±1．11％、26．43％±5．82％，与对照组比较有统计学意义（P〈0．01）。AO染色见高浓度TNF-α（10、100ng／ml）组出现明显的细胞凋亡。结论TNF-α对Leydig细胞睾酮的基础分泌具有抑制作用，在较高浓度时该作用可能与其抑制Leydig细胞增殖并促进细胞凋亡有相关。

Objective To explore the effects of TNF-α on the function of Leydig cells, in order to elucidate the possible mechanisms involved. Methods Highly pure primary Leydig cells were obtained by Percoll discontinuous density gradient method. HE stain was used to observe the morphology of the cultured cells. The Leydig cells were treated with different doses of rat TNF-α （0. 1, 1, 10, 100 ng/ml） for 48h, and then the supernatants of culture medium were collected every 24 hours at the 1st, 2nd, 3rd and 4th day. The testosterone level in the supernatant was measured by mdioimmunoassay. The proliferation and TNF-α-induced apoptosis of Leydig cells were examined by MTT assay, flow cytometry （FCM） as well as acridine orange （AO） stain. Resulls The purity of Leydig cells was 70% -80% after purification with Percoll discontinuous density gradient method. The Leydig cells were rich in cytoplasm, which contained some secretory granules and round nucleus. After TNF-α treatment in different concentrations （0. 1, 1, 10, 100ng/ml） for 24h, the inhibition rates of TNF-α on testosterone secretion of Leydig cells were 22. 0%, 35.0%, 53.0% and 74. 8%, respectively, and the decrease showed a time-dependent manner, but no statistically significant difference was found in each group at every time point except for 100ng/ml group. High concentration of TNF-α （10 and 100 ng/ml） could inhibit the proliferation and promote apoptosis of Laydig cells. Compared to control group, the inhibition rate and apoptosis rate in 10ng/ml and 100ng/ml group showed significant difference （respectively 38. 4%±4. 1%, 76. 4%±8. 7% and 13.2%±1.1 %, 26. 4 %± 5.8 %）. In addition, significant apoptosis could be seen in the high concentration groups as shown with AO staining. Conclusion The present study suggests that TNF-α can inhibit the basal testosterone secretion of Leydig cells, which might be related to the inhibition and apopotosis-induced effects of high concentration of TNF-α.