TNF-α和ROS交互作用对受照射角质形成细胞CCL27分泌的影响

发布时间：2019-02-26 13:58

【摘要】：目的：放射性皮肤损伤是常见的由射线引起的皮肤炎性反应,其中急性放射性皮肤损伤是肿瘤放射治疗中最常见的并发症,约90%以上的放疗病人会出现红斑及以上皮肤反应,可引起局部感染,疼痛,严重情况下导致治疗中断,造成肿瘤控制率和治愈率的降低。在皮肤的免疫炎性反应中,趋化因子在T淋巴细胞归巢的多步模式中起到了重要作用。CCL27(CC chemokine ligand 27,CCL27)是一种小分子分泌蛋白趋化因子,其分布具有组织和细胞特异性,主要由皮肤组织中的角质形成细胞分泌。因此,本研究采用人角质形成细胞,HaCaT细胞,经X射线照射后,观察辐射对CCL27分泌的影响。方法：本研究采用HaCaT细胞,经2Gy和5Gy的X射线照射后,用DCFH-DA探针孵育,检测细胞内活性氧(reactive oxygen species,ROS)的水平。同时采用ELISA方法,检测细胞培养液中CCL27的浓度和肿瘤坏死因子(Tumor necrosis factor-a,TNF-a)的浓度。继而以活性氧(ROS)清除剂5mM的N-乙酰半胱氨酸(N-Acetylcysteine, NAC)预处理细胞,经2Gy和5Gy的X射线照射后,分别检测细胞内ROS的浓度以及培养液中TNF-α、CCL27的浓度。继而分别用H2O2、TNF-α预处理细胞,检测细胞培养液中CCL27的浓度。此外,以脂质体为载体,将TNF-α siRNA转染至HaCaT细胞中,经照射后,用DCFH-DA探针检测细胞内ROS的表达含量。同时,单独采用H2O2处理细胞,经ELISA检测细胞培养液中TNF-α的浓度。最后,以外源性的TNF-α处理细胞,检测细胞内ROS的含量。结果：受照射细胞内ROS水平增高,培养液中的TNF-α和CCL27的浓度增加。经NAC预处理后,受照细胞培养液中的RCS、TNF-α以及CCL27的浓度下降。经H202和TNF-α分别处理后,细胞培养液中CCL27浓度升高。将siRNA转入细胞后,经X线照射,细胞内ROS的水平降低。H2O2可以促进细胞培养液中TNF-α浓度的增加。反之,用外源性的TNF-α处理细胞也可以导致细胞内ROS的水平升高。结论：辐射能够诱导角质形成细胞对CCL27分泌的增加。其主要作用机制与TNF-α与ROS的交互作用有关。对放射性皮肤损伤的研究具有一定价值。
[Abstract]:Objective: radiation-induced skin injury is a common skin inflammatory reaction caused by radiation, among which acute radiation-induced skin injury is the most common complication in radiotherapy of tumors. More than 90% of radiation-treated patients will have erythema or more skin reactions. Can cause local infection, pain, serious cases lead to treatment interruption, resulting in the reduction of tumor control rate and cure rate. Chemokine plays an important role in the multi-step pattern of T lymphocyte homing in skin immune inflammatory reaction. CCL 27 (CC chemokine ligand 27 (CCL 27) is a small molecule secretory protein chemokine whose distribution is tissue-and cell-specific. It is mainly secreted by keratinocytes in the skin tissue. In this study, human keratinocytes and HaCaT cells were irradiated by X-ray to observe the effect of radiation on CCL27 secretion. Methods: HaCaT cells were exposed to X-ray irradiation of 2Gy and 5Gy and incubated with DCFH-DA probe to detect the level of reactive oxygen species (reactive oxygen species,ROS) in the cells. At the same time, the concentration of CCL27 and tumor necrosis factor (Tumor necrosis factor-a,TNF-a (TNF-(Tumor necrosis factor-a,TNF-a) in cell culture medium were detected by ELISA. Then the cells were pretreated with N-acetylcysteine (NAC) of reactive oxygen species (ROS) scavenger 5mM. After X-ray irradiation with 2Gy and 5Gy, the concentration of ROS in the cells and the concentrations of TNF- 伪 and CCL27 in the culture medium were measured respectively. Then the cells were pretreated with H _ 2O _ 2 and TNF-伪 respectively to detect the concentration of CCL27 in the culture medium. In addition, TNF- 伪 siRNA was transfected into HaCaT cells with liposome as vector. After irradiation, the expression of ROS in HaCaT cells was detected by DCFH-DA probe. At the same time, the cells were treated with H2O2 alone, and the concentration of TNF- 伪 in the culture medium was detected by ELISA. Finally, the cells were treated with exogenous TNF- 伪 to detect the content of ROS in the cells. Results: the level of ROS in irradiated cells increased, and the concentration of TNF- 伪 and CCL27 in culture medium increased. After pretreatment with NAC, the concentration of RCS,TNF- 伪 and CCL27 in the culture medium of irradiated cells decreased. After treated with H2O2 and TNF- 伪 respectively, the concentration of CCL27 in the culture medium increased. After siRNA was transferred into the cells, the intracellular ROS level was decreased by X-ray irradiation. H2O2 could promote the increase of TNF- 伪 concentration in the cell culture medium. On the other hand, exogenous TNF- 伪 treatment can also lead to the increase of intracellular ROS level. Conclusion: radiation can induce the increase of CCL27 secretion in keratinocytes. The main mechanism of action is related to the interaction between TNF- 伪 and ROS. It has some value in the study of radiation skin injury.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R730.55

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