The Mechanisms Of Ionizing Radiation Induced Bystander Responses In Skin Fibroblast Cells WS1

Objective:Radiation-induced bystander effects(RIBEs) are defined as the biological changes of non-irradiated cells after receiving bystander signals from irradiated cells.Non-targeted radiation effects including bystander responses challenged the traditional target theory of radiobiology and have become new doctrines. RIBEs may play an important role in assessing health risks of low-dose radiation, the therapeutic efficacy of radiotherapy and the damage to normal tissues. Thus it has become one of the hot topics in the field of radiation biology. Right now, the study of RIBEs has been focusing on the underlying molecular mechanisms and the influence on the organisms. From the point view of the occurrence of bystander responses, it includes steps such as that the key signaling pathways are activated in the signaling cells upon irradiation, irradiated cells release bystander signals, and the receptor cells respond to the bystander signals and display biological changes. The main purpose of this thesis was to study the molecular mechanisms of bystander responses from the angle of the receptor cells. More specifically, the study was to verify whether X-rays can induce medium-mediated bystander responses in WS1 human embryonic skin fibroblasts first; Then investigate whether α-irradiated Ha Cat keratinocytes can induce bystander responses in WS1 cells and what are the underlying mechanisms if so; Finally, construct a simple human skin tissue model by using Ha Cat cells and WS1 cells, which can pave the way for the further study on RIBEs in the ex vivo 3D skin model.Methods:In this study, conditioned medium transfer method and co-culture method were used to study the medium-mediated RIBEs. In the first part of the study, using micronucleus formation, DNA damage, apoptosis, proliferation, the ROS levels and mi R-21 as endpoints, we have demonstrated that X-rays can induce medium-mediatedbystander responses in WS1 cells; We also measured the content of TGF-β1 in the conditioned medium by using ELISA. The aim of the second part of this study was to validate the dosimitry and biological efficiency of an user friendly α-irradiation equipment which was designed and built by ourselves. The endpoints such as the induction of 53BP1 foci, a surrogate marker of DNA double strand breaks and colony formation were used. In the third part of the study, by using the changes in intracellular ROS levels and 53BP1 foci induction as endpoints, we investigated the bystander responses in unirradiated WS1 cells when co-cultured with α-irradiated Ha Cat keratinocytes. More importantly, by using transfection method to change mi R-21 expression or increase SOD2 level in WS1 cells exogenously, we also explored the important roles of mi R-21 and SOD2 of bystander WS1 cells in the induction of bystander effects. In the last part, we used WS1 cells, Ha Cat cells and rat rail collagen to construct a simple human skin tissue model, and we also used HE staining and immunohistochemistry staining to verify the success.Results:1. X-rays can induce medium-mediated bystander responses in WS1 cells.Compared with fresh medium(Ctr) and sham-radiation-conditioned medium(RCM), the conditioned medium from irradiated WS1 cells collected 3 hours after 1 Gy of X-ray exposure(3 h RCM) could induce a 11% increase in the ROS levels of bystander WS1 cells after 30 min of treatment, a 38% increase in the percentage of 53BP1 positive cells after 1 h of treatment, a 1.38-fold increase in mi R-21 expression level and a 44%increase in micronucleus formation and about one fold increase in apoptosis after 24 h of treatment. In addition, the content of TGF-β1 in 3 h RCM was also increased by 18%.In the co-culture system, the percentage of positive cells with 53BP1 foci increased by46% after 1 h of co-culture, and the percentage of nuclei with micronucleus increased by about 34% after 24 h of co-culture.2. The α-irradiation equipment was effective. Its dose rate was calculated as 0.14Gy/min. The colony formation in WS1 cells irradiation with α-particles and X-rays was measured, and the relative biological efficiency(RBE) was calculated as 1.71 at the cell survival of 0.5. Moreover, 53BP1 foci were used as the marker of DNA damage inα-irradiated WS1 cells. The results showed that the induction of 53BP1 foci increased0.38-fold and 1-fold in WS1 cells 30 min after exposed to 0.28 and 0.56 Gy,respectively. The results also suggest the ununiformity of α-induced DNA damage.3. The α-irradiated Ha Cat keratinocytes can induce medium-mediated bystander responses in WS1 cells. And mi R-21 and SOD2 in WS1 cells play key regulating roles in the occurrence of RIBEs. The ROS levels in unirradiated bystander WS1 cells were significantly elevated after 30 min of co-culture with α-irradiated Ha Cat cells, and53BP1 foci, a surrogate marker of DNA damage, were increased 1.4-fold after 3 h of co-culture. This indicates the occurrence of oxidative stress and DNA damage in bystander WS1 cells after co-culture with irradiated keratinocytes. Furthermore, the expression of mi R-21 was increased in bystander WS1 cells, downregulation of mi R-21 eliminated the bystander responses, overexpression of mi R-21 alone could induce bystander-like oxidative stress and DNA damage in WS1 cells. These data indicate an important mediating role of mi R-21 in RIBEs in this system. Besides, Mn SOD(SOD2)was also involved in the bystander responses, overexpressing SOD2 could abrogate the bystander oxidative stress and DNA damage in unirradiated WS1 cells, indicating that SOD2 was critical to the induction of RIBEs. Moreover, we found that mi R-21 regulated SOD2.4. Construction of three-dimensional(3D) model of human skin tissue. Using collagen, human embryonic skin fibroblasts WS1 and immortalized human keratinocytes Ha Cat we constructed an ex vivo 3D skin model system. HE staining showed that Ha Cat cells differentiated, and stratified, and immunohistochemical staining of 53BP1 foci showed DNA damage in the cells in skin model when irradiated with X-rays. In addition, a cell line with stably overexpressed SOD2 was established, so that it can be used in 3D constructs to study the roles of SOD2 in RIBEs in 3D model.Conclusions:This study has demonstrated that X-rays can induce medium-mediated bystander responses in WS1 cells. And WS1 cells can also respond to the bystander signals released from α-irradiated Ha Cat cells and display bystander responses. From the angle of non-irradiated cells, mi R-21 and SOD2 in bystander WS1 cells play key roles in the occurrence of RIBEs, and mi R-21 may mediate bystander responses through its regulation on SOD2. More specifically, the bystander signals from α-irradiated cellsinduce the up-regulation of mi R-21 levels in WS1 cells, the up-regulated mi R-21 can further down-regulate the expression of SOD2, thus leading to the bystander oxidative stress and DNA damage in WS1 cells. In the end, we have established a three-dimensional human skin model successfully and paved the way for the further study on RIBEs in an ex vivo 3D skin model.