C. Shao, M. Folkard, B. D. Michael, K. M. Prise, Targeted cytosplasmic irradiation induces bystander responses. Proc. Natl. Acad. Sci. U.S.A. 101, 13495-13500 (2004).
研究旁效应的常用方法

低通量粒子照射：
细胞受到照射的平均粒子数小于1
缺点：不能分辨核、质照射，通常不考虑细胞质效应

细胞共培养
bystander
IR
IR
Non-IR

辐射后条件培养基处理 条件培养基：受照射细胞培养一段时间后的培养 基
Conditioned medium (CM)
Nonirradiated cells
NO
细胞核照射 vs 细胞质照射
直接照射效应： 细胞核照射 > 细胞质照射
辐射诱导的旁效应： 细胞核照射 ≈ 细胞质照射
Relative YMN in T98G
3
**
*
2
Control 10 cells irradiated 100% irradiated
A
0.25 0.20 0.15
Control Nucleus irradiated Cytoplasm only irradiated
Relative ROS-Fluorescence
1.4
1）SOD、DMSO等ROS清除剂，可以 消除旁效应引起的姐妹染色体交换 （SCE）、基因突变； 2）ROS介导低剂量辐射诱导的蛋白表 达：p53、p21、p34、MAPK、NFkB等； 3）旁细胞中ROS的产生。细胞群体的 1%受到单离子的精确照射，导致 30%的细胞产生ROS，细胞群体的 ROS荧光强度显著增加。
Nuclear IR
Cytoplasmic IR
现有微束装置20多台(含在研)

Gray Cancer Institute, UK Columbia University, USA (RARAF) (H、 3He、4He, LET=30、100 keV/m)

JAERI-Takasaki Institute (TIARA) (Ar、Ne, LET=1200、430 keV/m)
0.20
OneHe FiveHe OnePTIO FivePTIO OneFilipin FiveFilipin
p<0.05
*
*
*
Filipin
Sphingomyelinase
*
Ceramide
0.15
iNOS
0.10 0 1 10
Cytoplasmic Irradiated T98G cell number
*
1.2
1.0
0.8
** **
0.6
Fraction of Irradiated Cells
O2∸
SOD
H2O2
CAT
H2O+O2

钙流、线粒体膜电位
1）条件培养基引起细胞内 钙流的产生、线粒体膜电 位的降低、以及相对应的 ROS的形成。 2）Cyclosporin 可以防止 线粒体膜电位的崩溃，具 有抑制旁效应的作用。
GJICD细胞中的旁效应
H3标记的 CHO细胞与AL细胞3D方式下共培养24h， AL细胞受ROS和 GJIC调节的旁效应突变率显著增加。 其它实验表明，比起ROS， GJIC对旁效应具有更大的贡献。
细胞质照射诱导的旁效应

微束实验表明，AL细胞之细胞质精确照射可以 引起细胞的致死与突变。
This Week in ST Home > Literature > This Week in ST > pp. tw333 Sci. STKE, Vol. 2004, Issue 251, pp. tw333, 21 September 2004. [DOI: 10.1126/stke.2512004tw333]
**
B
**
YMN
*
* *
10 T98G IR
0.10 0.05 0.00
1
* *
AG01522 T98G
0
Nuclear traversal
Cytoplasmic traversal
Irradiated T98G cells induce bystander micronucleus

All of Science's STKE
核照射
细胞质照射
细胞质照射
细胞质照射可以通过旁效应引起细胞致死损伤
单个T98G神经胶质瘤细胞的细胞质精确照射，可引起 周围成百个未受照射细胞的致死损伤

辐射诱导的NO自由基是该旁效应的信号因子之一 NO自由基通过细胞膜rafts介导的途径产生。
YMN of T98G population
单细胞照射： CHO未见旁效应 EM9 弱旁效应 Xrs5 显著旁效应
DNA双链断裂修复缺陷型细胞对旁 效应信号因子具有敏感性
细胞间隙通讯（GJIC）在旁效应中的作用

GJIC容许细胞间小分子物质的传输，是正常细胞胞间通讯的重要渠道，但 多数肿瘤细胞不具有GJIC。

GJIC调节旁效应的直接实验证据： 1）Lindane关闭了AG1522原初成纤维细胞的GJIC，也抑制了旁效应p21、 p53等的表达； 2）辐射诱导的旁效应（基因表达、突变、SCE等）在GJIC缺陷型细胞(WMaB1)中不能产生。


CANCER BIOLOGY:
Harming Innocent Bystanders
Irradiation of a subpopulation of cells is known to cause damage to neighboring cells that are not directly exposed to the radiation, the so-called bystander response. Bystander responses are important when considering the potential damage caused by environmental exposure to radiation or to radiation used in cancer treatment. Although it is known that cells whose nuclei were exposed to radiation can trigger the bystander response, Shao et al. now show that even radiation delivered to the cytoplasm of a single cell triggers bystander responses. To deliver such a targeted irradiation, the authors used a charged-particle microbeam to expose cultured human glioblastoma T98G cells to a controlled number of 3He2+ ions. The bystander response was based on the production of micronuclei (MN) (a measure of chromosomal damage) in the cell population. The abundance of MN increased when a single T98G cell in the population was exposed to a single 3He2+ ion, and the bystander response occurred in T98G cultures and in cocultures with human primary fibroblast AG0 cells. The bystander response was blocked by addition of a nitric oxide (NO) scavenger to the medium. Disruption of glycosphingolipid-enriched membrane microdomains (GEMs) with filipin also blocked the bystander response, which suggests that signals from the membrane are important. When a chemical assay for NO production was used, an increase in the number of cells positive for NO increased after a single T98G cell was exposed to a single 3He2+ ion through the cytoplasm, and this increase in NO was blocked if cells were first treated with filipin. Thus, NO, produced through a process that requires GEMs in response to cytoplasmic irradiation, appears to mediate a signal that causes chromosomal damage in neighboring cells.