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The aim of the study was to investigate the sensitivity of AHH-1 human lymphoblastoid cells to radiation and its relevance to intracellular events, specifically alteration in cellular energy-producing systems. AHH-1 human lymphoblastoid cells were irradiated with 6 Gy of gamma radiation, and then were collected at the indicated time points. Parallel studies were conducted to assess the effects of radiation on the cell proliferation and apoptotic index. Mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) production were monitored. A marked decrease of cell viability was observed as early as 12 h postirradiation and fraction of apoptotic cells was highest at 24 h. Intracellular ROS generation measured with 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) appeared to be highest as early as 30 min postirradiation and resumed to normal level at 6 h. Unexpectedly, the fluorescence intensity of Rhodamine 123 for measuring MMP did not change during the first 3h after radiation and exhibited an aberrant increase at 6 h. The results suggest that AHH-1 cells are sensitive to radiation-induced apoptosis and ROS generation is an early phase in the apoptosis process. Moreover, the results might cast doubts on those studies using Rhodamine 123 which hypothesized that the fall in MMP is one of the early events of apoptosis.

Bcl-xL belongs to a family of proteins which inhibit apoptosis in a number of stimuli including ionizing radiation. To better understand the effects and mechanisms of Bcl-xL on the apoptosis of lymphocytes and provide experimental basis to treat immune injury induced by radiation, we used normal human lymphoblastoid AHH-1 cells that were engineered to overexpress Bcl-xL proteins. Our results showed that overexpressed Bcl-xL reduced time-dependent increase of apoptosis induced by ionizing radiation. Reactive oxygen species (ROS) generation and Bax protein expression in the transfected AHH1-Bcl-xL cells were also lower compared to parental AHH-1 cells. Unexpectedly, the fluorescence intensity of Rhodomine 123 (Rh 123) for measuring mitochondrial membrane potential (MMP) did not change at all detected time points. These results possess a vital significance for insights into a new strategy for gene therapy of radiation-induced immune injury.

Objective: To observe the apoptotic characteristics of mouse spleen lymphocyte after lethal dose gamma-irradiation and its relationship to the expression of Bax and Bcl-XL proteins., Methods: Two hundred and twenty-five second-grade C57 mice were randomly divided into six groups of 0, 6, 9, 12, 15 and 20 Gy. They were sacrificed by dislocation and samples were taken on 1-28 days after whole body single gamma-irradiation. Lymphocyte apoptosis and necrosis were analyzed by TdT-mediated dUTP nick end labeling (TUNEL) and flow cytometry (FCM) techniques. The expression of Bax and Bcl-XL proteins were estimated by immunohistochemical method., Results: (1) The number of peripheral white blood cells of mice increased temporarily at 6 hours after radiation, thereafter, began to decrease rapidly, which reached the minimum on day 7 and recovered normal level basically one month after 6 Gy gamma-irradiation. (2) Apoptotic rate of spleen lymphocytes increased significantly, peaking at 6 hours after radiation, which was found to have a dose-response relationship during 6-24 hours after < or =12 Gy irradiation, but decreased after > or =15 Gy irradiation. (3) It was confirmed by FCM that the apoptotic rate of spleen lymphocytes increased along with the elevation of radiation dose. However, the apoptotic rate began to decrease and the necrotic rate rose distinctively after > or =15 Gy irradiation. The analysis of DNA gel electrophoresis supported above-mentioned results. (4) The expression of Bax protein in spleen lymphocyte enhanced at 6 hours after 6 Gy gamma-irradiation and peaked by 12 Gy-irradiation, showing a dose-dependent pattern, but which was not be found after > or =15 Gy gamma-irradiation. On the other hand, the expression of Bcl-XL protein reduced persistently with the increase of radiation dose, and also presented a better dose-dependent effect after < or =12 Gy irradiation., Conclusion: After 6-12 Gy gamma-irradiation, the apoptosis is the major death way of spleen lymphocyte, while both necrosis and apoptosis are important death pathways after > or =15 Gy irradiation. Pro-apoptotic Bax and anti-apoptotic Bcl-XL play an important role in the apoptotic regulation of spleen lymphocytes induced by lethal dose radiation.

Aim: To observe the relationship between apoptosis of mouse thymic lymphocytes and the expressions of bax, bcl-2 and bcl-X(L) after gamma-ray radiation with lethal dose and provide the basis for treatment of acute severe radiation sickness., Methods: 250 cleaning-grade C57 mice were randomly divided into 6 groups and treated with 0, 6, 9, 12, 15 and 20 Gy of whole body single gamma-irradiation, respectivety. The mice were killed by dislocation and then the thymus and peripheral blood samples were taken at 1-28 days and 3-12 months after irradiation. The lymphocytic apoptosis was analyzed by TUNEL. The expressions of bax, bcl-2 and bcl-X(L) were detected by in situ hybridization and immunohistochemical staining., Results: (1)Mouse peripheral leucocytes showed a transient elevation at 6 h after radiation and then decreased rapidly. The leucocyte's number reduced to minimal level at day 7 after 6 Gy gamma-irradiation and returned to basic normal value until one month after radiation. (2)24 h after irradiation the apoptotic rate of thymic lymphocytes increased swiftly, and apoptotic rate was positively correlated with radiation dose in the range of 6-12 Gy. There was no such a correlation after >/=15 Gy irradiation. (3)24 h after 6 Gy irradiation the apoptotic rate of thymic lymphocytes reached the maximal level. Afterwards it began to decrease, but still higher than that of control group even after 12 months. (4)3 h after 6 Gy radiation the expression of Bax protein in thymic lymphocytes increased immediately, and reached the highest value at 24 h. On the other hand, the expressions of Bcl-2 and Bcl-X(L) proteins reduced evidently at 3 h after radiation, reaching the lowest level at 24 h. Analysis of bax and bcl-2 mRNAs was concordant with the protein expression results., Conclusion: After 6-12 Gy gamma-ray irradiation, the apoptotic rate of thymic lymphocytes is positively correlated with the radiation doses, suggesting that apoptosis is a major way of thymic lymphocyte death after

Aim: To observe the effects of large dose of gamma-irradiation on immune function of mice., Methods: 225 cleaning-grad C57 mice, weighing(20+/-2.0) g, were randomly divided into 6 groups, and treated with 0, 6, 9, 12, 15 and 20 Gy gamma-irradiation. At different times after irradiation, lymphocytes were collected and lymphocytic apoptosis and T cell subsets were analyzed by TUNEL, May-Grunwald Giemsa (MGG) staining and flow cytometry., Results: (1)At early stage(1-14) d after radiation, the apoptotic rate of peripheral blood lymphocytes increased significantly and 12 Gy radiation resulted in the highest apoptotic rate. The number of T lymphocytes and T cell subsets decreased continuously in a dose-dependent manner. CD8(+) T cells were the most sensitive in T cell subsets to irradiation. These results suggested that early severe injury might be one of the important features of immune injury caused by acute radiation. (2) One month after radiation, the apoptotic rate of lymphocytes began to decrease and T lymphocytes and their subsets recovered gradually. However, neither the lymphocytic apoptotic rate nor the number of CD3(+) T cells and CD8(+) T cells, recovered to normal level, indicating that large dose of radiation had severe remote effects on immune function., Conclusion: Apoptosis of a large number of peripheral blood lymphocytes in early stage after radiation may result in sharp reduction of T cell number and late immune function depression.

Objective: To observe the radioprotection of recombinant human interleukin-3 (rhIL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF) and rhIL-3 (rhIL-3+GM-CSF) on peripheral lymphocytes of rhesus monkey irradiated by 3.0 Gy gamma-rays, and attempt to provide evidence of cytokines used effectively in the therapy of acute radiation sickness., Methods: Thirty rhesus monkey used in the experiment were randomly divided into six groups of rhIL-3 20 microg.kg -1.d -1, 60 microg.kg -1.d -1 GM-CSF 10 microg.kg -1.d -1 IL-3 20 microg.kg -1.d -1 +GM-CSF 10 microg.kg -1.d -1 radiation control and normal control. 21 d after whole body gamma-irradiation and subcutaneous injection of cytokines, T lymphocyte and its subsets, Bax/Bcl-2 proteins in lymphocytes were determined by immunohistochemical staining with alkaline phosphatase, and lymphocyte apoptosis was detected by TdT-mediated dUTP nick end labeling (TUNEL) technique., Results: (1) After irradiation the quantities of peripheral lymphocyte, T cell and its subsets obviously decreased as compared with those of normal controls. For instance, the percentages of lymphocyte, T, T H and Ts cells in radiation control group reduced to 44 percent, 42 percent, 41 percent and 57 percent of normal controls, respectively. (2)After radiation the reduction of lymphocyte, T, T H and Ts cells were evidently improved by injection of GM-CSF and GM-CSF+IL-3, The T,T H cells in GM-CSF and GM-CSF+IL-3 groups were respectively elevated by 1.57 and 1.76 fold, as well as 1.48 and 1.72 fold of radiation controls. (3) A large amount of lymphocyte apoptosis was found after radiation, GM-CSF and GM-CSF+IL-3 treatment could distinctively inhibit abundant lymphocyte apoptosis induced by acute irradiation,the apoptotic rates of lymphocytes in GM-CSF and GM-CSF+IL-3 groups reduced to 41 percent and 48 percent respectively when compared with that of radiation controls., Conclusion: A definite dose of GM-CSF and GM-CSF+IL-3 could suppress the reduction of lymphocyte, T and T H cells and lymphocyte apoptosis induced by 3.0 Gy gamma-irradiation. It confirms that inhibition of GM-CSF and GM-CSF+IL-3 on lymphocyte reduction as well as apoptosis might be one of the major causes to alleviate radiation injury of lymphocytes and improve the immunological function.

We studied the apoptosis of mousecirculating lymphocytes and its mechanism induced by 2, 4, 6, and 8 Gy of whole-body gamma-irradiation and the expression of bax and bcl-2 gene products as related to apoptosis. We found that, in the early stage after irradiation (4th-7th day), the percentage of lymphocyte apoptosis increased rapidly. Four hours after 2, 4, 6, and 8 Gy of gamma-irradiation, the apoptotic lymphocytes were 2.6, 3.8, 5.5, and 10.4 times those in the controls, respectively. A good correlation was found between the intensity of apoptosis and the radiation dose. As the radiation dose increased, the absolute counts of peripheral lymphocytes decreased sharply; 4 hours after 2, 4, 6, and 8 Gy of gamma-irradiation, the lymphocyte counts were 82, 63, 47, and 22% of the controls, respectively. The peak value of lymphocyte apoptosis was observed on the 7th day after irradiation using the in situ terminal labeling method, and the apoptotic lymphocytes were found to be 16 times the number in the controls. These results were in accordance with those obtained by the May-Grünwald-Giemsa staining method. The absolute counts of peripheral lymphocytes dropped to their lowest value on the 7th day after irradiation, suggesting that lymphocyte apoptosis might be the major cause of lymphocytopenia in the early stage after irradiation. The abnormal expression of bax and bcl-2 gene products in irradiated lymphocytes was closely related to apoptosis in peripheral lymphocytes.