| Objective: Deinococcus radiodurans is one of the prokaryotes strongest resistance to radiation on Earth. The extreme radiation resistance of the bacterium is closely related to the protein expression of DNA damage response(DDR) and repair system, in which the Ppr I protein is a key repair protein as aregulatory factor. The subject group recent studies show that Deinococcus radioduransppr I gene transfection on radiation injury of human and mammalian cells in vitro has a very significant radiation protective effect, indicating that the prokaryotic protein may become an effective radiation protection drug of higher eukaryotes radiation injury. On this basis,we successfully expressed and isolated the Ppr I protein in Escherichia coli and Pichia pastoris, and confirmed that Ppr I protein and Ppr I protein with TAT transmembrane structure(TAT-Ppr I protein) had significant anti-radiation effect on human isolated cells irradiated with lethal dose. However, the protective effect and mechanism of TAT-Ppr I protein expressed by eukaryotic system on acute radiation injury in mammals has not been reported at home and abroad. Therefore, we mainly study the effect and mechanism of TAT-Ppr I protein on acute radiation injury in mammals in this study, in order to provide valuable experimental data for clinical application of acute radiation injury.Materials and methods: The experiment cultured the two kinds of Pichia yeast strains containing p HBM905A-His-Ppr I carrier and p PICZ alpha A-TAT-Ppr I vector preserved by the subject group, in order to obtain a large number of Deinococcus radiodurans Ppr I protein and TAT-Ppr I protein expressed by eukaryotic system. Thepurified Ppr I protein and TAT-Ppr I protein were injected into pure BALB/C mice, and the mice were injected with normal saline as the control group, using gamma ray irradiation. To observe changes of body weight, blood cell count, blood biochemistry and survival rate in irradiated mice. To observe the pathological changes of liver, lung,small intestine and testis tissue of irradiated mice. to observe the distribution of TAT-Ppr I protein in mice using a small animal imaging instrument. To observe the apoptosis rate of bone marrow cells and splenic lymphocytes in irradiated mice by flow cytometry. To observe superoxide dismutase(SOD) activity, catalase(CAT)activity and malondialdehyde(MDA) content in irradiated mice by using antioxidant ELISA kit.Result:1. A large number of purified Ppr I fusion protein and TAT-Ppr I fusion protein of Deinococcus radiodurans were successfully obtained through the expression of eukaryotic system.2. Screening results of Ppr I protein injected dose : On the 7th days after irradiation, when injection dose of Ppr I protein was 600ug/kg, the weight of the mice recovered quickly, and the total number of white blood cells, lymphocytes, platelets in peripheral blood of mice were respectively 1.07×109/L, 0.71×109/L, 164.33×109/L,which was significantly higher than that of the control group(P< 0.05). The apoptosis rate of mouse bone marrow cells and the apoptosis rate of mouse spleen cells were6.92% and 30.38% respectively,which was significantly lower than that of the control group(P< 0.05).3. Screening results of TAT-Ppr I protein injected dose: On the 7th days after irradiation, when injection dose of TAT-Ppr I protein was 800ug/kg, the weight of the mice recovered quickly, and the total number of white blood cells, lymphocytes,platelets in peripheral blood of mice were respectively 0.84×109/L, 0.18×109/L,178.67×109/L, which was significantly higher than that of the control group(P < 0.05).The apoptosis rate of mouse bone marrow cells were 31.41% respectively, which was significantly lower than that of the control group(P < 0.05). Compared with thecontrol group, there was no significant difference in the rate of spleen cell apoptosis between the two groups(P>0.05).4. Mortality variation of irradiated mice. The death of irradiated mice was more concentrated in 10~20 days, and the mortality of the mice in protein injection group before and after irradiation were 50% and 70% respectively, and the mortality were significantly lower than that of the normal saline injection group 90%.5. Changes in body weight in mice. The body weight of mice was increased from fourth days after irradiation. Compared with the normal saline injection group, the weight of the mice in protein injection group before and after irradiation was higher,and the increase in protein injection group before irradiation was more obvious.6. Changes of peripheral blood cells. Seventh days after irradiation, the total number of white blood cells, lymphocytes, platelets protein injection group before irradiation were respectively 0.61×109/L, 0.26×109/L, 211.00×109/L, which was significantly higher than that of the normal saline injection group(P< 0.05). Fourteenth days after irradiation, the total number of white blood cells, lymphocytes, platelets in the protein injection group before irradiation were respectively 0.95×109/L, 0.69×109/L,257.33×109/L, which was significantly higher than that of the normal saline injection group(P < 0.001). On the 14 th days after irradiation, the total number of white blood cells, lymphocytes, platelets in the protein injection group after irradiation were respectively 0.70×109/L, 0.62×109/L, 482.25×109/, which was significantly higher than that of the normal saline injection group(P< 0.01). On the 7th days after irradiation, the total number of blood white cells, platelets in the protein injection group before irradiation were significantly higher than that in the protein injection group after irradiation.7. Changes of liver function in peripheral blood. On the 28 th days after irradiation, ALT content and AST content of the protein injection group before irradiation were respectively45.41 IU/L, 133.92 IU/L, which was significantly lower than that of the normal saline injection group and the protein injection group after irradiation(P< 0.05).8. Changes of renal function in peripheral blood. UREA content of the protein injection group before irradiation on the 14 th days and 28 th days after irradiation were respectively 6.00mmol/L, 6.43mmol/L, which was obviously decreased, compared with saline injection group(P < 0.05). CREA content of the protein injection group after irradiation on 7th days after irradiation was 7.03mmol/L, and was lower than the saline injection group and the protein injection group before irradiation(P<0.05).UREA content of the protein injection group before irradiation on the 28 th days after irradiation was significantly lower than that of t the protein injection group after irradiation(P< 0.01)9. Observation of the pathology of organs and tissue. Whether the protein injection group before irradiation or the protein injection group after irradiation reduced the acute radiation injury of the mouse liver, lung, small intestine, testicle tissue and accelerated the recovery of the organization.10. Observation of small animal imaging. The distribution of fluorescence signal can be seen in various tissues and organs of mice at 5min to 24 h after the injection of TAT-Ppr I protein.11. Changes of apoptosis rate of bone marrow cells and splenic lymphocytes. On3~28th days after irradiation, compared with the normal saline group, whether the protein injection group before irradiation or the protein injection group after irradiation decreased the apoptosis rate of mice bone marrow cells(P< 0.01). On the 7th days, the apoptosis rate of mice splenic lymphocytes of the protein injection group before and after irradiation were lower than the normal saline group(P<0.001). On the 3th days and 7th days after irradiation, compared with the protein injection group after irradiation, the apoptosis rate of mice bone marrow cells of the protein injection group before irradiation injection group was lower(P<0.05).12. Changes of SOD, CAT activity and MDA content in liver. On the 7th days after irradiation, the activity of SOD in liver tissue of the protein injection group before irradiation was higher than that of the saline injection group(P < 0.05), and the content of MDA were significantly decreased at all time points, compared with the normalsaline group. On the 7th days and 14 th days after irradiation, the activity of CAT in liver tissue of the protein injection group after irradiation were higher than that of normal saline injection group(P<0.01), and the content of MDA were lower than that of the saline injection group, on the 3th days and 14 th days after irradiation(P<0.05).The activity of SOD in liver tissue of the protein injection group before irradiation was higher than that of the protein injection group after irradiation, on the 3th days and 28 th days after irradiation(P<0.05).Conclusion:1. Deinococcus radiodurans Ppr I fusion protein and TAT-Ppr I fusion protein,which were expressed by eukaryotic system, and a large number of proteins were successfully obtained by purification.2. The effective injection dose range of Ppr I protein and TAT-Ppr I protein was400-600 g/kg, 600-800 g/kg, and the best injection dose was 600 g/kg, 800 g/kg.3. Injection of TAT-Ppr I protein can significantly reduce the mortality of the irradiated mice, and the effect of injection of TAT-Ppr I protein before irradiationis more obvious.4. Injection of TAT-Ppr I protein can significantly increase the body weight of mice with acute radiation injury.5. Injection of TAT-Ppr I protein can significantly reduce the degree of peripheral blood leukocytes, lymphocytes, platelets of mice with acute radiation injury, and the effect of injection of TAT-Ppr I protein before irradiation is more obvious.6. Injection of TAT-Ppr I protein has little effect on the blood ALT, AST content of mice with acute radiation injury. Injection of TAT-Ppr I protein before irradiation can accelerate the recovery of ALT and AST content, and improve the liver function in a certain extent.7. Injection of TAT-Ppr I protein before irradiation can reduce the blood UREA content of mice with acute radiation injury. Injection of TAT-Ppr I protein after irradiation can reduce the blood CREA content of mice with acute radiation injury. The renal function was affected in a certain extent.8. Injection of TAT-Ppr I protein significantly reduced the acute radiation injury of liver, lung, small intestine and testis tissue of mice, and accelerated the repair of injury,and the effect of injection of TAT-Ppr I protein before irradiation is more obvious.9. The TAT-Ppr I protein can be quickly distributed to various tissues and organs of mice by intravenous or intramuscular injection.10. TAT-Ppr I protein significantly reduced the apoptosis rate of bone marrow cells and splenic lymphocytes of mice with acute radiation injury, and the antiapoptotic effect of injection of TAT-Ppr I protein before irradiation is better.11. Injection of TAT-Ppr I protein increased the activity of SOD and CAT in liver tissue of mice with acute radiation injury, and reduced the content of MDA in liver tissue of mice with the acute radiation injury, but has a little effect on activity of SOD,and the antioxidant effect of injection of TAT-Ppr I protein before irradiation is better.The results of this study suggest that, Ppr I and TAT-Ppr I prokaryotic protein expressed by eukaryotic system have good protective and therapeutic effects on mammalian with acute radiation injury. These effects are closely related to their ant-i oxidation and anti-apoptotic mechanism. The protective effect of injection of TAT-Ppr I protein before irradiation is more significant than that injection of TAT-Ppr I protein after irradiation. The results provided valuable experimental data for further study of the role and mechanism of Ppr I protein and its application in clinical medicine. |
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