耐辐射球菌PprI蛋白对细胞增殖、毒性及动物免疫毒性的研究
发布时间：2018-07-18 09:36
【摘要】：目的：耐辐射球菌（Deinococcus radiodurans）（简称DR）是迄今为止地球上发现的辐射抗性最强的生物之一，该菌对电离辐射、紫外线、干燥氧化剂等因素引起的致死和突变性DNA损伤具有极端抗性。目前对于这种超强抗性的具体机制，学界至今尚无定论。在耐辐射球菌的修复机制中，多种修复蛋白对其特异的辐射抗性具有重要的作用。PprI是耐辐射球菌中的特有基因，又名irrE，该被认为是耐辐射球菌DNA修复与保护途径的总开关，基因的表达产物PprI蛋白是一种促进DNA修复的多效蛋白的诱导物。近年来我科室研究人员研究证实pprI基因转染对哺乳动物中子和γ射线急性放射损伤具有显著的防治作用，并构建重组原核表达质粒pET-28a-His-PprI转化入E. coli BL21(DE3）RP，诱导表达并纯化得到了耐辐射球菌PprI融合蛋白，且研究证实了此蛋白对γ射线辐射损伤小鼠具有一定的抗放作用。本课题在此蛋白作用机制的基础上，对其细胞毒性及免疫毒性作了初步的研究，为其以后的临床应用前安全性评价提供依据，目前关于此蛋白的毒性研究，尚未见国内外文献报道。 材料与方法：PprI蛋白由本实验室张永芹硕士通过原核表达纯化,以人脐静脉血管内皮细胞为作用对象，应用MTT法检测此蛋白对细胞增殖的影响，采用细胞克隆形成法检测对细胞存活的影响，并绘制出细胞存活曲线。以ICR小鼠作为免疫对象，实验组分为生理盐水免疫组和PprI蛋白免疫组，通过免疫器官脏器系数，，淋巴细胞转化能力（MTT法），细胞因子释放（ELISA试剂盒），T淋巴细胞亚群变化（流式细胞仪）进一步观察此蛋白对小鼠免疫功能的影响。 结果：本课题初步研究得出，PprI蛋白浓度为2.5、5、10ug/mL范围内对细胞产生抑制作用，且抑制作用与蛋白浓度呈正相关，细胞密度0.5×104作用时间24h的细胞半抑制率IC50为6.40ug/mL。PprI蛋白浓度为100、200、400、600ng/mL范围内对细胞产生增殖作用，蛋白浓度400ng/mL作用36h时增殖作用最为显著。由多靶单击模型拟合的细胞存活曲线得知，PprI蛋白组D0值为1.33，Dq值为1.17，N值为2.41，SF2值为0.51，各值均高于单纯照射组。在小鼠免疫及免疫后三周的观察期间内，由免疫器官脏器系数计算可知，免疫初期即初次免疫后14天，高剂量组肾脏重量系数低于生理盐水对照组，有显著性差异（P＜0.05)，中、高剂量组胸腺重量系数高于生理盐水对照组，有显著性差异（P＜0.05)；免疫末期即末次免疫后3天，低剂量组肾脏重量系数低于生理盐水对照组，有显著性差异（P＜0.05)，高剂量组胸腺重量系数高于生理盐水对照组，有显著性差异（P＜0.05)；恢复期即末次免疫后3周，高剂量组肝脏重量系数高于生理盐水对照组，有显著性差异（P＜0.05)，低剂量组胸腺重量系数高于生理盐水对照组，有显著性差异（P＜0.05)。免疫功能检测方面，免疫剂量达到中、高剂量时，增强淋巴细胞转化功能；中剂量时脾脏CD3+T淋巴细胞增加，低、中、高剂量时胸腺CD3+T淋巴细胞降低；高剂量时，与生理盐水对照组组相比，免疫末期PprI蛋白免疫组IL-4抗体分泌增加，有显著性差异（P＜0.05)；IFN-γ抗体分泌水平无明显差别。 结论： 1. PprI蛋白浓度达到2.5、5、10ug/mL时对细胞增殖产生抑制作用，且抑制作用与蛋白浓度呈正相关，细胞密度0.5×104作用时间24h的细胞半抑制浓度IC50为6.40ug/mL。PprI蛋白浓度为100、200、400、600ng/mL时对细胞产生增殖作用，蛋白浓度400ng/mL作用36h时增殖作用最为显著。 2.由多靶单击模型拟合出细胞存活曲线得知，PprI蛋白在一定程度上可以使细胞的平均致死剂量增大，放射抗拒性增强，细胞修复能力增强。 3. PprI蛋白对小鼠免疫器官的影响主要是中枢性免疫器官胸腺及主要脏器肾脏，且其刺激是可逆的。对小鼠脾脏、肝脏基本无毒性影响。 4. PprI蛋白免疫剂量达到100μg/kg、200μg/kg时，PprI蛋白可以增强小鼠脾脏、胸腺淋巴细胞转化功能。 5. PprI蛋白刺激机体的免疫方式后期主要为体液免疫应答，恢复期主要为细胞免疫应答（迟发型免疫应答）。 6. PprI蛋白可以升高小鼠脾脏CD3+T细胞在T细胞亚群中的比例，CD4／CD8比值一直处于正常值范围内；降低小鼠胸腺CD3+T细胞在T细胞亚群中的比例，在免疫恢复期小鼠主要表现为细胞免疫。 7.综上所述，小鼠基本表现为免疫功能增强，但对免疫器官和免疫功能无不良影响，基本无免疫毒性作用，安全性良好，为以后此蛋白的临床应用提供了一定的参考基础。
[Abstract]:Objective: Deinococcus radiodurans (DR) is one of the most highly resistant organisms found on earth so far. It has extreme resistance to fatal and mutant DNA damage caused by ionizing radiation, ultraviolet radiation and dry oxidant. In the repair mechanism of S. radiococcus, multiple repair proteins play an important role in its specific radiation resistance..PprI is a unique gene in radiococcus, also known as irrE. It is considered to be the total switch of the DNA repair and protection pathway of S. radioformans, and the gene expression product PprI protein is a multi effect to promote the DNA repair. In recent years, our department researchers have studied that pprI gene transfection has a significant effect on the acute radiation damage of mammalian neutrons and gamma rays, and the Recombinant Prokaryotic expression plasmid pET-28a-His-PprI was transformed into E. coli BL21 (DE3) RP and was induced to express and purify the PprI fusion protein of S. radioformans. The study confirmed that the protein has a certain anti radiation effect on gamma ray radiation injury in mice. On the basis of this protein action mechanism, the cytotoxicity and immuno toxicity of the protein have been preliminarily studied, which provides the basis for the safety evaluation before its clinical application. At present, the toxicity study on this protein has not yet been seen in the domestic and foreign languages. Give a report.
Materials and methods: PprI protein was purified by the prokaryotic expression of PprI in our laboratory, and the human umbilical vein endothelial cells were used as the object. The effect of this protein on cell proliferation was detected by MTT method. Cell survival was detected by cell clone formation, and the cell survival curve was plotted. ICR mice were used as immunization. Subjects, the experimental group was divided into the physiological saline immunization group and the PprI protein immunization group. The effect of the protein on the immune function of mice was further observed through the immune organ organ coefficient, the lymphocyte transformation ability (MTT), the cytokine release (ELISA Kit) and the T lymphocyte subgroup change (flow cytometry).
Results: the preliminary study showed that the concentration of PprI protein was inhibited in the range of 2.5,5,10ug/mL, and the inhibitory effect was positively correlated with the protein concentration. The cell density of the cell density of the cell density of 0.5 x 104 time 24h was IC50 in the range of 100200400600ng/mL in the range of 6.40ug/mL.PprI protein. The proliferation effect of protein concentration 400ng/mL was the most significant when 36h was used. The cell survival curve fitted by the multi target click model showed that the D0 value of the PprI protein group was 1.33, the Dq value was 1.17, the N value was 2.41, and the SF2 value was 0.51. All the values were higher than those of the simple irradiation group. In the observation period of the immune and immune three weeks after the immunization, the immune organ coefficient was calculated. The renal weight coefficient of the high dose group was lower than that of the normal saline control group (P < 0.05). In the high dose group, the weight coefficient of the thymus gland was higher than that of the normal saline control group (P < 0.05). There was a significant difference (P < 0.05). The renal weight coefficient of the low dose group was lower than the physiological salt in the low dose group. There were significant differences in the water control group (P < 0.05). The weight coefficient of the thymus gland in the high dose group was higher than that in the normal saline control group (P < 0.05). The recovery period was 3 weeks after the last immunization, the liver weight coefficient of the high dose group was higher than that of the normal saline control group (P < 0.05). The weight coefficient of the thymus gland in the low dose group was higher than that of the normal saline group. In the control group, there was a significant difference (P < 0.05). In the immunological function test, the immune dose was reached, the lymphocyte transformation was enhanced at high dose, and the spleen CD3+T lymphocyte increased at the middle dose, low, middle, and high dose of CD3+T lymphocyte in the thymus. At high dose, compared with the saline control group, the PprI protein immunized at the end of the immune system. There was a significant difference in IL-4 antibody secretion in the epidemic group (P < 0.05), and there was no significant difference in the secretion level of IFN- gamma antibody.
Conclusion:
When the concentration of 1. PprI protein reached 2.5,5,10ug/mL, the cell proliferation was inhibited, and the inhibitory effect was positively correlated with the protein concentration. The cell density of the cell density of the cell density of 0.5 x 104 time 24h was the proliferation of the cells when the concentration of 6.40ug/mL.PprI protein was 100200400600ng/mL, and the protein concentration 400ng/mL action 36h increased. Colonization is the most significant.
2. by fitting the cell survival curve by multi target clicking model, PprI protein can increase the average lethal dose of the cell to a certain extent, enhance the radiological resistance and enhance the cell repair ability.
The effect of 3. PprI protein on the immune organs of mice is mainly the central immune organ thymus and the main organs and kidney, and its stimulation is reversible. It has no toxic effect on the spleen and liver of mice.
4. when the immunization dose of PprI reached 100 mu g/kg and 200 g/kg, PprI protein could enhance the function of spleen and thymus lymphocyte transformation in mice.
5. PprI protein stimulates the immune response of the body in the later stage, which is mainly humoral immune response. The recovery stage is mainly cellular immune response (delayed immune response).
6. PprI protein can increase the proportion of spleen CD3+T cells in T cell subsets, and the ratio of CD4 / CD8 is always in the normal range, and the proportion of CD3+T cells in the thymus of mice is reduced in the T cell subgroup, and the main expression in the immune recovery stage is cell immunity.
7. to sum up, the mice basically showed that the immune function was enhanced, but it had no adverse effects on immune organs and immune functions, and had no immune toxicity, and had good safety. It provided a reference basis for the future clinical application of this protein.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R378
本文编号：2131499
[Abstract]:Objective: Deinococcus radiodurans (DR) is one of the most highly resistant organisms found on earth so far. It has extreme resistance to fatal and mutant DNA damage caused by ionizing radiation, ultraviolet radiation and dry oxidant. In the repair mechanism of S. radiococcus, multiple repair proteins play an important role in its specific radiation resistance..PprI is a unique gene in radiococcus, also known as irrE. It is considered to be the total switch of the DNA repair and protection pathway of S. radioformans, and the gene expression product PprI protein is a multi effect to promote the DNA repair. In recent years, our department researchers have studied that pprI gene transfection has a significant effect on the acute radiation damage of mammalian neutrons and gamma rays, and the Recombinant Prokaryotic expression plasmid pET-28a-His-PprI was transformed into E. coli BL21 (DE3) RP and was induced to express and purify the PprI fusion protein of S. radioformans. The study confirmed that the protein has a certain anti radiation effect on gamma ray radiation injury in mice. On the basis of this protein action mechanism, the cytotoxicity and immuno toxicity of the protein have been preliminarily studied, which provides the basis for the safety evaluation before its clinical application. At present, the toxicity study on this protein has not yet been seen in the domestic and foreign languages. Give a report.
Materials and methods: PprI protein was purified by the prokaryotic expression of PprI in our laboratory, and the human umbilical vein endothelial cells were used as the object. The effect of this protein on cell proliferation was detected by MTT method. Cell survival was detected by cell clone formation, and the cell survival curve was plotted. ICR mice were used as immunization. Subjects, the experimental group was divided into the physiological saline immunization group and the PprI protein immunization group. The effect of the protein on the immune function of mice was further observed through the immune organ organ coefficient, the lymphocyte transformation ability (MTT), the cytokine release (ELISA Kit) and the T lymphocyte subgroup change (flow cytometry).
Results: the preliminary study showed that the concentration of PprI protein was inhibited in the range of 2.5,5,10ug/mL, and the inhibitory effect was positively correlated with the protein concentration. The cell density of the cell density of the cell density of 0.5 x 104 time 24h was IC50 in the range of 100200400600ng/mL in the range of 6.40ug/mL.PprI protein. The proliferation effect of protein concentration 400ng/mL was the most significant when 36h was used. The cell survival curve fitted by the multi target click model showed that the D0 value of the PprI protein group was 1.33, the Dq value was 1.17, the N value was 2.41, and the SF2 value was 0.51. All the values were higher than those of the simple irradiation group. In the observation period of the immune and immune three weeks after the immunization, the immune organ coefficient was calculated. The renal weight coefficient of the high dose group was lower than that of the normal saline control group (P < 0.05). In the high dose group, the weight coefficient of the thymus gland was higher than that of the normal saline control group (P < 0.05). There was a significant difference (P < 0.05). The renal weight coefficient of the low dose group was lower than the physiological salt in the low dose group. There were significant differences in the water control group (P < 0.05). The weight coefficient of the thymus gland in the high dose group was higher than that in the normal saline control group (P < 0.05). The recovery period was 3 weeks after the last immunization, the liver weight coefficient of the high dose group was higher than that of the normal saline control group (P < 0.05). The weight coefficient of the thymus gland in the low dose group was higher than that of the normal saline group. In the control group, there was a significant difference (P < 0.05). In the immunological function test, the immune dose was reached, the lymphocyte transformation was enhanced at high dose, and the spleen CD3+T lymphocyte increased at the middle dose, low, middle, and high dose of CD3+T lymphocyte in the thymus. At high dose, compared with the saline control group, the PprI protein immunized at the end of the immune system. There was a significant difference in IL-4 antibody secretion in the epidemic group (P < 0.05), and there was no significant difference in the secretion level of IFN- gamma antibody.
Conclusion:
When the concentration of 1. PprI protein reached 2.5,5,10ug/mL, the cell proliferation was inhibited, and the inhibitory effect was positively correlated with the protein concentration. The cell density of the cell density of the cell density of 0.5 x 104 time 24h was the proliferation of the cells when the concentration of 6.40ug/mL.PprI protein was 100200400600ng/mL, and the protein concentration 400ng/mL action 36h increased. Colonization is the most significant.
2. by fitting the cell survival curve by multi target clicking model, PprI protein can increase the average lethal dose of the cell to a certain extent, enhance the radiological resistance and enhance the cell repair ability.
The effect of 3. PprI protein on the immune organs of mice is mainly the central immune organ thymus and the main organs and kidney, and its stimulation is reversible. It has no toxic effect on the spleen and liver of mice.
4. when the immunization dose of PprI reached 100 mu g/kg and 200 g/kg, PprI protein could enhance the function of spleen and thymus lymphocyte transformation in mice.
5. PprI protein stimulates the immune response of the body in the later stage, which is mainly humoral immune response. The recovery stage is mainly cellular immune response (delayed immune response).
6. PprI protein can increase the proportion of spleen CD3+T cells in T cell subsets, and the ratio of CD4 / CD8 is always in the normal range, and the proportion of CD3+T cells in the thymus of mice is reduced in the T cell subgroup, and the main expression in the immune recovery stage is cell immunity.
7. to sum up, the mice basically showed that the immune function was enhanced, but it had no adverse effects on immune organs and immune functions, and had no immune toxicity, and had good safety. It provided a reference basis for the future clinical application of this protein.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R378
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