耐輻射球菌PprI蛋白對(duì)細(xì)胞增殖、毒性及動(dòng)物免疫毒性的研究
發(fā)布時(shí)間：2018-07-18 09:36
【摘要】：目的：耐輻射球菌（Deinococcus radiodurans）（簡(jiǎn)稱(chēng)DR）是迄今為止地球上發(fā)現(xiàn)的輻射抗性最強(qiáng)的生物之一，該菌對(duì)電離輻射、紫外線、干燥氧化劑等因素引起的致死和突變性DNA損傷具有極端抗性。目前對(duì)于這種超強(qiáng)抗性的具體機(jī)制，學(xué)界至今尚無(wú)定論。在耐輻射球菌的修復(fù)機(jī)制中，多種修復(fù)蛋白對(duì)其特異的輻射抗性具有重要的作用。PprI是耐輻射球菌中的特有基因，又名irrE，該被認(rèn)為是耐輻射球菌DNA修復(fù)與保護(hù)途徑的總開(kāi)關(guān)，基因的表達(dá)產(chǎn)物PprI蛋白是一種促進(jìn)DNA修復(fù)的多效蛋白的誘導(dǎo)物。近年來(lái)我科室研究人員研究證實(shí)pprI基因轉(zhuǎn)染對(duì)哺乳動(dòng)物中子和γ射線急性放射損傷具有顯著的防治作用，并構(gòu)建重組原核表達(dá)質(zhì)粒pET-28a-His-PprI轉(zhuǎn)化入E. coli BL21(DE3）RP，誘導(dǎo)表達(dá)并純化得到了耐輻射球菌PprI融合蛋白，且研究證實(shí)了此蛋白對(duì)γ射線輻射損傷小鼠具有一定的抗放作用。本課題在此蛋白作用機(jī)制的基礎(chǔ)上，對(duì)其細(xì)胞毒性及免疫毒性作了初步的研究，為其以后的臨床應(yīng)用前安全性評(píng)價(jià)提供依據(jù)，目前關(guān)于此蛋白的毒性研究，尚未見(jiàn)國(guó)內(nèi)外文獻(xiàn)報(bào)道。 材料與方法：PprI蛋白由本實(shí)驗(yàn)室張永芹碩士通過(guò)原核表達(dá)純化,以人臍靜脈血管內(nèi)皮細(xì)胞為作用對(duì)象，應(yīng)用MTT法檢測(cè)此蛋白對(duì)細(xì)胞增殖的影響，采用細(xì)胞克隆形成法檢測(cè)對(duì)細(xì)胞存活的影響，并繪制出細(xì)胞存活曲線。以ICR小鼠作為免疫對(duì)象，實(shí)驗(yàn)組分為生理鹽水免疫組和PprI蛋白免疫組，通過(guò)免疫器官臟器系數(shù)，，淋巴細(xì)胞轉(zhuǎn)化能力（MTT法），細(xì)胞因子釋放（ELISA試劑盒），T淋巴細(xì)胞亞群變化（流式細(xì)胞儀）進(jìn)一步觀察此蛋白對(duì)小鼠免疫功能的影響。 結(jié)果：本課題初步研究得出，PprI蛋白濃度為2.5、5、10ug/mL范圍內(nèi)對(duì)細(xì)胞產(chǎn)生抑制作用，且抑制作用與蛋白濃度呈正相關(guān)，細(xì)胞密度0.5×104作用時(shí)間24h的細(xì)胞半抑制率IC50為6.40ug/mL。PprI蛋白濃度為100、200、400、600ng/mL范圍內(nèi)對(duì)細(xì)胞產(chǎn)生增殖作用，蛋白濃度400ng/mL作用36h時(shí)增殖作用最為顯著。由多靶單擊模型擬合的細(xì)胞存活曲線得知，PprI蛋白組D0值為1.33，Dq值為1.17，N值為2.41，SF2值為0.51，各值均高于單純照射組。在小鼠免疫及免疫后三周的觀察期間內(nèi)，由免疫器官臟器系數(shù)計(jì)算可知，免疫初期即初次免疫后14天，高劑量組腎臟重量系數(shù)低于生理鹽水對(duì)照組，有顯著性差異（P＜0.05)，中、高劑量組胸腺重量系數(shù)高于生理鹽水對(duì)照組，有顯著性差異（P＜0.05)；免疫末期即末次免疫后3天，低劑量組腎臟重量系數(shù)低于生理鹽水對(duì)照組，有顯著性差異（P＜0.05)，高劑量組胸腺重量系數(shù)高于生理鹽水對(duì)照組，有顯著性差異（P＜0.05)；恢復(fù)期即末次免疫后3周，高劑量組肝臟重量系數(shù)高于生理鹽水對(duì)照組，有顯著性差異（P＜0.05)，低劑量組胸腺重量系數(shù)高于生理鹽水對(duì)照組，有顯著性差異（P＜0.05)。免疫功能檢測(cè)方面，免疫劑量達(dá)到中、高劑量時(shí)，增強(qiáng)淋巴細(xì)胞轉(zhuǎn)化功能；中劑量時(shí)脾臟CD3+T淋巴細(xì)胞增加，低、中、高劑量時(shí)胸腺CD3+T淋巴細(xì)胞降低；高劑量時(shí)，與生理鹽水對(duì)照組組相比，免疫末期PprI蛋白免疫組IL-4抗體分泌增加，有顯著性差異（P＜0.05)；IFN-γ抗體分泌水平無(wú)明顯差別。 結(jié)論： 1. PprI蛋白濃度達(dá)到2.5、5、10ug/mL時(shí)對(duì)細(xì)胞增殖產(chǎn)生抑制作用，且抑制作用與蛋白濃度呈正相關(guān)，細(xì)胞密度0.5×104作用時(shí)間24h的細(xì)胞半抑制濃度IC50為6.40ug/mL。PprI蛋白濃度為100、200、400、600ng/mL時(shí)對(duì)細(xì)胞產(chǎn)生增殖作用，蛋白濃度400ng/mL作用36h時(shí)增殖作用最為顯著。 2.由多靶單擊模型擬合出細(xì)胞存活曲線得知，PprI蛋白在一定程度上可以使細(xì)胞的平均致死劑量增大，放射抗拒性增強(qiáng)，細(xì)胞修復(fù)能力增強(qiáng)。 3. PprI蛋白對(duì)小鼠免疫器官的影響主要是中樞性免疫器官胸腺及主要臟器腎臟，且其刺激是可逆的。對(duì)小鼠脾臟、肝臟基本無(wú)毒性影響。 4. PprI蛋白免疫劑量達(dá)到100μg/kg、200μg/kg時(shí)，PprI蛋白可以增強(qiáng)小鼠脾臟、胸腺淋巴細(xì)胞轉(zhuǎn)化功能。 5. PprI蛋白刺激機(jī)體的免疫方式后期主要為體液免疫應(yīng)答，恢復(fù)期主要為細(xì)胞免疫應(yīng)答（遲發(fā)型免疫應(yīng)答）。 6. PprI蛋白可以升高小鼠脾臟CD3+T細(xì)胞在T細(xì)胞亞群中的比例，CD4／CD8比值一直處于正常值范圍內(nèi)；降低小鼠胸腺CD3+T細(xì)胞在T細(xì)胞亞群中的比例，在免疫恢復(fù)期小鼠主要表現(xiàn)為細(xì)胞免疫。 7.綜上所述，小鼠基本表現(xiàn)為免疫功能增強(qiáng)，但對(duì)免疫器官和免疫功能無(wú)不良影響，基本無(wú)免疫毒性作用，安全性良好，為以后此蛋白的臨床應(yīng)用提供了一定的參考基礎(chǔ)。
[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.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R378
本文編號(hào)：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.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R378
【參考文獻(xiàn)】
相關(guān)期刊論文 前4條
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