鉛暴露環(huán)境下干預HO-1調控HMGB1表達的研究
發(fā)布時間：2018-08-16 10:44
【摘要】：本論文研究了鉛暴露環(huán)境下HMGB1和HO-1表達的變化,以及干預HO-1對鉛暴露環(huán)境中HMGB1表達的影響。第一部分鉛暴露對斷乳仔鼠海馬組織HMGB1和HO-1表達的影響目的建立鉛暴露斷乳仔鼠模型,檢測斷乳仔鼠血液和海馬組織中的鉛含量,以及斷乳仔鼠海馬組織中HMGB1和HO-1的表達。方法1、動物建模:在檢測到雌鼠受孕當天,將其隨機分成3個組:(1)C組:空白對照組,給予雙蒸水;(2)L組:低劑量鉛暴露組,給予0.05%(0.5 g/L)醋酸鉛水溶液;(3)H組:高劑量鉛暴露組,給予0.2%(2.0 g/L)醋酸鉛溶液。采用自由飲水方式進行鉛暴露,直至仔鼠斷乳(出生后21d)。2、實驗方法:(1)采用Morris水迷宮對斷乳仔鼠進行行為學測試;(2)采用電感耦合等離子體發(fā)射光譜儀(Inductively coupled plasma-atomic emission spectroscopy,ICP-AES)檢測斷乳仔鼠全血和海馬組織中鉛含量;(3)采用逆轉錄-聚合酶鏈反應(Reverse transcriptase polymerase chain reaction,RT-PCR)檢測斷乳仔鼠海馬組織HMGB1和HO-1 mRNA水平;(4)采用蛋白免疫印跡法(Western blot,WB)檢測斷乳仔鼠海馬組織HMGB1和HO-1蛋白水平;(5)采用免疫熒光法(Immunofluorescent,IF)檢測HMGB1和HO-1在斷乳仔鼠在海馬組織各區(qū)域的蛋白水平。結果1、Mirros水迷宮實驗結果:鉛暴露會導致斷乳仔鼠的學習記憶能力下降。(1)逃避潛伏期:與C組比較,L組和H組的潛伏期時間明顯延長(P0.05);(2)穿越平臺次數:與C組比較,L組和H組的穿越平臺次數明顯減少(P0.05),且H組的次數明顯低于L組(P0.05)。2、血鉛和海馬組織鉛含量的檢測結果:鉛暴露會引起斷乳仔鼠的血鉛和海馬鉛水平的上升。ICP-AES檢測表明,斷乳仔鼠血鉛濃度和海馬鉛含量均隨著鉛暴露濃度的升高明顯增高(P0.05)。3、鉛暴露對斷乳仔鼠HMGB1表達的影響:(1)RT-PCR實驗發(fā)現,斷乳仔鼠海馬組織HMGB1 mRNA水平,會隨著鉛暴露濃度的升高明顯增高(P0.05);(2)WB實驗發(fā)現,HMGB1水平,隨著鉛暴露濃度的升高明顯增高(P0.05);(3)IF實驗發(fā)現,海馬組織CA1、CA3以及DG區(qū)HMGB1蛋白水平,隨著鉛暴露濃度的升高明顯增高(P0.05)。4、鉛暴露對斷乳仔鼠HO-1表達的影響:(1)RT-PCR實驗發(fā)現,斷乳仔鼠海馬組織HO-1 mRNA水平,會隨著鉛暴露濃度的升高明顯增高(P0.05);(2)WB實驗發(fā)現,斷乳仔鼠HO-1蛋白水平,隨著鉛暴露濃度的升高明顯增高(P0.05);(3)IF實驗發(fā)現,海馬組織CA1、CA3以及DG區(qū)HO-1蛋白水平,隨著鉛暴露濃度的升高明顯增高(P0.05)。第二部分鉛暴露對PC12細胞HMGB1和HO-1表達的影響目的建立鉛暴露PC12細胞模型,檢測HMGB1和HO-1的表達。方法1、細胞建模:將PC12細胞分為3組:0μM(空白對照組)、1μM和100μM的醋酸鉛,進行染鉛處理24 h。2、實驗方法:(1)采用RT-PCR檢測HMGB1和HO-1在PC12細胞的mRNA水平;(2)采用WB檢測HMGB1和HO-1在PC12細胞的蛋白水平;(3)采用IF檢測鉛暴露不同時間段(24h、48h、72h)HMGB1和HO-1在PC12細胞的蛋白水平。結果1、鉛暴露對PC12細胞HMGB1表達的影響:(1)RT-PCR實驗發(fā)現,HMGB1mRNA水平明顯增高,與鉛暴露濃度呈濃度依賴性關系(P0.05);(2)WB實驗發(fā)現:HMGB1蛋白水平明顯增高,與鉛暴露濃度呈濃度依賴性關系(P0.05);(3)IF實驗發(fā)現,鉛暴露不同時間(24h、48h、72h)PC12細胞HMGB1蛋白水平明顯增高,與鉛暴露濃度呈濃度依賴性關系(P0.05)。2、鉛暴露對PC12細胞HO-1表達的影響:(1)RT-PCR實驗發(fā)現,HO-1 mRNA水平明顯增高,與鉛暴露濃度呈濃度依賴性關系(P0.05);(2)WB實驗發(fā)現:HO-1蛋白水平明顯增高,與鉛暴露濃度呈濃度依賴性關系(P0.05);(3)IF實驗發(fā)現,鉛暴露不同時間(24h、48h、72h)PC12細胞HO-1蛋白水平明顯增高,與鉛暴露濃度呈濃度依賴性關系(P0.05)。第三部分鉛暴露環(huán)境下干預HO-1的表達對HMGB1表達的影響目的在鉛暴露細胞模型中加入HO-1的抑制劑ZnPP-Ⅸ或激動劑CoPP,觀察HMGB1表達的變化。方法1、細胞建模:將PC12細胞分為4組:(1)S組:空白對照組,0μM PbAc;(2)PA組:PbAc(1μM);(3)Zn組:PbAc(1μM)+ZnPP(20μM)和(4)Co組:PbAc(1μM)+CoPP(20μM)。鉛暴露處理12h時再加入干擾劑處理12h。2、實驗方法:(1)采用RT-PCR檢測HO-1和HMGB1在PC12細胞的mRNA水平;(2)采用WB檢測HO-1和HMGB1在PC12細胞的蛋白水平。結果1、(1)與S組比,PA組和Co組HO-1 mRNA水平和蛋白水平明顯增高(P0.05);(2)與PA組相比,Zn組HO-1表達明顯下降(P0.05),Co組HO-1表達明顯增高(P0.05)。2、(1)與S組比,PA組和Zn組HMGB1 mRNA水平和蛋白水平明顯增高(P0.05);(2)與PA組相比,Zn組HMGB1表達明顯增高(P0.05),Co組HMGB1表達明顯下降(P0.05)。結論:1、鉛暴露可以上調HMGB1和HO-1的表達。2、上調HO-1可以抑制鉛暴露環(huán)境下HMGB1的表達。
[Abstract]:In this paper, we studied the changes of HMGB1 and HO-1 expression in lead-exposed environment, and the effect of HO-1 intervention on HMGB1 expression in lead-exposed environment. Part I Effects of lead exposure on HMGB1 and HO-1 expression in hippocampus of weaned offspring. Objective To establish a lead-exposed weaned offspring model, detect the lead content in blood and hippocampus, and Methods 1. Animal modeling: On the day of gestation, female rats were randomly divided into three groups: (1) C group: blank control group, given double-steamed water; (2) L group: low-dose lead exposure group, given 0.05% (0.5 g/L) lead acetate solution; (3) H group: high-dose lead exposure group, given 0.2% (2.0 g/L) lead acetate solution. (2) Inductively coupled plasma-atomic emission spectroscopy (ICP-AES) was used to detect the whole blood and hippocampus of weaned offspring. (3) Reverse transcriptase polymerase chain reaction (RT-PCR) was used to detect the levels of HMGB1 and HO-1 mRNA in the hippocampus of weaned offspring; (4) Western blot (WB) was used to detect the levels of HMGB1 and HO-1 protein in the hippocampus of weaned offspring; (5) Immunofluorescence assay (Immuno) Fluorescent, IF) was used to detect the protein levels of HMGB1 and HO-1 in the hippocampus of weaned offspring. Results 1. Mirros water maze experiment showed that lead exposure could lead to the decline of learning and memory ability of weaned offspring. (1) Evasive latency: Compared with group C, the latency of group L and group H was significantly prolonged (P 0.05); (2) The number of crossing platforms: compared with group C. The number of plateau crossing in group L and group H was significantly decreased (P 0.05), and the number of plateau crossing in group H was significantly lower than that in group L (P 0.05). The results of blood lead and hippocampal lead content detection showed that lead exposure could cause the increase of blood lead and hippocampal lead levels in weaned offspring. The expression of HMGB1 in the hippocampus of weaned offspring was significantly increased with the increase of lead exposure (P 0.05). The levels of HMGB1 protein in CA1, CA3 and DG regions increased significantly with the increase of lead exposure concentration (P 0.05). 4. The effect of lead exposure on the expression of HO-1 in the hippocampus of weaned offspring rats: (1) RT-PCR showed that the level of HO-1 mRNA in the hippocampus of weaned offspring rats increased significantly with the increase of lead exposure concentration (P 0.05); (2) WB showed that the level of HO-1 protein in weaned offspring rats increased with the increase of lead exposure concentration (P 0.05). (3) IF assay showed that the levels of CA1, CA3 and HO-1 protein in hippocampus increased significantly with the increase of lead exposure (P 0.05). (2) Effects of lead exposure on the expression of HMGB1 and HO-1 in PC12 cells Objective To establish a lead-exposed PC12 cell model and detect the expression of HMGB1 and HO-1. Cell modeling: PC12 cells were divided into three groups: 0 mu M (blank control group), 1 mu M and 100 mu M lead acetate, and treated with lead for 24 h.2. Methods: (1) The mRNA levels of HMGB1 and HO-1 in PC12 cells were detected by RT-PCR; (2) The protein levels of HMGB1 and HO-1 in PC12 cells were detected by WB; (3) IF was used to detect HMGB in different lead exposure periods (24h, 48h, 72h). Results (1) The expression of HMGB1 in PC12 cells was significantly increased by lead exposure. (1) The expression of HMGB1 mRNA was significantly increased by RT-PCR, which was in a concentration-dependent relationship with lead exposure (P 0.05); (2) The level of HMGB1 protein was significantly increased by WB, and was in a concentration-dependent relationship with lead exposure (P 0.05); (3) IF test. It was found that the HMGB1 protein level in PC12 cells increased significantly at different lead exposure time (24h, 48h, 72h) and was in a concentration-dependent relationship with lead exposure (P 0.05). The level of HO-1 protein in PC12 cells increased significantly at different lead exposure time (24h, 48h, 72h) and showed a concentration-dependent relationship with lead exposure concentration (P 0.05). The third part was the effect of intervention of HO-1 expression on HMGB1 expression in lead exposure environment. Methods 1. Cell modeling: PC12 cells were divided into 4 groups: (1) S group: blank control group, 0 mu M PbAc; (2) PA group: PbAc (1 mu M); and (3) Zn group: PbAc (1 mu M) + ZnPP (20 mu M) and (4) Co group: PbAc (1 mu M) + CoPP (20 mu) after 12 hours of lead exposure. Methods: (1) The mRNA levels of HO-1 and HMGB1 in PC12 cells were detected by RT-PCR; (2) The protein levels of HO-1 and HMGB1 in PC12 cells were detected by WB. Results (1) Compared with S group, HO-1 mRNA and protein levels in PA group and Co group were significantly increased (P 0.05); (2) Compared with PA group, HO-1 expression in Zn group was significantly decreased (P 0.05), and HO-1 expression in Co group was significantly increased (P 0.05). HMGB1 mRNA and protein levels in PA group and Zn group were significantly higher than those in S group (P 0.05). (2) Compared with PA group, HMGB1 expression in Zn group was significantly higher (P 0.05) and HMGB1 expression in Co group was significantly lower (P 0.05). Conclusion: 1. Lead exposure can up-regulate the expression of HMGB1 and HO-1.
【學位授予單位】：南昌大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R114
本文編號：2185748
[Abstract]:In this paper, we studied the changes of HMGB1 and HO-1 expression in lead-exposed environment, and the effect of HO-1 intervention on HMGB1 expression in lead-exposed environment. Part I Effects of lead exposure on HMGB1 and HO-1 expression in hippocampus of weaned offspring. Objective To establish a lead-exposed weaned offspring model, detect the lead content in blood and hippocampus, and Methods 1. Animal modeling: On the day of gestation, female rats were randomly divided into three groups: (1) C group: blank control group, given double-steamed water; (2) L group: low-dose lead exposure group, given 0.05% (0.5 g/L) lead acetate solution; (3) H group: high-dose lead exposure group, given 0.2% (2.0 g/L) lead acetate solution. (2) Inductively coupled plasma-atomic emission spectroscopy (ICP-AES) was used to detect the whole blood and hippocampus of weaned offspring. (3) Reverse transcriptase polymerase chain reaction (RT-PCR) was used to detect the levels of HMGB1 and HO-1 mRNA in the hippocampus of weaned offspring; (4) Western blot (WB) was used to detect the levels of HMGB1 and HO-1 protein in the hippocampus of weaned offspring; (5) Immunofluorescence assay (Immuno) Fluorescent, IF) was used to detect the protein levels of HMGB1 and HO-1 in the hippocampus of weaned offspring. Results 1. Mirros water maze experiment showed that lead exposure could lead to the decline of learning and memory ability of weaned offspring. (1) Evasive latency: Compared with group C, the latency of group L and group H was significantly prolonged (P 0.05); (2) The number of crossing platforms: compared with group C. The number of plateau crossing in group L and group H was significantly decreased (P 0.05), and the number of plateau crossing in group H was significantly lower than that in group L (P 0.05). The results of blood lead and hippocampal lead content detection showed that lead exposure could cause the increase of blood lead and hippocampal lead levels in weaned offspring. The expression of HMGB1 in the hippocampus of weaned offspring was significantly increased with the increase of lead exposure (P 0.05). The levels of HMGB1 protein in CA1, CA3 and DG regions increased significantly with the increase of lead exposure concentration (P 0.05). 4. The effect of lead exposure on the expression of HO-1 in the hippocampus of weaned offspring rats: (1) RT-PCR showed that the level of HO-1 mRNA in the hippocampus of weaned offspring rats increased significantly with the increase of lead exposure concentration (P 0.05); (2) WB showed that the level of HO-1 protein in weaned offspring rats increased with the increase of lead exposure concentration (P 0.05). (3) IF assay showed that the levels of CA1, CA3 and HO-1 protein in hippocampus increased significantly with the increase of lead exposure (P 0.05). (2) Effects of lead exposure on the expression of HMGB1 and HO-1 in PC12 cells Objective To establish a lead-exposed PC12 cell model and detect the expression of HMGB1 and HO-1. Cell modeling: PC12 cells were divided into three groups: 0 mu M (blank control group), 1 mu M and 100 mu M lead acetate, and treated with lead for 24 h.2. Methods: (1) The mRNA levels of HMGB1 and HO-1 in PC12 cells were detected by RT-PCR; (2) The protein levels of HMGB1 and HO-1 in PC12 cells were detected by WB; (3) IF was used to detect HMGB in different lead exposure periods (24h, 48h, 72h). Results (1) The expression of HMGB1 in PC12 cells was significantly increased by lead exposure. (1) The expression of HMGB1 mRNA was significantly increased by RT-PCR, which was in a concentration-dependent relationship with lead exposure (P 0.05); (2) The level of HMGB1 protein was significantly increased by WB, and was in a concentration-dependent relationship with lead exposure (P 0.05); (3) IF test. It was found that the HMGB1 protein level in PC12 cells increased significantly at different lead exposure time (24h, 48h, 72h) and was in a concentration-dependent relationship with lead exposure (P 0.05). The level of HO-1 protein in PC12 cells increased significantly at different lead exposure time (24h, 48h, 72h) and showed a concentration-dependent relationship with lead exposure concentration (P 0.05). The third part was the effect of intervention of HO-1 expression on HMGB1 expression in lead exposure environment. Methods 1. Cell modeling: PC12 cells were divided into 4 groups: (1) S group: blank control group, 0 mu M PbAc; (2) PA group: PbAc (1 mu M); and (3) Zn group: PbAc (1 mu M) + ZnPP (20 mu M) and (4) Co group: PbAc (1 mu M) + CoPP (20 mu) after 12 hours of lead exposure. Methods: (1) The mRNA levels of HO-1 and HMGB1 in PC12 cells were detected by RT-PCR; (2) The protein levels of HO-1 and HMGB1 in PC12 cells were detected by WB. Results (1) Compared with S group, HO-1 mRNA and protein levels in PA group and Co group were significantly increased (P 0.05); (2) Compared with PA group, HO-1 expression in Zn group was significantly decreased (P 0.05), and HO-1 expression in Co group was significantly increased (P 0.05). HMGB1 mRNA and protein levels in PA group and Zn group were significantly higher than those in S group (P 0.05). (2) Compared with PA group, HMGB1 expression in Zn group was significantly higher (P 0.05) and HMGB1 expression in Co group was significantly lower (P 0.05). Conclusion: 1. Lead exposure can up-regulate the expression of HMGB1 and HO-1.
【學位授予單位】：南昌大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R114
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