MT2在小鼠内毒素耐受中的作用及机制研究
发布时间：2018-09-03 20:04
【摘要】：目的应用CRISPR技术构建MT2基因缺陷鼠(MT2-/-)。用标准内毒耐受程序处理MT2-/-小鼠和野生型小鼠。观察内毒素攻击时两组小鼠血清炎症因子水平、肝脏病理性损伤的差异,以及小鼠肝细胞内p38蛋白激酶的磷酸化情况,探讨MT2对内毒素耐受现象的影响及作用机制,为进一步认识MT2的抗炎作用机制及脓毒症治疗方法提供实验依据。方法1.MT2-/-小鼠的构建利用CRISPR/Cas9技术获得杂合子MT2+/-小鼠(委托南方模式生物技术公司完成),通过杂交和回交(遗传背景均为C57BL/6J)获得子代,应用PCR方法、WB法及基因测序验证小鼠基因型,获得足够数量的MT2-/-小鼠与野生型小鼠用于后续实验。2.内毒素耐受实验挑选6-8周龄不同基因型小鼠,雌雄对半,按照随机数字法分为:野生型对照组(WT Ctrl组)、MT2-/-对照组(MT2-/-Ctrl组)、野生型高剂量LPS刺激组(WT LPS组)、MT2-/-高剂量LPS刺激组(MT2-/-LPS组)、野生型耐受组(WT ET组)、MT2-/-内毒素耐受组(MT2-/-ET组),观察每组小鼠生存状态及存活数目,记录并分析。3.内毒素耐受效应观察重复前述内毒素耐受程序,条件同前,高剂量内毒素(50 mg/kg)刺激小鼠后12 h、1 d、2 d、3 d、4 d、5 d的6个时间节点,分别取外周血,ELISA测定炎症因子(TNF-α、IL-10)水平及丙氨酸氨基转移酶(ALT)含量;内毒素耐受组小鼠高剂量内毒素(50 mg/kg)刺激处理后第5 d,剖取肝组织,ELISA测定肝组织匀浆中丙二醛(MDA)及超氧化物歧化酶(SOD)值,HE染色观察肝组织病理损伤情况,TUNEL法计算肝细胞凋亡指数;蛋白印记法及免疫组化法检测不同基因型内毒素耐受的小鼠肝脏中p38丝裂原激活蛋白激酶(p38MAPK)磷酸化水平。结果1.经过PCR、WB及基因测序验证,MT2敲基因小鼠构建成功;2.MT2敲基因小鼠及野生型小鼠均产生内毒素耐受现象,MT2敲基因小鼠需要5d从内毒素打击中恢复正常,野生型小鼠需要3 d即可恢复常态;高剂量内毒素打击后,MT2-/-LPS组小鼠死亡多集中在36 h内,WT LPS组集中在48 h以内;比较同一时间节点,MT2敲基因小鼠死亡率高(P0.05);3.MT2-/-ET组小鼠促炎因子TNF-α水平和ALT含量高于WT ET组(P0.05),且出现峰值时间短,而抗炎因子IL-10水平低于WT ET组,差异有统计学意义;4.MT2-/-ET组小鼠血清脂质过氧化产物(MDA)的水平与对照组对比明显升高(P0.001),抗氧化酶(SOD)活性明显降低(P0.001),WT ET组MDA水平低于MT2-/-ET组(P0.05);5.MT2-/-ET组小鼠肝脏病理性损伤重、凋亡小体多、p38 MAPK信号通路的磷酸化水平高(P0.05vs.WT ET组)。结论成功构建MT2-/-小鼠,MT2缺失的小鼠仍可产生内毒素耐受,但被高剂量内毒素打击后需要更长的时间恢复;MT2能抑制内毒素耐受小鼠促炎因子TNF-α释放,促进抗炎因子IL-10释放;同时MT2可能通过减少肝脏组织中p-p38蛋白的表达,减轻肝脏的病理性损伤,MT2发挥抗炎作用可能与抑制p38MAPK信号通路有关。
[Abstract]:Objective to construct MT2 gene deficient mice (MT2-/-) by CRISPR. MT2-/- mice and wild-type mice were treated with standard internal toxicity tolerance program. To investigate the effect of MT2 on the phenomenon of endotoxin tolerance and to observe the difference of serum inflammatory factor level, hepatic pathological injury and phosphorylation of p38 protein kinase in liver cells between the two groups during endotoxin attack, and to explore the mechanism of the effect of MT2 on the phenomenon of endotoxin tolerance. To further understand the anti-inflammatory mechanism of MT2 and the treatment of sepsis to provide experimental basis. MT2-r-mice were constructed using CRISPR/Cas9 technique to obtain heterozygote MT2 / -mice (commissioned by Southern Model Biotechnology Company), and offspring were obtained by hybridization and backcrossing (both genetic background was C57BL/6J). 2. A sufficient number of MT2-/- mice and wild type mice were obtained for further experiment using PCR method and gene sequencing. Different genotypes of mice aged 6-8 weeks were selected by endotoxin tolerance test, and half of them were male and female. According to the random number method: wild type control group (WT Ctrl group), wild type LPS stimulation group (WT LPS group), wild type control group (WT Ctrl group), wild type high dose LPS stimulation group (WT LPS group), MT2-r-high dose LPS stimulation group (MT2-/-LPS group), wild type tolerance group (WT ET group), MT2-r-endotoxin tolerance group (MT2-/-ET group), observe. To observe the survival status and number of mice in each group, Record and analyze. The effects of endotoxin tolerance were observed by repeating the previous procedure of endotoxin tolerance. Under the same conditions, the mice were stimulated with high dose of endotoxin (50 mg/kg) at 6 time nodes at 12 h, 1 day, 2 d, 3 d, 4 d and 5 d after stimulation. The levels of TNF- 伪 -IL-10 and the content of alanine aminotransferase (ALT) were measured by Elisa. On the 5th day after high dose endotoxin (50 mg/kg) stimulation in endotoxin tolerance group, liver tissue was taken to detect malondialdehyde (MDA) (MDA) and superoxide dismutase (SOD) (SOD) value in liver tissue homogenate by Elisa and HE staining was used to observe the pathological injury of liver tissue. Hepatocyte apoptosis index; The phosphorylation of p38 mitogen-activated protein kinase (p38MAPK) in the liver of mice with different genotypes of endotoxin tolerance was detected by protein imprinting and immunohistochemistry. Result 1. PCR,WB and gene sequencing proved that MT2 knockout mice were successfully constructed. 2. Both MT2 knockout mice and wild type mice produced endotoxin tolerance. It took 5 days for MT2 knockout mice to return to normal during endotoxin attack. It took 3 days for wild-type mice to return to normal, and the death rate of mice in MT2-p-LPS group was mainly within 36 hours after endotoxin treatment, and that in WT LPS group was within 48 hours. The levels of proinflammatory factor TNF- 伪 and ALT in MT2 knockout mice in MT2-ET group were higher than those in WT ET group at the same time (P0.05), and the peak time was shorter, and the IL-10 level of anti-inflammatory factor was lower than that of WT ET group. The level of serum lipid peroxidation product (MDA) in MT2-ET group was significantly higher than that in control group (P0.001), and the level of MDA in WT ET group was significantly lower than that in MT2-/-ET group (P0.001). The phosphorylation level of apoptotic polymorphic p38 MAPK signaling pathway was high (P0.05vs.WT ET group). Conclusion MT2-/- mice without MT2 can still produce endotoxin tolerance, but it takes a longer time to recover after being hit by high dose endotoxin. It can inhibit the release of TNF- 伪 and promote the release of anti-inflammatory factor IL-10 in endotoxin tolerant mice. At the same time, MT2 may reduce the expression of p-p38 protein in liver tissue and reduce the pathological injury of liver. The anti-inflammatory effect of MT2 may be related to the inhibition of p38MAPK signaling pathway.
【学位授予单位】：成都医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R459.7
[Abstract]:Objective to construct MT2 gene deficient mice (MT2-/-) by CRISPR. MT2-/- mice and wild-type mice were treated with standard internal toxicity tolerance program. To investigate the effect of MT2 on the phenomenon of endotoxin tolerance and to observe the difference of serum inflammatory factor level, hepatic pathological injury and phosphorylation of p38 protein kinase in liver cells between the two groups during endotoxin attack, and to explore the mechanism of the effect of MT2 on the phenomenon of endotoxin tolerance. To further understand the anti-inflammatory mechanism of MT2 and the treatment of sepsis to provide experimental basis. MT2-r-mice were constructed using CRISPR/Cas9 technique to obtain heterozygote MT2 / -mice (commissioned by Southern Model Biotechnology Company), and offspring were obtained by hybridization and backcrossing (both genetic background was C57BL/6J). 2. A sufficient number of MT2-/- mice and wild type mice were obtained for further experiment using PCR method and gene sequencing. Different genotypes of mice aged 6-8 weeks were selected by endotoxin tolerance test, and half of them were male and female. According to the random number method: wild type control group (WT Ctrl group), wild type LPS stimulation group (WT LPS group), wild type control group (WT Ctrl group), wild type high dose LPS stimulation group (WT LPS group), MT2-r-high dose LPS stimulation group (MT2-/-LPS group), wild type tolerance group (WT ET group), MT2-r-endotoxin tolerance group (MT2-/-ET group), observe. To observe the survival status and number of mice in each group, Record and analyze. The effects of endotoxin tolerance were observed by repeating the previous procedure of endotoxin tolerance. Under the same conditions, the mice were stimulated with high dose of endotoxin (50 mg/kg) at 6 time nodes at 12 h, 1 day, 2 d, 3 d, 4 d and 5 d after stimulation. The levels of TNF- 伪 -IL-10 and the content of alanine aminotransferase (ALT) were measured by Elisa. On the 5th day after high dose endotoxin (50 mg/kg) stimulation in endotoxin tolerance group, liver tissue was taken to detect malondialdehyde (MDA) (MDA) and superoxide dismutase (SOD) (SOD) value in liver tissue homogenate by Elisa and HE staining was used to observe the pathological injury of liver tissue. Hepatocyte apoptosis index; The phosphorylation of p38 mitogen-activated protein kinase (p38MAPK) in the liver of mice with different genotypes of endotoxin tolerance was detected by protein imprinting and immunohistochemistry. Result 1. PCR,WB and gene sequencing proved that MT2 knockout mice were successfully constructed. 2. Both MT2 knockout mice and wild type mice produced endotoxin tolerance. It took 5 days for MT2 knockout mice to return to normal during endotoxin attack. It took 3 days for wild-type mice to return to normal, and the death rate of mice in MT2-p-LPS group was mainly within 36 hours after endotoxin treatment, and that in WT LPS group was within 48 hours. The levels of proinflammatory factor TNF- 伪 and ALT in MT2 knockout mice in MT2-ET group were higher than those in WT ET group at the same time (P0.05), and the peak time was shorter, and the IL-10 level of anti-inflammatory factor was lower than that of WT ET group. The level of serum lipid peroxidation product (MDA) in MT2-ET group was significantly higher than that in control group (P0.001), and the level of MDA in WT ET group was significantly lower than that in MT2-/-ET group (P0.001). The phosphorylation level of apoptotic polymorphic p38 MAPK signaling pathway was high (P0.05vs.WT ET group). Conclusion MT2-/- mice without MT2 can still produce endotoxin tolerance, but it takes a longer time to recover after being hit by high dose endotoxin. It can inhibit the release of TNF- 伪 and promote the release of anti-inflammatory factor IL-10 in endotoxin tolerant mice. At the same time, MT2 may reduce the expression of p-p38 protein in liver tissue and reduce the pathological injury of liver. The anti-inflammatory effect of MT2 may be related to the inhibition of p38MAPK signaling pathway.
【学位授予单位】：成都医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R459.7
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