MT2在小鼠內(nèi)毒素耐受中的作用及機制研究
發(fā)布時間：2018-09-03 20:04
【摘要】：目的應用CRISPR技術構(gòu)建MT2基因缺陷鼠(MT2-/-)。用標準內(nèi)毒耐受程序處理MT2-/-小鼠和野生型小鼠。觀察內(nèi)毒素攻擊時兩組小鼠血清炎癥因子水平、肝臟病理性損傷的差異,以及小鼠肝細胞內(nèi)p38蛋白激酶的磷酸化情況,探討MT2對內(nèi)毒素耐受現(xiàn)象的影響及作用機制,為進一步認識MT2的抗炎作用機制及膿毒癥治療方法提供實驗依據(jù)。方法1.MT2-/-小鼠的構(gòu)建利用CRISPR/Cas9技術獲得雜合子MT2+/-小鼠(委托南方模式生物技術公司完成),通過雜交和回交(遺傳背景均為C57BL/6J)獲得子代,應用PCR方法、WB法及基因測序驗證小鼠基因型,獲得足夠數(shù)量的MT2-/-小鼠與野生型小鼠用于后續(xù)實驗。2.內(nèi)毒素耐受實驗挑選6-8周齡不同基因型小鼠,雌雄對半,按照隨機數(shù)字法分為:野生型對照組(WT Ctrl組)、MT2-/-對照組(MT2-/-Ctrl組)、野生型高劑量LPS刺激組(WT LPS組)、MT2-/-高劑量LPS刺激組(MT2-/-LPS組)、野生型耐受組(WT ET組)、MT2-/-內(nèi)毒素耐受組(MT2-/-ET組),觀察每組小鼠生存狀態(tài)及存活數(shù)目,記錄并分析。3.內(nèi)毒素耐受效應觀察重復前述內(nèi)毒素耐受程序,條件同前,高劑量內(nèi)毒素(50 mg/kg)刺激小鼠后12 h、1 d、2 d、3 d、4 d、5 d的6個時間節(jié)點,分別取外周血,ELISA測定炎癥因子(TNF-α、IL-10)水平及丙氨酸氨基轉(zhuǎn)移酶(ALT)含量;內(nèi)毒素耐受組小鼠高劑量內(nèi)毒素(50 mg/kg)刺激處理后第5 d,剖取肝組織,ELISA測定肝組織勻漿中丙二醛(MDA)及超氧化物歧化酶(SOD)值,HE染色觀察肝組織病理損傷情況,TUNEL法計算肝細胞凋亡指數(shù);蛋白印記法及免疫組化法檢測不同基因型內(nèi)毒素耐受的小鼠肝臟中p38絲裂原激活蛋白激酶(p38MAPK)磷酸化水平。結(jié)果1.經(jīng)過PCR、WB及基因測序驗證,MT2敲基因小鼠構(gòu)建成功;2.MT2敲基因小鼠及野生型小鼠均產(chǎn)生內(nèi)毒素耐受現(xiàn)象,MT2敲基因小鼠需要5d從內(nèi)毒素打擊中恢復正常,野生型小鼠需要3 d即可恢復常態(tài);高劑量內(nèi)毒素打擊后,MT2-/-LPS組小鼠死亡多集中在36 h內(nèi),WT LPS組集中在48 h以內(nèi);比較同一時間節(jié)點,MT2敲基因小鼠死亡率高(P0.05);3.MT2-/-ET組小鼠促炎因子TNF-α水平和ALT含量高于WT ET組(P0.05),且出現(xiàn)峰值時間短,而抗炎因子IL-10水平低于WT ET組,差異有統(tǒng)計學意義;4.MT2-/-ET組小鼠血清脂質(zhì)過氧化產(chǎn)物(MDA)的水平與對照組對比明顯升高(P0.001),抗氧化酶(SOD)活性明顯降低(P0.001),WT ET組MDA水平低于MT2-/-ET組(P0.05);5.MT2-/-ET組小鼠肝臟病理性損傷重、凋亡小體多、p38 MAPK信號通路的磷酸化水平高(P0.05vs.WT ET組)。結(jié)論成功構(gòu)建MT2-/-小鼠,MT2缺失的小鼠仍可產(chǎn)生內(nèi)毒素耐受,但被高劑量內(nèi)毒素打擊后需要更長的時間恢復;MT2能抑制內(nèi)毒素耐受小鼠促炎因子TNF-α釋放,促進抗炎因子IL-10釋放;同時MT2可能通過減少肝臟組織中p-p38蛋白的表達,減輕肝臟的病理性損傷,MT2發(fā)揮抗炎作用可能與抑制p38MAPK信號通路有關。
[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- 偽 -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- 偽 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- 偽 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.
【學位授予單位】：成都醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：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- 偽 -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- 偽 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- 偽 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.
【學位授予單位】：成都醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R459.7
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