硫化氢对烧伤大鼠体外培养皮肤巨噬细胞MAPK信号传导通路ERK、JNK及p38蛋白作用的影响

发布时间：2018-03-08 19:49

本文选题：硫化氢　切入点：巨噬细胞　出处：《青海大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:探究H_2S对烧伤大鼠体外培养皮肤巨噬细胞MAPK信号传导通路ERK、JNK及p38蛋白作用的影响。方法:将大鼠背部创面皮肤巨噬细胞当作实验对象,随机挑选30只健康的SD大鼠(雌雄不限),其中5只正常饲养,余下25只大鼠行烧伤造模后喂养,从正常饲养的5只大鼠中选1只设为正常对照组,从烧伤大鼠中选12只再随机分为4组(每组3只)设为实验对照组(烧伤对照组,NaHS(H_2S供体)组1h、6h、12h,格列本脲(K~+通道阻滞剂)组1h、6h、12h,NaHS+格列本脲组1h、6h、12h。正常对照组不做处理,余各组大鼠背部全部进行深II°5%TBSA损伤处理,分离背部创面皮肤基底巨噬细胞至78个培养瓶中,体外培养稳定后,正常对照组及烧伤对照组均加入200ul的PBS液,NaHS组中将终浓度调整为200umol/L的NaHS加入到培养基中,格列本脲组中将终浓度调整为200umol/L的GLBN加入到培养基中,NaHS+格列本脲组中加入相同终浓度为200umol/L的NaHS和格列本脲,以上每组作用时间均设定为1h、6h、12h。分别在对应时间点对ERK、JNK、p38蛋白进行Western Blot免疫印迹分析。结果:1、各组间ERK蛋白表达量之间比较:(1)烧伤对照组与正常对照组ERK蛋白表达量差别之间比较有统计学意义(P0.05),(2)在同一时相点,NaHS组与各组别ERK蛋白表达量差别之间比较有统计学意义(P0.05),(3)NaHS+GLBN组与GLBN组ERK蛋白表达量差异比较有统计学意义(P0.05),(4)GLBN组与烧伤对照组ERK蛋白表达量差别之间比较有统计学意义(P0.05)。2、各组间JNK蛋白表达量之间比较:(1)烧伤对照组与正常对照组JNK蛋白表达量差别之间比较有统计学意义(P0.05),(2)在同一时间点,NaHS组与各组别JNK蛋白表达量差别之间比较有统计学意义(P0.05),(3)NaHS+GLBN组与GLBN组JNK蛋白表达量差异比较有统计学意义(P0.05),(4)GLBN组与烧伤对照组JNK蛋白表达量差别之间比较有统计学意义(P0.05)。3、各组间p38蛋白表达量之间比较:(1)烧伤对照组与正常对照组p38蛋白表达量差别之间比较有统计学意义(P0.05),(2)在同一时相点,NaHS组与各组别p38蛋白表达量差别之间比较有统计学意义(P0.05),(3)NaHS+GLBN组与GLBN组p38蛋白表达量差异比较有统计学意义(P0.05),(4)GLBN组与烧伤对照组p38蛋白表达量差别之间比较有统计学意义(P0.05)。结论:(1)烧伤后ERK表达减少而JNK和p38表达增多;(2)烧伤后给予微量的H_2S使ERK蛋白相对表达量升高,JNK和p38蛋白相对表达量下降;(3)格列本脲能够抑制K~+通道开放,使ERK蛋白相对表达量下降,JNK和p38蛋白相对表达量提高;(4)外源性H_2S能够通过K~+通道提高ERK表达且减低JNK和p38表达,从而对巨噬细胞MAPK信号通路产生影响。
[Abstract]:Objective: to investigate the effect of H2S on the MAPK signal transduction pathway of skin macrophages (ERKN JNK) and p38 protein in cultured skin macrophages of burned rats. Methods: the skin macrophages of rat back wounds were used as experimental objects. Thirty healthy Sprague-Dawley rats (male and female) were randomly selected. Among them, 5 rats were fed normally, the remaining 25 rats were fed after burn modeling, and one of the 5 healthy rats was selected as the normal control group. Twelve burn rats were randomly divided into 4 groups (3 rats in each group) as experimental control group (NaHS H2S donor) group (burn control group: 1 h, 6 h, 12 h, glibenclamide K ~ + channel blocker) group, 1 h, 6 h, 12 h, NaHS glibenclamide group, 1 h 6 h and 12 h, normal control group were not treated. All rats in the remaining groups were treated with TBSA injury in the deep II 掳5 and the skin basal macrophages were separated into 78 culture flasks. After in vitro culture, the skin macrophages were cultured stably. The normal control group and burn control group were added to 200ul PBS solution NaHS group. The final concentration of NaHS was adjusted to 200umoll / L in the medium. In glibenclamide group, the final concentration of GLBN adjusted to 200umoll / L was added to the culture medium. The same final concentration of NaHS and glibenclamide was added to the culture medium of glibenclamide group with the same final concentration. The time of action of each group was 1 h, 6 h and 12 h. The Western Blot immunoblotting analysis of ERKN JNKK p38 protein was carried out at the corresponding time points. The results showed that the expression of ERK protein was compared between the burn control group and the normal control group, and the ERK protein expression level was compared between the burn control group and the normal control group. There were significant differences in the expression of ERK protein between nahs group and each group at the same time point. There was significant difference in ERK protein expression between nahs GLBN group and GLBN group. There was significant difference in ERK protein expression between nahs group and burn group. There were significant differences in the expression of ERK protein between the control group and the control group (P0.05 / 2, respectively) and the expression of JNK protein between the burn control group and the normal control group (P0.05 / 2) at the same time, there was a significant difference in the expression of JNK protein between the burn control group and the normal control group. There were significant differences in the expression of JNK protein between the nahs group and the different groups. There were significant differences in the expression of JNK protein between the nahs GLBN group and the GLBN group. There were significant differences in the expression of JNK protein between the two groups and the burn control group. The difference of p38 protein expression between burn control group and normal control group was statistically significant (P 0.05) the expression of p38 protein in NaHS group was different from that in each group at the same time point. There were significant differences in the expression of p38 protein between the two groups. Conclusion the expression of p38 protein in the GLBN group and the GLBN group was significantly lower than that in the burn group and the burn control group. Conclusion the expression of p38 protein in the burn group is lower than that in the control group. Conclusion the expression of p38 protein in the burn group is significantly lower than that in the control group. Conclusion the expression of p38 protein in the GLBN group is significantly lower than that in the control group (P < 0.05). The expression of JNK and p38 was increased after burn. The relative expression of ERK and p38 protein decreased after burn.) glibenclamide could inhibit the opening of K ~ channel. The relative expression of ERK and p38 protein were decreased. (4) exogenous H2s could increase the expression of ERK and decrease the expression of JNK and p38 through K ~ channel, thus affecting the MAPK signal pathway of macrophages.
【学位授予单位】：青海大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R644

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