与ZNF446同源的LT-7在布鲁菌侵染THP-1中对MAPK通路影响的研究
发布时间：2018-12-15 19:47
【摘要】：目的：布病（Brucellosis）是一种严重危害人和动物健康的人兽共患传染病。巨噬细胞是布鲁菌生存和繁殖的靶细胞之一，布鲁菌的胞内生存与抑制宿主细胞凋亡有关，外膜蛋白OMP25(OuterMembrane Protein25)在布鲁菌的毒力及胞内寄生中起重要作用。前期，本课题组应用噬菌体展示技术筛选出与布鲁菌OMP25特异性结合的42个环形七肽，通过生物信息学分析，LT-7C与锌指蛋白ZNF446（Zinc Finger Protein Peptide446）对应区域有100%的同源性。本研究以布鲁菌RB51(Brucellaabortus strain RB51)侵染THP-1(human leukemia cell line)细胞，研究LT-7C在不同阶段对MAPK通路(MAPK signal pathway)的影响，进而探讨LT-7C对细胞凋亡的影响。 方法：本研究利用合成的环形七肽与THP-1共孵育，采用MTT法进行多肽的毒性检测，选择合适的七肽浓度与THP-1共孵育，实验设未感染组和感染组，包括未感染对照组、未感染线性七肽组、未感染环形七肽组、感染对照组、感染线性七肽组和感染环形七肽组。以MOI(multiplicity of infection)为50:1建立RB51感染THP-1细胞的感染模型。分别收集4、8、12、24h和48h的感染细胞，利用流式细胞仪检测不同时间段的早期凋亡率；感染4h和48h时，曲拉通裂解细胞，进行细胞内布鲁菌计数；采用qRT-PCR技术和Western blot检测4h和48h MAPK通路中ERK1/2、JNK1/2、P38的mRNA水平和蛋白的表达变化。 结果：(1)合成的环形七肽对THP-1无毒副作用；(2)流式细胞术检测显示：在未感染组中环形七肽组和线性七肽组与对照组比较早期凋亡率随着时间的变化呈上升趋势，统计学分析各组差异无统计学意义（P0.05）；在感染组中环形七肽组和线性七肽组与感染对照组比较12h前呈上升趋势，12h后呈下降趋势，其中4h时感染环形组高于对照组，差异有统计学意义（P0.05）；48h时感染环形组低于对照组，，差异有统计学意义（P0.05）。(3)CFU计数结果显示感染环形七肽组早期4h时胞内细菌量低于感染对照组，并且差异有统计学意义(P0.05)；线性七肽组4h胞内含菌量高于感染对照组，差异无统计学意义；48h时各组差异无统计学意义。(4) qRT-PCR检测结果显示4h时感染线性组LT-7组和感染环形LT-7C组erk1的拷贝数低于感染对照组，ekr2、p38、jnk1和jnk2拷贝数在各感染组无变化，未感染组4h时各基因的拷贝数无变化；48h时感染环形组中jnk1/2拷贝数低于感染对照组；erk1、ekr2、p38拷贝数在各感染组无变化，未感染组48h时各基因的拷贝数无变化；（5）Western blot结果与qRT-PCR一致。 结论：(1)以MOI为50:1的RB51感染的环形七肽组中，环形七肽4h时有抑制胞内入侵作用，48h时其对胞内入侵无作用。(2)环形七肽组4h时ERK1/2的低表达与凋亡率增高有关，48h时JNK1/2的低表达与凋亡率降低有关。
[Abstract]:Objective: (Brucellosis) is a zoonotic infectious disease which seriously endangers human and animal health. Macrophages are one of the target cells for brucellosis survival and reproduction. The intracellular survival of Brucella is related to the inhibition of host cell apoptosis. The outer membrane protein OMP25 (OuterMembrane Protein25) plays an important role in the virulence and intracellular parasitism of Brucella. In the early stage, the phage display technique was used to screen 42 cyclic heptapeptides specifically bound to Brucella OMP25. By bioinformatics analysis, 100% homology was found between LT-7C and zinc finger protein (ZNF446 (Zinc Finger Protein Peptide446). In this study, THP-1 (human leukemia cell line) cells were infected with brucellosis (RB51 (Brucellaabortus strain RB51) to study the effect of LT-7C on (MAPK signal pathway) of MAPK pathway at different stages, and then to explore the effect of LT-7C on apoptosis. Methods: in this study, the synthetic ring heptapeptide was co-incubated with THP-1, the toxicity of the peptide was detected by MTT method, and the appropriate concentration of heptapeptide was co-incubated with THP-1. The experiment was divided into two groups: uninfected group and infected group, including uninfected control group. No infection linear heptapeptide group, no infection ring heptapeptide group, infection control group, infection linear heptapeptide group and infection ring heptapeptide group. The infection model of THP-1 cells infected with RB51 was established by using MOI (multiplicity of infection) as 50:1. The rate of early apoptosis was detected by flow cytometry at 24 h and 48 h, respectively, and at 4 h and 48 h after infection, the cells were lysed by traitone, and the intracellular brucellosis was counted. QRT-PCR and Western blot were used to detect the expression of ERK1/2,JNK1/2,P38 mRNA and protein in MAPK pathway at 4h and 48h. Results: (1) the synthetic ring heptapeptide had no side effect on THP-1; (2) flow cytometry showed that in the uninfected group, the early apoptosis rate of the ring heptapeptide group and the linear heptapeptide group showed an increasing trend with the change of time compared with the control group, and there was no significant difference between the two groups (P0.05). In the infection group, the ring heptapeptide group and the linear heptapeptide group showed an upward trend before 12 h and a downward trend after 12 h, and the infection ring group was higher than the control group at 4 h, the difference was statistically significant (P0.05). 48 h infection ring group was lower than the control group, the difference was statistically significant (P0.05). (3) CFU count results showed that the number of intracellular bacteria in the infection ring heptapeptide group was lower than that in the infected control group at 4 h, and the difference was statistically significant (P0.05). The intracellular bacterial content in the linear heptapeptide group was higher than that in the infected control group at 4 h, and the difference was not statistically significant. (4) the results of qRT-PCR detection showed that the copy number of erk1 in LT-7 group and ring LT-7C group was lower than that in infected control group at 4 h, but the copy numbers of ekr2,p38,jnk1 and jnk2 were not changed in each infection group. The number of copies of each gene did not change at 4 hours in the uninfected group. The copy number of jnk1/2 in the infection ring group was lower than that in the infected control group at 48 h, but the copy number of erk1,ekr2,p38 did not change in each infection group, but the copy number of each gene in the non-infected group did not change at 48 h. (5) the result of) Western blot was consistent with that of qRT-PCR. Conclusion: (1) in the ring heptapeptide group infected with MOI 50:1 RB51, the circular heptapeptide can inhibit the invasion of the cells at 4 h. (2) the low expression of ERK1/2 was related to the increase of apoptosis rate at 4 h, and the low expression of JNK1/2 was related to the decrease of apoptosis rate at 48 h.
【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R516.7
[Abstract]:Objective: (Brucellosis) is a zoonotic infectious disease which seriously endangers human and animal health. Macrophages are one of the target cells for brucellosis survival and reproduction. The intracellular survival of Brucella is related to the inhibition of host cell apoptosis. The outer membrane protein OMP25 (OuterMembrane Protein25) plays an important role in the virulence and intracellular parasitism of Brucella. In the early stage, the phage display technique was used to screen 42 cyclic heptapeptides specifically bound to Brucella OMP25. By bioinformatics analysis, 100% homology was found between LT-7C and zinc finger protein (ZNF446 (Zinc Finger Protein Peptide446). In this study, THP-1 (human leukemia cell line) cells were infected with brucellosis (RB51 (Brucellaabortus strain RB51) to study the effect of LT-7C on (MAPK signal pathway) of MAPK pathway at different stages, and then to explore the effect of LT-7C on apoptosis. Methods: in this study, the synthetic ring heptapeptide was co-incubated with THP-1, the toxicity of the peptide was detected by MTT method, and the appropriate concentration of heptapeptide was co-incubated with THP-1. The experiment was divided into two groups: uninfected group and infected group, including uninfected control group. No infection linear heptapeptide group, no infection ring heptapeptide group, infection control group, infection linear heptapeptide group and infection ring heptapeptide group. The infection model of THP-1 cells infected with RB51 was established by using MOI (multiplicity of infection) as 50:1. The rate of early apoptosis was detected by flow cytometry at 24 h and 48 h, respectively, and at 4 h and 48 h after infection, the cells were lysed by traitone, and the intracellular brucellosis was counted. QRT-PCR and Western blot were used to detect the expression of ERK1/2,JNK1/2,P38 mRNA and protein in MAPK pathway at 4h and 48h. Results: (1) the synthetic ring heptapeptide had no side effect on THP-1; (2) flow cytometry showed that in the uninfected group, the early apoptosis rate of the ring heptapeptide group and the linear heptapeptide group showed an increasing trend with the change of time compared with the control group, and there was no significant difference between the two groups (P0.05). In the infection group, the ring heptapeptide group and the linear heptapeptide group showed an upward trend before 12 h and a downward trend after 12 h, and the infection ring group was higher than the control group at 4 h, the difference was statistically significant (P0.05). 48 h infection ring group was lower than the control group, the difference was statistically significant (P0.05). (3) CFU count results showed that the number of intracellular bacteria in the infection ring heptapeptide group was lower than that in the infected control group at 4 h, and the difference was statistically significant (P0.05). The intracellular bacterial content in the linear heptapeptide group was higher than that in the infected control group at 4 h, and the difference was not statistically significant. (4) the results of qRT-PCR detection showed that the copy number of erk1 in LT-7 group and ring LT-7C group was lower than that in infected control group at 4 h, but the copy numbers of ekr2,p38,jnk1 and jnk2 were not changed in each infection group. The number of copies of each gene did not change at 4 hours in the uninfected group. The copy number of jnk1/2 in the infection ring group was lower than that in the infected control group at 48 h, but the copy number of erk1,ekr2,p38 did not change in each infection group, but the copy number of each gene in the non-infected group did not change at 48 h. (5) the result of) Western blot was consistent with that of qRT-PCR. Conclusion: (1) in the ring heptapeptide group infected with MOI 50:1 RB51, the circular heptapeptide can inhibit the invasion of the cells at 4 h. (2) the low expression of ERK1/2 was related to the increase of apoptosis rate at 4 h, and the low expression of JNK1/2 was related to the decrease of apoptosis rate at 48 h.
【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R516.7
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