與ZNF446同源的LT-7在布魯菌侵染THP-1中對MAPK通路影響的研究
發(fā)布時間：2018-12-15 19:47
【摘要】：目的：布病（Brucellosis）是一種嚴重危害人和動物健康的人獸共患傳染病。巨噬細胞是布魯菌生存和繁殖的靶細胞之一，布魯菌的胞內生存與抑制宿主細胞凋亡有關，外膜蛋白OMP25(OuterMembrane Protein25)在布魯菌的毒力及胞內寄生中起重要作用。前期，本課題組應用噬菌體展示技術篩選出與布魯菌OMP25特異性結合的42個環(huán)形七肽，通過生物信息學分析，LT-7C與鋅指蛋白ZNF446（Zinc Finger Protein Peptide446）對應區(qū)域有100%的同源性。本研究以布魯菌RB51(Brucellaabortus strain RB51)侵染THP-1(human leukemia cell line)細胞，研究LT-7C在不同階段對MAPK通路(MAPK signal pathway)的影響，進而探討LT-7C對細胞凋亡的影響。 方法：本研究利用合成的環(huán)形七肽與THP-1共孵育，采用MTT法進行多肽的毒性檢測，選擇合適的七肽濃度與THP-1共孵育，實驗設未感染組和感染組，包括未感染對照組、未感染線性七肽組、未感染環(huán)形七肽組、感染對照組、感染線性七肽組和感染環(huán)形七肽組。以MOI(multiplicity of infection)為50:1建立RB51感染THP-1細胞的感染模型。分別收集4、8、12、24h和48h的感染細胞，利用流式細胞儀檢測不同時間段的早期凋亡率；感染4h和48h時，曲拉通裂解細胞，進行細胞內布魯菌計數(shù)；采用qRT-PCR技術和Western blot檢測4h和48h MAPK通路中ERK1/2、JNK1/2、P38的mRNA水平和蛋白的表達變化。 結果：(1)合成的環(huán)形七肽對THP-1無毒副作用；(2)流式細胞術檢測顯示：在未感染組中環(huán)形七肽組和線性七肽組與對照組比較早期凋亡率隨著時間的變化呈上升趨勢，統(tǒng)計學分析各組差異無統(tǒng)計學意義（P0.05）；在感染組中環(huán)形七肽組和線性七肽組與感染對照組比較12h前呈上升趨勢，12h后呈下降趨勢，其中4h時感染環(huán)形組高于對照組，差異有統(tǒng)計學意義（P0.05）；48h時感染環(huán)形組低于對照組，，差異有統(tǒng)計學意義（P0.05）。(3)CFU計數(shù)結果顯示感染環(huán)形七肽組早期4h時胞內細菌量低于感染對照組，并且差異有統(tǒng)計學意義(P0.05)；線性七肽組4h胞內含菌量高于感染對照組，差異無統(tǒng)計學意義；48h時各組差異無統(tǒng)計學意義。(4) qRT-PCR檢測結果顯示4h時感染線性組LT-7組和感染環(huán)形LT-7C組erk1的拷貝數(shù)低于感染對照組，ekr2、p38、jnk1和jnk2拷貝數(shù)在各感染組無變化，未感染組4h時各基因的拷貝數(shù)無變化；48h時感染環(huán)形組中jnk1/2拷貝數(shù)低于感染對照組；erk1、ekr2、p38拷貝數(shù)在各感染組無變化，未感染組48h時各基因的拷貝數(shù)無變化；（5）Western blot結果與qRT-PCR一致。 結論：(1)以MOI為50:1的RB51感染的環(huán)形七肽組中，環(huán)形七肽4h時有抑制胞內入侵作用，48h時其對胞內入侵無作用。(2)環(huán)形七肽組4h時ERK1/2的低表達與凋亡率增高有關，48h時JNK1/2的低表達與凋亡率降低有關。
[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.
【學位授予單位】：石河子大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：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.
【學位授予單位】：石河子大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R516.7
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