β防御素124沉默通过p38MAPK/AP1通路调控山羊附睾头细胞趋化因子和细胞因子的表达

doi:10.11843/j.issn.0366-6964.2020.009

摘要/Abstract

摘要： 旨在研究山羊β防御素124（goat beta-defensin 124，gBD124）沉默对p38MAPK/AP1通路及下游细胞趋化因子和细胞因子表达的影响。本研究将设计的3条gBD124-shRNAs载入LV10-U6/RFP&Puro质粒载体，与包装质粒共转染到293T细胞中，用梯度稀释法测定病毒原液的滴度；将构建的3个LV10-gBD124和LV10-gBD124-NC载体转染到山羊附睾头细胞中，筛选有效沉默载体；然后用有效沉默载体联合转染附睾头细胞，设置了空白细胞对照组、LV10-NC组、有效沉默载体组，用2 μg·mL^-1嘌呤霉素筛选后分别收集细胞和培养液，采用qRT-PCR、Western blot和ELISA检测gBD124、MAPK信号通路关键蛋白、细胞因子及趋化因子基因及蛋白表达情况。本研究构建了gBD124沉默慢病毒载体，筛选出LV10-gBD124-51和LV10-gBD124-161两个有效载体，并成功构建了gBD124沉默稳定转染的附睾头细胞株。与NC对照组相比，gBD124沉默显著降低了山羊附睾头细胞MAPK通路中MAPK1以及AP1的两个亚型c-JUN和c-FOS基因表达（P<0.05），显著增加了RASA1基因表达（P<0.05）；gBD124沉默显著降低了总p38MAPK、总c-JUN和总c-FOS蛋白表达，以及磷酸化p38MAPK和c-JUN蛋白表达（P<0.05）；gBD124沉默显著上调了MAPK通路下游IL-1β及其受体IL-1R2、IL-8和趋化因子CCL6和CCL21基因表达（P<0.05），下调了CCL5和IL-1α基因表达（P<0.05）；gBD124沉默显著降低了细胞培养液中CCL5浓度（P<0.05）。与空白对照组相比，gBD124沉默显著增加了培养液中IL-1β和IL-8浓度，降低了IL-1α浓度（P<0.05）。gBD124基因沉默通过抑制p38MAPK/AP1信号通路调控附睾头细胞趋化因子和细胞因子的表达。

关键词: 附睾头细胞, β防御素124, shRNA慢病毒载体, p38MAPK/AP1通路, 山羊

Abstract: The study was conducted to investigate the effects of silencing goat beta-defensin 124 (gBD124) on the expression of key genes in p38MAPK/AP1 pathway and their downstream target genes of the cytokines and chemokines. Three designed gBD124-shRNAs were loaded into LV10-U6/RFP&Puro shuttle plasmid, then cotransfected into 293T cell with packaging plasmid for lentivirus packaging. Virus titer was detected using the method of suspension gradient dilution. Three recombinant LV10-gBD124 vectors and LV10-gBD124-NC were transfected into epididymal caput cells of Taihang goat, respectively for screening the effective silent vectors. The effective recombinant LV10-gBD124 vectors were co-transfected into epididymal caput cells, and the blank cell group, LV10-NC group and effective silent vector group were setup, respectively. The epididymal caput cells and culture mediums were collected separately after screening by 2 μg·mL^-1 puromycin. The mRNA and protein expression levels of gBD124, some key genes in MAPK signaling pathway, cytokines and chemokines were detected by qRT-PCR, Western blot and the high-specific ELISA kits, respectively. The results showed that LV10-gBD124 recombinant vectors were constructed and two valid recombinant vectors of LV10-gBD124-51 and LV10-gBD124-161 were screened for silencing gBD124. The epididymal caput cells with gBD124 silenced were successfully constructed. The results of qRT-PCR indicated that silencing of gBD124 in epididymal caput cells significantly decreased the expressions of MAPK1, c-JUN and c-FOS(P<0.05),and significantly increased the expression of RASA1 in the MAPK signaling pathway(P<0.05). The result of Western blot showed that protein levels of total p38MAPK, total c-JUN, total c-FOS, phosphor-p38MAPK and phosphor-c-JUN were significantly reduced in epididymal caput cells with gBD124 silenced (P<0.05). The expressions of IL-1β, IL-1R2, IL-8, CCL6, CCL21 were markedly promoted (P<0.05), the expressions of CCL5 and IL-1α were significantly reduced in epididymal caput cells with gBD124 silenced (P<0.05). The concentration of CCL5 in the culture medium was significantly reduced in the group of silencing gBD124 (P<0.05). Compared with the blank control group, the concentrations of IL-1β and IL-8 in the culture medium were enhanced, and the concentration of IL-1α was significantly decreased in the group of silencing gBD124(P<0.05). The silencing of gBD124 in epididymal caput cells could regulate the expression of cytokines and chemokines through inhibiting the p38MAPK/AP1 signaling pathway.

Key words: epididymal caput cell, beta-defensin 124, shRNA-lentivirus vector, p38MAPK/AP1 pathway, goat

中图分类号:

S826.2

孟繁荣, 邰苗苗, 董复成, 任有蛇, 刘文忠, 乔利英, 张春香. β防御素124沉默通过p38MAPK/AP1通路调控山羊附睾头细胞趋化因子和细胞因子的表达[J]. 畜牧兽医学报, 2020, 51(6): 1248-1259.

MENG Fanrong, TAI Miaomiao, DONG Fucheng, REN Youshe, LIU Wenzhong, QIAO Liying, ZHANG Chunxiang. Silencing of Goat Beta-defensin 124 Regulate the Expression of Cytokines and Chemokines in Epididymal Caput Cell by the p38MAPK/AP1 Signaling Pathway[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(6): 1248-1259.

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