【摘要】：目的明確TOLL樣受體2(TLR2)與micro RNA144(mi R-144)作用之間的關(guān)系。方法利用不同濃度的mi R-144的模擬物(mimics)和抑制劑(inhibitor)瞬時轉(zhuǎn)染大鼠巨噬細(xì)胞系NR8383細(xì)胞,RT-q PCR檢測mi R-144和TLR2及其下游分子TNF-α的表達。利用大鼠肝臟c DNA為模板,PCR獲取目的片段(即含mi R-144野生及突變結(jié)合位點的TLR2 m RNA的3'UTR區(qū));用SacⅠ、XbaⅠ雙酶切pmir GLO報告基因載體和含mi R-144野生及突變結(jié)合位點的TLR2 m RNA的3'UTR區(qū),構(gòu)建攜帶上述片段的雙熒光素酶報告基因,并通過PCR、雙酶切、DNA測序鑒定構(gòu)建的重組質(zhì)粒,即pmir-TLR2-3'UTR及pmir-mutant-TLR2-3'UTR;并用mi R-144的mimics與雙熒光素酶報告基因共轉(zhuǎn)染明確mi R-144與TLR2 m RNA的3'UTR區(qū)的靶向關(guān)系。結(jié)果瞬時轉(zhuǎn)染100 nmol/L mi R-144 mimics后,NR8383細(xì)胞中mi R-144表達顯著升高,而TLR2及其下游分子TNF-α表達顯著下降;而100 nmol/L mi R-144 inhibitor作用則相反。PCR和雙酶切DNA測序結(jié)果證實pmir-TLR2-3'UTR及pmir-mutant-TLR2-3'UTR重組載體構(gòu)建成功;用100 nmol/L mi R-144 mimics與空載體及上述兩個構(gòu)建體分別共轉(zhuǎn)染HEK 293T細(xì)胞后,pmir-TLR2-3'UTR轉(zhuǎn)染組相對熒光素酶活性顯著降低。結(jié)論 mi R-144通過靶向結(jié)合TLR2 m RNA的3'UTR區(qū)負(fù)性調(diào)節(jié)TLR2及其下游促炎因子的表達。
[Abstract]:Objective to investigate the relationship between TOLL like receptor 2 (TLR2) and micro RNA144 (mi R-144). Methods Rat macrophage cell line NR8383 was transiently transfected with different concentrations of mi R-144 mimic (mimics) and inhibitor (inhibitor). The expression of mi R-144, TLR2 and its downstream TNF- 偽 was detected by RT-q PCR. Using c DNA of rat liver as template, PCR was used to obtain the target fragment (that is, the 3'UTR region of TLR2 m RNA containing mi R-144 wild and mutant binding sites). A double luciferase reporter gene containing Sac 鈪，

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