口服IL12重组双歧杆菌对柯萨奇B3病毒诱导BLAB/c小鼠心肌炎的影响

Bifidobacterium as an oral delivery carrier of interleukin12 for the treatment of coxsackievirus B3induced myocarditis in the BALB/c mice

摘要:

目的构建白细胞介素（IL）12基因重组双歧杆菌(pBBADsIL12转化双歧杆菌)，观察pBBADsIL12转化双歧杆菌是否对柯萨奇B3病毒(CVB3)诱导的小鼠心肌炎具有治疗作用。方法构建小鼠IL12(mIL12)基因的pBBADsIL12表达载体，转化双歧杆菌，体外通过酶联免疫吸附试验及Western免疫印迹验证重组双歧杆菌工程菌在L阿拉伯糖诱导下mIL12的表达。选取BLAB/c小鼠30只，腹腔内注射CVB3感染剂量，14 d后形成病毒性心肌炎，将感染的小鼠随机分成IL12组、绿荧光蛋白(GFP)组及生理盐水组。IL12组给予口服pBBADsIL12转化双歧杆菌；GFP组给予口服pBBADsGFP转化双歧杆菌；生理盐水组给予腹腔注射无菌PBS（1次/d）。所有小鼠均在治疗14 d后取心脏标本观察心肌病理变化，检测心肌病毒滴度，荧光定量PCR分析Th1细胞因子水平。结果治疗14 d后，HE染色显示IL12组小鼠心肌炎症程度较GFP组和生理盐水组明显减轻；IL12组小鼠心脏炎症病变百分比为（18±5）%，心肌的病毒滴度为（2.89±0.18）pfu/g，显著低于GFP组［分别为（31±6）%和（4.83±0.14）pfu/g］及生理盐水组［分别为（32±9）%和（4.80±0.15）pfu/g］，差异有统计学意义(均P＜0.01）；IL12组小鼠心脏γ干扰素［（2.27±0.15）pg/mL］和肿瘤坏死因子α［（3.05±0.17）pg/mL］水平显著高于GFP组［分别为（1.32±0.11）pg/mL和（2.37±0.16）pg/mL］及生理盐水组［分别为（1.38±0.11）pg/mL和(2.37±0.12)pg/mL］，差异有统计学意义（均P<0.01）。结论此次研究成功构建了一种新型表达mIL12的双歧杆菌载体，口服IL12转化双歧杆菌对CVB3病毒诱导的小鼠心肌炎具有较好疗效。

Abstract:

ObjectiveTo develop a novel oral delivery system for interleukin12 (IL12) using genetically engineered Bifidobacterium longum(B. longum) as the carrier and further evaluate the efficacy of IL12expressed B. longum on the coxsackievirus B3 (CVB3)induced myocarditis in mice.MethodsA mIL12 gene expression vector pBBADsIL12 for B. longum was constructed and transformed into Bifidobacterium, the expression of mIL12 in the engineered B. longum was identified in vitro by Western Blot and enzymelinked immunosorbent assay (ELISA) after Larabinose induction. BLAB/c mice were inoculated i.p. with infectious dose of CVB3 for fourteen days and were divided randomly into three groups. The IL12 group and green fluorescent protein group (GFP group) were orally administered with pBBADsIL12 and pBBADsGFP transformed B. longum for fourteen days respectively after the inoculation of the virus； saline group was administered i.p. with sterile PBS. All animals were killed in day 14 of treatment, and the murine hearts were dissected aseptically for hematoxylineosin (HE) staining, viral titer and RNA extraction for Th1 cell cytokines quantification.ResultsAfter 14 days of treatment, HE staining revealed that the severity of virusinduced myocarditis in IL12 group was reduced compared with that of GFP group and saline group; the percentage of cardiac pathological lesions and CVB3 titers in IL12 group was （18±5）% and (2.89±0.10)pfu/g respectively,which was significantly lower than that of GFP group (［31±6］%, ［4.83±0.14］pfu/g) and saline group (［32±9］%, ［4.80±0.15］pfu/g), respectively (all P<0.01); levels of interferonγ(IFNγ) and tumor necrosis factorα(TNFα) in cardiac tissue and supernatants of IL12 group was（2.27±0.15）pg/mL and （3.05±0.17）pg/mL, respectively, which was significantly higher than that of GFP group (［1.32±0.11］pg/mL,［2.37±0.16］pg/mL) and saline group (［1.38±0.11］pg/mL, ［2.37±0.12］pg/mL）, respectively (all P<0.01).ConclusionA novel oral delivery system of Bifidobacterium for murine IL12 has been successfully established. Oral administration of mIL12transformed B. longum may play a therapeutic role in the treatment of CVB3induced myocarditis in the mice.