【摘要】：目的:探討白細(xì)胞介素4(IL-4)基因修飾骨髓間充質(zhì)干細(xì)胞(bone-marrow mesenchymal stem cells, BMMSCs)內(nèi)耳局部植入對免疫性感音神經(jīng)性聾動物的內(nèi)耳病理損傷和生理功能障礙的調(diào)節(jié)與治療作用。 方法:采用鑰孔嘁血藍(lán)蛋白(KLH)抗原在已致敏的豚鼠圓窗龕局部免疫,造成免疫性感音神經(jīng)性聾動物模型33只。分為IL-4基因修飾BMMSCs組(A組)、空載慢病毒感染的BMMSCs對照組(B組,即BMMSCs空載對照組)和模擬手術(shù)對照組(C組),均將各組細(xì)胞懸液經(jīng)鼓階開窗植入內(nèi)耳。提取豚鼠BMMSCs,重組IL-4基因的慢病毒載體體外轉(zhuǎn)染BMMSCs,成功后鼓階開窗植入內(nèi)耳。采用免疫熒光組織化學(xué)試驗(yàn)法觀察導(dǎo)入內(nèi)耳的BMMSCs和免疫酶組織化學(xué)試驗(yàn)法觀察IL-4基因產(chǎn)物在內(nèi)耳組織結(jié)構(gòu)中的分布和表達(dá)情況。應(yīng)用酶聯(lián)免疫吸附試驗(yàn)(ELISA)和聽性腦干誘發(fā)電位(ABR)測試觀察血清抗KLH特異性抗體水平和聽覺功能變化。并行內(nèi)耳石蠟切片和光鏡觀察。 結(jié)果:局部KLH免疫后與內(nèi)耳局部BMMSCs植入后2周對比,特異性抗KLH抗體水平無顯著性差異;A組和B組ABRⅢ波閾值不同程度降低,但前者閾值降低更為明顯,兩組間比較差異有統(tǒng)計學(xué)意義(P0.05);免疫組織化學(xué)結(jié)果顯示BMMSCs(熒光反應(yīng)陽性細(xì)胞)主要分布在鼓階、前庭階。酶反應(yīng)陽性物(IL-4基因產(chǎn)物)部位主要集中于螺旋神經(jīng)節(jié)、骨螺旋板唇部、Corti器、血管紋、耳蝸骨壁、以及蝸管中的BMMSCs的細(xì)胞和其周圍。內(nèi)耳光鏡觀察結(jié)果顯示:A組和B組僅在鼓階內(nèi)有絮狀物、注射部位有少量紅細(xì)胞和白細(xì)胞;C組對照組可見螺旋神經(jīng)節(jié)和蝸軸小血管周圍有單個核細(xì)胞浸潤,部分聽損耳還可見螺旋神經(jīng)節(jié)細(xì)胞數(shù)目減少,不同程度的膜迷路積水,蝸管內(nèi)有絮狀物和漂浮細(xì)胞。 結(jié)論:重組IL-4基因和空載慢病毒在體外可成功地轉(zhuǎn)染BMMSCs。經(jīng)鼓階途徑行IL-4基因修飾BMMSCs和空載慢病毒轉(zhuǎn)染BMMSCs內(nèi)耳移植,均可明顯減輕免疫性感音神經(jīng)性聾動物的內(nèi)耳免疫炎性反應(yīng)和聽覺功能損傷,前者作用更為顯著。從而提示BMMSCs(包括經(jīng)IL-4基因修飾的BMMSCs)局部應(yīng)用可對免疫性內(nèi)耳病的免疫炎性損傷產(chǎn)生一定程度的調(diào)節(jié)和治療作用,并有向病變部位遷移、聚集的傾向。
[Abstract]:Aim: to investigate the effects of interleukin-4 (IL-4) gene modified bone marrow mesenchymal stem cells (bone-marrow mesenchymal stem cells, BMMSCs) implanted locally on the pathological injury and physiological dysfunction of the inner ear in rats with immune sensorineural hearing loss. Methods: 33 animal models of immune sensorineural deafness were established by local immunization of keyhole chirin (KLH) antigen in the sensitized rounding window niche of guinea pigs. They were divided into IL-4 gene modified BMMSCs group (group A), BMMSCs control group (group B) and simulated operation control group (group C). The lentivirus vector containing recombinant IL-4 gene from guinea pig BMMSCs, was successfully transfected into BMMSCs, in vitro. The distribution and expression of IL-4 gene products in the inner ear were observed by immunofluorescence histochemical assay and immunohistochemical method respectively. The changes of serum anti KLH specific antibody and auditory function were observed by enzyme linked immunosorbent assay (ELISA) and auditory brainstem evoked potential (ABR). Paraffin sections of inner ear and light microscope were observed. Results: there was no significant difference in the level of specific anti KLH antibody between two weeks after local KLH immunization and 2 weeks after BMMSCs implantation in the inner ear, but the threshold of ABR 鈪，

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