【作者】 王璐；

【导师】 孙虹； 丁大连；

【作者基本信息】 中南大学 ， 临床医学， 2014， 博士

【摘要】 目的：(1)通过构建锰(manganese, Mn)的耳毒性动物模型,观察氯化锰(manganese chloride, MnCl2)对离体培养的新生大鼠耳蜗组织的毒性作用及其剂量反应关系。(2)评估烟酰胺腺嘌呤二核苷酸(nicotinamide adenine dinucleotide, NAD)治疗Mn耳毒性的有效性,观察MnCl2所致的耳蜗细胞凋亡情况及NAD在抗细胞凋亡中的作用。(3)评估携带XIAP基因(X-linked inhibitor of apoptosis protein,凋亡抑制蛋白家族的成员之一)的改良型纳米羟基磷灰石载体复合物(PEG-PEI-nHAT-pEGFPN1-XIAP)治疗Mn耳毒性的安全性和有效性。方法：(1)采用出生后第3天的SASCO-SD大鼠作为本实验研究对象,处死后小心分离其耳蜗组织,培养过夜后,实验组以MnCl2浓度分别为0.5mM、1.0mM口3.0mM的培养液培养48h,对照组则以不含MnCl2的培养液培养。免疫荧光染色后于激光共聚焦倒置荧光显微镜下观察各组病理改变,并绘制耳蜗图。(2)采用上述大鼠,经解剖分离、培养过夜后,对照组以仅含0.5-3.0mM MnCl2的培养液处理,实验组以含0.5-30mM MnCl2和20mM NAD的培养液处理,空白对照组以不含MnCl2和NAD的培养液处理,均培养48h。免疫荧光染色后于激光共聚焦倒置荧光显微镜下观察各组病理改变,并绘制耳蜗图。对耳蜗基底膜单位长度内各组存活的听神经纤维(auditory nerve fiber, ANF)和耳蜗神经节单位体积内存活的螺旋神经节细胞(spiral ganglion neuron, SGN)进行计数和统计学分析。(3)采用上述大鼠,经解剖分离、培养过夜后,对照组以仅含1.0mM MnC12的培养液处理,实验组以含1.0mM MnCl2和20mM NAD的培养液处理,空白对照组以不含MnCl2和NAD的培养液处理,各组培养6h后用于caspase染色,培养9h后用于TUNEL染色。于激光共聚焦倒置荧光显微镜下观察各组TUNEL和caspase染色的情况。对单位体积内凋亡细胞标记阳性的SGN及存活的SGN进行计数和统计学分析。(4)采用化学共沉淀—水热合成法制备改良型纳米羟基磷灰石载体(PEG-PEI-nHAT),并与治疗基因XIAP结合,制备纳米基因载体复合物PEG-PEI-nHAT-pEGFPNl-XIAP。对纳米基因载体复合物的安全性进行评估时,对照组以不含PEG-PEI-nHAT-pEGFPN1-XIAP的培养液处理,实验组以含500μ1浓度为1μg/μl的PEG-PEI-nHAT-pEGFPN1-XIAP的培养液处理；进行有效性评估时,对照组以仅含0.5～3.0mM MnCl2的培养液处理,实验组以含0.5-3.0mM MnCl2加500μ1浓度为1gg/μl的PEG-PEI-nHAT-pEGFPN1-XIAP的培养液处理,均培养48h。于激光共聚焦倒置荧光显微镜下观察各组SGN的存活情况,对单位体积内存活的SGN进行计数和统计学分析。结果：(1)对照组中耳蜗组织内、外毛细胞排列整齐有序,未见损伤或缺失,ANF由SGN发出,向毛细胞(hair cell, HC)呈放射状分布,排列平整,密集成束,SGN具有大而圆的胞体。MnCl2处理组(0.5-30mM MnCl2处理48h)中,随MnCl2浓度增高,HC、ANF和SGN的密度不断降低,HC的表皮板和静纤毛中的肌动蛋白也有所减少,ANF开始呈稀薄泡状,SGN胞体收缩碎裂,呈现凋亡细胞的特征。耳蜗图显示,随MnCl2浓度增高,HC缺失比例从20%逐渐升至70%。(2)NAD保护组(0.5-3.0mM MnCl2+20mM NAD处理48h)和MnCl2处理组(0.5-3.0mM MnCl2处理48h)相比,耳蜗组织的损伤得到了显著改善。在相应的MnCl：浓度下,加入20mM NAD后残存的ANF和SGN均有提高,由SGN辐射状向HC发出的ANF形态较完整,断裂成泡状的情况减少。同一MnCl2浓度下,NAD保护组中存活的ANF和SGN数均高于MnCl2处理组,差异有统计学意义(p<0.05)。(3)空白对照组中,TUNEL染色阳性的SGN不明显；MnCl2处理组(1.0mM MnCl2处理9h)中,TUNEL染色阳性的SGN频繁可见；NAD保护组(1.0mM MnCl2+20mM NAD处理9h)中,TUNEL染色阳性的SGN相对于MnCl2处理组有所减少。MnCl2处理组(1.0mM MnCl2处理6h)中,较多的SGN被活化的caspase-3,-8,-9探针标记,尤其是胞体固缩、变形的SGN；NAD保护组(1.0mMMnCl2+20mM NAD处理6h)中,被活化的caspase-3,-8,-9探针标记的SGN明显少于MnCl2处理组。NAD保护组和MnCl2处理组中TUNEL和caspase染色呈阳性的细胞的比例差异均有统计学意义(p<0.05)。(4)纳米基因载体复合物的安全性评价中,对照组中正常的SGN数量多,其胞体大而圆；实验组(500μl PEG-PEI-nHAT-pEGFPN1-XIAP处理48h)中SGN无明显的固缩和破碎,胞体大而圆,XIAP基因的绿色荧光蛋白在SGN及周围细胞内有表达而呈绿色荧光；两组存活的SGN计数无统计学差异(p>0.05)。有效性评价中,随着MnCl2浓度上升,对照组(0.5-3.0mM MnCl2处理48h)中多数SGN胞体破碎、细胞核固缩、细胞严重缺失；实验组中(0.5-3.0mM MnCl2+500μl PEG-PEI-nHAT-pEGFPN1-XIAP处理48h),随着MnCl2浓度上升,SGN仍不可避免出现核固缩、破碎和细胞缺失,但严重程度低于对照组,且XIAP基因中的绿色荧光蛋白在SGN及周围细胞内有表达而呈绿色荧光；同一浓度下,实验组中存活的SGN数均高于对照组,差异有统计学意义(p<0.05)。提示XIAP基因对锰中毒导致的耳毒性损伤可能具有保护作用。结论：(1)Mn可引起SD大鼠耳蜗HC.ANF和SGN的病理损伤,毒性作用呈量效关系,病理损害程度随Mn浓度增加而增大。(2)NAD可减轻Mn对SD大鼠耳蜗ANF和SGN的损伤作用,在低浓度Mn条件下,NAD可减轻Mn对SD大鼠耳蜗HC的损伤作用。(3)Mn耳蜗毒性的病理生理学机制与caspase-3,-8,-9的活化、DNA碎裂以及细胞凋亡有关,NAD可抑制Mn诱导的caspase活化、DNA碎裂和细胞凋亡。(4)纳米基因载体复合物PEG-PEI-nHAT-pEGFPN1-XIAP对耳蜗SGN无明显急性毒性作用,XIAP基因对Mn所致的耳蜗SGN损伤可能具有保护作用。图22幅,表9个,参考文献182篇。更多还原

【Abstract】 Objectives:(1) To establish the animal model of manganese (Mn) ototoxicity and observe the toxic effect of manganese chloride (MnCl2) on rat cochlea in vitro as well as its does-response relationship.(2) To evaluate the effectiveness of nicotinamide adenine dinucleotide (NAD) in treatment of Mn induced ototoxicity and to observe apoptosis of cochlear cells induced by MnCl2and performance of NAD in anti-apoptosis process.(3) To evaluate the safety and effectiveness of an improved hydroxyapatite nanoparticle vector mediated delivery of the XIAP gene (X-linked inhibitor of apoptosis protein, a member of inhibitor of apoptosis protein family) in treatment of Mn induced ototoxicity.Methods:(1) Postnatal day3SASCO Sprague-Dawley rats were used for this study. The cochleae were carefully dissected out and cultured overnight. On the second day, the cultures were treated with or without MnCl2at concentration from0.5mM to3.0mM for48h. Specimens were immune stained and examined under the laser confocal inverted fluorescence microscope, and cochleograms were prepared from cochlea cultures.(2) After dissection and overnight culture, the cultures were exposed to MnCl2at concentrations from0.5mM to3.0mM in the presence or absence of20mM NAD for48h; and the blank control group was treated without MnCl2or NAD. Specimens were immune stained and examined under the laser confocal inverted fluorescence microscope, and cochleograms were prepared from cochlea cultures. The number of surviving auditory nerve fiber (ANF) per unit length of cochlear basilar membrane and spiral ganglion neuron (SGN) per unit volume of cochlear ganglion in each group was calculated and performed with statistical tests.(3) After dissection and overnight culture, the cultures were exposed to MnCl2in the presence or absence of20mM NAD; and the blank control group was treated without MnCl2or NAD.6hours following treatment, the unfixed specimens were labeled with caspase; and9hours after treatment, TUNEL staining was performed. Specimens were then examined under the laser confocal inverted fluorescence microscope, and the ratio of caspase or TUNEL labeled SGN in each group was calculated and performed with statistical tests.(4) Improved nano-hydroxyapatite (PEG-PEI-nHAT) were synthesized by chemical coprecipitation-hydrothermal synthesis method and combined to the therapeutic gene XIAP to prepare the nano gene-vector compound PEG-PEI-nHAT-pEGFPNl-XIAP. In safety assessment, cultures were treated in the present or absence of500μl PEG-PEI-nHAT-pEGFPN1-XIAP at concentration of1μg/μl for48h; in effectiveness evaluation, cultures were exposed to MnCl2at concentrations from0.5mM to3.0mM in the presence or absence of500μl PEG-PEI-nHAT-pEGFPN1-XIAP at concentration of1μg/ul for48h. Specimens were immune stained and examined under confocal microscope, and the number of surviving SGN in each group was calculated and performed with statistical tests.Results:(1) In controlled group, outer hair cell (OHC) and inner hair cell (IHC) were arranged in orderly and there was no hair cell loss or damage; ANF of SGN radiating outward towards the hair cell (HC) were organized into smooth, thick fascicles; and normal SGN have large and oval-shaped soma. In treatment group, the density of HC, ANF and SGN were decreasing as the concentrations of Mn increased; the actin in the cuticular plate and the stereocilia of HC were reduced, the ANF started to appear thin and fragment, and SGN soma were shrunken and fragmented, illustrating characteristics of cell apoptosis. Cochleograms showed that the ratio of HC loss was increasing from20%to70%approximately.(2) Compared with the control (cochlear cultures treated with0.5-3.0mM MnCl2for48h), NAD clearly prevented the toxic actions of Mn on cochlear culture in treatment group (treated with0.5-3.0mM MnCl2combined with20mM NAD for48h). The addition of20mM NAD to the Mn treated cultures resulted in a substantial increase in ANF survival along with reduced blebbing and fragmentation of the fibers. Soma and nucleolus shrinkage and SGN losses were less severe than in cultures treated with a similar concentration on Mn alone. Under each Mn concentration, the survival rate of SGN and ANF in treatment group was significantly greater than in the control group (p<0.05).(3) TUNEL staining was minimal in control cultures but was frequently observed in SGN cultured for9h in1.0mM Mn. Adding20mM NAD to cultures treated with1.0mM Mn reduced the number of TUNEL positive SGN. Caspase-3, caspase-8and casepase-9labeling was seldom observed in SGN in control samples; in contrast, caspase labeling was seen in many SGN in cultures treated with1.0mM Mn for6h; caspase labeling occurred predominantly in SGN with shrunken and/or distorted soma; with1.0mM Mn plus20mM NAD, there was a noticeable reduction in caspase-3, caspase-8and caspase-9labeling compared to1.0mM Mn alone. The ratios of TUNEL and caspase positive SGN in treatment group were both significantly less than in the control group (p<0.05).(4) In security assessment of PEG-PEI-nHAT-pEGFPN1-XIAP, SGN have large and oval-shaped soma in both group, and there was no significant SGN loss or damage in culture treated with PEG-PEI-nHAT-pEGFPN1-XIAP for48h. XIAP was expressed in SGN and peripheral cells, presenting green fluorescence. There was no statistical difference between the number of survived SGN in control and treatment group (p>0.05). In effectiveness evaluation, SGN soma were shrunken and fragmented and cell loss was servere in control group as MnCl2concentration increased from0.5to3.0mM. In contrast, karyopyknosis, soma fragment and cell loss still existed but was less severe in culture treated with0.5-3.0mM MnCl2plus500μl PEG-PEI-nHAT-pEGFPN1-XIAP compared with the control, and XIAP was expressed in SGN and labeled. Under each concentration of Mn, the survival rate of SGN in culture treated with PEG-PEI-nHAT-pEGFPN1-XIAP and Mn was significantly greater than in the control group (p<0.05), indicating that XIAP gene may protect cochlear culture from Mn-induced ototoxicity.Conclusions:(1) Mn causes significant toxicity on HC, ANF and SGN in SD rat in does-dependent manner. The extent of pathological damage exacerbates as the concentration of Mn increases.(2) NAD can prevent toxicity of Mn on ANF and SGN of rat cochlea. In lower concentration, NAD can prevent toxicity of Mn on HC of rat cochlea.(3) The pathophysiology of Mn toxicity on cochlea is due in part to the upregulation of caspase-3,-8and-9, DNA fragmentation and induction of apoptosis. NAD suppresses Mn-induced upregulation of caspase, DNA fragmentation and apoptosis.(4) The improved nano-hydroxyapatite vector mediated delivery of the XIAP gene (PEG-PEI-nHAT-pEGFPN1-XIAP) demonstrates no apparent acute toxic effect on SGN and XIAP gene can potentially prevents toxicity of Mn on SGN.更多还原