巨细胞病毒感染导致听力损失的发病机制及宫内基因治疗的初步探索
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摘要
第一部分MCMV感染导致听力损失的发病机制研究
【目的】建立小鼠巨细胞病毒（MCMV）感染导致听力损失的小鼠动物模型，研究高迁移率族蛋白B1（HMGB-1）细胞信号通路在其发病机制中的作用。
【方法】体外细胞培养法制备MCMV Smith株，细胞病变法（CPE）测定病毒致半数细胞感染量（TCID50）。ELISA法筛选MCMV IgM、IgG均阴性的BALB/c小鼠，雌雄小鼠交配受孕，待分娩后随机选择乳鼠分为病毒感染组和实验对照组，于生后24h内颅内注射病毒悬液100TCID50/10μL和等量无菌生理盐水。观察各组幼鼠的生存发育情况；测定2周龄幼鼠的听性脑干反应（ABR）；取幼鼠内耳组织分别采用RT-PCR和Western Blot法检测MCMV和HMGB-1 mRNA、糖基化终产物受体（RAGE）和NF-κB抑制物（IκBα）蛋白的表达。
【结果】MCMV TCID_(50)为10~(5.22)/0.1mL。病毒感染组幼鼠存活44/121只，存在明显生长发育异常；实验对照组幼鼠存活47/47只，无显著异常。ABR测定听阈显示病毒感染组为28.42±3.03 dB，实验对照组为15.66±0.38 dB，差异有非常显著意义（P<0.01）。病毒感染组MCMV mRNA检测阳性，实验对照组阴性；HMGB-1 mRNA病毒感染组显著高于实验对照组（P<0.05）；病毒感染组RAGE蛋白表达较实验对照组明显升高，而IκBα明显降低，差异均有非常显著意义（P<0.01）。
【结论】成功建立了MCMV感染导致听力损失的动物模型；HMGB-1/RAGE/NF-κB通路可能在CMV感染导致的听力障碍发病机制中占据重要地位。
第二部分pshHMGB-1-EGFP双基因载体的构建和体外表达
【目的】构建HMGB-1短发夹状RNA（shRNA）和增强型绿色荧光蛋白（EGFP）的双基因真核表达载体，并转染NIH/3T3细胞，以筛选出最有效的干扰质粒用于宫内基因治疗（IUGT）活体实验。
【方法】设计并合成5种DNA寡核苷酸双链，分别含有被9bp茎环（loop）序列间隔的4个HMGB-1基因片段的反向重复序列以及阴性对照序列NC，与携带EGFP基因的pGenesil-1质粒载体连接，构建重组质粒pshHMGB-1-EGFP a、b、c、d和阴性对照质粒pNC-EGFP。对重组质粒酶切鉴定和测序后采用脂质体转染法转染NIH/3T3细胞，采用荧光显微镜和流式细胞仪检测转染效率，RT-PCR和Western Blot测定HMGB-1mRNA和蛋白水平。
【结果】Sal I酶切鉴定和测序结果表明重组质粒构建正确；在荧光显微镜下可以观察到NIH/3T3细胞内的绿色荧光，流式细胞技术检测转染率达57.25％；分别转染重组质粒pshHMGB-1-EGFP a、b、d的3组细胞HMGB-1 mRNA水平均呈不同程度下降，以质粒d最为明显，与其余各组差异具有显著意义（P<0.05）；转染重组质粒pshHMGB-1-EGFP a、b、d的3组细胞HMGB-1蛋白水平均降低，以d最为明显，与转染阴性对照质粒NC组和空白细胞组的差异具有非常显著意义（P<0.01）。
【结论】成功构建了pshHMGB-1-EGFP双基因真核表达载体；重组干扰质粒能有效转染NIH/3T3细胞，并产生RNAi效应抑制HMGB-1表达，为RNAi用于IUGT的动物实验奠定了基础。
第三部分pshHMGB-1-EGFP用于宫内基因治疗的初步研究
【目的】观察pshHMGB-1-EGFP重组质粒通过胎盘转染胎儿的效率和胎儿体内的RNAi效应，探索RNAi联合IUGT治疗先天性疾病的可能性。
【方法】将孕12.5d BALB/c小鼠随机分为4组：pshHMGB-1-EGFP＋脂质体组（1组），pNC-EGFP＋脂质体组（2组），pNC-EGFP组（3组），生理盐水组（4组）。分别经尾静脉注射DNA-脂质体复合物（或质粒DNA或无菌生理盐水）。72h后处死孕鼠，分离胎盘和胎鼠各脏器，荧光显微镜检测胎盘和胎鼠体内EGFP表达；RT-PCR检测胎鼠HMGB-1 mRNA水平；电泳迁移率改变分析（EMSA）测定胎鼠NF-κB活性。
【结果】借助脂质体转染的1、2组孕鼠所有胎仔体内均可见绿色荧光，单纯质粒注射的3组孕鼠所产41只胎仔均未检出荧光。1、2组胎盘绒毛膜板和迷路区、胚胎肝、肾、脑组织局部尤其脑室、心肌纤维之间小血管、大血管壁、肺间质血管壁、脾皮质及髓质血窦均可见较强烈绿色荧光表达，3、4组相应区域荧光表达微弱或未见明显荧光。1组HMGB-1 mRNA较其余3组明显降低，差异有显著意义（P<0.05）；NF-κB活性较其余3组明显降低，差异有非常显著意义（P<0.01）。
【结论】pshHMGB-1-EGFP能在脂质体辅助下通过胎盘转染胎仔，并在胎仔体内发挥RNAi效应抑制HMGB-1表达和NF-κB活性，为CMV宫内感染导致的先天性疾病提示了一条可能的治疗途径。
PART ONEPathogenesis of Hearing Loss Induced by Infection of MCMV【Objective】To establish a murine model of hearing loss due to MCMV infection and probeinto the role of HMGB-1 cellular signal pathway in the mechanism of CMV-inducedhearing handicap.【Methods】The Smith strain of MCMV was passaged in NIH/3T3 cells in vitro and TCID_(50)of the virus was quantitated in the light of CPE. MCMV-free BALB/c mice were screenedby ELISA assay of MCMV-specific IgM/IgG antibodies. After overnight mating，pregnantmice were separately bred. The newborn pups were randomly divided into two groups:MCMV-infected group(infected group)，which was intracerebrally inoculated with viralsuspension in the first 24 hours after birth，and experimental control group(control group)，which was injected with sterile 0.9% sodium chloride of equal volume. We recorded theliving and developing condition of the pups，measured the ABR thresholds of the mice twoweeks later. MCMV and HMGB-1 mRNA in the cochleae were assayed by RT-PCR，in themeantime the expression of RAGE and IκBαwere measured by Western Blot analysis.【Result】TCID_(50) of the virus was 10~(5.22)/0.1mL. 44 in the 121 pups of the infected groupsurvived with notable anomaly 2 weeks after birth，while all the pups of the control groupnormally developed. The ABR thresholds of the two groups were 28.42±3.03 dB (infected)and 15.66±0.38 dB (control)，respectively(P<0.01). In the cochlear extract，MCMV mRNAwas detected in the infected group and the relative abundance of HMGB-1 mRNA were 3.124±1.836 and 1.077±0.085，respectively(P<0.05). According to the results of WesternBlot，the RAGE level was conspicuously higher while the IκBαapparently decreased in theinfected group(P<0.01).
【Conclusion】The murine model of hearing loss induced by MCMV was successfullyestablished. The HMGB-1/RAGE/NF-Κb pathway might play an important role in thepathogenesis of hearing handicap caused by CMV infection.
PART TWO Construction and In Vitro Expression of Double-geneVector PshHMGB-1-EGFP
【Objective】To construct an eukaryotic vector co-expressing shRNA specific to HMGB-1and EGFP，and transfect NIH/3T3 cells to screen out the most effective interfering plasmidfor IUGT in vivo.
【Methods】We designed and synthesized five double-strand oligoneucleiotides of DNA，respectively including inverted repeat sequences of four different gene fragments ofHMGB-1 spaced by a 9bp loop sequence and a negative control sequence NC. The doublestrands were inserted into the plasimid pGenesil-1 carrying EGFP gene to constructrecombinant plasmid vectors: pshHMGB-1-EGFP a、b、c、d，and pNC-EGFP. Therecombinant plasmids were digested by Sal I and identified by electrophoresis on agarosegel，meanwhile the plasmid DNA was sequenced. Then we transfected NIH/3T3 cells withthe plasmid/liposome complex. The transfection efficiency was estimated by fluorescencemicroscope and flow cytometer，and the HMGB-1 level was assessed by RT-PCR andWestern Blot analysis.
【Result】The restrictive digestion and identification as well as DNA sequencing allindicated that the recombinant vectors were correctly constructed. The green fluorescenceof EGFP could be observed in transfected NIH/3T3 cells，and the transfecting efficacy is57.25% according to flow cytometry assay. The mRNA and protein level of HMGB-1 in theNIH/3T3 cells transfected by pshHMGB-1-EGFP a、b、d obviously decreased，among whichpshHMGB-1-EGFP d displayed the strongest inhibitory effect(P<0.05).
【Conclusion】The double-gene eukaryotic expression vector pshHMGB-1-EGFP wassuccessfully constructed. The recombinant interfering plasmid could efficaciously transfectNIH/3T3 cells and perform RNAi effect，which set up foundation for applying RNAi inIUGT in vivo.
PART THREE Preliminary Research on Application of the RecombinantPlasmid Vector PshHMGB-1-EGFP in IUGT
【Objective】To probe into the possibility of treating congenital diseases combining RNAitechnology and IUGT by virtue of observing and evaluating the capability of passingthrough the placenta and transfecting the fetuses of pshHMGB-1-EGFP，as well as theRNAi effect in the fetuses.
【Methods】The dams at day 12.5 post coitus were randomly divided into four groups:Group 1 inoculated with pshHMGB-1-EGFP+liposome via the tail vein; Group 2 withpNC-EGFP+liposome; Group 3 with pNC-EGFP alone; Group 4 with aseptic normal saline.The dams were sacrificed 72 hours after inoculation，meanwhile the placenta weredissected together with the fetal organs. EGFP expression in the placenta and fetal organswas examined by fluorescence microscope; HMGB-1 mRNA was measured by RT-PCR;the activity of NF-κB was determined by EMSA.
【Result】Green fluorescence could be seen in all the offsprings of the dams in Group 1 and2; none of the fetuses in Group 3 and 4 presented visible fluorescence. Fluorescence inGroup 1 and 2 was particularly obvious in the chorionic plate and labyrinth zone of theplacenta，fetal liver and kidney，local cerebral region especially proximal to the ventricles，capillaries among cardiac fibers，the wall of main vessels，the interstitial vessels of thelung，the cortex and sinus of spleen. Fluorescence was rather feeble or even could not beseen in corresponding regions of Group 3 and 4. The HMGB-1 mRNA and the NF-κBactivity were clearly lower in Group 1 compared with the other three groups(P<0.05).
【Conclusion】PshHMGB-1-EGFP could efficaciously get through the placenta andtransfect the fetues with the help of liposome. HMGB-1 expression and NF-κB activitywere inhibited by RNAi effect caused by the plasmid，which presented a novel potentialremedy for congenital diseases due to intrauterine infection of CMV.
【目的】建立小鼠巨细胞病毒（MCMV）感染导致听力损失的小鼠动物模型，研究高迁移率族蛋白B1（HMGB-1）细胞信号通路在其发病机制中的作用。
【方法】体外细胞培养法制备MCMV Smith株，细胞病变法（CPE）测定病毒致半数细胞感染量（TCID50）。ELISA法筛选MCMV IgM、IgG均阴性的BALB/c小鼠，雌雄小鼠交配受孕，待分娩后随机选择乳鼠分为病毒感染组和实验对照组，于生后24h内颅内注射病毒悬液100TCID50/10μL和等量无菌生理盐水。观察各组幼鼠的生存发育情况；测定2周龄幼鼠的听性脑干反应（ABR）；取幼鼠内耳组织分别采用RT-PCR和Western Blot法检测MCMV和HMGB-1 mRNA、糖基化终产物受体（RAGE）和NF-κB抑制物（IκBα）蛋白的表达。
【结果】MCMV TCID_(50)为10~(5.22)/0.1mL。病毒感染组幼鼠存活44/121只，存在明显生长发育异常；实验对照组幼鼠存活47/47只，无显著异常。ABR测定听阈显示病毒感染组为28.42±3.03 dB，实验对照组为15.66±0.38 dB，差异有非常显著意义（P<0.01）。病毒感染组MCMV mRNA检测阳性，实验对照组阴性；HMGB-1 mRNA病毒感染组显著高于实验对照组（P<0.05）；病毒感染组RAGE蛋白表达较实验对照组明显升高，而IκBα明显降低，差异均有非常显著意义（P<0.01）。
【结论】成功建立了MCMV感染导致听力损失的动物模型；HMGB-1/RAGE/NF-κB通路可能在CMV感染导致的听力障碍发病机制中占据重要地位。
第二部分pshHMGB-1-EGFP双基因载体的构建和体外表达
【目的】构建HMGB-1短发夹状RNA（shRNA）和增强型绿色荧光蛋白（EGFP）的双基因真核表达载体，并转染NIH/3T3细胞，以筛选出最有效的干扰质粒用于宫内基因治疗（IUGT）活体实验。
【方法】设计并合成5种DNA寡核苷酸双链，分别含有被9bp茎环（loop）序列间隔的4个HMGB-1基因片段的反向重复序列以及阴性对照序列NC，与携带EGFP基因的pGenesil-1质粒载体连接，构建重组质粒pshHMGB-1-EGFP a、b、c、d和阴性对照质粒pNC-EGFP。对重组质粒酶切鉴定和测序后采用脂质体转染法转染NIH/3T3细胞，采用荧光显微镜和流式细胞仪检测转染效率，RT-PCR和Western Blot测定HMGB-1mRNA和蛋白水平。
【结果】Sal I酶切鉴定和测序结果表明重组质粒构建正确；在荧光显微镜下可以观察到NIH/3T3细胞内的绿色荧光，流式细胞技术检测转染率达57.25％；分别转染重组质粒pshHMGB-1-EGFP a、b、d的3组细胞HMGB-1 mRNA水平均呈不同程度下降，以质粒d最为明显，与其余各组差异具有显著意义（P<0.05）；转染重组质粒pshHMGB-1-EGFP a、b、d的3组细胞HMGB-1蛋白水平均降低，以d最为明显，与转染阴性对照质粒NC组和空白细胞组的差异具有非常显著意义（P<0.01）。
【结论】成功构建了pshHMGB-1-EGFP双基因真核表达载体；重组干扰质粒能有效转染NIH/3T3细胞，并产生RNAi效应抑制HMGB-1表达，为RNAi用于IUGT的动物实验奠定了基础。
第三部分pshHMGB-1-EGFP用于宫内基因治疗的初步研究
【目的】观察pshHMGB-1-EGFP重组质粒通过胎盘转染胎儿的效率和胎儿体内的RNAi效应，探索RNAi联合IUGT治疗先天性疾病的可能性。
【方法】将孕12.5d BALB/c小鼠随机分为4组：pshHMGB-1-EGFP＋脂质体组（1组），pNC-EGFP＋脂质体组（2组），pNC-EGFP组（3组），生理盐水组（4组）。分别经尾静脉注射DNA-脂质体复合物（或质粒DNA或无菌生理盐水）。72h后处死孕鼠，分离胎盘和胎鼠各脏器，荧光显微镜检测胎盘和胎鼠体内EGFP表达；RT-PCR检测胎鼠HMGB-1 mRNA水平；电泳迁移率改变分析（EMSA）测定胎鼠NF-κB活性。
【结果】借助脂质体转染的1、2组孕鼠所有胎仔体内均可见绿色荧光，单纯质粒注射的3组孕鼠所产41只胎仔均未检出荧光。1、2组胎盘绒毛膜板和迷路区、胚胎肝、肾、脑组织局部尤其脑室、心肌纤维之间小血管、大血管壁、肺间质血管壁、脾皮质及髓质血窦均可见较强烈绿色荧光表达，3、4组相应区域荧光表达微弱或未见明显荧光。1组HMGB-1 mRNA较其余3组明显降低，差异有显著意义（P<0.05）；NF-κB活性较其余3组明显降低，差异有非常显著意义（P<0.01）。
【结论】pshHMGB-1-EGFP能在脂质体辅助下通过胎盘转染胎仔，并在胎仔体内发挥RNAi效应抑制HMGB-1表达和NF-κB活性，为CMV宫内感染导致的先天性疾病提示了一条可能的治疗途径。
PART ONEPathogenesis of Hearing Loss Induced by Infection of MCMV【Objective】To establish a murine model of hearing loss due to MCMV infection and probeinto the role of HMGB-1 cellular signal pathway in the mechanism of CMV-inducedhearing handicap.【Methods】The Smith strain of MCMV was passaged in NIH/3T3 cells in vitro and TCID_(50)of the virus was quantitated in the light of CPE. MCMV-free BALB/c mice were screenedby ELISA assay of MCMV-specific IgM/IgG antibodies. After overnight mating，pregnantmice were separately bred. The newborn pups were randomly divided into two groups:MCMV-infected group(infected group)，which was intracerebrally inoculated with viralsuspension in the first 24 hours after birth，and experimental control group(control group)，which was injected with sterile 0.9% sodium chloride of equal volume. We recorded theliving and developing condition of the pups，measured the ABR thresholds of the mice twoweeks later. MCMV and HMGB-1 mRNA in the cochleae were assayed by RT-PCR，in themeantime the expression of RAGE and IκBαwere measured by Western Blot analysis.【Result】TCID_(50) of the virus was 10~(5.22)/0.1mL. 44 in the 121 pups of the infected groupsurvived with notable anomaly 2 weeks after birth，while all the pups of the control groupnormally developed. The ABR thresholds of the two groups were 28.42±3.03 dB (infected)and 15.66±0.38 dB (control)，respectively(P<0.01). In the cochlear extract，MCMV mRNAwas detected in the infected group and the relative abundance of HMGB-1 mRNA were 3.124±1.836 and 1.077±0.085，respectively(P<0.05). According to the results of WesternBlot，the RAGE level was conspicuously higher while the IκBαapparently decreased in theinfected group(P<0.01).
【Conclusion】The murine model of hearing loss induced by MCMV was successfullyestablished. The HMGB-1/RAGE/NF-Κb pathway might play an important role in thepathogenesis of hearing handicap caused by CMV infection.
PART TWO Construction and In Vitro Expression of Double-geneVector PshHMGB-1-EGFP
【Objective】To construct an eukaryotic vector co-expressing shRNA specific to HMGB-1and EGFP，and transfect NIH/3T3 cells to screen out the most effective interfering plasmidfor IUGT in vivo.
【Methods】We designed and synthesized five double-strand oligoneucleiotides of DNA，respectively including inverted repeat sequences of four different gene fragments ofHMGB-1 spaced by a 9bp loop sequence and a negative control sequence NC. The doublestrands were inserted into the plasimid pGenesil-1 carrying EGFP gene to constructrecombinant plasmid vectors: pshHMGB-1-EGFP a、b、c、d，and pNC-EGFP. Therecombinant plasmids were digested by Sal I and identified by electrophoresis on agarosegel，meanwhile the plasmid DNA was sequenced. Then we transfected NIH/3T3 cells withthe plasmid/liposome complex. The transfection efficiency was estimated by fluorescencemicroscope and flow cytometer，and the HMGB-1 level was assessed by RT-PCR andWestern Blot analysis.
【Result】The restrictive digestion and identification as well as DNA sequencing allindicated that the recombinant vectors were correctly constructed. The green fluorescenceof EGFP could be observed in transfected NIH/3T3 cells，and the transfecting efficacy is57.25% according to flow cytometry assay. The mRNA and protein level of HMGB-1 in theNIH/3T3 cells transfected by pshHMGB-1-EGFP a、b、d obviously decreased，among whichpshHMGB-1-EGFP d displayed the strongest inhibitory effect(P<0.05).
【Conclusion】The double-gene eukaryotic expression vector pshHMGB-1-EGFP wassuccessfully constructed. The recombinant interfering plasmid could efficaciously transfectNIH/3T3 cells and perform RNAi effect，which set up foundation for applying RNAi inIUGT in vivo.
PART THREE Preliminary Research on Application of the RecombinantPlasmid Vector PshHMGB-1-EGFP in IUGT
【Objective】To probe into the possibility of treating congenital diseases combining RNAitechnology and IUGT by virtue of observing and evaluating the capability of passingthrough the placenta and transfecting the fetuses of pshHMGB-1-EGFP，as well as theRNAi effect in the fetuses.
【Methods】The dams at day 12.5 post coitus were randomly divided into four groups:Group 1 inoculated with pshHMGB-1-EGFP+liposome via the tail vein; Group 2 withpNC-EGFP+liposome; Group 3 with pNC-EGFP alone; Group 4 with aseptic normal saline.The dams were sacrificed 72 hours after inoculation，meanwhile the placenta weredissected together with the fetal organs. EGFP expression in the placenta and fetal organswas examined by fluorescence microscope; HMGB-1 mRNA was measured by RT-PCR;the activity of NF-κB was determined by EMSA.
【Result】Green fluorescence could be seen in all the offsprings of the dams in Group 1 and2; none of the fetuses in Group 3 and 4 presented visible fluorescence. Fluorescence inGroup 1 and 2 was particularly obvious in the chorionic plate and labyrinth zone of theplacenta，fetal liver and kidney，local cerebral region especially proximal to the ventricles，capillaries among cardiac fibers，the wall of main vessels，the interstitial vessels of thelung，the cortex and sinus of spleen. Fluorescence was rather feeble or even could not beseen in corresponding regions of Group 3 and 4. The HMGB-1 mRNA and the NF-κBactivity were clearly lower in Group 1 compared with the other three groups(P<0.05).
【Conclusion】PshHMGB-1-EGFP could efficaciously get through the placenta andtransfect the fetues with the help of liposome. HMGB-1 expression and NF-κB activitywere inhibited by RNAi effect caused by the plasmid，which presented a novel potentialremedy for congenital diseases due to intrauterine infection of CMV.
引文
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14.张英，闻良珍.巨细胞病毒感染小鼠行为学及脑皮质CaMmRNA表达的改变.中国优生与遗传杂志，2005，13（10）：19-20.
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16. Tsukamoto M，Ochiya T，et al. Gene transfer and expression in progeny afterintravenous DNA injection into pregnant mice. Nature Genetics，1995，9：243-248.
17. Okuda K，Xin KQ，et al. Transplacental genetic immunization after intravenous deliveryof plasmid DNA to pregnant mice. J Immunol，2001，167：5478-5484.
1. Van den Pol AN，Mocarski E，Saederup N，et al. Cytomegalovirus cell tropism，replication，and gene transfer in brain. J Neurosci，1999，19(24)：10948-10965.
2. Holtappels R，Podlech J，Grzimek NK，et al. Experimental preemptive immunotherapyof murine cytomegalovirus disease with CD8 T-cell lines specific for ppM83 andpM84，the two homologs of human cytomegalovirus tegument protein ppUL83 (pp65).J Virol. 2001，75(14)：6584-6600.
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6. Coan PM，Ferguson-Smith AC，Burton GJ. Developmental Dynamics of the DefinitiveMouse Placenta Assessed by Stereology. Biology of Reproduction，2004，70：1806–1813.
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8. Davis GL，Hawrisiak MM. Experimental cytomegalovirus infection and the developingmouse inner ear. Lab Invest，1977，37(1)：20-29.
9. Davis GL，Strauss M. Viral disease of the labyrinth. II. An experimental model usingmouse cytomegalovirus. Ann Otol Rhinol Laryngol，1973，82(4)：584-594.
10.董民声，董明敏，娄卫华.内耳疾病研究进展.郑州：河南医科大学出版社，1999.310-331.
11.张英，闻良珍.巨细胞病毒感染小鼠行为学及脑皮质CaMmRNA表达的改变.中国优生与遗传杂志，2005，13（10）：19-20.
12. Saiko Sugiura，Tetsushi Yoshikawa，Yukihiro Nishiyama，et al. Detection of HumanCytomegalovirus DNA in Perilymph of Patients With Sensorineural Hearing LossUsing Real-Time PCR. Journal of Medical Virology，2003，69：72–75.
13. Stagno S，Reynolds DW，Amos CS，et al. Auditory and visual defects resulting fromsymptomatic and subclinical congenital cytomegaloviral and toxoplasma infections.Pediatrics，1977，59（5）：669-678.
14. Davis LE，et al. Cytomegalovirus isolation from a human inner ear. Ann Otol，1979，88：424-426.
15. Fowler KB，Faye P，Mccollister，et al. Progressive and fluctuating senso rineuralhearing loss in children with asymptomatic congenital cytomegalovirus infection. JPediatr，1997，130 (4) : 624-630.
16. Rarey KE，Davis LE. Temporal bone histopathology 14 years after cytomegalicinclusion disease: a case study. Laryngoscope，1993，103(8): 904-909.
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1. Van den Pol AN，Mocarski E，Saederup N，et al. Cytomegalovirus cell tropism，replication，and gene transfer in brain. J Neurosci，1999，19(24)：10948-10965.
2. Holtappels R，Podlech J，Grzimek NK，et al. Experimental preemptive immunotherapyof murine cytomegalovirus disease with CD8 T-cell lines specific for ppM83 andpM84，the two homologs of human cytomegalovirus tegument protein ppUL83 (pp65).J Virol. 2001，75(14)：6584-6600.
3. Wang H，Bloom O，Zhang MH，et al. HMG-1 as a late mediator of endotoxin lethalityin mice. Science，1999，285(5425)：248–251.
4.任传路，刘善荣，杨玲等.发育相关转录因子GATA4在小鼠肝癌细胞中的表达及其意义.第二军医大学学报，2005，26 (4)：361-365.
5. Rawlinson WD，Farrell HE，Barrell BG. Analysis of the complete DNA sequence ofmurine cytomegalovirus. J Virol，1996，70：8833-8849.
6. Coan PM，Ferguson-Smith AC，Burton GJ. Developmental Dynamics of the DefinitiveMouse Placenta Assessed by Stereology. Biology of Reproduction，2004，70：1806–1813.
7. Watson ED，Cross JC. Development of Structures and Transport Functions in theMouse Placenta. Physiology，2005，20：180-193.
8. Davis GL，Hawrisiak MM. Experimental cytomegalovirus infection and the developingmouse inner ear. Lab Invest，1977，37(1)：20-29.
9. Davis GL，Strauss M. Viral disease of the labyrinth. II. An experimental model usingmouse cytomegalovirus. Ann Otol Rhinol Laryngol，1973，82(4)：584-594.
10.董民声，董明敏，娄卫华.内耳疾病研究进展.郑州：河南医科大学出版社，1999.310-331.
11.张英，闻良珍.巨细胞病毒感染小鼠行为学及脑皮质CaMmRNA表达的改变.中国优生与遗传杂志，2005，13（10）：19-20.
12. Saiko Sugiura，Tetsushi Yoshikawa，Yukihiro Nishiyama，et al. Detection of HumanCytomegalovirus DNA in Perilymph of Patients With Sensorineural Hearing LossUsing Real-Time PCR. Journal of Medical Virology，2003，69：72–75.
13. Stagno S，Reynolds DW，Amos CS，et al. Auditory and visual defects resulting fromsymptomatic and subclinical congenital cytomegaloviral and toxoplasma infections.Pediatrics，1977，59（5）：669-678.
14. Davis LE，et al. Cytomegalovirus isolation from a human inner ear. Ann Otol，1979，88：424-426.
15. Fowler KB，Faye P，Mccollister，et al. Progressive and fluctuating senso rineuralhearing loss in children with asymptomatic congenital cytomegalovirus infection. JPediatr，1997，130 (4) : 624-630.
16. Rarey KE，Davis LE. Temporal bone histopathology 14 years after cytomegalicinclusion disease: a case study. Laryngoscope，1993，103(8): 904-909.
17. Goodwin GH，Sanders C，Johns EW. A new group of chromatin-associated proteinswith a high content of acidic and basic amino acids. Eur J Biochem，1973，38(1)：14–19.
18. Dumitriu IE，Baruah P，Manfredi AA，et al. HMGB1: guiding immunity fromwithin. Trends Immunol. 2005，26(7)：381-387.
19. Scaffidi P，Misteli T，Bianchi ME. Release of chromatin protein HMGB1 by necroticcells triggers inflammation. Nature，2002，418(6894)：191-195.
20. Andersson U，Wang H，Palmblad K，et al. High mobility group 1 protein (HMG-1)stimulates proinflammatory cytokine synthesis in human monocytes. J Exp Med，2000，192(4)：565–570.
21. Schmidt AM，Yan SD，Yan SF，et al. The multiligand receptor RAGE as a progressionfactor amplifying immune and inflammatory responses. J Clin Invest，2001，108：949-955.
22. Yeh CH，Sturgis L，Haidacher J，et al. Requirement for p38 and p44/p42 mitogenactivatedprotein kinases in RAGE-mediated nuclear factor-kappaB transcriptionalactivation and cytokine secretion. Diabetes，2001，50：1495–1504.
23. Schwedler S，Schinzel R，Vaith P，Wanner C: Inflammation and advanced glycationend products in uremia: simple coexistence，potentiation or causal relationship? KidneyInt，2001，59(Suppl 78)：S32–S36.
24. Fiuza C，Bustin M，Talwar S，et al. Inflammation-promoting activity of HMGB1 onhuman microvascular endothelial cells. Blood，2003，101(7)：2652-2660.
25. Treutiger CJ，Mullins GE，Johansson AS，et al. High mobility group 1 B - box mediatesactivation of human endothelium. J Intern Med，2003，254(4)：375-385.
26. Belal A Jr. Pathology of vascular sensorineural hearing impairment. Laryngoscope，1980，90(11 Pt 1)：1831-9.
27. Grahame-Clarke C. Human cytomegalovirus，endothelial function and atherosclerosis.Herpes，2005，12(2)：42-5.
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