目的 了解铜离子转运蛋白1（copper transport protein 1,CTR1）在大鼠内耳的表达情况及硫酸铜和顺铂鼓室给药对CTR1表达的影响。方法 Wistar雄性大鼠24只,随机分为4组,每组6只,Ⅰ组为正常对照组（非处理组）;Ⅱ组为双耳圆窗龛放置顺铂...目的 了解铜离子转运蛋白1（copper transport protein 1,CTR1）在大鼠内耳的表达情况及硫酸铜和顺铂鼓室给药对CTR1表达的影响。方法 Wistar雄性大鼠24只,随机分为4组,每组6只,Ⅰ组为正常对照组（非处理组）;Ⅱ组为双耳圆窗龛放置顺铂溶液（0.5mg/ml）的明胶海绵;Ⅲ组为双耳圆窗龛放置顺铂溶液（1mg/ml）的明胶海绵;Ⅳ组双耳圆窗龛放置硫酸铜溶液（0.02mg/kg）的明胶海绵。采用免疫组织化学技术,对各组耳蜗冰冻切片行CTR1蛋白的免疫组织化学染色,光学显微镜下观察耳蜗组织中CTR1的表达情况;提取各组耳蜗组织总蛋白,应用Western-blot技术检测各组CTR1蛋白表达水平;提取耳蜗组织的总RNA,应用RT-PCR技术检测各组CTR1mRNA的表达水平。结果 Ⅰ-Ⅳ组大鼠的耳蜗各回Corti器、螺旋神经节、血管纹的细胞质和细胞膜上均有CTR1表达,CTR1的平均光密度值呈降低趋势;各组耳蜗组织均有明显的CTR1表达,Ⅰ-Ⅳ组CTR1蛋白的光密度值分别为0.532±0.031、0.394±0.024、0.234±0.030和0.191±0.015,呈下降趋势,各组间比较差异有统计学意义（P〈0.05）;各组耳蜗组织均有明显的CTR1mRNA表达,Ⅰ-Ⅳ组光密度值分别为0.508±0.035、0.391±0.022、0.240±0.023和0.186±0.021,呈下降趋势,各组间比较差异均有统计学意义（P〈0.05）。结论 CTR1在大鼠内耳有丰富的表达,其表达量可随鼓室内顺铂浓度增高而下降,且鼓室内给予硫酸酮其表达量下降。展开更多
Objective:The purinergic receptor P2X2(P2RX2)encodes an ATP-gated ion channel permeable to Na+,K+,and especially Ca²⁺.Loss-of-function mutations in P2RX2 are known to cause autosomal dominant nonsyndromic deafnes...Objective:The purinergic receptor P2X2(P2RX2)encodes an ATP-gated ion channel permeable to Na+,K+,and especially Ca²⁺.Loss-of-function mutations in P2RX2 are known to cause autosomal dominant nonsyndromic deafness 41(DFNA41),which manifests as high-frequency hearing loss,accelerated presbycusis,and increased susceptibility to noise-induced damage.Zebrafish,owing to their small size,rapid development,high fecundity,transparent embryos,and high gene conservation with humans,provide an ideal model for studying human diseases and developmental mechanisms.This study aims to generate a p2rx2 knockout zebrafish model using CRISPR/Cas9 gene editing system to investigate the effect of p2rx2 deficiency on the auditory system,providing a basis for understanding P2RX2-related hearing loss and developing gene therapy strategies.Methods:Two CRISPR targets(sgRNA1 and sgRNA2)spaced 47 bp apart were designed within the zebrafish p2rx2 gene.Synthesized sgRNAs and Cas9 protein were microinjected into single-cell stage Tübingen(TU)-strain zebrafish embryos.PCR and gel electrophoresis verified editing efficiency at 36 hours post-fertilization(hpf).Surviving embryos were raised to adulthood(F0),tail-clipped,genotyped,and screened for positive mosaics.F1 heterozygotes were generated by outcrossing,and F2 homozygous mutants were obtained by intercrossing.Polymerase chain reaction(PCR)combined with sequencing verified mutation type and heritability.At 5 days post-fertilization(dpf),YO-PRO-1 staining was used to examine hair cell morphology and count in lateral line neuromasts and the otolith region.Auditory evoked potential(AEP)thresholds at 600,800,1000,and 2000 Hz were measured in nine 4-month-old wild type and mutant zebrafish per group.Results:A stable p2rx2 knockout zebrafish line was successfully established.Sequencing revealed a 66 bp insertion at the first target site introducing a premature stop codon(TAA),leading to early termination of protein translation and loss of function.Embryos developed normally with no gross malformations.At 5 dpf,mutants exhibited significantly reduced hair cell density in the otolith region compared with wild type,although lateral line neuromasts were unaffected.AEP testing showed significantly elevated auditory thresholds at all 4 frequencies in homozygous mutants compared with wild type(all P<0.001),indicating reduced hearing sensitivity.Conclusion:We successfully generated a p2rx2 loss-of-function zebrafish model using CRISPR/Cas9 technology.p2rx2 deficiency caused hair cell defects in the otolith region and increased auditory thresholds across frequencies,indicating its key role in maintaining zebrafish auditory hair cell function and hearing perception.The phenotype’s restriction to the otolith region suggests tissue-specific roles of p2rx2 in sensory organs.This model provides a valuable tool for elucidating the molecular mechanisms of P2RX2-related hearing loss and for screening otoprotective drugs and developing gene therapies.展开更多
目的特异性免疫治疗(specific immune therapy,SIT)变应性鼻炎(allergic rhinitis,AR)疗程一般在2年以上,但患者依从性差,影响疗效。文中通过比较标准化屋尘螨疫苗SIT AR 1年与2年的临床疗效的差异,探讨AR SIT适宜的维持治疗时间。方法...目的特异性免疫治疗(specific immune therapy,SIT)变应性鼻炎(allergic rhinitis,AR)疗程一般在2年以上,但患者依从性差,影响疗效。文中通过比较标准化屋尘螨疫苗SIT AR 1年与2年的临床疗效的差异,探讨AR SIT适宜的维持治疗时间。方法收集2009年1月至2011年12月南昌大学第二附属医院耳鼻咽喉头颈外科,98例接受SIT的AR患者,分为2组:SIT 1年组(46例)、SIT 2年组(52例),随访记录2组患者鼻部症状评分及药物用量评分情况,采用国际常用的鼻-结膜炎生活质量调查量表(rhinoconjunctivitis quality of life questionnaire,RQLQ)评估2组患者2年生活质量的变化。结果第1年SIT 1年组和SIT 2年组患者鼻部症状评分情况(7.00±1.08 vs 6.63±0.79)及药物用量评分(1.50±0.51 vs0.41±0.42)差异无统计学意义(P>0.05);RQLQ量表评分均下降,差异无统计学意义(345.00±31.89 vs 344.42±32.26)(P>0.05)。第2年SIT 1年组和SIT 2年组患者鼻部症状评分(6.20±0.78 vs 4.29±0.64)及药物用量评分(0.53±0.43 vs0.21±0.34),差异有统计学意义(P<0.05);SIT 2年组RQLQ量表评分(223.08±30.39)显著低于SIT 1年组(319.13±36.38),差异有统计学意义(P<0.05)。结论 AR SIT适宜的维持治疗时间以2年为好。展开更多
文摘目的 了解铜离子转运蛋白1（copper transport protein 1,CTR1）在大鼠内耳的表达情况及硫酸铜和顺铂鼓室给药对CTR1表达的影响。方法 Wistar雄性大鼠24只,随机分为4组,每组6只,Ⅰ组为正常对照组（非处理组）;Ⅱ组为双耳圆窗龛放置顺铂溶液（0.5mg/ml）的明胶海绵;Ⅲ组为双耳圆窗龛放置顺铂溶液（1mg/ml）的明胶海绵;Ⅳ组双耳圆窗龛放置硫酸铜溶液（0.02mg/kg）的明胶海绵。采用免疫组织化学技术,对各组耳蜗冰冻切片行CTR1蛋白的免疫组织化学染色,光学显微镜下观察耳蜗组织中CTR1的表达情况;提取各组耳蜗组织总蛋白,应用Western-blot技术检测各组CTR1蛋白表达水平;提取耳蜗组织的总RNA,应用RT-PCR技术检测各组CTR1mRNA的表达水平。结果 Ⅰ-Ⅳ组大鼠的耳蜗各回Corti器、螺旋神经节、血管纹的细胞质和细胞膜上均有CTR1表达,CTR1的平均光密度值呈降低趋势;各组耳蜗组织均有明显的CTR1表达,Ⅰ-Ⅳ组CTR1蛋白的光密度值分别为0.532±0.031、0.394±0.024、0.234±0.030和0.191±0.015,呈下降趋势,各组间比较差异有统计学意义（P〈0.05）;各组耳蜗组织均有明显的CTR1mRNA表达,Ⅰ-Ⅳ组光密度值分别为0.508±0.035、0.391±0.022、0.240±0.023和0.186±0.021,呈下降趋势,各组间比较差异均有统计学意义（P〈0.05）。结论 CTR1在大鼠内耳有丰富的表达,其表达量可随鼓室内顺铂浓度增高而下降,且鼓室内给予硫酸酮其表达量下降。
基金supported by the Natural Science Foundation of Hunan Province(2023JJ30753)the Innovative Construction Foundation of Hunan Province(2023SK4030)the Natural Science Foundation of Changsha(kq2208326),China。
文摘Objective:The purinergic receptor P2X2(P2RX2)encodes an ATP-gated ion channel permeable to Na+,K+,and especially Ca²⁺.Loss-of-function mutations in P2RX2 are known to cause autosomal dominant nonsyndromic deafness 41(DFNA41),which manifests as high-frequency hearing loss,accelerated presbycusis,and increased susceptibility to noise-induced damage.Zebrafish,owing to their small size,rapid development,high fecundity,transparent embryos,and high gene conservation with humans,provide an ideal model for studying human diseases and developmental mechanisms.This study aims to generate a p2rx2 knockout zebrafish model using CRISPR/Cas9 gene editing system to investigate the effect of p2rx2 deficiency on the auditory system,providing a basis for understanding P2RX2-related hearing loss and developing gene therapy strategies.Methods:Two CRISPR targets(sgRNA1 and sgRNA2)spaced 47 bp apart were designed within the zebrafish p2rx2 gene.Synthesized sgRNAs and Cas9 protein were microinjected into single-cell stage Tübingen(TU)-strain zebrafish embryos.PCR and gel electrophoresis verified editing efficiency at 36 hours post-fertilization(hpf).Surviving embryos were raised to adulthood(F0),tail-clipped,genotyped,and screened for positive mosaics.F1 heterozygotes were generated by outcrossing,and F2 homozygous mutants were obtained by intercrossing.Polymerase chain reaction(PCR)combined with sequencing verified mutation type and heritability.At 5 days post-fertilization(dpf),YO-PRO-1 staining was used to examine hair cell morphology and count in lateral line neuromasts and the otolith region.Auditory evoked potential(AEP)thresholds at 600,800,1000,and 2000 Hz were measured in nine 4-month-old wild type and mutant zebrafish per group.Results:A stable p2rx2 knockout zebrafish line was successfully established.Sequencing revealed a 66 bp insertion at the first target site introducing a premature stop codon(TAA),leading to early termination of protein translation and loss of function.Embryos developed normally with no gross malformations.At 5 dpf,mutants exhibited significantly reduced hair cell density in the otolith region compared with wild type,although lateral line neuromasts were unaffected.AEP testing showed significantly elevated auditory thresholds at all 4 frequencies in homozygous mutants compared with wild type(all P<0.001),indicating reduced hearing sensitivity.Conclusion:We successfully generated a p2rx2 loss-of-function zebrafish model using CRISPR/Cas9 technology.p2rx2 deficiency caused hair cell defects in the otolith region and increased auditory thresholds across frequencies,indicating its key role in maintaining zebrafish auditory hair cell function and hearing perception.The phenotype’s restriction to the otolith region suggests tissue-specific roles of p2rx2 in sensory organs.This model provides a valuable tool for elucidating the molecular mechanisms of P2RX2-related hearing loss and for screening otoprotective drugs and developing gene therapies.
文摘目的特异性免疫治疗(specific immune therapy,SIT)变应性鼻炎(allergic rhinitis,AR)疗程一般在2年以上,但患者依从性差,影响疗效。文中通过比较标准化屋尘螨疫苗SIT AR 1年与2年的临床疗效的差异,探讨AR SIT适宜的维持治疗时间。方法收集2009年1月至2011年12月南昌大学第二附属医院耳鼻咽喉头颈外科,98例接受SIT的AR患者,分为2组:SIT 1年组(46例)、SIT 2年组(52例),随访记录2组患者鼻部症状评分及药物用量评分情况,采用国际常用的鼻-结膜炎生活质量调查量表(rhinoconjunctivitis quality of life questionnaire,RQLQ)评估2组患者2年生活质量的变化。结果第1年SIT 1年组和SIT 2年组患者鼻部症状评分情况(7.00±1.08 vs 6.63±0.79)及药物用量评分(1.50±0.51 vs0.41±0.42)差异无统计学意义(P>0.05);RQLQ量表评分均下降,差异无统计学意义(345.00±31.89 vs 344.42±32.26)(P>0.05)。第2年SIT 1年组和SIT 2年组患者鼻部症状评分(6.20±0.78 vs 4.29±0.64)及药物用量评分(0.53±0.43 vs0.21±0.34),差异有统计学意义(P<0.05);SIT 2年组RQLQ量表评分(223.08±30.39)显著低于SIT 1年组(319.13±36.38),差异有统计学意义(P<0.05)。结论 AR SIT适宜的维持治疗时间以2年为好。
