The purpose of the present study was to determine protectivie effects of basic fibroblast growth factor (bFGF) on cochlear neurons and hair cells in vitro and in vivo. In experiment I, cultured spiral ganglion neurons...The purpose of the present study was to determine protectivie effects of basic fibroblast growth factor (bFGF) on cochlear neurons and hair cells in vitro and in vivo. In experiment I, cultured spiral ganglion neurons (SGNs) prepared from P3 mice were exposed to 20mM glutamate for 2 hours before the culture medium was replaced with fresh medium containing 0, 25, 50, and 100 ng/ml bFGF, respectively. Fourteen days later, all cultures were fixed with 4% paraformaldehyde, and stained with 1% toluidine blue. The number of surviving SGNs were counted and the length of SGNs neurites were measured. Exposure to 20 mM glutamate for 24 hours resulted in an inhibition on neurite outgrowth of SGNs and elevated cell death. Treatment of the cultures with bFGF led to promotion of neurite outgrowth and elevated number of surviving SGNs. Effects of bFGF were dose dependent with the highest potency at 100 ng/ml. In experiment Ⅱ, in vivo studies were carried out with guinea pigs in which bFGF or artificial perilymph was perfused into the cochlea to assess possible protective effects of bFGF on cochlear hair cells and compound action potentials(CAP). The CAPs were measured before, immediatly and 48 hours after exposure to noise. Significant differences in CAP were observed (p＜0. 05 ) among the bFGF perfused group, control group(t =3. 896 ) and artificial perilymph perfused group (t =2. 520) at 48 hours after noise exposure, Cochleae were removed and hair cell Loss was analyzed in surface preparations prepared from all experimental animals. Acoustic trauma caused loss of 651 and 687 inner hair cells in the control and artificial perilymph perfused group, respectively. In sharp contrast, only 31 inner hair cells were lost in the bFGF perfused ears. Similarly, more outer hair cells died in the control and perilymph perfuesed group (41830 and 41968, respectively) than in the group treated with bFGF (34258). Our results demonstrate that bFGF protected SGNs against glutmate neurotoxicity in vitro. In addition, treatment with bFGF also protected hair cells from acoustic trauma.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
旨在对基质金属蛋白酶组织抑制剂(tissue inhibitor of matrix metalloproteinases,TIMPs)家族成员中的TIMP1进行克隆和分析,通过过表达与敲减TIMP1,探究TIMP1对毛囊毛乳头细胞(dermal papilla cells,DPCs)毛囊生长相关基因的调控作用...旨在对基质金属蛋白酶组织抑制剂(tissue inhibitor of matrix metalloproteinases,TIMPs)家族成员中的TIMP1进行克隆和分析,通过过表达与敲减TIMP1,探究TIMP1对毛囊毛乳头细胞(dermal papilla cells,DPCs)毛囊生长相关基因的调控作用。本研究通过分子克隆TIMP1编码区(coding sequence,CDS)序列,并对其进行生物信息学功能分析。之后,构建过表达载体pcDNA 3.1-TIMP1并设计合成siRNA,在DPCs中过表达和敲低TIMP1,检测毛囊生长发育相关基因的表达,并通过EdU和CCK-8检测DPCs的增殖水平。结果显示,兔TIMP1基因的CDS区全长624 bp,共编码207个氨基酸。生物信息学分析表明TIMP1蛋白存在信号肽,不包含跨膜结构域,在不同哺乳动物中存在同源性。在DPCs中过表达TIMP1能够极显著上调BMP2(bone morphogenetic protein 2)、SFRP2(secreted frizzled-related protein 2)、TGFβ1(tranforming growth factorβ1)基因的mRNA表达水平(P<0.01),极显著下调WNT2(wnt family member 2)基因的mRNA表达水平(P<0.01),敲减TIMP1能显著下调BMP2、SFRP2基因的mRNA的表达(P<0.05),极显著下调TGFβ1基因的mRNA表达(P<0.01)。此外,EdU和CCK-8结果显示过表达TIMP1能够抑制DPCs的增殖,敲低TIMP1能够促进DPCs的增殖。本研究成功克隆家兔TIMP1基因CDS序列,并对其生物信息学功能进行预测,初步分析TIMP1对毛囊生长相关基因的调控作用,验证了其抑制DPCs增殖的作用,为阐明家兔毛囊生长发育的理论研究提供参考。展开更多
文摘The purpose of the present study was to determine protectivie effects of basic fibroblast growth factor (bFGF) on cochlear neurons and hair cells in vitro and in vivo. In experiment I, cultured spiral ganglion neurons (SGNs) prepared from P3 mice were exposed to 20mM glutamate for 2 hours before the culture medium was replaced with fresh medium containing 0, 25, 50, and 100 ng/ml bFGF, respectively. Fourteen days later, all cultures were fixed with 4% paraformaldehyde, and stained with 1% toluidine blue. The number of surviving SGNs were counted and the length of SGNs neurites were measured. Exposure to 20 mM glutamate for 24 hours resulted in an inhibition on neurite outgrowth of SGNs and elevated cell death. Treatment of the cultures with bFGF led to promotion of neurite outgrowth and elevated number of surviving SGNs. Effects of bFGF were dose dependent with the highest potency at 100 ng/ml. In experiment Ⅱ, in vivo studies were carried out with guinea pigs in which bFGF or artificial perilymph was perfused into the cochlea to assess possible protective effects of bFGF on cochlear hair cells and compound action potentials(CAP). The CAPs were measured before, immediatly and 48 hours after exposure to noise. Significant differences in CAP were observed (p＜0. 05 ) among the bFGF perfused group, control group(t =3. 896 ) and artificial perilymph perfused group (t =2. 520) at 48 hours after noise exposure, Cochleae were removed and hair cell Loss was analyzed in surface preparations prepared from all experimental animals. Acoustic trauma caused loss of 651 and 687 inner hair cells in the control and artificial perilymph perfused group, respectively. In sharp contrast, only 31 inner hair cells were lost in the bFGF perfused ears. Similarly, more outer hair cells died in the control and perilymph perfuesed group (41830 and 41968, respectively) than in the group treated with bFGF (34258). Our results demonstrate that bFGF protected SGNs against glutmate neurotoxicity in vitro. In addition, treatment with bFGF also protected hair cells from acoustic trauma.
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.
文摘旨在对基质金属蛋白酶组织抑制剂(tissue inhibitor of matrix metalloproteinases,TIMPs)家族成员中的TIMP1进行克隆和分析,通过过表达与敲减TIMP1,探究TIMP1对毛囊毛乳头细胞(dermal papilla cells,DPCs)毛囊生长相关基因的调控作用。本研究通过分子克隆TIMP1编码区(coding sequence,CDS)序列,并对其进行生物信息学功能分析。之后,构建过表达载体pcDNA 3.1-TIMP1并设计合成siRNA,在DPCs中过表达和敲低TIMP1,检测毛囊生长发育相关基因的表达,并通过EdU和CCK-8检测DPCs的增殖水平。结果显示,兔TIMP1基因的CDS区全长624 bp,共编码207个氨基酸。生物信息学分析表明TIMP1蛋白存在信号肽,不包含跨膜结构域,在不同哺乳动物中存在同源性。在DPCs中过表达TIMP1能够极显著上调BMP2(bone morphogenetic protein 2)、SFRP2(secreted frizzled-related protein 2)、TGFβ1(tranforming growth factorβ1)基因的mRNA表达水平(P<0.01),极显著下调WNT2(wnt family member 2)基因的mRNA表达水平(P<0.01),敲减TIMP1能显著下调BMP2、SFRP2基因的mRNA的表达(P<0.05),极显著下调TGFβ1基因的mRNA表达(P<0.01)。此外,EdU和CCK-8结果显示过表达TIMP1能够抑制DPCs的增殖,敲低TIMP1能够促进DPCs的增殖。本研究成功克隆家兔TIMP1基因CDS序列,并对其生物信息学功能进行预测,初步分析TIMP1对毛囊生长相关基因的调控作用,验证了其抑制DPCs增殖的作用,为阐明家兔毛囊生长发育的理论研究提供参考。
