大鼠核转录因子Atoh1的克隆表达及鉴定

目的利用基因工程方法克隆SD大鼠Atoh1基因CDS区序列,构建大鼠核转录因子Atoh1的真核表达载体并在真核细胞中表达。方法从两只SD大鼠结肠黏膜提取总RNA,采用逆转录PCR法扩增Atoh1基因CDS区序列并亚克隆于PMD-19T载体中。测序鉴定后将Atoh1基因连接于含有EGFP和内部核糖体转入位点(IRES)的真核细胞表达载体pIRES2-EGFP中,对重组质粒进行酶切鉴定和测序鉴定后,以脂质体介导法转染至293T细胞,荧光显微镜和Western blot检测其在293T细胞中的表达。结果扩增得到大鼠Atoh1 CDS区长1 056 bp,编码351个氨基酸,与GeneBank公布的参考序列对比,有两处碱基发生突变,但克隆序列编码的氨基酸序列与参考序列完全一致,两处碱基应为单核苷酸多态性(SNP),突变为无义突变,不影响蛋白表达。双酶切和测序结果证明Atoh1已正确地克隆到真核表达载体pIRES2-EGFP中,荧光显微镜和Western blot证实Atoh1目的蛋白能在293T细胞中稳定表达。结论基因工程方法可成功克隆出Atoh1编码序列,真核表达载体pAtoh1-IRES2-EGFP构建成功并可以在293T细胞中表达。     

大鼠Atoh1真核表达载体pAtoh1-IRES2-EGFP的构建及转染293T细胞的研究

目的:利用基因工程方法克隆SD大鼠Atoh1基因编码区序列,构建Atoh1的真核表达载体pA-toh1-IRES2-EGFP,并验证其在293T细胞中的表达。方法:通过RT-PCR从SD大鼠结肠组织内扩增出Atoh1基因全长CDS序列,并TA克隆于PMD-19T载体中。纯化回收的目的片段测序后连接于真核细胞表达载体pIRES2-EGFP中,构建pAtoh1-IRES2-EGFP真核表达载体。再次测序后脂质体介导重组质粒转染293T细胞,荧光显微镜下观察绿色荧光蛋白的表达。结果:测序后将扩增得到的大鼠Atoh1CDS序列与GeneBank公布的参考序列对比,有两处碱基发生突变,但克隆序列编码的氨基酸序列与参考序列完全一致,两处碱基应为单核苷酸多态性,突变为无义突变,不影响蛋白表达。双酶切和测序结果证明Atoh1已正确地克隆到真核表达载体pIRES2-EGFP中,重组质粒转染293T细胞24h后荧光显微镜下观察到绿色荧光蛋白表达。结论:获得正确Atoh1编码序列,真核表达载体pAtoh1-IRES2-EGFP构建成功并可以在293T细胞中表达,为进一步对感音神经性聋的基因治疗奠定了基础。     

不同类型载体携带Math1-EGFP基因对293T细胞转染效率及细胞毒性的研究

目的比较腺病毒、脂质体、纳米材料三种不同类型的载体携带目的基因对293T细胞的转染效率及细胞毒性的大小,筛选理想的基因载体。方法用重组腺病毒、Lipofectamine^TM 2000、Entranster^TM—D纳米材料为转染载体,携带含有增强型绿色荧光蛋白（EGFP）报告基因的Math1-EGFP基因及真核表达质粒pRK5-Math1—EGFP,按照转染试剂盒说明优化其转染条件,分别转染293T细胞。24小时后在共聚焦显微镜下观察转染结果并计数阳性细胞率,采用RT—PCR法检测转染细胞中Math1基因mRNA的表达,用MTT法检测不同转染条件下,不同转染载体对293T细胞的细胞毒性。结果经优化转染条件,重组腺病毒载体转导的细胞中荧光蛋白表达几乎为95％以上,Lipofectamine^TM 2000、Entranste^TM-D纳米材料载体转染的细胞中荧光蛋白表达也分别达到70％和80％以上,而Entranste^rTM-D纳米材料载体在最高转染效率的剂量下仍然保持80％以上的细胞生存率。RT-PCR进一步证实,三种载体转染细胞均有Math1基因mRNA的表达。结论Entranster^TM—D纳米载体作为一种新型纳米聚合物转染试剂,能成功携带内耳基因治疗关键基因Math1基因转染293T细胞,并表现了低毒、高效性能。     

三种方法转染Math1基因效率和细胞毒性研究

目的比较腺病毒、脂质体、纳米材料三种不同类型的载体体外携带目的基因Math1对HEK293T细胞的转染效率及细胞毒性的大小,以期筛选理想的基因转染载体材料。方法选用重组腺病毒、LipofectamineTM2000、Superfect纳米材料作为转染载体,携带含有增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)报告基因的Math1-EGFP基因及真核表达质粒pRK5-Math1-EGFP,并且根据不同转染载体说明步骤优化体外转染条件,分别转染293T细胞。在一定的时间内利用荧光显微镜观察细胞转染结果并计数阳性细胞,采用MTT(噻唑蓝)法检测三种不同载体体外细胞毒性,应用RT-PCR(逆转录聚合酶链反应)技术检测转染阳性细胞中目的基因Math1的mRNA表达情况。结果在优化的体外转染条件下,重组腺病毒载体介导的细胞转染率达到了94%以上,脂质体和商品化Superfect纳米材料转染的细胞中,荧光蛋白表达率分别为73%和80%以上,同时,商品化Superfect纳米材料在达到80%转染率的条件下,细胞存活率为90%。应用RT-PCR方法证实,三种载体转染细胞均有Math1基因的mRNA的表达。结论商品化Superfect纳米材料作为一种新型的、安全有效的纳米转染材料,能够成功携带耳聋治疗关键基因Math1转染293T细胞,以期在内耳基因治疗的研究当中得到有效的应用。     

阿霉素对豚鼠耳蜗外侧壁中多耐药基因1基因表达的影响

目的:探讨豚鼠耳蜗外侧壁中多耐药基因1(mdr1)是否对外源性的阿霉素(ADM)发生应激反应;ADM在耳蜗内淋巴液中积聚浓度的变化。方法:经静脉注射中毒剂量的ADM后,利用RTPCR技术半定量mdr1mRNA;采用高效液相色谱法(HLPC)检测内淋巴液中的ADM浓度。结果:①注射后24h时相点ADM组mdr1mRNA的量明显高于正常对照组,差异有统计学意义(P<0.05);而其4h时相点,与空白组相比差异无统计学意义;生理盐水组各时相点mdr1mRNA的量与空白组间差异无统计学意义;②内淋巴液中的ADM浓度4h时相点较24h时相点高,差异有统计学意义(P<0.05)。结论:mdr1是一种防御或应激基因,可被外源性ADM所诱导;其在耳蜗外侧壁中的表达,可能与减少耳毒性药物在内淋巴液中的积聚,增强耳蜗自我保护功能有关。     

Math1基因内耳导入后噪声性聋豚鼠听功能改变观察

目的观察Math1基因内耳导入对噪声性聋豚鼠听功能的影响,探讨Math1基因过表达对噪声损伤耳蜗的生物学效应,为内耳基因治疗提供实验基础和理论依据。方法经脉冲噪声致聋的豚鼠45只（各频率ABR阈值均≥95dB SPL）,雌雄不限,实验开始时体草250～300g。随机分为3组：Ad—Math1-EGFP组（30只）;Ad—EGFP组（5只）;空白组（10只）。各组豚鼠在基因转导后4周、8周分别测试双耳ABR。测试完毕后处死动物,观察听泡及耳蜗尤炎性病变者记录听阈结果。结果Math1导入后4周,导入耳各频率ABR阈值低于对照耳（右耳）,也低于Ad—EGFP组及空内组,平均达到85dBSPL。Math1导入后8周,导入耳各频率ABR阂值低于对照耳（右耳）,也低于Ad—EGFP组及空白组,与4周时比较,进一步好转,平均达到75dB SPL。结论Math1基因内耳导入可使噪声导致全聋的豚鼠听功能部分恢复,为噪声性聋的治疗打开了新的思路和手段。     

X射线交错互补修复基因1多态性和EB病毒感染对鼻咽癌细胞基本特性的影响

目的　探讨X射线交错互补修复基因1（X-ray repair cross-complementing gene 1,XRCC1）多态性与EB病毒感染对鼻咽癌细胞基本特性的影响。方法　将鼻咽癌细胞株分组,使之分别表达不同的XRCC1基因型,转染EB病毒,比较转染前后不同基因型鼻咽癌细胞的基本特性,并检测XRCC1蛋白的表达水平。结果　XRCC1不同基因型的鼻咽癌细胞之间基本特性无明显差异,XRCC1蛋白水平无差异。EB病毒感染后,各组鼻咽癌细胞生长加快、增殖能力变强、迁移速度加快、凋亡率降低,XRCC1蛋白水平无明显改变。EB病毒感染后,194位点突变型的鼻咽癌细胞与280、399位点突变型细胞相比,细胞增殖和迁移能力改变程度较小,差异有统计学意义。其他基因型细胞基本特性及XRCC1蛋白水平无差异。结论　EB病毒感染可增加鼻咽癌易感性,EB病毒感染后XRCC1 194位点的Trp突变型基因可能对鼻咽癌有保护作用。     

病毒与非病毒载体转染新生大鼠血管纹边缘细胞的比较研究

目的通过病毒与非病毒载体对原代培养的新生大鼠内耳血管纹边缘细胞转染情况的比较，选择出适合边缘细胞的基因治疗载体。方法用质粒（pEGFP—N1）、血清5型重组腺病毒（rAd5）以及血清2型重组腺相关病毒（rAAV2）三种载体转染新生大鼠（≤72小时）耳边缘细胞，通过对转染效率、细胞凋亡以及细胞活性的检测将三种载体加以比较。结果对边缘细胞的转导效率依次为rAd5〉rAAV2〉pEGFP—N1，AnnexinV—APC／7-AAD双染法检测转染阳性细胞的损伤情况依次为rAAV2〈rAd5〈pEGFP—N1，CCK一8法检测转染后的细胞活性依次为rAAV2〉rAd5〉pEGFP—N1。结论病毒载体相对于非病毒载体以其高转导效率、低细胞毒性在对原代培养的新生大鼠耳边缘细胞的转染中表现出明显优势。与血清2型重组腺相关病毒比较，相对高效、低毒性的血清5型重组腺病毒更适用于原代培养的新生大鼠血管纹边缘细胞的基因转染研究。     

Absence of Atoh1 induced partially different cell fates of cochlear and vestibular sensory epithelial cells in mice

Abstract

Background： Atoh1, also named Math1, is essential for the development of inner ear hair cells. Many studies have confirmed that the absence of Atoh1 resulted in a total loss of inner ear hair cells, which indicates that Atoh1 plays very similar roles in the development of hair cells in the cochlea and vestibule.

Objective： The aim of this study was to evaluate whether Atoh1 plays different roles in the cochlea and vestibule.

Material and methods: We generated Atoh1-null mice by inbreeding Atoh1^cre/+ heterozygous mice and compared with the epithelial cell status of the cochlea and vestibule.

Results: We found that no inner ear hair cells were detected in Atoh1-null mice. However, a different cell status was found in the mutant cochlea and vestibule on the last embryonic day (E18.5). In the Atoh1-null cochlea, the epithelial cells that should develop into hair cells were totally absent, while in the Atoh1-null vestibule, most of the epithelial cells that should develop into hair cells still survived.

Conclusions: Our data indicate that Atoh1 may have similar but partially different functions in the development of hair cells in the cochlea and vestibule.     

转基因诱导新生大鼠耳蜗大、小上皮嵴细胞分化为毛细胞样细胞实验

目的通过转染Hathl基因诱导大鼠耳蜗大上皮嵴细胞（greater epithelial ridge,GER）和小上皮嵴细胞（1esser epithelial ridge,LER）分化为毛细胞样细胞。方法取出生后第l天的大鼠耳蜗,利用机械分离和酶消化相结合的方法分别分离出纯的GER和LER细胞,并转染Hathl基因,培养后做免疫组化染色观察。结果GER和LER体外培养并转染Hathl基因后,免疫组化检测显示肌球蛋白（myosinVIIa）染色阳性,表达毛细胞的特异标记物。提示GER和LER已分化为毛细胞样细胞。结论GER和LER细胞作为毛细胞前体细胞可以实现体外培养,转染Hathl后．可分化为毛细胞样细胞。     

In vivo imaging of mammalian cochlear blood flow using fluorescence microendoscopy.

AIMS: We sought to develop techniques for visualizing cochlear blood flow in live mammalian subjects using fluorescence microendoscopy. BACKGROUND: Inner ear microcirculation appears to be intimately involved in cochlear function. Blood velocity measurements suggest that intense sounds can alter cochlear blood flow. Disruption of cochlear blood flow may be a significant cause of hearing impairment, including sudden sensorineural hearing loss. However, inability to image cochlear blood flow in a nondestructive manner has limited investigation of the role of inner ear microcirculation in hearing function. Present techniques for imaging cochlear microcirculation using intravital light microscopy involve extensive perturbations to cochlear structure, precluding application in human patients. The few previous endoscopy studies of the cochlea have suffered from optical resolution insufficient for visualizing cochlear microvasculature. Fluorescence microendoscopy is an emerging minimally invasive imaging modality that provides micron-scale resolution in tissues inaccessible to light microscopy. In this article, we describe the use of fluorescence microendoscopy in live guinea pigs to image capillary blood flow and movements of individual red blood cells within the basal turn of the cochlea. METHODS: We anesthetized eight adult guinea pigs and accessed the inner ear through the mastoid bulla. After intravenous injection of fluorescein dye, we made a limited cochleostomy and introduced a compound doublet gradient refractive index endoscope probe 1 mm in diameter into the inner ear. We then imaged cochlear blood flow within individual vessels in an epifluorescence configuration using one-photon fluorescence microendoscopy. RESULTS: We observed single red blood cells passing through individual capillaries in several cochlear structures, including the round window membrane, spiral ligament, osseous spiral lamina, and basilar membrane. Blood flow velocities within inner ear capillaries varied widely, with observed speeds reaching up to approximately 500 microm/s. CONCLUSION: Fluorescence microendoscopy permits visualization of cochlear microcirculation with micron-scale optical resolution and determination of blood flow velocities through analysis of video sequences.     

慢病毒载体和腺病毒载体转染原代培养的螺旋神经节细胞的比较

目的:比较慢病毒载体和腺病毒载体转染新生大鼠耳蜗螺旋神经节细胞(SGCs)的不同转染特性.方法:体外培养新生大鼠SGCs,适当滴度的慢病毒载体和腺病毒载体转染SGCs,于病毒转染后3 d(培养后6 d)和转染后7 d(培养后10 d),荧光显微镜下观察绿色荧光蛋白报告基因(GFP)表达情况和SGCs的细胞形态.行SGC...     

The effects of ototoxic drugs on mechano-electric transduction channels in chick cochlear hair cells

The effects of ototoxic drugs on mechano-electrical transduction (MET) currents were investigated in dissociated cochlear hair cells of the chick, using a wholecell patch-electrode voltage-clamp technique. Dihydrostreptomycin (DHSM) and cisplatin (cis-dichlorodiammine platinum II, CDDP) blocked the MET channel in a dose- and voltage-dependent manner. In contrast, acetyl salicylate did not suppress the MET current. At ?50 mV, DHSM and CDDP blocked the MET channel with a Hill coefficient of 0.93 and 2.1, respectively. These findings suggest that a single DHSM molecule or more than one CDDP molecule binds to a single MET channel.     

Retinoic acid induces differentiation of cochlear neural progenitor cells into hair cells

IntroductionInner ear progenitor cells have the potential for multi-directional differentiation. Retinoic acid is an important requirement for the development of the inner ear. Blocking the Curtyr's retinoic acid signaling pathway can significantly reduce the number of hair cells. Therefore, we believe that retinoic acid may induce the regeneration of inner ear hair cells.ObjectiveTo investigate whether the cochlear neural progenitor cells maintain the characteristics of stem cells during recovery and subculture, whether retinoic acid can induce cochlear neural progenitor cells into hair cells in vitro, and whether retinoic acid promotes or inhibits the proliferation of cochlear neural progenitor cells during differentiation.MethodsCochlear neural progenitor cells were cultured and induced in DMEM/F12 + RA (10^?6 M) and then detected the expressions of hair cell markers (Math1 and MyosinVIIa) by immunofluorescence cytochemistry and realtime-polymerase chain reaction, and the proliferation of cochlear neural progenitor cells was detected by Brdu.ResultsThe nestin of cochlear neural progenitor cells was positively expressed. The ratios of Math1-positive cells in the control group and experimental group were 1.5% and 63%, respectively; the ratios of MyosinVIIa-positive cells in the control group and experimental group were 0.96% and 56%, respectively (p < 0.05). The ratios of Brdu⁺-labeled cells in retinoic acid group, group PBS, and group FBS were 20.6%, 29.9%, and 54.3%, respectively; however, the proliferation rate in the experimental group decreased.ConclusionRetinoic acid can promote cochlear neural progenitor cells to differentiate into the hair cells.