【作者】 高雪；

【导师】 邱建华；

【作者基本信息】 第四军医大学 ， 耳鼻喉， 2008， 博士

【摘要】 多种疾病、感染、理化因素（噪声、毒物、耳毒性药物等）以及年龄等因素均可引起内耳毛细胞受到损伤,从而导致感音神经性聋。感音神经性聋是严重影响人们健康及生存质量的致残疾病,临床上尚无理想的治疗方法,目前关于感音神经性聋的防治研究已成为国际听力学和耳科学研究的热点和难点之一。耳蜗毛细胞作为声音的感受器将声刺激转化为神经冲动,对于听觉的产生有着不可替代的作用,鸟类和低等动物毛细胞具有较强的再生修复能力,而哺乳动物的毛细胞长期以来被人们认为是终末细胞。然而,近来研究发现哺乳动物毛细胞损伤后亦可通过其前体细胞增殖和分化进行部分修复,具有一定的再生修复能力。因此,诱导哺乳动物耳蜗毛细胞的再生和修复有望为治疗感音神经性耳聋开辟崭新的途径。LIM蛋白通过介导蛋白质-蛋白质相互作用,参与很多生物学功能,如细胞命运选择,细胞分化,细胞骨架形成等。因此,LIM蛋白可被看作是形成更高层次的蛋白质复合物的脚手架。CRP2和CRIP2分别由csrp2和crip2基因编码,CRP2蛋白包含了两个LIM结构域,它作为体内重要的转录因子,参与多种细胞的增殖分化,促进细胞进入有丝分裂,它对于细胞功能的维持也发挥着非常重要的作用。CRIP2是2003年新发现的LIM蛋白,它与CRP2蛋白的空间结构较为相似,均含有两个LIM结构域,但是与CRP2相比,它无明确的核定位序列,因此,它大部分在胞浆中表达,胞核中表达缺乏或很少。CRP2与CRIP2在蛋白水平上有42%的相似性。尽管,研究显示LIM蛋白在细胞增殖分化中发挥着非常重要的作用,目前还没有关于CRP2和CRIP2在内耳表达的报道。实验目的:本实验以LIM蛋白CRP2及CRIP2为研究对象,制备了特异性多克隆抗体,分析了它们在大鼠内耳及嗅球前体细胞中的表达及分布情况,并且从细胞水平观察了细菌脂多糖刺激后CRIP2的表达变化。为进一步研究CRP2及CRIP2在内耳发育及毛细胞再生中的作用奠定基础。实验方法和结果:1.CRP2及CRIP2原核表达载体的构建及多克隆抗体的制备根据GenBank公布的大鼠CRP2 （NM₁77425）、CRIP2 （NM₀22501）全长cDNA序列,借助计算机软件Primer Premier5.0、Oligo6.5设计了含有不同酶切位点的8条引物。采用RT-PCR技术从大鼠主动脉组织中提取总cDNA并扩增出相应大小的目的片段。将不同的目的基因片段分别克隆入载体pGEM-T-easy中进行测序,结果与GenBank报道的完全一致。将测序正确的csrp2、crip2按照BamHI和HindIII酶切位点克隆入原核表达载体pRSET-A中,将连接产物转化入E.coli BL21,挑出阳性克隆,IPTG诱导表达重组的6×His融合蛋白。SDS-PAGE分析的结果表明成功地表达出了相应的2种融合蛋白;用6×His的单克隆抗体进行Western Blot分析的结果也显示在相应条带处有特异性的阳性反应,其分子量与各自蛋白的预计大小相符。随后采用Ni-NTA亲和色谱柱纯化得到了2种融合蛋白,定量后保存于-20℃。采用获得的融合蛋白作为免疫原,常规免疫家兔及Bab/c小鼠制备特异性多克隆抗体。ELISA法检测制备抗体效价为1:3200至1:6400。Western Blot结果显示两株抗体可分别特异性识别CRP2及CRIP2融合蛋白。为了进一步鉴定抗体的特异性,我们将csrp2及crip2 DNA片段测序正确后克隆入真核表达载体pcDNA 3.1（-）,重组质粒酶切鉴定正确后以阳离子聚合物转染Cos-7细胞,分别以RT-PCR方法检测mRNA表达和间接免疫荧光技术检测目的蛋白的表达。结果证明csrp2及crip2可在Cos-7细胞中表达,采用制备抗体可以特异性结合内源性CRP2及CRIP2蛋白。2.CRP2及CRIP2在内耳的表达及分布2.1 RT-PCR分析mRNA表达将实验大鼠按照年龄分组,时间点选为大鼠内耳发育成熟的重要时间,分别为胚胎15天（E15）,出生后1天（P1）、3天（P3）、6天（P6）、9天（P9）以及25 d（P25）。分别取材后提取总RNA,随后进行反转录,获得模板cDNA,PCR扩增csrp2及crip2基因片段,对结果进行观察,用SPSS11.0对实验数据进行分析。结果表明CRP2 mRNA的表达只存在于E15胚胎大鼠内耳,出生后CRP2的表达消失。CRIP2 mRNA则具有不同的表达模式,出生前和出生后各时间点均有表达,并且表达水平相对稳定,各组间无明显差别。2.2 Western Blot分析蛋白表达为了检测CRP2及CRIP2蛋白在内耳的表达,采用制备的兔源性多抗作为一抗进行Western Blot分析,结果与RT-PCR结果相似,CRP2蛋白的表达只存在于E15胚胎大鼠内耳,出生后该蛋白的表达消失。CRIP2蛋白则具有不同的表达模式,出生前和出生后各时间点均有表达,并且表达水平相对稳定,各组间无明显差别。2.3 CRP2及CRIP2在内耳的分布研究因为CRP2蛋白在大鼠出生后内耳中无表达,因此我们研究了CRIP2在成熟大鼠内耳的分布情况,免疫组化分析显示CRIP2蛋白在耳蜗的基底膜、血管纹、螺旋神经节细胞中均有表达。3.LPS上调体外培养大鼠内耳血管纹边缘细胞中CRIP2的表达。我们首先采用免疫荧光法证实了CRIP2蛋白主要分布于MC的胞浆中。同时,我们发现所有CRIP2蛋白阳性的细胞均表达CK18,证实了血管纹表达CRIP2蛋白的细胞为MC。然后,我们采用不同浓度的LPS刺激体外培养的MC。结果显示:LPS可以上调体外培养的MC表达CRIP2蛋白,并且随着LPS浓度增加,CRIP2表达也相应增加。4.CRP2及CRIP2在大鼠嗅球神经前体细胞中的表达与定位我们利用制备的多抗检测了CRP2及CRIP2在大鼠嗅球神经前体细胞中的表达与定位,结果显示CRP2在未分化的嗅球神经前体细胞中有表达,并且在胞浆和胞核中都有分布。而CRIP2蛋白具有不同的表达及分布模式,它在分化前和和分化后的嗅球前体细胞中均有表达,只分布于胞浆,胞核中未发现表达。结论:LIM蛋白CRP2、CRIP2蛋白均在内耳中有表达,但表达模式不同。CRP2蛋白只在出生前大鼠内耳中有表达,而出生后则消失,提示该蛋白有可能参与了内耳的发育,而CRIP2在内耳发育、成熟中表达稳定,提示其与内耳的功能成熟有关,并且可能参与了G-感染所引起的内耳免疫反应。为了进一步鉴定CRP2、CRIP2蛋白的生物学特性,我们检测了它们在嗅球前体细胞中的表达,发现在具有增殖能力的嗅球前体细胞内CRP2及CRIP2的表达均为阳性,而分化后的细胞中只能检测到CRIP2的表达。结果显示CRP2蛋白可能参与了细胞的增殖;CRIP2可能与细胞的某些基本生理功能相关。本试验为研究LIM蛋白在内耳发育成熟中的作用奠定了一定的基础。更多还原

【Abstract】 The mammalian inner ear possesses a very limited ability for self-repair and regeneration. For this reason, the loss of sensory hair cells from the ears of mature mammalians usually leads to permanent deficits in hearing as consequence of acoustic trauma, treatment with ototoxic drugs, infections or autoimmune pathologies, or as part of the aging process. In contrast, the ears of most non-mammalian vertebrates (fish, amphibians and avian) can regenerate hair cells after injury. The process of hair cell regeneration has been characterized most completely in the avian inner ear, where lost sensory cells are quickly replaced via a process of regenerative cell proliferation. Thus, the factors that permit sensory regeneration in the ears of non-mammals but inhibit such regeneration in mammals are of great biological and clinical interest.LIM protein family is shown to play myriad roles in cell fate specification, differentiation, and cytoskeletal organization. They likely act to promote the formation of multimeric transcription regulatory complexes with bridging factors such as basic belic-loop-helix (bHLH) and GATA proteins. Additionally, they could function antagonistically toward LIM homeodomain proteins by competing for binding to the essential cofactor LIM-domain-binding protein.CRP2 and CRIP2 are encoded by csrp2 and crip2, respectively. CRP2 contains two tandemLy arranged LIM domains and is implicated in diverse processes linked to cellular differentiation and growth control. Conditional cell transformation reveals that CRP2 is essential to the maintenance of normal cell function. CRIP2 is a novel LIM protein and was first characterized in 2003. It consists of two LIM domains spaced by a short amino acid stretch. It serves as a novel LIM-only protein that is highly homologous to rat ESP1 and CRP2 sequences. CRIP2 display a wide, overlapping tissue distribution with protein tyrosine phosphatase which have a potential role in the dynamics of the actin cytoskeleton. The CRIP2 displays amino sequence identities of 50% compared with the sequence of CRP2. On the protein level, the two proteins share 42% similarity. It has been reported that these two proteins have distinct tissue distribution in various tissues, suggesting that each might serve related but specific roles in tissue organization or function. Although LIM proteins have been found to play essential role in cell proliferation and differentiation, there was no report showing the expression of Group 2 LIM proteins in the inner earAIMSystematically examined the expression of CRIP2 and CRP2 in the rat inner ear and olfactory precursor cells and investigate the relationship between LPS stimulation and CRIP2 expression in the cultured MC.Methods and Results1.Preparation and identification of anti-CRP2 and anti-CRIP2 sera Cloning of csrp2 and crip2 and Construction of expression vector. Complete sequences of csrp2 and crip2 were amplified using cDNA synthesized from undifferentiated olfactory precursor cells. BamHI and HindIII sites were designed in the primers to facilitate cloning into pRSET A expression vector. The PCR products were first cloned into pGEM-T-Easy vector and the BamHI-HindIII fragments were then subcloned in the pRSET A expression vector, respectively. The insert sizes were confirmed by digestion and further verified by sequencing. Expression and purification of the recombinant protein. Under the control of IPTG-inducible phage T7 promoter, csrp2 cDNA in pRSET A is predicted to encode a recombinant protein of 193 aa with a molecular weight of ~24 kDa. The crip2 cDNA in pRSET A is predicted to encode a recombinant protein of 208 aa with a molecular weight of ~27 kDa. Small-scale cultures of the positive clones were subjected to IPTG induction to identify clones capable of expressing the predicted recombinant proteins. Recombinant proteins migrated at apparent molecular weight that matched the prediction. The results confirmed that the CRP2 and CRIP2 could be efficiently expressed in E. coli host cells. Expression was not detected in uninduced recombinant strains. Western Blot results confirmed the expressed proteins were the target proteins.To examine the relative distribution of the expressed recombinant protein in the soluble and insoluble fractions, both the supernatant and the pellet of the cell lysate were examined to detect the recombinant proteins. The expression of both recombinant proteins was detected predominantly in the soluble fraction.After purifying the recombinant proteins by affinity chromatography, predominantly one band of the recombinant protein could be seen in the SDS-PAGE and Western Blot, indicating that the recombinant proteins were highly purified. The protein yields were 0.7 mg/mL and 0.6 mg/mL for CRP2 and CRIP2, respectively.Characterization of the antisera. After immunizing rabbits and mice with His-tagged CRP2 and CRIP2, respectively, anti-CRP2 and anti-CRIP2 sera were purified by protein G affinity chromatography. Titers of both antibodies were analyzed by ELISA using purified CRP2 or CRIP2 as antigen and were showed to be approximately 1:1600. There was no cross-reactivity between two antibodies. Titers of mice antibody were approximately 1:6400.It is known that antibodies that recognize proteins in their denatured form (SDS-PAGE gel) are not always able to detect the same proteins in a native form, and vice versa. To determine if the prepared antibodies detect endogenic CRP2 and CRIP2 in eukaryotic cells, the antibodies were used in lysate of Cos-7 cells transfected with pcDNA3.1(-)-csrp2.and Cos-7 cells transfected with pcDNA3.1(-)-crip2. The results presented a protein of ~21 kDa was detected by Western Blot analysis using rabbit anti-CRP2 antibody. Similarly, a protein of ~24 kDa was blotted with the rabbit anti-CRIP2 antibody. In contrast, no band was visualized in the Cos-7 cells transfected with pcDNA3.1(-) using either antibody. The results indicated that these two antibodies are specific to CRP2 and CRIP2, respectively. Same results were achieved using mice antibodies that we prepared (Data not shown).2.Expression of CRP2 and CRIP2 in rat inner earAnalysis CRIP2 and CRP2 mRNA by RT-PCR. We analyzed the expression of crip2 and csrp2 genes at several key developmental stages, RT-PCR analysis of mRNA extracted from cochleae showed that crip2 are expressed in rat inner ears of different age while the expression of csrp2 can be detected in the embryonic rat inner ear. The nucleic acid sequences of all these PCR products were verified by sequencing and are identical to the CRIP2 sequence previous reported. The RT-PCR products were compared and all 6 groups’cochlea specimens expressed CRIP2 mRNA at similar levels.CRIP2 protein expression. The expression levels of CRIP2 and CRP2 were further determined at protein level. CRIP2 and CRP2 protein expression within the inner ear were initially determined in the rat using Western Blot analysis. Both 6 groups’cochlea expressed CRIP2 protein (Fig. 2). Anti-CRIP2 antibody detected a single band of ~25kDa within the protein extract from the cochlea. Compared withβ-actin expression, the expression of CRIP2 in different cochlea has no significant difference, indicating no significant correlation between the level of CRIP2 and ages tested. The expression of CRP2 can be detected in the embryonic rat inner ear. This result is consistent with mRNA levels at different development stages.Immunofluorescent localization. In order to understand the functional relationship between CRIP2 and development of hearing organs, we tried to determine the presence and expression pattern of CRIP2 in the cochlea of rats. The immunofluorescence results showed that CRIP2 was detected in the spiral ganglion cells, basal membrane and stria vascularis in rat cochlea tissue. Moreover, the expression of CRIP2 was found in hair cells and supporting cells of the organ of Corti. In contrast, CRIP2 expression was not detected at the peripheral auditory nerve. A similar CRIP2 expression pattern was found in the cochlea of newborn rat (data not shown). No specific fluorescence was detected in negative controls.3. LPS upregulates the expression of CRIP2 in the cultured MC.In order to understand the functional relationship between CRIP2 and immunity of inner ear, we tried to determine the presence and expression pattern of CRIP2 in the cultured MC. Hochest 33258 was used as a marker for the nuclei. The immunofluorescence results showed that CRIP2 was localized predominantly in the cytoplasm. The expression of CRIP2 is upregualted by the stimulation of LPS. And as the concentration of LPS increases, the stimulation became stronger. RT-PCR and Western Blot analysis showed the similar results.4. Sub-cellular localization and expression analysis of CRP2 and CRIP2 proteins in rat olfactory precursor cells.With the prepared CRP2 and CRIP2 antibodies, we detected the subcellular localization of CRIP2 and CRP2 in the undifferentiated olfactory precursor cells and in the differentiated end cells by immunofluoence staining. Hochest 33258 was used as a marker for the nuclei. The results show that in the undifferentiated olfactory precursor cells, CRP2 was localized both in the nucleus and cytoplasm, which is consistent with previous reports that CRP2 are localized in the cytoplasm along the cytoskeleton and in the nucleus. In contrast, CRIP2 was localized predominantly in the cytoplasm, which is consistent with a previous report that CRIP2 is present in the cytoplasm of epithelial cells. In the differentiated end cells, only the expression of CRIP2 in the cytoplasm can be detected and anti-CRP2 does not detect any protein. Western Blot analysis showed the similar results.ConclusionIn this study we confirmed the expression of CRIP2 and CRP2 in the rat inner ear and olfactory precursor cells by RT-PCR, Western Blot and immunofluorescence analysis. The expression profile of CRIP2, which has the characteristic of important transcriptional regulator, suggests that CRIP2 plays a role in the development and formation of cochlea. The expression profile of CRP2 suggests that CRP2 may play a role in the inner ear development. This study is the first report confirming the expression of CRIP2 and CRP2 in rat inner ear and olfactory precursor cells other than hearts and other organs.更多还原