【作者】 钟翠萍；

【导师】 邱建华；

【作者基本信息】 第四军医大学 ， 耳鼻咽喉科学， 2010， 博士

【摘要】 在成年哺乳动物耳蜗中,听觉毛细胞和神经元损伤后不能再生,这成为感音神经性聋难以治愈的主要原因。近年来,自新生鼠耳蜗分离出前体细胞,在体外可定向分化为表达毛细胞和螺旋神经元表型的细胞。我们在体外比较了P1、P7和P14的大鼠耳蜗前体细胞的数量、增殖能力及超微结构,结果表明出生后耳蜗前体细胞大部分处于细胞周期的静止期,并逐步通过分化和凋亡彻底地退出了细胞周期,耳蜗前体细胞也并非真正意义上的干细胞,而是处于干细胞与成熟子代细胞之间的中间细胞或是处于干细胞未端的细胞。通过实验发现c-Myc可能参与调控耳蜗的发育以及前体细胞的增殖、分化。结合之前的工作基础,我们利用腺病毒技术将c-Myc和cyclin A2共转染至新生鼠耳蜗前体细胞,可使其增殖,并促进前体细胞重新进入细胞周期。初步研究其调控机制,表明为经典的CKI-cyclin-CDK途径。实验一大鼠耳蜗前体细胞的分离、培养目的对新生大鼠耳蜗前体细胞进行分离、培养。方法从P7大鼠耳蜗中分离、培养前体细胞,用免疫细胞化学的方法对前体细胞进行鉴定;血清诱导分化后鉴定分化潜能,进一步了解其多向分化特性。结果原代培养的细胞,培养1天后即可见“细胞球”,随着培养天数的增加,可见细胞球体积增大,所含细胞增多;培养5天后,少部分细胞球内的细胞出现分化及贴壁现象。细胞球内大部分细胞呈nestin、musashi1和BrdU阳性,表明其具有自我更新及有丝分裂的能力。细胞球经诱导分化14 d后,对分化细胞行免疫细胞化学鉴定,发现分化细胞表达毛细胞标志物myosin VIIA和phalloidin,表达成熟神经元标志物NeuN,表达不成熟神经元标志物Tuj1,表达星形胶质细胞标志物GFAP,表达少突胶质细胞标志物galactocerebroside,以及谷氨酸能神经元标志物GluR-1,证明其具有多向分化潜能。结论本部分实验为研究耳蜗前体细胞成年后“沉默”的机制奠定基础,为研究通过基因治疗的方法促进前体细胞增殖提供了一种体外模型。实验二不同日龄大鼠耳蜗前体细胞增殖能力及超微结构的比较目的耳蜗前体细胞在成年后沉默或消失的具体机制尚不清楚,为探讨其原因,我们在体外比较了P1、P7和P14的大鼠耳蜗前体细胞的数量、增殖能力及超微结构,观察耳蜗前体细胞的转归。方法对P1、P7和P14分离出的耳蜗前体细胞,经体外培养7天后进行计数,检测其数量变化,并每隔5天传代观察传代数;采用流式细胞技术检测细胞周期,来评估不同日龄新生大鼠耳蜗前体细胞增殖能力的变化;应用透射电镜观察不同日龄新生大鼠耳蜗前体细胞的超微结构。结果出生1周后耳蜗前体细胞的数量急剧下降,出生2周后的耳蜗内未能分离出前体细胞;P7前体细胞的传代数较P1减少;P7较P1更多的前体细胞处于有丝分裂的静止期,增殖指数下降;P7的前体细胞超微结构中有更多分化细胞的特点。结论耳蜗前体细胞出生后大部分处于细胞周期的静止期,并逐步通过分化和凋亡彻底地退出了细胞周期,他们并非真正意义上的干细胞,而是处于干细胞与成熟子代细胞之间的中间细胞或是处于干细胞未端的细胞。实验三c-Myc在大鼠耳蜗组织发育和耳蜗前体细胞分化过程中的表达变化目的原癌基因c-Myc具有调控G0/G1转换和去分化的双重作用。目前对于c-Myc在内耳中的功能尚未见报道。我们拟通过检测c-Myc在大鼠耳蜗组织发育及前体细胞分化过程中的表达情况,探讨其在哺乳动物耳蜗发育中的作用。方法1、取E10、E15、P1、P7和P14的SD大鼠耳蜗组织,应用RT-PCR、Western blot的方法检测c-Myc在大鼠耳蜗组织发育过程中的表达情况。2、取耳蜗前体细胞和分化7天后的分化细胞,用RT-PCR、免疫细胞化学染色和Western blot的方法检测c-Myc在耳蜗前体细胞分化过程中的表达情况。结果从胚胎至出生后的耳蜗发育过程中,c-Myc表达量呈现逐渐下降趋势;在前体细胞的分化过程中c-Myc的表达量也呈现下降。结论c-Myc的表达量在大鼠耳蜗组织发育及耳蜗前体细胞分化的过程中逐渐下降,这表明c-Myc可能参与调控大鼠耳蜗组织的发育及前体细胞的分化。实验四Ad-c-Myc-EGFP和Ad-CyclinA2-EGFP的构建和转染耳蜗前体细胞的浓度确定目的构建目的基因分别为c-Myc和CyclinA2的腺病毒载体,体外转染大鼠耳蜗前体细胞,观察转染效率及细胞活性,确定适当的转染浓度。方法1、构建复制缺陷重组腺病毒: Ad-CyclinA2-EGFP、Ad-c-Myc-EGFP及Ad-EGFP;2、在不同效靶比浓度下(MOI=50、100、150、200、250),Ad-EGFP转染耳蜗前体细胞,观察不同浓度转染时细胞形态变化,荧光显微镜观察EGFP表达情况,计算阳性细胞百分率;3、应用MTT观察细胞活性确定增殖点;4、确定MOI值,应用流式细胞仪检测转染效率。结果毒种上清液经PCR鉴定,能够特异地扩增出预期大小的条带,证明该重组腺病毒携带目的片段。在不同效靶比浓度下,EGFP阳性细胞百分率随MOI值的升高而增大;当MOI=50,100,150时重组腺病毒对细胞的毒性作用与对照组相比无显著差异;当MOI值=200时,细胞状态不佳,出现生长抑制。根据光镜下细胞形态学证据,确定本实验采用MOI值为150。经MTT法测定,细胞生长曲线呈S形,转染后36小时做为增殖点,应用流式细胞技术检测转染效率为25.2%。结论复制缺陷型腺病毒可有效转染原代培养的耳蜗前体细胞,为进一步研究c-Myc和CyclinA2基因对耳蜗前体细胞增殖能力的影响奠定了基础。实验五c-Myc、CyclinA2基因对耳蜗前体细胞增殖能力的影响目的耳蜗前体细胞在体外具有一定的增殖能力,可定向分化为表达毛细胞和神经元表型的细胞,但耳蜗前体细胞增殖能力差,并处于细胞周期的“静止”状态。我们用编码目的基因为c-Myc和CyclinA2的腺病毒表达载体转染耳蜗前体细胞,促进其增殖并重返细胞周期,从而为诱导耳蜗受损细胞再生提供新的思路和依据。方法分别设立Ad-CyclinA2-EGFP转染组、Ad-c-Myc-EGFP转染组、Ad-EGFP转染组及Ad-CyclinA2-EGFP、Ad-c-Myc-EGFP共转染组;用复制缺陷腺病毒转染大鼠耳蜗前体细胞,MOI值为150,用流式细胞技术检测c-Myc和CyclinA2基因对耳蜗前体细胞周期的影响;BrdU孵育观察转染后有丝分裂能力的变化;观察各组传代能力变化,采用方差分析对数据进行统计学处理。结果流式结果表明Ad-CyclinA2-EGFP转染组、Ad-c-Myc-EGFP转染组、Ad-EGFP转染组对耳蜗前体细胞周期无明显影响;而Ad-CyclinA2-EGFP、Ad-c-Myc-EGFP共转染组可使部分耳蜗前体细胞进入G1/S期和G2/M期,共转染组与对照组G0/G1期细胞比例分别为62.03±2.02%、78.27±6.09%(P﹤0.05);增殖指数分别为37.97±2.015、20.41±7.16(P﹤0.05)。共转染组中共表达绿色荧光(EGFP)、红色荧光(BrdU阳性)及蓝色荧光(Hoechst)的细胞球数量增多,表明共转染c-Myc、CyclinA2使更多的耳蜗前体细胞具有有丝分裂的能力;各组间传代能力无明显变化。结论c-Myc基因和CyclinA2基因单独作用于耳蜗前体细胞,在体外对细胞增殖与细胞周期无明显影响。而c-Myc和CyclinA2基因共同作用于耳蜗前体细胞,可促进细胞增殖,使部分前体细胞重返细胞周期。实验六c-Myc与CyclinA2基因促进耳蜗前体细胞增殖的作用机制目的研究c-Myc和CyclinA2基因共同转染耳蜗前体细胞后,检测目的基因表达情况以及促进细胞增殖的机制。方法应用real-time PCR和Western blot的方法观察转染Ad-EGFP与共转染Ad-CyclinA2-EGFP、Ad-c-Myc-EGFP时目的基因、PCNA、CDK2以及P27KIP1在细胞中的表达水平变化。结果real-time PCR和Western blot结果证实携带目的基因的腺病毒转染耳蜗前体细胞后能有效表达出相应的mRNA和蛋白;并可升高PCNA与CDK2的表达水平,降低P27KIP1的表达水平。结论复制缺陷型腺病毒可有效介导c-Myc和CyclinA2基因转染原代培养的耳蜗前体细胞,并通过上调细胞周期蛋白依赖激酶CDK2,下调CDK抑制蛋白P27KIP1,进而上调细胞DNA合成期标志蛋白PCNA来实现耳蜗前体细胞增殖的作用,其作用机制为经典的CKI-cyclin-CDK途径。更多还原

【Abstract】 In adult mammalian cochlea, hair cells and spiral ganglion cells can not regenerate spontaneously after damaged. This is the major cause of permanent hearing loss. Cochlear progenitor cells have been isolated from the early postnatal rats and mice in previous studies and can differentiate into neurons, astrocytes, hair cells and supporting cells in vitro. We isolated the progenitor cells from the cochleae of 1-, 7-, and 14-day-old rats, and compared with the proliferative capacity and ultrastructure of the cells from each age group using flow cytometry and transmission electron microscopy, respectively. Our study suggests that the dormant state of the cochlear progenitor cells after birth. Although these progenitor cells displayed some features of stem cells, they lost their‘‘stemness’’and the capacity to robustly generate spheres. The cochlear progenitor cells are remnants of the stem cells that originally gave rise to the sensory epithelium. The disappearance of the cochlear progenitor cells in adult mammalian cochleae might result from their differentiation and/or apoptosis.Our study suggests that it may be possible to keep these cells in a progenitor cell state or to activate the progenitor cells by triggering the cell cycle with the expression of cell cycle-related molecules. Hence, several cell cycle regulators, including c-myc and cyclin A2, may be involved in this progress. For better understanding of cochlea development mechanism, the level of c-myc was assessed in the embryonic and postnatal cochleae, the cochlear progenitor cells and the differentiated cells. Then, we established the adenoviral vector of c-myc and cyclinA2 gene and successfully delivered the target genes into the cochlear progenitor cells in vitro. The changes of the proliferative capacity were observed and the corresponding mechanism was further studied. We found c-myc and cyclinA2 gene may induce the progenitor cells proliferation and reenter into cell cycle. The mechanism is the classic way to CKI-cyclin-CDK.1. Isolation and culture of cochlear progenitor cells from newborn ratsObjective: To isolate and culture the CPCs. Methods: The CPCs were isolated from the P7 rat cochlea tissues and cultured. We identified progenitor cells in postnatal rat cochlea and their potential to differentiate into multiple lineages using immunocytochemistry. Results: After 1 day of culture, the acutely isolated cells formed floating otospheres. After a 3-day culture, the floating otospheres expanded gradually in both volume and cell number. Five days later, adherent and differential cells were found in some parts of the spheres. Acutely dissociated cells from the postnatal rat cochleae expressed nestin and musashi1 and incorporated BrdU after 7 days of culture. This demonstrates of these spheres not only expressed the specific markers of stem cells but also were actively undergoing mitosis. The progenitor cell-derived differentiated cells expressed myosin VIIA, phalloidin, NeuN, Tuj1, GFAP, galactocerebroside and GluR-1. The cells generated from the cochlea-derived spheres contained hair cells, neurons, neuroglial cells, and glutamic neurons. Conclusions: The progenitor cells were present in the cochlea of newborn SD rats. They had the capacity of self-renew and possess potential to differentiate into cell types of the inner ear in vitro. This experiment provided a good model in vitro for studying the mechanisms controlling the domant state of the CPCs in adult. 2. A comparison of the proliferative capacity and ultrastructure of progenitor cells from the cochleae of newborn rats of different agesObjective: The progenitor cells that are capable of proliferation and regeneration are present in mammalian cochleae. However, none progenitor cells has been isolated from the adult cochlea. We examined the proliferative potential of cells derived from neonatal rats of various ages. The determination of the differences between the progenitor cells from rats of different ages may provide clues to the mechanisms controlling the destiny of these cells. Methods: The progenitor cells were isolated from the cochleae of 1-, 7-, and 14-day-old rats, and total cells and spheres were counted after 7 days of culture. The proliferative capacity and ultrastructure of the cells from each age group were assessed using flow cytometry and transmission electron microscopy, respectively. Results: During the first two postnatal weeks, the number of progenitor cells gradually fell to zero. This decrease occurred in parallel with the impairment of the proliferative capacity of the cells and the accumulation of progenitor cells in G0/G1. In addition, some of the cells exited the cell cycle by means of gradual maturity and apoptosis. Conclusions: Our study suggests that CPCs are remnants of the stem cells that originally gave rise to the sensory epithelium. The disappearance of the CPCs in adult mammalian cochleae may result from their differentiation and/or apoptosis.3. Decreased level of c-myc in rat cochlea development and cochlear progenitor cell differentiationObjective: The c-myc oncogene is a major regulator for cell proliferation, growth, and apoptosis. However, the role of this gene in the mammalian cochlea development is still unclear. To explore the role of the gene in cochleae, the c-myc expression was assessed in rat cochlea tissues of different ages, the CPCs and the progenitor cell-derived differentiated cells. Methods: We used RT-PCR to detect c-myc mRNA expression in the cochlea tissues, the CPCs and the differentiated cells. The cochlea tissues were excised from E10, E15, P1, P7 and P14 rats respectively. The differentiated cells were cultured for 7 days and the cell spheres were collected for further assay. Western blotting was used to detect c-myc protein expression in the CPCs, the differentiated cells cultured for 7 days, and the cochlea tissues (E10, E15, P1, P7 and P14). We also used immunocytochemistry to detect c-myc protein expression in the differentiated cells and the cell spheres. Results: The results indicated that c-myc level in cochlea tissues decreased gradually during embryo to newborn. Furthermore, c-myc level fell down after differentiation of CPCs. Conclusions: It was suggested that c-myc might be involved in the modulation of rat cochlea development and CPCs differentiation.4. Construction and identification of recombinant adenovirus and determinating the suitable MOI in the transfection of the CPCsObjective: We respectively established the adenoviral vector of c-myc and cyclinA2 gene. The adenovirus was used to transfect the CPCs in vitro. By observing the transfection efficiency and the cell viability, the suitable MOI was determined. Methods: Recombinant adenovirus were constructed. The complementary DNA sequence of c-myc and cyclinA2 gene was respectively obtained from GenBank. The sequence was subcloned into pDC316-CMV-EGFP and recombined with backbone pAdEasy-1 in BJ5183 bacteria. The adenovirus generation, amplifcation, and titer process was completed sequentially. The vector containing EGFP alone was also constructed. The CPCs were infected by the recombinant adenovirus with Enhance Green fluorescence protein (Ad-EGFP) in different MOI (50,100,150,200,250). After the CPCs re-cultured, we observed the expression of EGFP and the cell morphology through fluorescent microscopy. To determine the proliferation point, we used MTT to assess the cell viability. The MOI was ultimately determined and the infection rate was observed through Flow cytometry. Results: The recombinant adenovirus construction was testified through the detection of the purpose fragment in PCR. A positive dose-response relationship was observed between the expression of EGFP gene ranging from 50 ~250 MOI. The virus infecting rat CPCs at a MOI of 50, 100 and 150 can not impair the cell vitality and morphology. However, the cell growth inhibition occurs at a MOI of 200 and the cells necrosis occurs at a MOI of 250. According to the morphological evidence, we ultimately determined the MOI of 150. Conclusions: Adenovirus can efficiently transfect rat CPCs with EGFP gene in vitro. It will be a powerful tool for gene therapy and cell therapy based on CPCs.5. The influence of c-myc and cyclinA2 on the gene-induced CPCs proliferation and cell cycle in VitroObjective: We established the adenoviral vector of c-myc and cyclinA2 gene and successfully delivered the target genes into the CPCs in vitro. Then, the changes of the proliferative capacity were observed. Methods: Plated on 6-well plates (Corning) at 2×106 cells/well, the CPCs were incubated with medium (1 ml) containing different adenoviruses (Ad-c-myc-EGFP, Ad-cyclinA2-EGFP, Ad-c-myc-EGFP and Ad-cyclinA2-EGFP, Ad-EGFP), each at the MOI of 150 using standard techniques. Flow cytometry was used for assessing the influence of the cell cycle and the capacity of proliferation. To assessing the capacity of mitosis, the CPCs cells after transfection were incorporated BrdU. Using immunocytochemistry we observed the expression of BrdU. We also observed the change of propagation. Results: Our data from flow cytometry analysis showed that the cell cycle distribution of the CPCs was significantly affected by the co-overexpression of cyclinA2 and c-myc protein. By contrast, those cells were transfected with Ad-EGFP, Ad-cyclinA2-EGFP and Ad-c-myc-EGFP respectively, and no significant differences were observed. The percentage of incorporated BrdU increased in the progenitor cells co-transfected with Ad-c-myc-EGFP and Ad-cyclinA2-EGFP. The capacity of propagation had no obviously change. Conclusions: These results suggested that cyclinA2 and c-myc induced the proliferation of CPCs and reentered into cell cycle together. However, these two genes had no alone significant effects on cell cycle and the capacity of proliferation.6. The mechanism of c-myc and cyclinA2 inducing the CPCs proliferationObjective: In order to examine the target gene expression and explore the underlying molecular mechanism of cyclinA2 and c-myc inducing the CPCs to reenter into the cell cycle, we detected the expression of target gene, cell cycle-related molecules and PCNA. Methods: We used real-time PCR and westen bloting to detect c-myc, cyclinA2, PCNA, CDK2 and P27kip1 mRNA and protein expression between the Ad-EGFP transfection group and the co-transfect c-myc and cyclinA2 group. Results: The expression of c-myc and cylinA2 protein and the mRNA levels in the Ad-c-myc-EGFP and the Ad-cyclinA2-EGFP infected the CPCs also showed obviously increasing trend than that of the in Ad-EGFP infected cells. At the same time, the up-regulation of cyclinA2 and c-myc protein was associated with the exaltation of CDK2 and PCNA but with the reduction of p27Kip1 in mRNA level and protein expression. Conclusions: These results suggested a successful transfection to the progenitor cells and a significant expression of target gene in these cells, including mRNA level and protein levels. The mechanism of cyclinA2 and c-myc gene inducing the CPCs proliferation and reentering into the cell cycle is the classic way through CKI-cyclin-CDK.更多还原