出生后哺乳动物胃肠道Cajal细胞的发育生物学特性研究

发布时间：2018-01-15 18:27

  本文关键词：出生后哺乳动物胃肠道Cajal细胞的发育生物学特性研究　出处：《第三军医大学》2009年博士论文　论文类型：学位论文

  更多相关文章： Cajal细胞 Kit受体 发育 增殖 CD44 免疫荧光染色

【摘要】： Cajal细胞(Interstitial cells of Cajal, ICCs)作为一种特殊的间质细胞,以细胞网络的形式分布于整个胃肠道,能产生自发性电慢波、参与神经信息传递,在调节胃肠道平滑肌运动中具有重要作用。近年临床研究发现ICCs与一些新生儿胃肠运动功能障碍性疾病:如婴儿肥厚性幽门狭窄(Infantile hypertrophic pyloric stenosis, IHPS)、新生儿假性肠梗阻(neonatal pseudo-obstruction)、先天性巨结肠(Hirschsprung’s disease, HD)及胃肠术后一过性肠麻痹等密切相关。尽管上述疾病的发生机制尚不清楚,但均有一个共同的特点:病变部位胃肠壁内ICCs数量呈不同程度减少,甚至缺如,ICCs彼此间不能形成完整的细胞网络,说明ICCs发育过程的延迟或异常可能会导致新生儿胃肠运动出现障碍。因此阐明ICCs的发育规律及增殖再生潜能,将对胃肠运动功能障碍性疾病的发病机制的认识和治疗提供一定的指导作用。 目前关于ICCs细胞的发育学研究主要集中在胚胎期,研究结果显示胚胎期ICCs的数量随着胎龄增加而增多,突起逐渐延长,出生前后已形成与成人相似的细胞网络。而且认为该细胞只在胚胎期及新生早期具有增殖能力。但关于哺乳动物出生后ICCs的发育变化规律及正常生理条件下的增殖特性尚未见文献报道。 此外,c-kit原癌基因及Kit蛋白作为ICCs的特异性标记物不仅能在ICCs持续表达,而且Kit信号通路对其发育、分化、增殖、存活、及正常功能的维持等均具有重要调控作用。但在上述疾病中,随ICCs数量的减少,Kit蛋白表达亦明显下调,无法对ICCs进行有效标记,所以ICCs确切的命运转归如何并不清楚。 因此,本论文首先研究出生后ICCs发育的时空变化规律,然后观察成体ICCs是否仍然能够增殖及不同年龄阶段ICCs的增殖特点,最后研究CD44在胃肠道内的表达模式及其与ICCs的关系。主要结果如下: 1.出生后,小肠的长度、直径及表面积不断增加,P32达到成年水平。应用Kit免疫荧光染色观察到出生时肠壁内ICCs主要位于肌间神经丛附近,细胞呈星形,核周胞质少,通过2-3个细小的突起及分支彼此相连形成较稀疏的细胞网络。随着年龄的增长,IC-MY突起及分支增粗延长,数量明显增多,P32达P0的16倍并形成与成年动物相似的完整致密的细胞网络。但肠道各部位ICCs的发育并不同步,存在从头端到尾端的时空差异,回肠IC-MY细胞网络的发育明显滞后于十二指肠和空肠。 2.通过给不同年龄的小鼠注射BrdU并结合Kit/ BrdU免疫荧光双重染色发现出生后ICCs仍具有增殖能力,但随年龄的增长其增殖能力会逐渐降低。成体小肠中存在Kit+/BrdU+/CD44+/CD34+/IGF-IR+细胞,可能是ICCs的前体细胞,可增殖分化为成熟的ICCs。 3.运用CD44、Kit和vimentin(ICCs另一种标记物)三重免疫荧光染色发现CD44阳性细胞与成年哺乳动物消化管各段肌层内Kit和vimentin阳性的ICCs均基本重合,并紧紧包绕在NF200阳性的神经纤维周围,而平滑肌细胞及成纤维细胞中未见该蛋白表达,说明CD44可作为ICCs细胞的特异标志物。胃肠壁内也存在极少量(0.6%)Kit-/CD44+的细胞,呈圆形,突起短而少,其分布部位及形态与胚胎期ICCs前体细胞相似,推测可能为成年动物中的ICCs前体细胞。 综上所述,本研究初步证明出生后随小肠结构的不断完善ICCs亦可进一步发育,P32左右才形成与成年动物相似的细胞网络。新生期到成年的ICCs仍保持增殖能力,但其增殖能力会随年龄的增长逐渐降低。成体动物小肠壁内存在Kit+/CD44+/CD34+/IGF-IR+的ICCs前体细胞,可增殖分化为成熟ICCs。CD44蛋白在成年哺乳动物胃肠道肌层内的分布与Kit阳性的ICCs基本一致,可作为鉴别该细胞的特异性标记物。
[Abstract]:Cajal cells (Interstitial cells of Cajal, ICCs) as a kind of special interstitial cells distributed throughout the gastrointestinal cellular network form, can generate spontaneous electric slow wave, involved in neurotransmission, plays an important role in regulating the motility of gastrointestinal smooth muscle. In recent years the clinical study found that ICCs and some neonatal gastrointestinal motility disorders such as infantile hypertrophic pyloric stenosis (Infantile hypertrophic pyloric stenosis, IHPS), neonatal intestinal pseudo obstruction (neonatal pseudo-obstruction), congenital megacolon (Hirschsprung 's disease, HD) and gastrointestinal surgery after intestinal paralysis is closely related to the disease. Although the occurrence mechanism is unclear, but have a common characteristic: the number of lesions in the gastrointestinal wall ICCs were reduced or absent, ICCs each other can not form a complete network of cells, ICCs Delayed or abnormal development process may lead to dysfunctional gastrointestinal motility in neonates. Therefore, elucidating the developmental regularity of ICCs and the potential of proliferation and regeneration will provide some guidance for the understanding and treatment of the pathogenesis of gastrointestinal motor dysfunction.
The current researches on ICCs cell development mainly concentrated in the embryonic stage, the results showed that the number of embryos of ICCs increased with the increase of gestational age, raised gradually extended, before and after birth and adult has formed a similar cellular network. And that the cells have the ability of proliferation in the early embryonic and neonatal development. But the proliferation characteristics of the changing rules of ICCs mammals after birth and normal physiological conditions has not been reported.
In addition, the original cancer gene and Kit protein c-kit as a specific marker of ICCs not only can continuously express in ICCs, and the Kit signal pathway in the development, differentiation, proliferation, survival, and maintenance of the normal functions have important regulatory roles. But in these diseases, with the reduction in the number of ICCs, Kit protein expression was also down, can not be effective markers for ICCs ICCs, so the exact outcome of how fate is not clear.
Therefore, we first study the temporal and spatial variation of postnatal ICCs development, then observe whether ICCs can still proliferate and proliferate at different ages. Finally, we study the expression pattern of CD44 in the gastrointestinal tract and its relationship with ICCs. The main results are as follows: ICCs
1. after birth, intestinal length, diameter and surface area increased, P32 reached the adult level. Application of Kit immunofluorescence staining was observed in the intestinal wall of ICCs was mainly located in the myenteric plexus near the cell showed star, cytoplasm, branches and tiny projections 2-3 are connected to each other to form a sparse through the network of cells. With the increase of age, IC-MY processes and branch thickening and extending, the number increased significantly, 16 times the P32 of P0 and the formation of resemblance to the adult animal complete compact cell network. But the gut of each part of ICCs development does not exist from the head end to synchronization, temporal and spatial differences of the tail end of the ileum IC-MY cellular network the development is obviously lagging behind in the duodenum and jejunum.
2. according to different age mice injected with BrdU combined with Kit/ BrdU double immunofluorescence staining indicated that after the birth of ICCs still have the ability of proliferation, but with the increase of age and its proliferation ability will be gradually reduced. Kit+/BrdU+/CD44+/CD34+/IGF-IR+ cells in small intestine, may have been the precursor of ICCs cells can proliferate and differentiate into mature ICCs.
3. the use of CD44, Kit and vimentin (ICCs another marker) three immunofluorescence staining showed that CD44 positive cells and adult mammalian digestive tube Kit and vimentin muscular layer of each section in the positive ICCs were coincident, and firmly wrapped around NF200 positive fibers and smooth muscle cells, and the expression of the protein no fiber cells, indicating that CD44 can be used as specific markers of ICCs cells. The gastrointestinal wall also has a very small amount of (0.6%) Kit-/CD44+ cells were round, protruding short and small, the distribution and morphology of ICCs and embryonic precursor cells are similar, presumably as an adult animal in ICCs precursor cells.
In summary, this study demonstrated that after birth with continuous improvement of intestinal structure of the further development of ICCs, before the formation of resemblance to the adult animal cell network P32. Neonatal to adult ICCs remains proliferation, but the proliferation ability will be gradually reduced with the increase of age. Adult animal small intestine Kit+/CD44+/CD34+/IGF-IR+ ICCs precursor cells can proliferate and differentiate into mature ICCs.CD44 protein in gastrointestinal tract of adult mammalian muscle and the distribution of Kit positive ICCs are basically the same, can be used as a specific marker to identify the cells.

【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R33

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本文编号：1429547