本文關(guān)鍵詞：出生后哺乳動(dòng)物胃腸道Cajal細(xì)胞的發(fā)育生物學(xué)特性研究　出處：《第三軍醫(yī)大學(xué)》2009年博士論文　論文類型：學(xué)位論文

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【摘要】： Cajal細(xì)胞(Interstitial cells of Cajal, ICCs)作為一種特殊的間質(zhì)細(xì)胞,以細(xì)胞網(wǎng)絡(luò)的形式分布于整個(gè)胃腸道,能產(chǎn)生自發(fā)性電慢波、參與神經(jīng)信息傳遞,在調(diào)節(jié)胃腸道平滑肌運(yùn)動(dòng)中具有重要作用。近年臨床研究發(fā)現(xiàn)ICCs與一些新生兒胃腸運(yùn)動(dòng)功能障礙性疾病:如嬰兒肥厚性幽門狹窄(Infantile hypertrophic pyloric stenosis, IHPS)、新生兒假性腸梗阻(neonatal pseudo-obstruction)、先天性巨結(jié)腸(Hirschsprung’s disease, HD)及胃腸術(shù)后一過性腸麻痹等密切相關(guān)。盡管上述疾病的發(fā)生機(jī)制尚不清楚,但均有一個(gè)共同的特點(diǎn):病變部位胃腸壁內(nèi)ICCs數(shù)量呈不同程度減少,甚至缺如,ICCs彼此間不能形成完整的細(xì)胞網(wǎng)絡(luò),說明ICCs發(fā)育過程的延遲或異�？赡軙�(huì)導(dǎo)致新生兒胃腸運(yùn)動(dòng)出現(xiàn)障礙。因此闡明ICCs的發(fā)育規(guī)律及增殖再生潛能,將對(duì)胃腸運(yùn)動(dòng)功能障礙性疾病的發(fā)病機(jī)制的認(rèn)識(shí)和治療提供一定的指導(dǎo)作用。 目前關(guān)于ICCs細(xì)胞的發(fā)育學(xué)研究主要集中在胚胎期,研究結(jié)果顯示胚胎期ICCs的數(shù)量隨著胎齡增加而增多,突起逐漸延長(zhǎng),出生前后已形成與成人相似的細(xì)胞網(wǎng)絡(luò)。而且認(rèn)為該細(xì)胞只在胚胎期及新生早期具有增殖能力。但關(guān)于哺乳動(dòng)物出生后ICCs的發(fā)育變化規(guī)律及正常生理?xiàng)l件下的增殖特性尚未見文獻(xiàn)報(bào)道。 此外,c-kit原癌基因及Kit蛋白作為ICCs的特異性標(biāo)記物不僅能在ICCs持續(xù)表達(dá),而且Kit信號(hào)通路對(duì)其發(fā)育、分化、增殖、存活、及正常功能的維持等均具有重要調(diào)控作用。但在上述疾病中,隨ICCs數(shù)量的減少,Kit蛋白表達(dá)亦明顯下調(diào),無法對(duì)ICCs進(jìn)行有效標(biāo)記,所以ICCs確切的命運(yùn)轉(zhuǎn)歸如何并不清楚。 因此,本論文首先研究出生后ICCs發(fā)育的時(shí)空變化規(guī)律,然后觀察成體ICCs是否仍然能夠增殖及不同年齡階段ICCs的增殖特點(diǎn),最后研究CD44在胃腸道內(nèi)的表達(dá)模式及其與ICCs的關(guān)系。主要結(jié)果如下: 1.出生后,小腸的長(zhǎng)度、直徑及表面積不斷增加,P32達(dá)到成年水平。應(yīng)用Kit免疫熒光染色觀察到出生時(shí)腸壁內(nèi)ICCs主要位于肌間神經(jīng)叢附近,細(xì)胞呈星形,核周胞質(zhì)少,通過2-3個(gè)細(xì)小的突起及分支彼此相連形成較稀疏的細(xì)胞網(wǎng)絡(luò)。隨著年齡的增長(zhǎng),IC-MY突起及分支增粗延長(zhǎng),數(shù)量明顯增多,P32達(dá)P0的16倍并形成與成年動(dòng)物相似的完整致密的細(xì)胞網(wǎng)絡(luò)。但腸道各部位ICCs的發(fā)育并不同步,存在從頭端到尾端的時(shí)空差異,回腸IC-MY細(xì)胞網(wǎng)絡(luò)的發(fā)育明顯滯后于十二指腸和空腸。 2.通過給不同年齡的小鼠注射BrdU并結(jié)合Kit/ BrdU免疫熒光雙重染色發(fā)現(xiàn)出生后ICCs仍具有增殖能力,但隨年齡的增長(zhǎng)其增殖能力會(huì)逐漸降低。成體小腸中存在Kit+/BrdU+/CD44+/CD34+/IGF-IR+細(xì)胞,可能是ICCs的前體細(xì)胞,可增殖分化為成熟的ICCs。 3.運(yùn)用CD44、Kit和vimentin(ICCs另一種標(biāo)記物)三重免疫熒光染色發(fā)現(xiàn)CD44陽性細(xì)胞與成年哺乳動(dòng)物消化管各段肌層內(nèi)Kit和vimentin陽性的ICCs均基本重合,并緊緊包繞在NF200陽性的神經(jīng)纖維周圍,而平滑肌細(xì)胞及成纖維細(xì)胞中未見該蛋白表達(dá),說明CD44可作為ICCs細(xì)胞的特異標(biāo)志物。胃腸壁內(nèi)也存在極少量(0.6%)Kit-/CD44+的細(xì)胞,呈圓形,突起短而少,其分布部位及形態(tài)與胚胎期ICCs前體細(xì)胞相似,推測(cè)可能為成年動(dòng)物中的ICCs前體細(xì)胞。 綜上所述,本研究初步證明出生后隨小腸結(jié)構(gòu)的不斷完善ICCs亦可進(jìn)一步發(fā)育,P32左右才形成與成年動(dòng)物相似的細(xì)胞網(wǎng)絡(luò)。新生期到成年的ICCs仍保持增殖能力,但其增殖能力會(huì)隨年齡的增長(zhǎng)逐漸降低。成體動(dòng)物小腸壁內(nèi)存在Kit+/CD44+/CD34+/IGF-IR+的ICCs前體細(xì)胞,可增殖分化為成熟ICCs。CD44蛋白在成年哺乳動(dòng)物胃腸道肌層內(nèi)的分布與Kit陽性的ICCs基本一致,可作為鑒別該細(xì)胞的特異性標(biāo)記物。
[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.

【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2009
【分類號(hào)】：R33

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相關(guān)期刊論文 前4條

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本文編號(hào)：1429547