本文關(guān)鍵詞： Cajal細胞 Kit受體 BrdU 干細胞因子 胰島素相關(guān)因子-受體I 甲磺酸伊馬替尼　出處：《第三軍醫(yī)大學》2010年博士論文　論文類型：學位論文

【摘要】： Cajal細胞(Interstitial cells of Cajal, ICCs)是一種特殊的間質(zhì)細胞,以細胞網(wǎng)絡(luò)的形式分布于整個胃腸道。它能自發(fā)性產(chǎn)生電慢波、整合并放大膽堿能和硝基能神經(jīng)遞質(zhì)的傳遞,同時也是胃腸道張力感受器,在調(diào)節(jié)胃腸道平滑肌運動中具有重要作用。近年臨床研究發(fā)現(xiàn)ICCs與一些新生兒胃腸運動功能障礙性疾病:如嬰兒肥厚性幽門狹窄(Infantile hypert- rophic pyloric stenosis, IHPS)、先天性巨結(jié)腸(Hirschsprung’s disease, HD)、新生兒假性腸梗阻(neonatal pseudo-obstruction)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)病機制目前仍不十分清楚,但是均有相似的病理改變,即消化管壁內(nèi)ICCs不同程度的減少,甚至缺如,同時ICCs彼此間及其與平滑肌細胞間不能形成完整的細胞網(wǎng)絡(luò),說明ICCs發(fā)育過程的延遲或異�？赡苁俏改c運動障礙性疾病的重要原因。因此,探討新生小鼠消化管壁內(nèi)ICCs的發(fā)育規(guī)律,將對闡明胃腸運動功能障礙性疾病的發(fā)病機制提供實驗基礎(chǔ),并為治療提供一定的指導作用。 結(jié)腸壁內(nèi)的ICCs根據(jù)分布的位置而分為四個不同的亞群:1)分布在肌間神經(jīng)叢周圍的ICCs(ICC-MY);2)環(huán)形平滑肌和縱形平滑肌內(nèi)的ICCs(ICC-IM);3)漿膜下ICCs(ICC-SS)和4)粘膜下層靠近環(huán)肌面的ICCs(ICC-SM)。不同亞群的ICCs的功能存在差別,如ICC-SM是結(jié)腸的起搏細胞,ICC-IM介導興奮性和抑制性神經(jīng)遞質(zhì)的傳遞,而ICC-SS可能具有感受張力變化的作用。而且不同亞群的ICCs的發(fā)育過程也存在較大差異。因此,首先了解新生小鼠結(jié)腸不同亞群ICCs的發(fā)育過程是十分必要的。此外,在我們前期的實驗結(jié)果顯示,小鼠小腸ICCs從出生到成年細胞數(shù)量增加了近30倍,而且增加的細胞可能是由胰島素樣生長因子受體1(insulin-like growth factor 1 receptor, IGF-1R)陽性的ICCs前體細胞的增殖而來。那么結(jié)腸內(nèi)的ICCs是否有增殖?是否也存在前體細胞?也是我們關(guān)注的問題。 ICCs持續(xù)表達由原癌基因c-kit編碼的III型受體酪氨酸激酶受體Kit蛋白,它能夠與平滑肌細胞Sl基因編碼的干細胞因子(stem cell factor,SCF)結(jié)合,對ICCs的發(fā)育、存活、增殖和表型維持有重要的調(diào)控作用。在c-Kit突變的W/W~V小鼠、W/W~ S大鼠,或者Sl基因突變的Sl/Sl~d小鼠動物模型中;或者在胚胎晚期或生后早期,用Kit中和性抗體或Kit受體阻斷劑阻斷Kit受體功能;以及在體外培養(yǎng)體系中加入Kit中和性抗體或去除外源性SCF,都將導致胃腸壁內(nèi)ICCs發(fā)育受阻,其數(shù)量明顯減少,細胞網(wǎng)絡(luò)被破壞,并伴有胃潴留、反流性食道炎、十二指腸內(nèi)容物逆流等胃腸功能障礙的癥狀。我們前期對成年小鼠和豚鼠研究表明: Kit信號通路對小腸壁內(nèi)ICCs的存活和自主節(jié)律性運動都具有重要作用。近年來的研究認為,出生后小鼠胃腸壁內(nèi)同樣存在ICCs前體細胞,但是不具有產(chǎn)生慢波的能力。在病理因素導致ICCs數(shù)量減少時,這些前體細胞在胰島素(insulin)/胰島素生長因子-I(insulin growth factor,IGF-I)信號和Kit/SCF信號的共同作用下,大量增殖再分化為成熟的,能產(chǎn)生自發(fā)性電活動的ICCs。那么Kit信號是否對結(jié)腸各亞型ICCs的發(fā)育和存活是否具有調(diào)控作用?各亞型ICCs的反應(yīng)是否相同?Kit信號通路和IGF-1R信號通路對ICCs的增殖有何調(diào)控作用?這些問題也是我們關(guān)注的問題。 本實驗分為以下三個部分: 第一部分:新生小鼠結(jié)腸ICCs的發(fā)育 通過Kit蛋白免疫熒光染色特異性標記結(jié)腸ICCs,闡明不同部位不同亞型的結(jié)腸ICCs在生后(P0)到成年(P56)的過程中,細胞位置、形態(tài)、密度和總數(shù)的變化。主要結(jié)果如下: 1. ICC-MY,ICC-IM,ICC-SS是出生0天即形成,ICC-SM是生后6天才出現(xiàn)于近端結(jié)腸,提示不同亞型的ICCs的發(fā)育過程有一定的時間差異。 2. ICC-SM在P6出現(xiàn)于近端結(jié)腸,P8才見于遠端結(jié)腸,提示同一亞型的ICCs在結(jié)腸不同部位的發(fā)育,存在時間順序性。且ICC-MY在近端結(jié)腸的發(fā)育早于中間段,遠端結(jié)腸的發(fā)育最晚。提示結(jié)腸ICCs是從頭端向尾端的方向發(fā)育成熟的。 3.出生后,隨著結(jié)腸的長度、直徑及表面積不斷增加,各亞型ICCs的總數(shù)均不斷增加。 上述結(jié)果表明新生小鼠結(jié)腸ICCs逐漸發(fā)育成熟且數(shù)量不斷增加,各亞型ICCs的發(fā)育存在一定的時間和空間順序性,表現(xiàn)為結(jié)腸靠近頭端的ICCs先發(fā)育,而各亞群中ICC-SM發(fā)育最遲。 第二部分:新生小鼠結(jié)腸ICCs的增殖特點 本研究通過BrdU標記和追蹤增殖細胞,并結(jié)合抗核增殖抗原Ki67標記以及多重免疫熒光染色的實驗方法,探討出生后結(jié)腸ICCs的增殖潛能和規(guī)律。主要結(jié)果如下: 1. ICC-MY,ICC-IM和ICC-SS具有增殖能力,在新生早期最旺盛,但隨年齡的增長,增殖能力逐漸降低。 2.新生小鼠結(jié)腸中存在Kit~+/IGF-IR~+/Ki67~+細胞,這些ICCs前體細胞,可能是增殖ICCs的主要來源。 3. ICC-SM具有與其他亞群ICCs不同的增殖模式,其數(shù)目的增加可能是由Kit~-的前體細胞增殖隨后開始表達Kit蛋白并分化為成熟的ICCs。 上述結(jié)果表明結(jié)腸成體ICC-IM,ICC-MY,ICC-SS均具有增殖的潛能,在結(jié)腸生后發(fā)育過程中大量增加的細胞,可能是來源于Kit~+/IGF-IR~+前體細胞。Kit陽性的ICC-SM不具有增殖能力,其數(shù)量的增加的過程可能是Kit~-的前體細胞增殖后再分化為Kit~+的ICC。 第三部分:Kit信號通路對新生小鼠結(jié)腸ICCs發(fā)育和增殖的調(diào)控 利用Imatinib灌胃構(gòu)建Kit信號通路被抑制的動物模型,BrdU腹腔注射,結(jié)合多重免疫熒光組織化學,激光共聚焦顯微鏡,western印跡的研究方法,探討Kit信號通路和IGF-IR信號通路對小腸和結(jié)腸ICCs存活和增殖的調(diào)控機制,以及ICCs增殖的模式。主要結(jié)果如下: 1. Imatinib能夠有效在體抑制Kit信號通路的激活。 2. Kit信號通路抑制后,除ICC-SM外的各亞群ICCs細胞數(shù)量明顯減少,細胞網(wǎng)絡(luò)不完整,說明Kit信號通路參與調(diào)節(jié)新生期小鼠腸道ICCs的存活,但是ICC-SM對該信號不敏感。 3.阻斷Kit信號通路,Kit~+ICCs和/或前體細胞的增殖完全停止,而抑制IGF-IR信號通路,增殖ICCs的細胞數(shù)量減少,證明ICCs的增殖依賴于Kit信號通路,而IGF-1R對ICCs增殖也具有一定的調(diào)控作用。 4.停止Imatinib處理后,ICCs細胞數(shù)量迅速恢復正常,而增殖的Kit~+ICCs的數(shù)量較少且出現(xiàn)較晚。提示Kit陰性的ICCs前體細胞的增殖可能是大量增加的ICCs來源之一。 上述結(jié)果表明新生期小鼠結(jié)腸的存活、增殖都依賴于Kit信號通路。當某些病理因素導致ICCs丟失時,Kit陰性的ICCs前體細胞可大量增殖分化為成熟的ICCs,維持腸道正常的生理功能。IGF-IR信號通路參與調(diào)節(jié)ICCs的增殖。 綜上所述,本研究初步證明出生后隨結(jié)腸結(jié)構(gòu)的不斷完善ICCs亦可進一步發(fā)育。新生小鼠結(jié)腸ICCs具有增殖能力,且隨年齡的增長逐漸降低消失。而增殖ICCs的主要來源可能是Kit~+/CD44~+/CD34~+/IGF-IR~+的ICCs前體細胞。Kit信號通路對ICCs的存活和增殖具有重要的調(diào)控作用,但是ICC-SM對Kit信號通路不敏感。
[Abstract]:Cajal cells (Interstitial cells of Cajal, ICCs) is a kind of special interstitial cells distributed throughout the gastrointestinal cellular network form. It can generate spontaneous electric slow wave, integration and bold alkali and nitro can transfer the neurotransmitter, but also gastrointestinal tension receptors, play an important role in regulation of gastrointestinal smooth muscle. Recent clinical studies have indicated that ICCs and some neonatal gastrointestinal motility disorders such as infantile hypertrophic pyloric stenosis (Infantile hypert- rophic pyloric stenosis, IHPS), congenital megacolon (Hirschsprung 's disease, HD), neonatal intestinal pseudo obstruction (neonatal pseudo-obstruction) and after gastrointestinal surgery intestinal paralysis and other closely related. Although the pathogenesis of these diseases remains unclear, but had similar pathological changes of digestive canal with different degrees of ICCs Reduced or absent, while ICCs each other and smooth muscle cells could not form a complete description of the cellular network, delay or abnormal development of ICCs may be an important cause of gastrointestinal motility disorder. Therefore, to investigate the development of neonatal mouse digestive wall ICCs, will provide an experimental basis for elucidating the pathogenesis of gastrointestinal motility dysfunction, and provide some guidance for treatment.
In the colon in ICCs according to the distribution of the location and divided into four subgroups: 1) distributed in the myenteric plexus around ICCs (ICC-MY); 2) circular smooth muscle and longitudinal muscle in ICCs (ICC-IM); 3) ICCs (ICC-SS) and subserous submucosa 4) near ring muscle surface ICCs (ICC-SM) ICCs. Different subsets of features are different, such as ICC-SM is colonic pacemaker cells, ICC-IM mediated transfer of excitatory and inhibitory neurotransmitter, and ICC-SS may have feelings of tension change. And the development process of different subsets of ICCs are quite different. So first, understand the newborn mouse colon ICCs subsets of the development process is very necessary. In addition, in our previous experimental results show that the number of small intestine of mice from birth to adult ICCs cells increased by nearly 30 times, and the increase of cells may be caused by the insulin-like growth factor receptor 1 (insu Lin-like growth factor 1 receptor, IGF-1R) proliferation of ICCs positive precursor cells. Is there any proliferation of ICCs in the colon? Is there any precursor cell as well? It is also our concern.
ICCs expression by c-kit proto oncogene encoding the III receptor tyrosine kinase receptor Kit protein, it can smooth muscle cells and Sl gene encoding stem cell factor (stem cell factor, SCF) combined with ICCs on the survival, development, maintenance, proliferation and phenotype plays an important role. In the c-Kit mutant W/W~V mice. W/W~ S rats or Sl gene mutation in Sl/Sl~d mouse animal model; or in the late embryo or early postnatal, blockade of Kit receptor function by Kit neutralizing antibody or Kit receptor antagonist; and the culture system with Kit and antibody or removal of exogenous SCF in vitro, will lead to gastrointestinal wall ICCs development is blocked, the number of cells was significantly reduced, the destruction of the network, and associated with gastric retention, reflux esophagitis, reflux of duodenal contents such as gastrointestinal dysfunction symptoms. We show that the early adult mice and guinea pigs: K Has the important role of it signaling pathway in the small intestine ICCs survival and spontaneous rhythmic movement. Recent studies suggest that there are also ICCs progenitor cells in postnatal mouse GI, but does not have the ability to produce slow wave. The pathological factors which lead to the reduction of the number of ICCs, these precursor cells in insulin (insulin) / insulin-like growth factor -I (insulin growth factor, IGF-I) interaction and Kit/SCF signal, and then proliferate and mature differentiation, can produce spontaneous activity of ICCs. then Kit whether the signal of different subtype of colonic ICCs development and survival is regulated? Each subtype of ICCs reaction the same? The proliferation of Kit and IGF-1R signal pathways to ICCs what role? These problems is our concern.
The experiment is divided into three parts:
Part 1: the development of colonic ICCs in newborn mice
Through Kit protein immunofluorescence staining, the colon ICCs was specifically labeled, and the location, morphology, density and total number of colon ICCs in different parts and subtypes after birth (P0) to adult (P56) were elucidated.
1. ICC-MY, ICC-IM and ICC-SS were formed on the 0 day of birth. ICC-SM appeared on the proximal colon 6 days after birth, suggesting that there is a time difference in the development of ICCs of different subtypes.
In 2. ICC-SM P6 appeared in the proximal colon, distal colon was found in P8, suggesting that the same subtype of ICCs development in different parts of the colon, there is time sequence. And the ICC-MY in the proximal colon in the middle period of early development, the development of the most distal colon. Late colon ICCs is from the beginning to the end. The direction of development and maturation.
After 3. birth, with the length, diameter and surface area of the colon increasing, the total number of subtypes of ICCs increased.
The above results showed that the colonic ICCs of newborn mice gradually grew and matured, and the number of ICCs increased. There was temporal and spatial ordering in the development of each subtype of ICCs. It showed that the ICCs near the head of the colon developed first, while the development of the subgroup was the most late.
The second part: the characteristics of the proliferation of colonic ICCs in newborn mice
The aim of this study is to explore the potential and rule of colon ICCs proliferation after birth by means of BrdU markers and tracking of proliferating cells, combined with Ki67 markers and multiple immunofluorescence staining methods.
1. ICC-MY, ICC-IM and ICC-SS have the ability to proliferate, which are most exuberant in the early freshmen, but the proliferation ability gradually decreases with age.
There are Kit~+/IGF-IR~+/Ki67~+ cells in the colon of 2. newborn mice, and these ICCs precursor cells may be the main source of ICCs proliferation.
3. ICC-SM has different proliferation patterns compared with other subsets of ICCs. The increase in number may be due to the proliferation of precursor cells of Kit~-, then the expression of Kit protein and the differentiation into mature ICCs..
The results showed that the colon into ICC-IM, ICC-MY, ICC-SS have the potential to proliferate, a substantial increase in the development of colon after birth in cells, may be derived from Kit~+/IGF-IR~+.Kit positive precursor cells ICC-SM does not have the ability of proliferation, the increase in the number of the process may be a precursor for the proliferation of Kit~- cells after differentiation Kit~+ ICC.
The third part: the regulation of Kit signaling pathway on the development and proliferation of colonic ICCs in newborn mice
The use of Imatinib gavage animal model of Kit signaling pathway was inhibited by intraperitoneal injection of BrdU, combined with multiple fluorescent immunohistochemistry, confocal microscopy, Western blotting methods, to explore the regulatory mechanism of Kit signaling pathway and IGF-IR signaling pathway on the proliferation and survival of ICCs in small intestine and colon, and the pattern of ICCs proliferation. The main results the following:
1. Imatinib can effectively inhibit the activation of the Kit signaling pathway in the body.
After inhibition of 2. Kit signaling pathway, the number of ICCs cells in each subgroup decreased significantly and the cell network was incomplete except for ICC-SM, indicating that Kit signaling pathway was involved in regulating the survival of intestinal ICCs in neonatal mice, but ICC-SM was not sensitive to the signal.
3., blocking the Kit signaling pathway, the proliferation of Kit~+ICCs and / or precursor cells completely stopped, while the inhibition of IGF-IR signaling pathway and the proliferation of ICCs cells decreased. It is proved that the proliferation of ICCs depends on Kit signaling pathway, while IGF-1R also plays a regulatory role in ICCs proliferation.
4. after stopping Imatinib treatment, the number of ICCs cells returned to normal quickly, while the number of Kit~+ICCs proliferated less and later. It suggested that the proliferation of Kit negative ICCs precursor cells may be one of the sources of ICCs.
The results showed that the survival of neonatal mouse colon, proliferation depends on Kit signaling pathway. When some pathological factors lead to the loss of ICCs, Kit negative ICCs progenitor cells can proliferate and differentiate into mature ICCs, keeping the normal physiological functions of.IGF-IR signaling pathway involved in the regulation of the proliferation of ICCs.
In summary, this study demonstrated that after birth with the continuous improvement of the structure of colon in the further development of ICCs. ICCs has the proliferation of newborn mouse colon, and gradually decreased with age growing disappear. And main source of proliferation of ICCs may be Kit~+/CD44~+/CD34~+/IGF-IR~+ ICCs precursor cells of.Kit signal pathway on survival and proliferation of ICCs play an important role however, ICC-SM is not sensitive to the Kit signaling pathway.

【學位授予單位】：第三軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2010
【分類號】：R329

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本文編號：1480224