Kit信号通路调控新生小鼠结肠Cajal间质细胞发育和增殖的实验研究

发布时间：2018-01-31 23:04

  本文关键词： Cajal细胞 Kit受体 BrdU 干细胞因子 胰岛素相关因子-受体I 甲磺酸伊马替尼　出处：《第三军医大学》2010年博士论文　论文类型：学位论文

【摘要】： Cajal细胞(Interstitial cells of Cajal, ICCs)是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。它能自发性产生电慢波、整合并放大胆碱能和硝基能神经递质的传递,同时也是胃肠道张力感受器,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 hypert- rophic pyloric stenosis, IHPS)、先天性巨结肠(Hirschsprung’s disease, HD)、新生儿假性肠梗阻(neonatal pseudo-obstruction)及胃肠术后一过性肠麻痹等密切相关。尽管上述疾病的发病机制目前仍不十分清楚,但是均有相似的病理改变,即消化管壁内ICCs不同程度的减少,甚至缺如,同时ICCs彼此间及其与平滑肌细胞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是胃肠运动障碍性疾病的重要原因。因此,探讨新生小鼠消化管壁内ICCs的发育规律,将对阐明胃肠运动功能障碍性疾病的发病机制提供实验基础,并为治疗提供一定的指导作用。 结肠壁内的ICCs根据分布的位置而分为四个不同的亚群:1)分布在肌间神经丛周围的ICCs(ICC-MY);2)环形平滑肌和纵形平滑肌内的ICCs(ICC-IM);3)浆膜下ICCs(ICC-SS)和4)粘膜下层靠近环肌面的ICCs(ICC-SM)。不同亚群的ICCs的功能存在差别,如ICC-SM是结肠的起搏细胞,ICC-IM介导兴奋性和抑制性神经递质的传递,而ICC-SS可能具有感受张力变化的作用。而且不同亚群的ICCs的发育过程也存在较大差异。因此,首先了解新生小鼠结肠不同亚群ICCs的发育过程是十分必要的。此外,在我们前期的实验结果显示,小鼠小肠ICCs从出生到成年细胞数量增加了近30倍,而且增加的细胞可能是由胰岛素样生长因子受体1(insulin-like growth factor 1 receptor, IGF-1R)阳性的ICCs前体细胞的增殖而来。那么结肠内的ICCs是否有增殖?是否也存在前体细胞?也是我们关注的问题。 ICCs持续表达由原癌基因c-kit编码的III型受体酪氨酸激酶受体Kit蛋白,它能够与平滑肌细胞Sl基因编码的干细胞因子(stem cell factor,SCF)结合,对ICCs的发育、存活、增殖和表型维持有重要的调控作用。在c-Kit突变的W/W~V小鼠、W/W~ S大鼠,或者Sl基因突变的Sl/Sl~d小鼠动物模型中;或者在胚胎晚期或生后早期,用Kit中和性抗体或Kit受体阻断剂阻断Kit受体功能;以及在体外培养体系中加入Kit中和性抗体或去除外源性SCF,都将导致胃肠壁内ICCs发育受阻,其数量明显减少,细胞网络被破坏,并伴有胃潴留、反流性食道炎、十二指肠内容物逆流等胃肠功能障碍的症状。我们前期对成年小鼠和豚鼠研究表明: Kit信号通路对小肠壁内ICCs的存活和自主节律性运动都具有重要作用。近年来的研究认为,出生后小鼠胃肠壁内同样存在ICCs前体细胞,但是不具有产生慢波的能力。在病理因素导致ICCs数量减少时,这些前体细胞在胰岛素(insulin)/胰岛素生长因子-I(insulin growth factor,IGF-I)信号和Kit/SCF信号的共同作用下,大量增殖再分化为成熟的,能产生自发性电活动的ICCs。那么Kit信号是否对结肠各亚型ICCs的发育和存活是否具有调控作用?各亚型ICCs的反应是否相同?Kit信号通路和IGF-1R信号通路对ICCs的增殖有何调控作用?这些问题也是我们关注的问题。 本实验分为以下三个部分: 第一部分:新生小鼠结肠ICCs的发育 通过Kit蛋白免疫荧光染色特异性标记结肠ICCs,阐明不同部位不同亚型的结肠ICCs在生后(P0)到成年(P56)的过程中,细胞位置、形态、密度和总数的变化。主要结果如下: 1. ICC-MY,ICC-IM,ICC-SS是出生0天即形成,ICC-SM是生后6天才出现于近端结肠,提示不同亚型的ICCs的发育过程有一定的时间差异。 2. ICC-SM在P6出现于近端结肠,P8才见于远端结肠,提示同一亚型的ICCs在结肠不同部位的发育,存在时间顺序性。且ICC-MY在近端结肠的发育早于中间段,远端结肠的发育最晚。提示结肠ICCs是从头端向尾端的方向发育成熟的。 3.出生后,随着结肠的长度、直径及表面积不断增加,各亚型ICCs的总数均不断增加。 上述结果表明新生小鼠结肠ICCs逐渐发育成熟且数量不断增加,各亚型ICCs的发育存在一定的时间和空间顺序性,表现为结肠靠近头端的ICCs先发育,而各亚群中ICC-SM发育最迟。 第二部分:新生小鼠结肠ICCs的增殖特点 本研究通过BrdU标记和追踪增殖细胞,并结合抗核增殖抗原Ki67标记以及多重免疫荧光染色的实验方法,探讨出生后结肠ICCs的增殖潜能和规律。主要结果如下: 1. ICC-MY,ICC-IM和ICC-SS具有增殖能力,在新生早期最旺盛,但随年龄的增长,增殖能力逐渐降低。 2.新生小鼠结肠中存在Kit~+/IGF-IR~+/Ki67~+细胞,这些ICCs前体细胞,可能是增殖ICCs的主要来源。 3. ICC-SM具有与其他亚群ICCs不同的增殖模式,其数目的增加可能是由Kit~-的前体细胞增殖随后开始表达Kit蛋白并分化为成熟的ICCs。 上述结果表明结肠成体ICC-IM,ICC-MY,ICC-SS均具有增殖的潜能,在结肠生后发育过程中大量增加的细胞,可能是来源于Kit~+/IGF-IR~+前体细胞。Kit阳性的ICC-SM不具有增殖能力,其数量的增加的过程可能是Kit~-的前体细胞增殖后再分化为Kit~+的ICC。 第三部分:Kit信号通路对新生小鼠结肠ICCs发育和增殖的调控 利用Imatinib灌胃构建Kit信号通路被抑制的动物模型,BrdU腹腔注射,结合多重免疫荧光组织化学,激光共聚焦显微镜,western印迹的研究方法,探讨Kit信号通路和IGF-IR信号通路对小肠和结肠ICCs存活和增殖的调控机制,以及ICCs增殖的模式。主要结果如下: 1. Imatinib能够有效在体抑制Kit信号通路的激活。 2. Kit信号通路抑制后,除ICC-SM外的各亚群ICCs细胞数量明显减少,细胞网络不完整,说明Kit信号通路参与调节新生期小鼠肠道ICCs的存活,但是ICC-SM对该信号不敏感。 3.阻断Kit信号通路,Kit~+ICCs和/或前体细胞的增殖完全停止,而抑制IGF-IR信号通路,增殖ICCs的细胞数量减少,证明ICCs的增殖依赖于Kit信号通路,而IGF-1R对ICCs增殖也具有一定的调控作用。 4.停止Imatinib处理后,ICCs细胞数量迅速恢复正常,而增殖的Kit~+ICCs的数量较少且出现较晚。提示Kit阴性的ICCs前体细胞的增殖可能是大量增加的ICCs来源之一。 上述结果表明新生期小鼠结肠的存活、增殖都依赖于Kit信号通路。当某些病理因素导致ICCs丢失时,Kit阴性的ICCs前体细胞可大量增殖分化为成熟的ICCs,维持肠道正常的生理功能。IGF-IR信号通路参与调节ICCs的增殖。 综上所述,本研究初步证明出生后随结肠结构的不断完善ICCs亦可进一步发育。新生小鼠结肠ICCs具有增殖能力,且随年龄的增长逐渐降低消失。而增殖ICCs的主要来源可能是Kit~+/CD44~+/CD34~+/IGF-IR~+的ICCs前体细胞。Kit信号通路对ICCs的存活和增殖具有重要的调控作用,但是ICC-SM对Kit信号通路不敏感。
[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.

【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R329

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