【作者】 梅峰；

【导师】 周德山；

【作者基本信息】 第三军医大学 ， 人体解剖与组织胚胎学， 2008， 博士

【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。近二十余年,国内外学者通过对胃肠壁内ICCs的形态特征、超微结构、起源及生理功能等一系列研究表明:ICCs具有产生自发性电慢波、参与神经信息整合与传递等功能,对胃肠平滑肌运动具有重要调节作用。近年的临床研究发现ICCs数量减少与一些胃肠运动功能障碍性疾病:例如糖尿病胃轻瘫、慢传输型便秘、Crohn’s病、术后胃肠运动功能障碍等密切相关。尽管上述疾病的发生机制尚不清楚,但均有一个共同的病理变化:即病变部位胃肠壁内ICCs数量呈不同程度减少,甚至缺如,ICCs彼此间及其与平滑肌细胞间不能形成完整的细胞网络。但因对上述疾病的发病机理及ICCs减少、细胞网络完整性被破坏的原因等尚不清楚,一直是临床治疗的难题。设想如果能够阐明促进ICCs细胞网络完整性恢复的调控机制,势必为临床相关疾病的治疗提供有益的思路。ICCs表达c-kit基因产物,一种原癌基因编码的受体酪氨酸激酶(Kit)。它的配体是干细胞因子(stem cell factor,SCF),由平滑肌和神经产生。在Kit或SCF基因突变的小鼠和大鼠,以及用Kit中和性抗体或Kit受体阻断剂在胚胎晚期或生后早期阻断Kit受体功能,将引起ICCs数量明显减少或丢失,伴有胃肠运动功能障碍,表明胚胎期ICCs的发育、存活及形态的维持需要Kit/SCF信号通路功能的正常。体外研究亦发现,在外源性SCF的作用下,培养的小鼠小肠ICCs具有一定的增殖能力,但这种增殖仅见于生后6天前的新生鼠,提示ICCs的增殖具有时间依赖性和SCF依赖性。我们的研究发现,小肠半横断吻合术后,吻合部位丢失的ICCs随时间的经过能够逐渐恢复正常的数量和分布模式,提示成年动物ICCs仍具有一定的增殖能力,且与平滑肌表达SCF有关,提示Kit/SCF信号通路可能参与了这一过程。已知成年哺乳动物胃肠壁内ICCs持续表达c-kit及其产物KIT蛋白,KIT/SCF信号通路对发育成熟的ICCs是否具有与胚胎期相似的作用尚不清楚。抑制成年哺乳动物Kit信号通路后,ICCs是否减少?发生凋亡或者转分化?丢失后能否恢复正常分布?在此过程中Kit/SCF信号通路的作用如何均亟待阐明。此外,肠梗阻等胃肠运动功能障碍性疾病ICCs丢失后能否恢复正常分布以及Kit/SCF信号通路的是否参与这一过程的调节均不清楚。因此,探讨Kit/SCF信号通路对成熟ICCs可塑性的调控机制将有利于理解胃肠运动功能障碍性疾病的发病机制,同时为临床相关疾病的治疗开辟新的策略。本实验分为两个部分:第一部分:Imatinib是Kit受体的特异性抑制剂。本研究给成年小鼠和豚鼠连续口服Imatinib,利用Kit和Vimentin免疫荧光染色观察阻断Kit信号通路后,ICCs的数量和突起长度的变化,同时Kit/a-SMA双重免疫荧光标记观察ICCs是否向平滑肌表型转分化,以及体外灌流观察小肠功能变化;停Imatinib处理后观察ICCs恢复情况以及小肠功能变化,并用Kit/PCNA和Kit/BrdU双重染色对各实验组ICCs的增殖进行分析。主要结果如下:1、Imatinib处理后,成年豚鼠和小鼠小肠的ICCs数量逐渐减少,随时间的延长尤为明显,最多可减少1/2以上,同时自主节律性运动功能亦受到抑制。2、ICCs数量减少的同时,可见Kit/a-SMA双标阳性ICC和Kit(-)/a-SMA(+)ICC样细胞,提示ICCs可能向平滑肌的表型转分化,但未见ICC出现凋亡。3、停止Imatinib处理后,ICCs数量和功能逐渐恢复正常,与此同时可见大量的BrdU/Kit和PCNA/Kit双标阳性的细胞。上述结果表明成年哺乳动物ICCs具有较强的可塑性,Kit信号通路对成熟ICCs的存活和增殖具有重要的调控作用,该信号通路功能障碍可能导致ICCs的发生转分化,成熟ICCs具有较强的增殖能力。第二部分:制作小肠机械性肠梗阻模型,采用Kit及Kit/a-SMA单/双重免疫荧光染色观察梗阻期间和解除梗阻后ICCs的数量变化,Western Blot检测SCF和Kit磷酸化(p-Kit)的变化,用Kit/BrdU双重染色显示成熟ICCs的增殖以及体外灌流检测小肠运动功能变化,探讨ICCs的数量变化与SCF表达及Kit信号通路激活或抑制的相关性。主要结果如下:1、梗阻期ICCs数量逐渐减少,梗阻14天靠近梗阻部位最明显,并可见Kit/a-SMA双标阳性细胞,同时肠运动功能亦受到明显的抑制;2、在梗阻期SCF表达量和p-Kit均下降,靠近吻合口部位最明显,约为正常的1/2到1/3,解除梗阻后,SCF和p-Kit恢复正常。3、解除梗阻后,ICCs数量增加,逐渐恢复正常分布模式同时可见大量BrdU/Kit双标阳性细胞,与此同时小肠运动功能也恢复正常。上述结果表明SCF表达的变化与Kit信号通路的抑制和激活密切相关,同时Kit信号通路对成熟ICCs的存活、表型维持以及增殖等可塑性具有重要调节作用。综上所述,本研究初步证明了成熟ICCs具有较强的可塑性,这种可塑性与Kit/SCF信号通路密切相关;Kit活性被抑制后ICCs可能向平滑肌的表型转分化;解除抑制后,ICCs能够恢复正常的数量和分布模式,其数量的增加与ICCs的增殖有关;肠梗阻时,ICCs数量的减少和解除梗阻后数量的增加与平滑肌表达SCF的变化平行,提示胃肠运动功能障碍性疾病可能与平滑肌表达的SCF不足, Kit信号通路活性低下有关。靶向性提高SCF的表达或直接激活Kit信号通路可能是临床治疗相关疾病的策略之一。更多还原

【Abstract】 Interstitial cells of Cajal (ICCs) are a kind of speical interstial cells, and distribute throughout the entire gastrointestinal tract. In recent two decades, a lot of researches have been carried out to investigate the morphology, ultrastructure, origination and physiology of these cells, and it have been identified that ICCs are the pacemaker cells that generate and propagate the slow waves in the alimentary tract, and act as the mediators of inputs from the enteric nervous system to GI smooth muscles; they also serve as a stretch sensor of the GI tract.Clinical studies have indicated that quite a number of GI motility disorders, such as diabetic gastroparesis, slow transit constipation, Crohn’s disease and post-operational gut dysmotilities, are characterized by a reduction of ICC numbers and impairment of the cellular network. However, the underlying reasons for ICCs loss and impairment are not yet well-known. Clarification of the regulatory factors that control survival and proliferation of ICCs will further our understanding and treatment of GI motility disorders.ICCs express the gene product of c-kit, a proto-oncogene that encodes the receptor tyrosine kinase (Kit). Its ligand, stem cell factor (SCF), is produced by smooth muscle cells (SMCs) and neurons. Inactivation of Kit with neutralizing antibodies or Kit blocker in fetal and neonatal animals, or non-lethal mutations of Kit or SCF in rats and mice, leads to defects in ICC networks and pacemaker activity of GI movement. Therefore, the Kit/SCF signal pathway is considered essential for the development, proliferation, differentiation and survival of ICCs during the fetal and neonatal periods.ICC precusors begin to express Kit protein at E12, and day-2 post-partum these cells further develop in morphogy, ultrastructure and function almost same with the mature ones. In vitro experiments have also shown that only cultured ICCs from the mice before day-6 post-partum can proliferate in response to SCF, indicating that the proliferation of ICCs is time-limited and SCF-dependent under culture conditions. Our previous experiments have shown that after semi-transection and anastomosis, ICCs at the anastomotic site are lost and then ICCs adjacent could proliferate and gradually recover to normal distribution. This finding suggests that ICCs in adults still possess a proliferative ability. Besides that, we also have found that the new ICCs always emerge with SCF-expressing smooth muscle cells, which hinted that Kit/SCF signal pathway might play an important role in the regulation of the proliferation of ICCs in adults.Although ICCs keep expressing the c-kit gene and its product, Kit protein, the effects of Kit signal pathway on mature ICCs are still unknown. Besides that, serveal issues are still needed to be clarified, such as how mature ICCs response to the inhibition of Kit signal pathway? Apoptosis? Death? Whether ICCs could recover to normal after loss? Which method ICCs are dependent on during the recovery? What role is the Kit/SCF signal pathway plays? Furthermore, it is also unclear that how ICCs response to disease, and whether the alterations of ICCs is under the regulation of Kit/SCF signal pathway. Therefore, to investigate the effects of Kit/SCF signal pathway on mature ICCs would benefit the understanding of the underlying mechanism of gastrointestinal motility disorders and exploring possible therapy strategies.Supposing Kit/SCF signal pathway regulate the plasticity of ICCs in adult, this would benefit the understanding of gastrointestinal disorders, and provide possible therapic method. Therefore, this investigate is divided into two part:Part 1:Imatinib is a potent inhibitor of Kit and can used in vivo. We treated adult guinea pigs and mice with Imatinib, and observed the alterations of the cell numbers and process length of ICCs by immunoflourosent staining with anti-Kit and Vimentin antibodies after a peroid of drug treatment. Also Kit/a-SMA double labeling were carried out to investigate whether ICCs transdifferentiate toward a phenotype of smooth muscle cells after drug treatment, and the mechanical contractions of small intestine were measured under the condition of in vitro organ bath. After drug withdrawal, except the observation of ICCs numbers and process length, and the measurement of mechanical contraction, Kit/PCNA and Kit/BrdU double staining were used to detect the proliferative events of ICCs. All the methods were used to clarify the association between the plasticity of ICCs and Kit/SCF signal pathway.The results are the followings:1 After Imatinib treatment, ICCs reduced in cell number and process length, meanwhile the mechanical contractions of small intestine were inhibited.2 Both Kit/a-SMA double labeled cells and Kit(-)/a-SMA(+) ICC-like cells were observed, which suggest a possibility that ICCs might transdifferentiate toward a phenotype of smooth muscle cells.3 After Imatinib withdrawal, ICCs recovered in cell numbers and process length, along with a regain of mechanical contractions.4. At the same time, a number of Kit/PCNA and Kit/BrdU double stained cells were found out, which hinted a proliferative events of ICCs.The above result have indicated that Kit singal pathway is essential for the maintanence, survival and proliferation, under the condition of Kit inactivation ICCs might transdifferntiate towards a smooth muscle cell phenotype, and ICCs in adults could proliferate after lost.Part 2:Mice with mechanical intestinal obstruction were used as animal models. During the intestinal obstruction, the changes of ICCs, including cell numbers and transdifferentiation, were investigate by Kit and Kit/a-SMA single and double immunoflouresence, and the alterations of SCF and p-Kit were assayed by western blot, beside that intestinal mechanical contrations were measured in in vitro organ bath. After removal of the obstruction, besides the ICCs number and expression of SCF and p-Kit protein, Kit/BrdU double labeling were applied to detect the proliferative events of ICCs. The methods were used to investigate the alterations of ICCs during intestitnal obstruction and after removal of obstrution, meanwhile analysized the expression of SCF and associated activation/inactivation of Kit. These results might clarify the regulative effects of Kit/SCF signal pathway on the plasticity of ICCs, and provide possible therapic strategies.The results were the followings:、1. The mechanical intestinal obstruction led to a reduction of the cell numbers of ICCs which was most obvious adjacent to the obstructive site, meanwhile Kit/a-SMA double positive cells were observed, and intestinal motitlity were inhibited obviously.2. After removal of obstruction, ICCs increased in amounts, and BrdU/Kit double labeled cells were observed accompanying with the recovery of intestinal motility.、3. The expression of SCF and p-Kit were apperantly down-regulated to about 1/2 or 1/3 during obstruction, which suggested the Kit/SCF signal pathway was inhibited.4. After removal of obstruction, SCF and p-Kit restored to normal level, which indicated that the activation of Kit/SCF signaling was closely associated with the revovery and proliferation of ICCs.The second part revealed that the expressive alterations of SCF was closely associated with the activation/inactivation of Kit receptor which tightly linked with the proliferation and transdifferentiation of ICCs. These finding suggested Kit/SCF signal pathway regulated the plasticity of ICCs in adult mice.To sum up, the results above proved that ICCs in adults still possess vivid plasticity, in other word, these cells could transdifferentiate towards a smooth muscle cell phenotype and reduced in numbers, also could proliferate and restore normal amounts, distribution and function. The smooth muscle cells could regulate the the plasticity of ICCs by up/down regulation of SCF expression. Therefore the present study suggested that one of the mechanisms underlying the gastrointestinal motility was insufficience which would lead to inactivation of Kit receptor and reduction of ICCs, meanwhile up-regulating the expression of SCF or directly activating the Kit signaling might be one of the potential therapic strategies.更多还原