摘 要
研究背景：胃肠动力障碍是糖尿病(diabetes mellitus，DM)的一个慢性并发症，发病率较高。目前为止，其发病机制尚不明确，一般认为糖尿病胃动力障碍主要与DM自主神经病变和胃肠激素失衡有关, 而DM的代谢紊乱, 特别是糖代谢紊乱可能是产生本症的基础。现在其限于对症，效果欠佳。因此，进一步研究该病的发病机制及寻更有效的方法或药物意义重大。近年来越来越多的实验证实Cajal间质细胞（Interstitial Cells of Cajal，ICC）与其关系密切，引起人们广泛关注。而ICC的分离和体外培养一直是个难题，也就阻碍了相关研究的发展。嘌呤受体是一类非肾上腺能非胆碱能（non-adrenergic
受体是其中一个较为特殊的受体，它具有non-cholinergic，NANC）受体， P2X
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独特的结构和功能，已成为研究热点。
嘌呤能受体在体外培养目的：建立稳定的ICC分离和体外培养方法。观察P2X
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嘌呤能受体的影响，探讨的ICC的表达情况、高糖对ICC形态及其ICC上P2X
7怎么自制纳米胶带
嘌呤能受体在糖尿病胃肠功能紊乱发生机制中的作用。
P2X
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方法：无菌条件下取出小鼠小肠组织，联合使用机械分离法和酶解法分离细胞，将细胞悬液接种于含有干细胞因子（stem cell factor，SCF）的M199培养基中进行培养。倒置显微镜下观察其形态，ICC特异的c-Kit抗体进行免疫荧光染后，用激光共聚焦显微镜进行鉴定。在稳定培养ICC的基础上应用ICC特异的受体抗体进行免疫荧光染，在激光共聚焦显微镜下进行鉴定，
c-Kit抗体和P2X
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受体是否表达于ICC。实验分组：正常对照组(葡萄糖浓度为5.5 mmol 以确定P2X
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／L)，高糖组(葡萄糖浓度为33 mmol／L)。通过倒置显微镜观察细胞形态，RT-PCR
对P2X
和c-Kit受体表达进行比较。
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结果：倒置显微镜下观察到ICC核大，有多个突起并有次级分支，相互之间连接形
受体在c-Kit抗成网络,c-kit抗体荧光染呈阳性。激光共聚焦显微镜下P2X
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体阳性的细胞上的免疫荧光染是阳性的；与正常组比较，加入葡萄糖后的ICC 细胞变大，突起变短，与周围细胞的网络连接减少；RT-PCR结果显示，加入高糖
受体的表达是增强的。
后的ICC的c-Kit受体表达减弱，而P2X
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风光互补led路灯
结论：ICC的分离和培养是个难题，实验过程中我们对其方法、要点及注意事项进行了探讨，并获得了成功。P2X
受体表达于体外培养的ICC上，高糖使ICC的
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受体的表达。高糖可能是形态发生了改变、减弱了c-Kit的表达、加强了P2X
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通过P2X
受体对ICC的功能和形态造成影响，这可能在糖尿病并发胃肠动力障
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碍中发挥一定的作用。
关键词：Cajal间质细胞；免疫荧光技术；高糖; P2X
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Isolation and Culture of Intestinal Interstitial Cells of Cajal and The Expression of P2X7 in Interstitial Cells of Cajal in Vitro : a Role of high Concentration Glucose
Abstract
Gastrointestinal motility disorder is one of chronic complications of diabets mellitus(DM). It’s morbidity is high and the pathogenesis remains unclear. Generally speaking, gastrointestinal motility disorder has relation to autonomic neuropathy and the gastrointinal hormone imblances ,especially the metabolism disorder of glucose may be the base of the disease. Now the treatment is limited to symptoms and the effect is not good. So it is great significance to further explore of the pathogenesis of the disease and more effective treatments. In recent years, more and more experiments have confirmed that interstitial cells of Cajal (ICC) have close relation to it. But it is difficult to isolate and culture ICC in v itro. Purinergic receptors are one kind of non-adrenergic non-cholinergic（NANC）ones. The P2X7 is one of them ,with specific structure and function,and that it have become an important research focus.
Objective: To explore the method for isolation and culture of intestinal ICC in Balb/c mice ,observe t
he expression of P2X7 on ICC, and the effect of high concentrtion gluose on it in vitro, and investigate the cellular mechanism of gastrointestinal dysmotility in diabetic mice.
Methods: The intestine segments of mice were dissected in asepsis, then the mechanical and enzymatic dissociation were used to culture ICC. The cells were suspended in M199 medium containing SCF (stem cell factor),their appearance were observed in upside-down light microscope. The cultured ICCs were stained with c-kit immunofluorescence antibody and indentified under the confocal microscopy. The cultured ICCs were double-labeled and indentified with c-kit and P2X7
immunofluorescence antibodies under the confocal microscopy. The ICCs were divided into high glucose and control groups,cell appearance was observed in upside-down light microscopy, and expression of P2X7 and c-Kit mRNA in ICC was showed by RT-PCR.
箱式高温煅烧炉>苜蓿根
Results: There were large nucleus and more than one primary processes that branched into secondary processes in ICC cells ,and networks formed between them. Fluorescent staining with c- kit antibody confirmed that ICC culture was successful. The immunofluorescence antibodes of P2X7 were also positive in cells in which the immunofluorescence antibodes of c-Kit were positive under the confocal microscopy. After treatment of high glucose,the bodies of ICC were bigger and their pro
滚轮片cesses were shorter ，the network formed from the processes was less.From RT-PCR, we also proved that P2X7 expressed in ICC ,and found the expression of c-Kit was more weaker and the expression of P2X7 was stronger than that of control.
Conclusion: It is difficult to isolate and culture ICC. We succeed in finding the method and outlining some question which need to be attached importance to in the study. P2X7 receptors express in ICC. Hyperglycemia may change the appearance , decrease the expression of c-Kit, and enhance the expression of P2X7 in ICC. High glouse may impact the morphology and the function of ICC through P2X7.These may be play roles in the cellular mechanism of gastrointestinal dysmotility in diabetic mice.
Key words: Interstitial cells of Cajal; Immunofluorescence; high glucose; P2X7 receptor
英文缩略词表（Abbreviation）
英文缩写 英文全称 中文全称
DM diabetes mellitus 糖尿病
ICC interstitial Cells of Cajal Cajal间质细胞 NANC non-adrenergic non-cholinergic
非肾上腺能非胆碱能 ATP adenosine-triphosphate 三磷酸腺苷
STZ streptozotocin 链脲佐菌素
ENS enteric nervous system 肠神经系统
ICC-MY myenteric plexus ICC 肠肌丛ICC
ICC-SM submuscular plexus ICC 黏膜下丛ICC
ICC-DMP deep muscular plexus ICC 深肌层ICC
ICC-IM intra-muscular ICC 肌内ICC
VIP vasoactive intestinal peptide receptor血管活性肠肽受体 NK neurokinin receptor 神经激肽受体羽毛球柱
5-HT5-hydroxytryptamine receptor 五羟胺受体
mER Membrane estrogen receptor 膜雌激素受体
AH after hyperpolarization 后超级化
SCF stem cell factor 干细胞因子
DEPC diethypyrocarbonate 焦碳酸二乙酯
Tm melting temperature 解链温度
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