二甲基亚砜对体外大鼠结肠平滑肌收缩的影响

[目的]观察二甲基亚砜（DMSO）对体外大鼠结肠平滑肌收缩的影响，探讨其作为脂溶性中药成分助溶剂的可行浓度范围。[方法]制备SD大鼠结肠平滑肌肌条，以9g／L NaCl空白对照（NS）组和乙酰胆碱对照（Ach）组，分别观察不同浓度DMSO对肌条自发收缩以及对Ach引起的肌条收缩的影响。[结果]DMSO在0．2%～1．4%浓度范围，对肌条自发收缩振幅和面积的影响与NS组比较均无统计学意义（P〉0．05）。与Ach组比较，在0．2％～1．8％浓度范围，DMSO对Ach引起的肌条收缩振幅和面积的影响均无统计学意义（P〉0．05）。[结论]DMSO在浓度≤1．4％时对体外大鼠结肠平滑肌的收缩无明显影响，作为脂溶性中药的助溶剂有运用价值。     

大黄素对结肠癌血管内皮生长因子受体的影响及对结肠癌抑制作用的研究

[目的]研究大黄素对血管内皮生长因子(VEGF)受体(VEGFR)阻断作用及对结肠癌细胞抑制作用的机制.[方法]软琼脂集落实验法,流式细胞术检测大黄素对结肠癌细胞增殖和凋亡的影响;酪氨酸激酶活性分析,Western blot方法检测大黄素对VEGFR的抑制作用.[结果]大黄素抑制结肠癌细胞生长,引起细胞凋亡,凋亡率由对照组(0 μmol/L)的(8.1±2.7)%上升至大黄素40 μmol/L时的(27.8±10.9)%(P＜0.01),呈浓度依赖性.大黄素抑制VEGFR酪氨酸激酶活性,VEGFR-1相对活性由对照组的100%降至大黄素40μmol/L时的22.4%(P＜0.01),VEGFR-2降至58.5%(P＜0.01),VEGFR-3降至31.6%(P＜0.01),大黄素作用后,VEGFR酪氨酸磷酸化状态蛋白量减少.[结论]大黄素能够通过抑制VEGFR酪氨酸激酶活性而抑制结肠癌生长,可作为一种有效的肿瘤血管生成抑制剂.     

美罗培南不同输注时间治疗老年住院患者获得性肺炎的临床效果比较

医院获得性肺炎系之指入院48 h内发生肺炎类型,发病率位居医院感染首位[1]。同时是病死率较高的医院感染类型,是导致患者住院时间延长,增加医疗负担的主要原因[2-7]。美罗培南属第二代碳青霉烯类强效、广谱抗菌药物,对大部分厌氧菌、需氧菌皆有效,且神经毒性低,对肾功能影响小,用于重症感染治疗,对耐药菌感染亦有较高的药效[8]。研究已证实美罗培南属时间依赖性抗菌药物,给药期间游离药物浓度超目标微生物最低抑菌浓度时间(f T>MIC)可预测其最佳杀菌活性[9]。fT>MIC>40%即可获取杀菌效应,超过60%~70%即可实现最大杀菌效应,而对重症感染或预防耐药菌株需达90%~100%[10-15]。延长美罗培南输注时间可延长fT>MIC,减少抗菌药物用量[16]。但对美罗培养延长输注过程中药物稳定性及是否可能存在药效下降等情况尚未明确。故本文对美罗培南不同输注时间治疗医院获得性肺炎的可行性展开了研究,以期为老年医院获得性肺炎的防控及治疗提供依据。     

白藜芦醇、染料木黄酮、姜黄素和大黄素对2BS细胞生长增殖能力的影响

目的观察白藜芦醇、染料木黄酮、姜黄素和大黄素对细胞衰老及细胞生长增殖能力的影响。方法配置白藜芦醇、染料木黄酮、姜黄素和大黄素不同浓度的DMEM培养基,分别培养人胚肺二倍体成纤维细胞(2BS),观察2BS细胞形态、体外传代次数,以MTT法观察细胞生长增殖能力变化,以单细胞凝胶电泳(彗星试验)法观察细胞DNA单链断裂水平。结果低浓度白藜芦醇、染料木黄酮、姜黄素和大黄素DMEM培养基培养的细胞体外传代次数均较对照组细胞下降;上述抗氧化剂在低浓度时可促进细胞生长增殖,但在高浓度时抑制细胞生长增殖;四种抗氧化剂均可断裂单链DNA,且随浓度加大损伤作用愈大。结论白藜芦醇、染料木黄酮、姜黄素和大黄素具有促进细胞生长增殖和细胞毒性双重作用,其细胞毒性源于其对DNA损伤作用。     

大黄素对原代培养小鼠肝细胞非酒精性脂肪变的影响

[目的]观察大黄素对原代培养小鼠肝细胞非酒精性脂肪变的影响。[方法]采用改良的胶原酶原位灌流法分离小鼠肝细胞,以含50%胎牛血清的1640培养液造成肝细胞脂肪变,加入不同浓度大黄素,检测培养上清液肝酶活性并观察细胞形态学变化。[结果]以50%胎牛血清的1640培养液培养的肝细胞明显脂肪变性,大黄素干预组培养上清液丙氨酸氨基转移酶(ALT)、门冬氨酸转氨酶(AST)、谷氨酰转肽酶(GGT)、乳酸脱氢酶(LDH)活性较模型组显著降低,苏木精-伊红染色显示肝细胞脂肪变性减轻。[结论]大黄素可明显减轻原代培养小鼠肝细胞的脂肪变性。     

茉莉酸甲酯对人胰腺癌细胞株HS766T抑制作用的研究

[目的]观察茉莉酸甲酯对人胰腺癌细胞株HS766T的抑制增殖及诱导凋亡的作用。[方法]用0~10mmol/L浓度的茉莉酸甲酯体外处理人胰腺癌细胞株HS766T,分别在24h、48h、72h、96h用MTT法检测其细胞生长活性;PI单染色法检测其细胞周期;Annexin V/PI双染色法检测其细胞凋亡;RT-PCR、Western Blot检测基因Bcl-2、Bax及其蛋白的表达量。[结果]用0~10mmol/L茉莉酸甲酯处理胰腺癌细胞24h、48h、72h、96h后,细胞抑制率分别为:14.40%~71.41%、23.62%~89.68%、29.30%~94.76%、37.50%~94.74%;经0.001、0.01mmol/L的茉莉酸甲酯分别作用24h、48h后,与对照组相比,茉莉酸甲酯处理组处于G0/G1/S期的比例升高,G2/M期的比例降低;同时人胰腺癌细胞在0.001、0.01 mmol/L茉莉酸甲酯处理24h、48h后,对照组凋亡率为1.2%、1.6%,0.001mmol/L浓度组凋亡率为12.3%、16.8%,0.01mmol/L浓度组凋亡率为14.5%、23.2%;RTPCR检测显示Bax和Bcl-2在不同浓度和时间的扩增倍数分别为:1.18~1.48、0.92~0.33;Western Blot检测结果显示,Bax表达呈递增而Bcl-2表达呈递减的规律。[结论]不同浓度的茉莉酸甲酯对人胰腺癌细胞株HS766T细胞均有抑制作用,且呈明显的浓度依赖性,48h前细胞抑制率与药物浓度呈正相关,并可降低抑凋亡基因Bcl-2及升高促凋亡基因Bax的表达,从而抑制该细胞株体外生长和促进其凋亡。     

用于评价小球隐孢子虫卵囊感染力的NMRI乳鼠模型

[目的 ]评价一定数量的小球隐孢子虫卵囊的感染力。 [方法 ]4日龄 NMRI乳鼠经口腔管饲法接种不同数量的卵囊 ,7d后处死乳鼠 ,取消化道幽门至肛门段并匀成悬液 ,取此悬液置载玻片上 ,石碳酸品红染色 ,相差显微镜查见卵囊的为被感染乳鼠。以各组被感染乳鼠百分率评价卵囊的感染力。 [结果 ]接种 15 0 0或 2 0 0 0的乳鼠均被感染 ;接种 10 0 0、 5 0 0、2 5 0、 10 0和 5 0个卵囊的乳鼠 ,感染率分别为 88%、 74%、 5 1%、 2 8%和 9.5 %。 [结论 ]NMRI乳鼠模型接种卵囊数在 15 0 0～5 0范围内 ,其接种量与感染率呈正相关。该模型可用于评价一定数量小球隐孢子虫卵囊的感染力。     

健脾清肠方对DSS诱导结肠炎小鼠肠道动力学影响的研究

[目的]探讨健脾清肠方(JQD)对DSS诱导结肠炎小鼠肠道动力的作用机制。[方法]C57BL/6小鼠随机分为Control组、DSS组、JQD组、5-ASA组。除Control组外,余各组小鼠用5%DSS自由饮用诱导结肠炎模型,造模7d后灌胃给药。第15天处死小鼠收集结肠组织,检测结肠组织病理学,ICC的超微结构,结肠组织IL-1β、IL-10、IFN-γ、TNF-α含量,c-kit mRNA、LC3-II mRNA、Beclin-l mRNA、NF-κB p65表达和结肠平滑肌张力。[结果]与DSS组比较,JQD组结肠黏膜表面上皮细胞移行修复,少量炎症细胞浸润,黏膜腺体基本恢复正常;ICC与其他细胞间连接完好,内部细胞器结构相对完整,可见到少量自噬泡存在;结肠组织IL-1β、TNF-α含量降低,IL-10、IFN-γ含量上升;c-kit mRNA表达上调,LC-3II mRNA、Beclin-1mRNA、NF-κB p65表达下降;结肠平滑肌条收缩振幅上升、收缩频率减少。[结论]JQD调节DSS模型小鼠异常肠道动力,其机制可能是通过抑制NF-κB/TNF-α通路及调节细胞因子IL-1β、IL-10、IFN-γ表达,抑制了肠道炎症的级联放大反应;抑制ICC过度自噬,调控ICC/SMC网络通路。     

参麦注射液对大鼠神经元内质网应激诱导凋亡的影响

目的 探讨参麦注射液对大鼠皮层神经元内质网应激诱导凋亡的保护作用.方法 体外培养SD乳鼠皮层神经元细胞,免疫组织化学、免疫荧光染色鉴定神经元纯度,流式细胞术AnnexinV、PI双标检测凋亡率及活性caspase-3、-9表达,Western印迹免疫分析GRP78、细胞色素C蛋白、Bcl-2蛋白表达,Fura-2/AM法荧光分光光度计检测细胞内钙浓度([Ca~(2+)]i).结果 SD乳鼠皮层神经元可纯化体外培养,2 μmol/L毒胡萝卜素作用神经元24、48 h细胞凋亡率分别是17.88%、21.38%,参麦治疗组分别是7.42%、8.16%,两组差异显著(P<0.05).胡萝卜素诱导神经元糖调节蛋白GRP78表达上调,活化caspase-3、-9,使细胞色素C蛋白表达增加,Bcl-2表达减少.参麦注射液促进细胞Bcl-2表达,抑制细胞色素C释放,减少活性caspase-3、-9含量,降低[Ca~(2+)]i浓度.结论 参麦注射液能抑制体外培养神经元内质网应激所致的凋亡可能与其降低[Ca~(2+)]i浓度有关.     

黄芪总黄酮对心肌细胞内游离钙离子浓度的影响

目的研究黄芪总黄酮（Total flavonoids of Ast ragalus,TFA）对体外培养的乳鼠心肌细胞内游离钙离子浓度的作用。方法实验应用钙离子荧光指示剂Fluo-3AM负载原代培养的乳鼠心肌细胞,通过激光共聚焦扫描显微镜上机检测,记录其荧光强度变化,同时观察黄芪总黄酮对乳鼠心肌细胞及化学处理因素H2O2损伤大鼠心肌细胞的荧光强度的作用。结果①H2O2损伤的乳鼠心肌细胞游离钙离子[Ca^2＋]i平均荧光强度值为（1346．89±65．36）nmol／L,与对照组平均荧光强度值（743．15±34．78）nmol／L相比,差异有统计学意义（P〈0．01,n=7）;同时H2O2（0．3mmol／L）使予给TFA（20mg／kg）组乳鼠心肌细胞的[Ca^2＋]i平均荧光强度值由（668．29±41．15）nmol／L下降到（649．31±39．17）nmol／L,差异无统计学意义（P〉0．05,n=7）。结论H2O2可明显升高乳鼠心肌细胞游离钙离子浓度,黄芪总黄酮可对抗H2O2的这种作用。     

Tissue culture of human fetal pancreas: growth hormone stimulates the formation and insulin production of islet-like cell clusters

The human fetal pancreas (HFP) is a potential source of insulin-producing B-cells for transplantation to insulin-dependent diabetic patients. We recently described a technique for culturing HFP tissue in vitro which results in the development of islet-like cell clusters (ICC). These clusters exhibited (pro)insulin biosynthesis and a modest rate of insulin secretion, and immunocytochemical staining indicated the presence of insulin-positive cells in the cell clusters. In this study this technique was used to evaluate the effects of the addition of 1000 micrograms/L GH to HFP cultured in medium RPMI-1640 plus 10% human serum. ICCs developed in 21 of 33 consecutive cultures. GH increased the yield of ICC by 35% compared to explants supplemented with human serum alone. The insulin content of the ICCs also was increased, but the size of individual ICCs was not affected by GH, as reflected by an unchanged DNA content. GH also caused increased insulin release when the ICCs were stimulated with 16.7 mM glucose plus 5 mM theophylline. However, (pro)insulin biosynthesis was not affected by the addition of GH. These results suggest that GH stimulates the formation of both ICCs and insulin production within the explants. These observations are relevant both for the production of human fetal B-cells intended for transplantation into insulin-dependent diabetic patients and for our knowledge of the growth regulation of the HFP B-cell.     

Function of interstitial cells of Cajal in the rabbit portal vein

Interstitial cells of Cajal (ICCs) were identified in the intact fixed media of the rabbit portal vein (RPV) using c-kit staining. The following experiments were performed using single cell preparations of the enzyme-dispersed vessel. Surviving contacts between the processes of single ICCs and the bodies of smooth muscle cells (SMCs) were observed in electron micrographs and by confocal microscopy. Spontaneous rhythmical [Ca2+]i oscillations were observed in ICCs after loading with the calcium indicator fluo-3 and were associated with depolarizations of the ICCs recorded by tight-seal patch pipette. To investigate signal transmission from ICCs to SMCs in dispersed cell pairs, or within small surviving fragments of the ICC network, an ICC was stimulated under voltage-clamp, while changes in [Ca2+]i in the stimulated cell as well as in a closely adjacent SMC or ICCs were monitored using fast x-y confocal imaging of fluo-3 fluorescence. After stimulation of single voltage-clamped ICC by a depolarizing step similar in duration to depolarizations associated with spontaneous [Ca2+]i oscillations, a depolarization and transient elevation of [Ca2+]i was observed in a closely adjacent SMCs after a delay of up to 4 seconds. In contrast, signal transmission from ICC to ICC was much faster, the delay being less than 200 ms. These results suggest that the an ICC may, in addition to generating an electrical signal (such as a slow wave) and thereby acting as a pacemaker for vascular SMCs of RPV, also release some unknown diffusible substance, which depolarizes the SMCs.     

Comparison of gene expression profiles between Opisthorchis viverrini and non-Opisthorchis viverrini associated human intrahepatic cholangiocarcinoma

Intrahepatic cholangiocarcinoma (ICC) is the second most common primary cancer in the liver, and its incidence is highest in the northeastern part of Thailand. ICCs in this region are known to be associated with infection with liver flukes, particularly Opisthorchis viverrini (OV), as well as nitrosamines from food. To clarify molecular mechanisms of ICC associated with or without liver flukes, we analyzed gene expression profiles of OV-associated ICCs from 20 Thai patients and compared their profiles with those of 20 Japanese ICCs that were not associated with OV, by means of laser microbeam microdissection and a cDNA microarray containing 27,648 genes. We identified 77 commonly upregulated genes and 325 commonly downregulated genes in the two ICC groups. Unsupervised hierarchical cluster analysis separated the 40 ICCs into two major branches almost completely according to the fluke status. The putative signature of OV-associated ICC exhibited elevated expression of genes involved in xenobiotic metabolism (UGT2B11, UGT1A10, CHST4, SULT1C1), whereas that of non-OV-associated ICC represented enhanced expression of genes related to growth factor signaling (TGFBI, PGF, IGFBP1, IGFBP3). Additional random permutation tests identified a total of 49 genes whose expression levels were significantly different between the two groups. We also identified genes associated with macroscopic type of ICCs. In conclusion, these data may not only contribute to clarification of common and OV-specific mechanisms underlying ICC, but also may serve as a starting point for the identification of novel diagnostic markers or therapeutic targets for the disease.     

Pathogenesis and classification of intrahepatic cholangiocarcinoma: different characters of perihilar large duct type versus peripheral small duct type

Intrahepatic cholangiocarcinomas (ICCs) are made up of heterogenous carcinomas arising from different anatomical sites of the liver. Two types of candidate stem/progenitor cells of the biliary tree are postulated to exist at the peribiliary glands for large bile ducts and at the canals of Hering for small ducts and hepatocytes. According to the recent observations, ICCs can be subclassified into two types: tumors involving the large bile ducts comparable in size to the intrahepatic second branches and composed of a tubular or papillary component with tall columnar epithelium, and tumors involving the smaller duct than segmental branches and composed of small tubules with cuboidal epithelium. Perihilar large duct type ICCs can be interpreted as arising from large bile duct type ICCs, and peripheral small duct type ICCs may arise from small bile duct type or ductular type ICCs. Chronic biliary inflammation induces neoplastic change of the large bile ducts and thereby progression to the perihilar large duct type ICC, which can be grossly classified into periductal filtrating type ICC and intraductal growth type ICC, while chronic hepatitis or cirrhosis induces mass‐forming peripheral small duct type ICC. The different morphological and molecular features, including stromal components and tumor vasculature, support the hypothesis that perihilar large duct type ICCs and peripheral small duct type ICCs arise from different backgrounds, have different carcinogenetic pathways, and exhibit different biologic behaviors.     

Control of gallbladder contractions by cholecystokinin through cholecystokinin-A receptors on gallbladder interstitial cells of Cajal

AIM： To identify the cholecystokinin （CCK）-A receptors （CCK-AR） on the guniea pig gallbladder interstitial cells of cajal （ICC） and to study CCK-8 induced gallbladder muscle strip contractions through the CCK-AR.
METHODS： The existence of CCK-AR was examined by immunohistofluorescence on sectioned tissue and cultured cells. In vitro contractile response of guinea pig gallbladder muscle strips and the strips with ICC removed were also studied with CCK-8 receptors added.
RESULTS： In tissue sections, intensely CCKAR- immunoreactive interstitial cells were found mainly in the muscular layers. In cultured cell sections, distinctive double staining of C-kit and CCK-AR ICCs were found. When we removed the ICC of the gallbladder, CCK-8 induced muscle strip contraction dose response curve significantly shifted to the right.
CONCLUSION： We proved that both the existence of CCK-AR on the guinea pig gallbladder ICC and CCK evoked contraction are mediated through direct action on CCK-AR on the gallbladder ICC.     

Intrahepatic cholangiocarcinoma: new insights in pathology

Cholangiocarcinomas are malignant tumors that derive from cholangiocytes of small intrahepatic bile ducts or bile ductules (intrahepatic cholangiocarcinoma; ICC), or of large hilar or extrahepatic bile ducts (extrahepatic cholangiocarcinoma; ECC). ICC and ECC differ in morphology, pathogenesis, risk factors, treatment, and prognosis. This review focuses on ICC, which is rising in incidence with the emergence of hepatitis C virus (HCV) infection as a risk factor. The authors examined 73 ICC, which were resected at The Mount Sinai Medical Center in New York City, and reviewed the literature. The tumors were categorized into classical and nonclassical ICCs based on histopathology. Classical ICCs (54.8%) were characterized by a tubular, glandular, or nested pattern of growth, were significantly associated with tumor size of more than 5 cm and the absence of underlying liver disease and/or advanced fibrosis. Nonclassical ICCs (45.2%) consisted of tumors with trabecular architecture, tumors that exhibited features of extrahepatic carcinomas, and carcinomas considered to be derived from hepatic progenitor cells, i.e., combined hepatocellular/cholangiocarcinomas and cholangiolocellular carcinomas (ductular type of ICC). They were smaller and often arose in chronic liver disease, mostly HCV infection, and/or with significant fibrosis. The role of immunohistochemistry in the diagnosis of ICC and the importance of the new American Joint Committee on Cancer Staging System for ICC are also discussed.     

The role of overexpression and gene amplification of cyclin D1 in intrahepatic cholangiocarcinoma

BACKGROUND/AIMS: Intrahepatic cholangiocarcinoma (ICC) is a primary liver malignant tumor with an extremely poor prognosis, but less attention has been directed to factors related to molecular carcinogenesis, including cell cycle proteins. We examined the expression and gene amplification of cyclin D1, the cell cycle regulating protein. Our objective was to evaluate correlations with clinicopathological factors in ICC. METHODS: Cyclin D1 overexpression and cellular proliferative activity (Ki-67 labeling index) were investigated immunohistochemically, and 20 cases were further investigated for cyclin D1 gene amplification, using differential PCR. We examined the correlation between the expression and gene amplification of cyclin D1 and clinicopathological factors, including overall survival in patients with ICC. RESULTS: Immunohistochemical analysis revealed an overexpression of cyclin D1 protein in 28 of 66 subjects with ICCs (42%). The cyclin D1 overexpression was associated with poor histological differentiation (P = 0.04), high cellular proliferative activity (P < 0.01), and a poor prognosis (P = 0.02) by univariate analysis, although it is not an independent prognostic factor by multivariate analysis. Cyclin D1 gene amplification was confirmed in five of the 20 patients. Of those five cases of ICC, all had poor histological differentiation, and four of the five ICCs (80%) showed evidence of cyclin D1 immunoreactivity. CONCLUSIONS: Overexpression and gene amplification of cyclin D1 are frequent and contribute to dedifferentiation and cellular proliferative activity of ICCs, and overexpression also indicates a poor prognosis for patients with ICC.     

Homing of the bone marrow-derived interstitial cells of Cajal is decreased in diabetic mouse intestine

Background: Interstitial cells of Cajal (ICCs), which express c‐Kit receptor tyrosine kinase (KIT), play an important role in gastrointestinal motility. Loss of ICCs likely contributes to diabetic gastrointestinal motility disorder, however, the mechanism of attrition remains unknown. Here, we test the hypothesis that the bone marrow‐derived progenitors are an important source of intestinal ICCs and that decreased homing of these progenitors in diabetes contributes to ICC diminution. Methods: Wild type mice were X‐ray irradiated, transplanted with bone marrow (BMT) from green fluorescence protein (GFP)‐transgenic (TG)‐mice and subsequently made diabetic by streptozotocin (STZ) injection. Intestinal homing of GFP‐positive bone marrow‐derived cells was examined 2 or 5 months after STZ treatment. Results: In the BMT‐mice, we found many GFP‐positive bone marrow‐derived cells (BMDCs) in most parts of the intestinal area, the number of BMDCs was significantly decreased in diabetic mice compared with nondiabetic controls. As a representative area, we further examined the myenteric plexus of the proximal small intestine, and found that the cell numbers of ICCs marked by c‐Kit‐positive immunoreactivity were decreased by more than 40% in diabetic versus nondiabetic mice. Furthermore, numbers of c‐Kit+/GFP+ and c‐Kit+/GFP‐ cells were similar in nondiabetic mice, and decreased by 45.8% and 42.0%, respectively, in diabetic mice. Conclusion: These results suggest that the decreased homing from the bone marrow is a major cause of ICC loss in the intestine in diabetes mellitus.     

Stem Cell Factor/Kit Signal Insufficiency Contributes to Hypoxia-Induced Intestinal Motility Dysfunctions in Neonatal Mice

Background

Gastrointestinal (GI) motility disorders represent a group of problems that more constantly encountered in preterm infants. However, whether hypoxia exposure contributes to the GI dysfunctions is still unclear.

Methods

Newborn mice were exposed to hypoxia (10%) from P1 to P7. Intestinal motilities were examined by a strain gauge transducer. The proliferation of ICCs was detected by using immunostaining for BrdU, Ki67, Kit, Ano1, and insulin-like growth factor 1 receptor (IGF-1R+). Smooth muscle cells and enteric neurons were revealed by immunostaining for α-SMA and NF200, respectively. Apoptosis was assessed by TUNEL assay. Kit signal pathway was examined by western blot and qPCR.

Results

Intestinal motilities were found weakened significantly in the hypoxic small intestines as compared to controls on P8. Kit+ or Ano1+ interstitial cells of Cajal (ICCs) were found obviously decreased in the myenteric ICCs (ICC-MY) of neonatal mice after exposed to hypoxia. A large number of ICC progenitors (IGF-1R+) were found highly mitotic (BrdU+ Ki67+) to populate ICC during early postnatal development in the normoxic mice. We found the ICC proliferation was significantly inhibited upon hypoxia exposure, without increasing apoptosis (TUNEL+). We next identified that Kit phosphorylation was inhibited 3 days after hypoxia exposure. The inhibition of Kit signaling was largely due to decreased the expression of the ligand of Kit receptor, stem cell factor (SCF), in the intestinal walls. Exposure to imatinib, a Kit receptor inhibitor, for 3 days from P4 phenocopied the effect of hypoxia on the neonatal pups that resulted in inhibited intestinal motilities and decreased Kit+ ICC numbers.

Conclusion

All together, our findings indicate the SCF/Kit signaling insufficiency may contribute to the underdevelopment of ICCs and intestinal motility dysfunction upon hypoxia exposure. The decease in ICC density is likely due to the cell cycle arrest of ICC progenitor cells.

     

Interstitial cells of Cajal do not harbor c-kit or PDGFRA gene mutations in patients with sporadic gastrointestinal stromal tumors

Background  Gastrointestinal stromal tumors (GISTs) are believed to originate from the interstitial cells of Cajal (ICCs) or from the precursors of ICCs. Most GISTs show an activating mutation in either the c-kit or platelet-derived growth factor receptor α (PDGFRA) gene that results in a constitutive, ligand-independent activation of receptor tyrosine kinases. These gene mutations may play an important role in transforming a GIST progenitor cell into a tumor cell during the early phase of GIST tumorigenesis. However, the precise mechanism of the tumorigenesis is not known. Methods  We examined ten patients with sporadic GIST for mutations in the tumor and its adjacent ICC cells, and compared the mutational status of ICC cells with that of the GIST cells in each patient. All cases were screened for mutations in the c-kit gene (exons 9, 11, 13, and 17) and in the PDGFRA gene (exons 12 and 18). Samples were limited to GIST cases from the small intestine, where ICCs were present in bundles and were considered suitable for isolation by laser capture microdissection. Results  Of the ten tumors screened, eight had mutations in the c-kit gene (all in exon 11) and two were wild-type, whereas none of the ICCs exhibited mutations in these genes. Conclusions  Our results suggest that ICCs adjacent to overt GISTs did not have mutations in the c-kit or PDGFRA genes, and overt GISTs may develop after the local and sporadic acquisition of these gene mutations, together with additional events.