涎腺腺样囊性癌细胞株p16基因缺失、突变及表达意义

目的　研究 p16基因在涎腺腺样囊性癌细胞株中的缺失和突变等结构变化及其表达之间的关系。方法　应用聚合酶链反应 (PCR)和 PCR-单链构象多态性 (SSCP)对涎腺腺样囊性癌细胞株 ACC- 2和高转移细胞克隆 ACC- M进行 p16基因缺失、突变的检测 ,应用免疫组化方法检测 p16基因在细胞株中的蛋白表达。结果　涎腺腺样囊性癌细胞株 ACC- 2检测 p16基因阳性 ,高转移细胞克隆 ACC- M缺失 ,两个细胞克隆均无点突变 ,ACC- 2的p16蛋白表达阳性 ,而 ACC- M蛋白表达阴性。结论　 p16基因在高转移涎腺腺样囊性癌克隆中的缺失 ,表明 p16基因在涎腺腺样囊性癌的演进和转移中具有抑癌作用。     

涎腺腺样囊性癌 Acc-2和 Acc-3细胞系的建立及其形态学观察

本文报道了小涎腺源性腺样囊性癌 Acc-2细胞系和大涎腺(腮腺)源性腺样囊性癌 Acc-3细胞系的建立经过。作者着重对两株细胞系细胞的形态学用普通光镜、相差显微镜、微分干涉相差显微镜、扫描电镜、透射电镜、组织化学和 Keratin 免疫荧光染色等方法作了较全面的观察。作者根据观察结果认为涎腺腺样囊性癌起源于一种多能干细胞,而肌上皮细胞是一种分化的表现,癌细胞经体外培养后,在分化程度低下时,可不出现肌上皮细胞。对此本文作了较详细的讨论。Acc-2和 Acc-3细胞系的建立,为进一步研究涎腺腺样囊性癌的组织发生及其他各种特性提供了良好的模型。     

涎腺腺样囊性癌组织中神经生长因子及其受体的表达

目的　研究涎腺腺样囊性癌 (adenoidcysticcarcinoma ,ACC)组织中NGF及其受体 (p75 ,TrKA)的表达 ,旨在探讨调节ACC浸润生长及分化的机理。方法　应用免疫组织化学方法检测了 42例ACC标本及周围正常腺体的NGF、p75与TrKA的表达 ,并对不同病理类型ACC中NGF及其受体的表达进行统计学分析。结果　NGF及 p75在筛状型ACC和管状型ACC中的表达明显高于在实性型ACC的表达 ,差异有高度统计学意义 ,而筛状型ACC与管状型ACC中NGF及 p75的表达差异无统计学意义 ;TrKA在筛状型ACC和管状型ACC中的表达也高于其在实性型ACC中的表达 ,差异有统计学意义 ,筛状型ACC与管状型ACC间TrKA表达无显著差异。NGF在正常涎腺闰管细胞 ,排泄管上皮细胞均有高效表达。结论　①ACC可能通过自泌和旁泌NGF机制调节分化 ;②ACC组织中p75和NGF间相互作用可能是ACC高浸润性的生物学基础 ,同时可能也是ACC嗜神经生长机理。     

神经生长因子对腺样囊性癌细胞系增殖与凋亡的影响

目的:研究神经生长因子(nerve growth factor,NGF)对腺样囊性癌细胞系增殖和凋亡的影响,为进一步揭示腺样囊性癌嗜神经侵袭以及沿神经播散的机理提供理论依据。方法:将人腺样囊性癌细胞系(ACC-2)和作为对照的人舌癌细胞系(Tca-8113)分别与不同浓度的NGF共培养24、48和72h。用MTT法及Annexin V-FITC/PI双标记流式细胞术,研究不同浓度人重组神经生长因子β(hβNGF),对人腺样囊性癌细胞系ACC-2及对照组舌癌细胞系Tca-8113增殖与凋亡的影响。结果:40 ng/mL和80 ng/mL的NGF对ACC-2细胞作用48 h和72 h后,ACC-2细胞的增殖水平明显高于其他各组,而NGF对ACC-2细胞的凋亡则没有影响;NGF对Tca-8113的增殖和凋亡均没有作用。结论:NGF作用于腺样囊性癌细胞,能有效促进腺样囊性癌细胞的增殖,这可能是腺样囊性癌嗜神经侵袭和沿神经播散的理论基础。     

NGF及其受体Trk-A与唾液腺腺样囊性癌相关性研究

目的:通过检测神经生长因子(nerve growth factor,NGF)及其受体Trk-A在唾液腺腺样囊性癌(adenoid cysticcarcinoma,ACC)中的表达,研究NGF及其受体Trk-A在腺样囊性癌嗜神经性侵袭中的相互关系。方法:采用Envision免疫组织化学二步法,完成病理切片,作半定量病理分析。结果:NGF及其受体Trk-A在腺样囊性癌不同病理类型中阳性表达率有高度统计学差异(P≤0.01),在神经侵袭组明显高于无侵袭组,但在腺样囊性癌对周围组织的浸润和转移方面没有统计学差异(P>0.05)。结论:NGF与其受体Trk-A可能对腺样囊性癌分化起调节功能,二者相互作用可能是ACC强浸润性的生物学机制。     

细胞外基质与涎腺腺样囊性癌的远处转移

目的 研究涎腺腺样囊性癌与细胞外基质的关系。方法 通过肿瘤手术标本的免疫组化染色和超微结构观察、细胞系体外粘附实验及人工重组基底膜侵袭实验，并采用整合素受体抑制剂精氨酸-天冬氨酸进行体外及荷瘤动物体内实验，观察其预防及治疗肺转移的效果。结果 肿瘤团索周围的基膜样物质呈多层、溶解或断裂现象。出现腺样囊性癌远处转移患者的肿瘤标本组织蛋白酶D免疫组化阳性反应率(75％)明显高于无转移患者(43．8％)。肺高转移涎腺腺样囊性癌细胞株对人工重组基膜的侵袭细胞数明显高于其相应的非高转移细胞系。精氨酸-天冬氨酸在体外能抑制肺高转移涎腺腺样囊性癌细胞对人工重组基膜的侵袭，在体内能显著延长涎腺腺样囊性癌实验性肺转移动物的生存期。结论 涎腺腺样囊性癌的远处转移与细胞外基质有密切关系。     

白血病抑制因子受体在人涎腺腺样囊性癌细胞系中的表达

目的 :探讨白血病抑制因子受体 (LIFR)与人涎腺腺样囊性癌转移的相关性。方法 :以涎腺腺样囊性癌细胞系ACC 2及其肺高转移株ACC M作为研究肿瘤转移分子机制的模型 ,应用RT PCR技术和免疫组化方法检测LIFR的表达。结果 :RT PCR检测表明LIFRmRNA在肺高转移细胞株ACC M细胞中的表达低于在ACC 2细胞中的表达 ,免疫组化检测表明LIFR蛋白在ACC M细胞中表达降低。结论 :LIFR在涎腺腺样囊性癌转移过程中可能发挥抑制作用。     

DNApolβ启动子和CMV启动子调控的p53基因在涎腺腺样囊性癌细胞中表达的比较

目的检测DNA polβ启动子在涎腺腺样囊性癌细胞中的活性，探讨DNA polβ启动子对外源性野生型p53基因表达的影响。方法荧光素酶法测定DNA polβ启动子在涎腺腺样囊性癌细胞中的活性。构建携带人野生型p53基因的真核表达载体，以脂质体法转染涎腺腺样囊性癌SACC- 83细胞，逆转录聚合酶链反应（RT- PCR）检测p53基因mRNA的表达。博莱霉素、H2O2及紫外线刺激转染细胞，采用RT- PCR及Western blot法比较DNA损伤下DNA polβ启动子和CMV启动子调控的p53基因及P53蛋白的表达。结果荧光素酶活性分析显示，涎腺腺样囊性癌细胞中DNApolβ启动子活性增高。p53基因的导入使其在涎腺腺样囊性癌细胞中表达增强，DNA polβ启动子组较CMV启动子组更为明显。DNA损伤后，DNA polβ启动子组的p53基因和P53蛋白的表达较CMV启动子组增强（P<0.05）。结论在涎腺腺样囊性癌SACC- 83细胞中，DNA polβ启动子的活性增高，DNA polβ启动子能够增强外源性野生型p53基因在涎腺腺样囊性癌细胞中的表达。     

p53基因抑制涎腺腺样囊性癌细胞端粒酶及增殖活性

目的研究外源性野生型p53基因对涎腺腺样囊性癌细胞的抑制作用。方法构建携带野生型p53基因的腺病毒表达载体，以脂质体法转染涎腺腺样囊性癌SACC-83细胞，RT-PCR检测p53基因表达；采用TRAP—PCR—ELISA法检测转染细胞端粒酶活性，荧光素酶分析法检测人端粒酶逆转录酶基因（hTERT）肩动子的转录；流式细胞术、软琼脂集落实验及裸鼠成瘤实验观察细胞生物特性的变化。结果外源性野生型p53基因导入使p53基因在涎腺腺样囊性癌细胞SACC-83中表达增强，其端粒酶活性降低、hTERT启动子转录抑制；转染细胞出现G1期阻滞，软琼脂集落形成率减少，裸鼠成瘤能力降低。结论腺病毒载体介导的外源性野生型p53基因可以抑制涎腺腺样囊性癌细胞端粒酶活性及细胞恶性表型。     

维生素K3对腺样囊性癌细胞株的生长抑制作用及其与As2O3的联合效果

目的:探讨维生素K3(VitK3)对腺样囊性癌细胞的凋亡诱导作用及其与As2O3的联合效果。方法:体外培养人腺样囊性癌细胞株(SACC-83),分别以VitK3、As2O3以及两者联合孵育细胞,采用四甲基偶氮唑蓝(MTT)法检测对腺样囊性癌细胞的抑制率,Annexin-V/Pl双染色流式细胞技术检测凋亡细胞的百分数,倒置显微镜及AO/EB染色观察细胞凋亡后的形态。采用SPSS 13.0软件包对数据进行方差分析和t检验。结果:体外实验表明,VitK3能抑制SACC-83细胞生长并呈剂量和时间依赖性,细胞死亡方式以凋亡为主。VitK3与As2O3联合应用时,具有协同作用。结论:VitK3能抑制腺样囊性癌细胞生长,并且与As2O3联合时有协同作用。     

Nerve growth factor beta/pro-nerve growth factor and their receptors in normal human oral mucosa

Nerve growth factor beta (NGF-beta) and its precursor proNGF are important for the differentiation and survival of neurons and dermal keratinocytes. The aim of this study was to determine the role that NGF might play in the differentiation and wound healing of oral mucosa. Cultured normal human oral mucosal keratinocytes expressed mRNA for NGF-beta/proNGF and for their receptors TrkA and p75(NTR). Lysates from cultured oral mucosal keratinocytes did not contain detectable amounts of mature 14-kDa NGF-beta but did contain several NGF proforms with molecular weights between 32 and 114 kDa. Culture medium from oral mucosal keratinocytes contained 75 kDa proNGF. The addition of NGF-beta significantly enhanced the proliferation of oral mucosal keratinocyte cultures and in vitro scratch closure. Immunostaining of biopsies from normal oral mucosa showed the presence of proNGF in all epithelial layers. NGF staining was observed in the granular and upper spinous cell layers. TrkA immunoreactivity was detected in basal and parabasal cells, with weak to moderate staining in spinous and granular cell layers. p75(NTR) staining was seen in basal cell layers. These findings indicate that NGF-beta/proNGF have mitogenic and motogenic effects on oral mucosal keratinocytes and therefore may aid in the healing of oral wounds. Differential expression of NGF and NGF receptors throughout the epithelium suggests a role in epithelial differentiation.     

Nerve growth factor and tyrosine kinase A in human salivary adenoid cystic carcinoma: expression patterns and effects on in vitro invasive behavior.

PURPOSE: Perineural invasion is a frequent occurrence in salivary adenoid cystic carcinoma (ACC) and may prevent complete surgical resection. Studies have indicated that nerve growth factor (NGF) and its high-affinity receptor tyrosine kinase A (TrkA) may play a role in perineural invasion in several malignancies in which perineural invasion is observed. The present study was conducted to investigate the expression of NGF and TrkA in salivary ACC and to examine the effects of NGF on adhesion, migration and invasion capacities of a salivary ACC cell line (SACC-83) in vitro. PATIENTS AND METHODS: Expression of NGF and TrkA was explored using immunohistochemistry in paraffin-embedded tissues of 32 cases of salivary ACC. The effects of NGF on in vitro adhesion, migration, and invasion capacities of the SACC-83 cell line were examined using an MTT assay and a modified Boyden chamber assay respectively. RESULTS: In ACC specimens, 31 (96.9%) and 32 (100%) tumors showed immunoreactivity for NGF and TrkA respectively. Significant correlations were found between NGF/TrkA expression levels and perineural invasion (P < .05). In cell adhesion assay, the percent adherences of SACC-83 cells co-cultured with 25 ng/ml NGF at 1.5 hours and 5, 25 ng/ml NGF at 6 hours were significantly higher than that co-cultured with 0 ng/ml NGF (P < .05). However, high concentration of NGF (500 ng/ml) resulted in a significant inhibition of invasion (P < .05). CONCLUSION: Overexpression of NGF and TrkA in human salivary ACC tissues may constitute a reason for perineural invasion in salivary ACC.     

Neurotrophins in cultured cells from periodontal tissues

We review the basic functions of neurotrophins and their receptors and discuss the expression and functions of neurotrophins and their specific receptors based on recent data using cultured cells from human periodontal tissues. Neurotrophins, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) play crucial roles in the differentiation and survival of neural cells. Neurotrophins activate 2 different receptor classes: the tropomyosin-related kinase (Trk) family of receptor tyrosine kinases (TrkA, TrkB, and TrkC) and the p75 receptor, a member of the tumor necrosis factor receptor superfamily. Neurotrophins regulate both cell death and cell survival through activations of Trk receptors and/or p75 neurotrophin receptor. It has been reported that neurotrophins are also produced from non-neuronal cells, such as leukocytes, osteoblasts, or fibroblasts, and act in many other ways on non-neuronal cells. Neurotrophin expression during bone fracture healing is especially interesting, and neurotrophins are now implicated in hard tissue regeneration. It is well known that neurotrophins and their receptors are expressed in tooth development. Recent studies have found that neurotrophins and Trk receptors are expressed in mouse osteoblastic cell lines. Human periodontal ligament cells, human gingival fibroblasts, and human gingival keratinocytes expressed mRNA for NGF and TrkA. The secretion of bioactive NGF peptides from human periodontal ligament cells and human gingival keratinocytes was confirmed by bioassay using PC12 cells (rat adrenal pheochromocytoma cells). The expression of NGF and TrkA.mRNA was regulated by interleukin (IL)-1beta. NGF increased DNA synthesis and expressions of mRNA for bone-related proteins, alkaline phosphatase, and osteopontin in human periodontal ligament cells. Neurotrophins and Trk receptors expressed in human periodontal tissue may contribute to regeneration as well as innervation of periodontal tissue through local autocrine and paracrine pathways. Recent data suggest that some functions of neurotrophins and Trk receptors relate to periodontal disease and periodontal tissue regeneration. However, in vivo studies will be required to clarify the roles of neurotrophins and their receptors, including p75, in periodontal disease and periodontal tissue regeneration.     

神经生长因子对腺样囊性癌细胞增殖的影响

目的:检测神经生长因子(nerve growth factor,NGF)在体外培养腺样囊性癌(adenoid cystic carcinoma,ACC)细胞中的表达,探讨NGF对ACC细胞增殖的作用。方法:运用蛋白质印迹分析技术(Western blotting)检测体外培养增殖期ACC细胞中NGF的表达。体外培养ACC细胞分为9组:对照组(细胞浓度分别为2×104个/mL、1×104个/mL、5×103个/mL、2.5×103个/mL,为第1～4组)、实验组(细胞浓度为2×104/mL,加入NGF抗体的浓度分别为100μg/mL、20μg/mL、4μg/mL和0.8μg/mL,为第5～8组)、空白对照组(纯培养液,为第9组),采用MTT法检测孵育72 h后,计数ACC细胞增殖的数量;方差分析比较各组细胞增殖数量的差异。结果:ACC细胞中表达NGF;100μg/mL、20μg/mL和4μg/mL浓度的NGF抗体可明显抑制ACC细胞增殖的数量,P<0.01;随着NGF抗体浓度的递减,代表细胞增殖数量的OD值逐渐增大;当NGF抗体的浓度减至0.8μg/mL时,与对照组相比,ACC细胞增殖数量有所减少,但差别不显著,P>0.05;4种NGF抗体浓度的实验组之间细胞增殖的差异不显著,P=0.187。结论:NGF参与ACC细胞的增殖;NGF抗体通过中和NGF以剂量依赖的方式抑制ACC细胞增殖,反证了NGF能以剂量依赖的方式促进ACC增殖。这可能是NGF促进ACC神经侵袭的机制之一。     

NGF and its receptors TrkA and p75NTR in the epithelium of oral lichen

Background:  Nerve growth factor (NGF) can through its receptors TrkA and p75^NTR convey signals for cell survival, differentiation and death. The aim of this study was to examine whether NGF can play a role in the pathology of oral lichen (OL).
Methods:  Sections from biopsies taken from patients with erythematous (ERY) OL and from volunteers with normal oral mucosa (NOM) were immunostained with antibodies against NGF, proNGF, TrkA, phosphorylated Trk, p75^NTR and phosphorylated Akt (pAkt) and expression of RNA coding for proNGF/NGF was investigated by in situ hybridization.
Results:  Both in ERY OL and NOM, cytoplasmic staining for NGF was seen in granular and upper spinous cell layers of the epithelium, whereas proNGF staining was seen in all epithelial cell layers. In situ hybridization showed that the proNGF protein was produced in the same cell layers. In OL, strong cytoplasmic stainings for TrkA and activated Trk (pTrk) were observed in all epithelial cell layers while these stainings were only weak in NOM. Basal keratinocytes in OL showed no or only weak cytoplasmic staining for p75^NTR, but in NOM there was a clear cell membrane staining. In OL, strong cytoplasmic and intermittent nuclear staining for pAkt was observed in spinous, granular and superficial layers, while basal and parabasal keratinocytes were negative. This staining was weak or absent in the entire epithelium of NOM.
Conclusions:  TrkA upregulation and activation in OL is one of the pathways that can activate pAkt and thereby rescue epithelial cells from untimely cell death.     

神经生长因子对人牙髓细胞增殖与分化作用的研究

目的 研究神经生长因子对体外培养的人牙髓细胞增殖和分化作用的影响。方法 组织块法行人牙髓细胞的原代培养后，采用四唑盐比色法和酶动力学方法，测定不同浓度（1、10、100U／mL）的神经生长因子对体外培养的第5-8代人牙髓细胞增殖及碱性磷酸酶活性的作用。结果 与对照组相比，10U／mL的神经生长因子可显著促进人牙髓细胞的增殖（P〈0．05）；100U／mL的神经生长因子可显著促进碱性磷酸酶的活性（P〈0．01）。结论 不同浓度的神经生长因子可显著促进人牙髓细胞的增殖与分化。     

Distribution of nerve growth factor,pro‐nerve growth factor,and their receptors in human salivary glands

Nerve growth factor (NGF) is a pluripotent mediator that is present in a range of human tissues. Nerve growth factor was originally considered important only in neuronal homeostasis and pathophysiology, but later it was also implicated in the pathophysiology of inflammation, epithelial differentiation, and wound healing. In this study, the distribution of nerve growth factor beta (NGF‐β) and pro‐NGF, and their receptors – tyrosine kinase A (TrkA) and p75^NTR – was examined in human parotid, submandibular, sublingual, and labial salivary glands by immunohistochemistry. Intercalated, striated, and collecting‐ducts in all gland types showed strong staining for pro‐NGF but only weak cytoplasmic or sparse nuclear staining for NGF‐β. Tyrosine kinase A was strongly expressed in the ducts of all gland types, whereas p75^NTR expression was mainly confined to collecting ducts. In acini, no or only weak cytoplasmic staining was found for all markers, and some nuclei stained positive for NGF‐β, pro‐NGF, and TrkA. Western blotting of saliva showed secretion of several forms of pro‐NGF, while no mature NGF‐β was detected. Salivary pro‐NGF may play a role in oral wound healing.     

神经生长因子及其受体在下颌骨骨折愈合中的表达及意义

目的:探讨神经生长因子(nerve growth factor,NGF)及其受体(p75和TrkA)在骨折愈合过程的表达及其对骨折愈合的影响。方法:将32只白兔随机分为8组(每组4只),4只无骨折作为健康对照组2,8只制作右下颌骨骨折模型,立即行钛板固定。术后实验组动物分别在1、3、5、7、14、21、28 d各取4只处死。切取下颌骨脱钙后制作骨组织切片,行HE染色、NGF及受体的免疫组化染色。结果:NGF在骨折愈合过程中表达阳性3,～5 d达到高峰,在未骨折组仅有轻度阳性表达。NGFR-TrkA在骨折愈合过程中表达强阳性7,d达到高峰,到21 d时软骨细胞强阳性。NGFR-p75在骨折愈合过程中表达阳性,5～7 d达到高峰,到21 d时仍为阳性表达。结论:在骨折愈合过程中NGF、p75和TrkA共同调节骨细胞活性,对骨的修复重建起到重要作用。     

NGF及其受体P75与肝素酶在口腔腺样囊性癌嗜神经侵袭中的表达及相关性分析

目的：探讨腺样囊性癌（adenoidcysticcarcinoma,ACC）组织中肝素酶（heparanase,HPA）和神经生长因子（nervegrowthfactor,NGF）及其受体P75的表达和相互作用,以及与ACC嗜神经侵袭之间的关系。方法：应用免疫组化SP法检测42例ACC组织中HPA、NGF和P？5的表达,并对他们在不同病理类型和组织学部位的表达进行统计学分析。结果：NGF和P75在嗜神经（PNI）组和非嗜神经（NPNI）组的表达有统计学差异（P〈0．05）,两者呈正相关（r=0．429,P〈0．05）。嗜神经组HPA和P75的表达正相关（r=0．558,P〈0．05）。P75在神经周组织和远离神经部位的表达有显著性差异（P〈0．05）,NGF在神经组织和远离神经处的表达无明显差异。结论：在ACC的神经浸润中,NGF及其受体P75扮演着重要角色,但并非唯一因素,NGF可以通过和受体p75结合可以提高HPA的表达率和生物活性．进而促进ACC对神经组织的浸润。