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。     

人涎腺腺样囊性癌神经侵袭动物模型的建立

目的　建立人涎腺腺样囊性癌神经侵袭的动物模型 ,为研究腺样囊性癌神经侵袭的发生、发展提供实验依据和条件。　方法　将人涎腺腺样囊性癌细胞肺高转移株ACC M细胞注射入裸鼠眶下区皮下 ,分别于注射后7d、11d、14d、2 1d、2 8d、动物自行死亡或 5 6d时用HE染色方法观察肿瘤生长和神经侵袭的情况 ,并测量眶下神经管内神经侵袭的长度。结果　 85 7%的荷瘤裸鼠发生眶下管内神经侵袭。 7d、11d、14d、2 1d、2 8d和自行死亡组神经侵袭长度分别为 0 0 9mm、(0 2 7± 0 0 2 )mm、(0 38± 0 0 5 )mm、(0 6 8± 0 18)mm、(1 0 7± 0 36 )mm和 (2 1± 0 77)mm。结论　该模型是研究腺样囊性癌神经侵袭的良好模型 ,11～ 2 1d作为观察时限为宜 ,尤以 14d最佳     

神经营养因子-3在涎腺腺样囊性癌中的表达及其意义

目的 :通过检测神经营养因子 -3 (NT -3 )在涎腺腺样囊性癌 (ACC)的表达情况 ,探讨NT -3与ACC嗜神经侵袭的关系。方法 :以 3 2例ACC、7例正常腮腺及 3例腺泡细胞癌标本为研究对象 ,采用免疫组化法对NT -3进行检测。结果 :NT -3在正常腮腺的导管细胞表达为阳性 ;在腺泡细胞癌为阴性 ;在ACC病例中的阳性率为 93 .8% ( 3 0 /3 2 )。按病理学表现分组时 ,存在嗜神经现象组的NT -3表达水平明显高于未见嗜神经现象组 (P <0 .0 5 )。存在嗜神经现象组的大部分切片中神经束普遍较相应的正常腮腺组织或腺泡细胞癌密集 ,且有 4例ACC可见沿神经周分布的肿瘤细胞的染色强度明显高于远离神经者。结论 :NT -3可能作为ACC嗜神经侵袭的生物学标志。ACC中NT -3表达的增强可能是促进其嗜神经侵袭的原因之一     

神经生长因子、血管内皮生长因子在腺样囊性癌侵袭转移中的作用

目的:观察神经生长因子(nerve growth factor, NGF)、血管内皮生长因子(vascular endothelial growth factor, VEGF)在腺样囊性癌(adenoid cystic carcinoma, ACC)组织中的表达,探讨NGF和VEGF在ACC侵袭转移机制中的作用.方法:采用免疫组化SP法对NGF、VEGF在63 例ACC组织中的表达进行检测,并采用Cox比例风险回归模型对影响预后的多项临床因素进行分析.结果:NGF、VEGF在ACC组织中具有较高阳性表达,Cox模型提示NGF、VEGF、神经是否有侵袭、TNM分期、复发等是预后的危险因素,腺样-管状型的预后较实性型好.结论:ACC的预后与多因素相关,其中NGF、VEGF与ACC的侵袭转移有较大关系,可望作为判断预后的参考指标.     

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。     

腺样囊性癌嗜神经侵袭中神经生长因子、上皮钙黏素和钙结合蛋白A4的表达及意义

目的 探讨腺样囊性癌(adenoid cystic carcinoma,ACC)组织中神经生长因子(nerve growth factor,TGF)及上皮钙黏素(E-cadherin,E-cad)、钙结合蛋白A4(S100A4)的表达和临床意义,分析它们之间的相关性及其与ACC嗜神经侵袭(perineural invasion,PNI)现象的关系,进一步探讨NGF在PNI过程中对ACC细胞的黏附力和迁移运动能力的影响.方法 对42例ACC组织按年龄、性别、发病部位、临床分期及PNI和非PNI(NPNI)进行分组,42例中PNI组26例,NPNI组16例,应用免疫组化SP法检测其中NGF、E-cad和S100A4的表达与分布,分析三者与临床病理参数之间的关系,并对它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S100A4在PNI组的表达率分别为88%(23/26)和77%(20/26),较NPNI组(8/16和7/16)高,差异有统计学意义(P＜0.05),两者在PNI组的表达呈正相关(r=0.316,P＜0.05).PNI组中E-cad的表达为31%(8/26),较NPNI组(12/16)低(P＜0.05),与NGF的表达(r=-0.385,P＜0.05)和S100A4的表达呈明显负相关(r=-0.612,P=0.000).NGF在PNI组ACC神经周组织的表达[83%(25/30)]高于远离神经部位[47%(14/30)],差异有统计学意义(P＜0.05).结论 在ACC嗜神经侵袭过程中,NGF可能扮演着重要角色,但并非惟一因素,NGF可能通过和相应受体结合提高S100A4的表达率而降低E-cad 的表达和生物活性,使癌细胞间黏附力降低,运动能力增强进而促进ACC对神经组织的侵袭.     

Tip30基因修饰的ACC-M细胞裸鼠体内致瘤性观察

目的:观察肿瘤抑制基因Tip30修饰后的人涎腺腺样囊性癌高转移细胞(ACC鄄M)所致裸鼠移植瘤体内生长的变化。方法:BALB/Cnu/nu鼠30只(4周龄,体重18￣20g),随机分3组,各10只;Tip30基因修饰的ACC鄄M细胞(AdTIP30/ACC鄄M)、野生型ACC鄄M细胞(wt/ACC鄄M)及对照基因LacZ修饰的ACC鄄M细胞(AdLacZ/ACC鄄M)分别注射小鼠颌下区皮下,观察肿瘤生长情况及小鼠存活时间。结果:(1)经TIP30基因修饰组肿瘤出现的时间比野生型对照组及基因对照组晚3d,且肿瘤生长缓慢(P<0.01);(2)存活时间显示,基因修饰组(53.0±3.3)d,有2只长期存活;野生型对照组(26±2.2)d;对照基因组(29±2.4)d。结论:抑癌基因Tip30能够显著抑制人涎腺腺样囊性癌细胞在裸鼠体内的生长,延长荷瘤小鼠存活期。     

雪旺细胞标记物Leu-7(HNK-1)在涎腺腺样囊性癌嗜神经侵袭中的意义

目的检测Leu-7在涎腺腺样囊性癌(ACC)的表达情况,探讨Leu-7与ACC嗜神经侵袭的关系.方法采用免疫组织化学方法对28 例ACC,3例正常腮腺,3例正常颌下腺,3例正常舌下腺,5例肌上皮瘤及5例涎腺腺泡细胞癌标本组织切片中的Leu-7进行检测.结果 28例ACC中的嗜神经侵袭率为85.6%(24/28),Leu-7阳性率为96.4%(27/28), Leu-7的表达在正常大涎腺的导管细胞及其周围肌上皮细胞为阳性,在肌上皮瘤的阳性率为80.0%(4/5),而在腺泡细胞癌内均为阴性.结论腺样囊性癌中肿瘤性肌上皮细胞发生雪旺细胞分化进而侵袭神经可能是腺样囊性癌嗜神经侵袭的病理组织学基础.     

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。     

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。     

不同部位腺样囊性癌嗜神经侵袭动物模型的建立

目的：建立人涎腺腺样囊性癌嗜神经侵袭的动物模型,为研究腺样囊性癌在不同部位嗜神经侵袭的发生发展提供实验依据和条件。方法：将人涎腺腺样囊性癌肺高转移癌Acc—M细胞系接种于裸鼠臀部肌肉内及面后部皮下,分别于接种后7d,10d,16d,20d,28d 36d及动物自行死亡时用HE染色观察坐骨神经及面神经受肿瘤侵袭的对比情况,并同时观察肺转移情况。结果：面神经组荷瘤率为82．1％,神经侵袭率为17、9％,．未发现功能障碍：而坐骨神经组荷瘤率为71、4％,神经侵袭率为53．6％,出现功能障碍率10．7％,均未见肺转移。结论：该模型是研究人涎腺腺样囊性癌嗜神经侵袭的良好模型,为研究嗜神经侵袭机制及探索新的治疗途径提供了一个极为有用的工具。     

人涎腺腺样囊性癌嗜神经侵袭的动物实验研究

目的 探讨人涎腺腺样囊性癌对神经的侵袭情况.方法 将30只裸鼠平均分为三组,采用Acc-M,Tca-8113及Eca-109三种细胞系分别接种于10只裸鼠的坐骨神经附近,28天后同时处理所有被接种的裸鼠,以对比这三种细胞系成瘤后对神经的侵袭情况.结果 Acc-M组有90%发生神经侵袭,远远高于Tca-8113组15%及Eca-109组10%(P＜0.001).结论 腺样囊性癌嗜神经侵袭是其特征性生物学行为.     

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。     

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.     

Immunohistochemical detection of nerve growth factor and its receptors in the rat periodontal ligament during tooth movement

Objectives

Nerve growth factor (NGF) and its receptors, p75 and tyrosine receptor kinase A (Trk A), have been shown to increase following trauma. The aims of this study were to examine changes in the detection of NGF and its receptors during orthodontic tooth movement in the rat, and the effects of anti-NGF on these changes.

Design

Orthodontic separators were placed between the right maxillary first and second molars of Sprague-Dawley rats which were equally divided into two groups. Animals from the second group were injected with anti-NGF. The left sides served as controls, and animals were sacrificed at 0, 3, 7 and 14 days.

Results

Results of immunohistochemical localisation for p75, Trk A, calcitonin gene-related peptide (CGRP) and NGF showed staining intensity increased at day 3, with a peak at day 7 and decreasing intensity at day 14. Anti-NGF injected animals showed reduced staining at all observation periods.

Conclusion

Data suggest that orthodontic injury induces NGF production, leading to sprouting and invasion by CGRP-positive nerve fibers and that injection of anti-NGF reduces NGF tissue levels and prevents innervation by CGRP-positive fibers.     

Neural communication in the trigeminal ganglion contributes to ectopic orofacial pain

Ectopic orofacial pain develops with local inflammation in remote orofacial structures. However, the mechanism underlying the spreading of pain to remote orofacial areas after local inflammation is still unknown. We investigated the functional significance of neural communication within the trigeminal ganglion (TG) via nerve growth factor (NGF) or calcitonin gene-related peptide (CGRP) in relation to whisker pad skin hyperalgesia following complete Freund's adjuvant (CFA) injection into the lower lip. Heat hyperalgesia and mechanical allodynia were induced in the ipsilateral whisker pad skin following lower lip inflammation, and reversed by local injection of transient receptor potential vanilloid 1 (TRPV1) or P2X₃ receptor (P2X₃R) antagonist, respectively. The heat hyperalgesia was diminished by neutralizing anti-NGF antibody administration into the lower lip and intraganglionic administration of tyrosine kinase receptor inhibitor. TG neurons that innervate the whisker pad skin and lower lip both expressed labeled NGF, which was administrated into the lower lip. Moreover, NGF and CGRP expression in the TG was increased following lower lip inflammation. The number of TRPV1-positive neurons that innervate the whisker pad skin was increased following lower lip inflammation and this increase was annulled by anti-NGF administration. P2X₃R- and CGRP-positive TG neurons innervating whisker pad skin were also increased by lower lip inflammation. The present findings suggest that induced NGF and CGRP following local inflammation in the lower lip increases TRPV1 and P2X₃R in TG neurons, which may result in the development of the whisker pad ectopic pain.     

染料木黄酮抗涎腺腺样囊性癌远处转移的实验研究

目的　研究染料木黄酮抗涎腺腺样囊性癌 (ACC)实验性肺转移的作用。方法　分别用ACC M细胞及经染料木黄酮处理的ACC M细胞对 2 0只裸鼠尾静脉接种形成肺转移模型 ,6周后处死动物 ,比较治疗组与对照组裸鼠ACC肺转移率、瘤结节数目 ;观察肺转移灶血管内皮生长因子 (VEGF)、基质金属蛋白酶 (MMP 9)的表达及凋亡情况。结果　染料木黄酮治疗组瘤结节数目明显少于对照组(分别为 9 2 9± 1 80和 2 7 4 4± 13 5 5 ,P <0 0 5 ) ;治疗组转移灶凋亡指数 (AI)明显高于对照组 ( 15 37±3 96及 6 0 3± 3 36 ,P <0 0 5 ) ;治疗组VEGF及MMP 9表达明显弱于对照组 (P <0 0 5 )。结论　染料木黄酮有一定的抗ACC M远处转移的作用 ,这可能是多种机制作用的结果