趋化因子SDF-1α及其受体介导涎腺腺样囊性癌细胞的趋化与侵袭

探讨趋化因子SDF-1α及其受体CXCR4对涎腺腺样囊性癌细胞趋化与侵袭活性的促进作用。采用RT-PCR法检测涎腺腺样囊性癌肺高、低转移细胞株ACC-M和ACC-2中CXCR4 mRNA的表达;Boyden趋化小室法检测在SDF-1α作用下ACC-M细胞和ACC-2细胞的趋化与侵袭活性;检测CXCR4 mAb对ACC-M细胞和ACC-2细胞趋化活性和侵袭活性的抑制作用。结果显示:2株涎腺腺样囊性癌细胞中均存在不同程度CXCR4 mRNA的表达,其在肺高转移细胞株ACC-M中的表达显著高于肺低转移细胞株ACC-2;SDF-1α对2种细胞均具有趋化活性和侵袭活性,且对ACC-M细胞的趋化活性和侵袭活性显著强于ACC-2细胞;CXCR4 mAb能够抑制涎腺腺样囊性癌细胞的这种趋化活性和侵袭活性。趋化因子SDF-1α及其受体CXCR4能够介导涎腺腺样囊性癌细胞的趋化与侵袭。     

miR-98在涎腺腺样囊性癌组织中的表达及其对ACC-M细胞增殖、迁移的影响

目的探讨miR-98在涎腺腺样囊性癌组织中的表达及其对腺样囊性癌细胞系ACC-M增殖以及迁移能力的影响。方法通过实时PCR检测5例涎腺腺样囊性涎组织及癌旁正常组织miR-98的表达水平;将miR-98模拟物瞬时转入高转移腺样囊性癌细胞系(ACC-M)使miR-98过表达,并通过实时PCR方法验证;流式细胞仪分析转染后ACC-M细胞周期的改变,甲基噻唑基四唑比色法检测转染后ACC-M细胞的增殖,划痕实验检测转染后ACC-M细胞的迁移能力,免疫印迹法检测转染后ACC-M细胞细胞周期蛋白CyclinB与CDC2的表达变化。结果 miR-98在涎腺腺样囊性癌组织中的表达水平显著低于癌旁正常组织,P0.01。转染miR-98模拟物能够显著上调ACC-M细胞miR-98的表达水平,ACC-M细胞的S期比例明显下降,而G 0/G 1期的细胞明显增多,增殖受到明显抑制,迁移能力下降,显著降低ACC-M细胞CyclinB与CDC2的蛋白表达水平。结论过表达miR-98能够抑制涎腺腺样囊性癌细胞的增殖与迁移能力。     

miR-24-3p在涎腺腺样囊性癌组织中的表达及其对ACC-M细胞增殖、侵袭的影响

目的探讨miR-24-3p在涎腺腺样囊性癌组织中的表达及其对腺样囊性癌细胞系ACC-M增殖以及侵袭能力的影响。方法通过实时PCR检测5例涎腺腺样囊性涎组织及癌旁正常组织miR-24-3p的表达水平;将miR-24-3p模拟物瞬时转入高转移腺样囊性癌细胞系(ACC-M)使miR-24-3p过表达,并通过实时PCR方法验证;流式细胞仪分析转染后ACC-M细胞周期的改变,甲基噻唑基四唑比色法检测转染后ACC-M细胞的增殖,Transwell实验检测转染后ACC-M细胞的侵袭能力,蛋白质免疫印迹法检测转染后ACC-M细胞血小板源性生长因子受体B(PDGFRB)的表达变化。结果 miR-24-3p在涎腺腺样囊性癌组织中的表达水平显著低于癌旁正常组织。转染miR-24-3p mimics能够显著上调ACC-M细胞miR-24-3p的表达水平,ACC-M细胞的S期比例明显下降,而G_0/G_1期的细胞明显增多,增殖受到明显抑制,侵袭能力下降,显著降低ACC-M细胞PDGFRB的蛋白表达。结论 miR-24-3p在涎腺腺样囊性癌中低表达,过表达miR-24-3p能够抑制涎腺腺样囊性癌细胞的增殖与侵袭能力。     

力学刺激对腺样囊性癌细胞株侵袭力的影响

我们旨在观察周期性双轴力学刺激对腺样囊性癌高转移细胞株ACC-M,腺样囊性癌低转移细胞株ACC-2体外侵袭力以及MMP-9表达的影响,以探讨腺样囊性癌的侵袭机制。分别选取4000微应变和1000微应变加载,将ACC-M,ACC-2每天加载1次,加载时间分别为1、3和6h,共加载2d。以在相同培养小室中静态培养组作为对照。细胞在力学刺激后行MMP-9免疫荧光染色,用激光共聚焦显微镜和图像分析软件进行定量分析。用Transwell小室行ACC-M,ACC-2细胞体外侵袭力测定。结果表明:MMP-9在ACC-M细胞表达明显高于ACC-2细胞,提示MMP-9与ACC的侵袭有密切关系。在力学刺激下,ACC-M和ACC-2细胞的MMP-9的表达及侵袭能力随力学刺激的大小和作用时间的变化而变化。ACC-M,ACC-2细胞在力学刺激后出现侵袭能力的改变,同时还可改变其基质金属蛋白酶-9的表达,显示力学刺激可以调节肿瘤转移,我们认为有基质金属蛋白酶-9之外的蛋白在其中起作用。     

限制片段差异显示PCR分析金属基质蛋白酶家族在乳腺癌表达的差异

目的：建立乳腺癌差异表达谱,以筛选乳腺癌相关候选基因;比较金属基质蛋白酶家族(matrix metalloproteinases,MMPs)在乳腺癌中的表达差异。方法：采集乳腺癌组织、转移淋巴结及正常乳腺组织,用限制片段差异显示PCR技术(Restriction fragments differential display PCR,RFDD-PCR)建立表达谱。以电泳和荧光扫描成像对表达谱片段进行分离、显示。筛选分析MMPs及其相关基因的表达差异。结果：共获得5407个基因片段,差异片段占30％以上;共显示13个MMPs基因及其相关基因金属蛋白酶组织抑制因子和转化生长因子β。其中除MMP20外,其余均有量或质的差异。结论：有多个MMPs基因参与了乳腺癌的发生、发展过程,其中MMP2、MMP10、MMP11、MMP14、MMP15、MMP28基因的开启可能为肿瘤进程的早期事件。     

涎腺腺样囊性癌中Cyclin D1、MMP-7和PRB2/p130表达及意义

目的探讨Cyclin D1、MMP-7和PRB2/p130蛋白在涎腺腺样囊性癌组织中的表达及与涎腺腺样囊性癌生物学行为的关系。方法应用免疫组化SP法检测46例涎腺腺样囊性癌和30例正常涎腺组织中Cyclin D1、MMP-7和PRB2/p130蛋白的表达。结果 Cyclin D1、MMP-7和PRB2/p130在涎腺腺样囊性癌中的阳性率分别为71.74%、67.39%、63.04%;Cyclin D1和PRB2/p130蛋白在涎腺腺样囊性癌中的表达与病理类型有关(P<0.05),MMP-7在涎腺腺样囊性癌中的表达与病理类型无关(P>0.05);Cyclin D1、MMP-7和PRB2/p130蛋白在涎腺腺样囊性癌复发组中的阳性率与无复发组相比,差异有显著性(P<0.05);Cyclin D1与PRB2/p130在涎腺腺样囊性癌中的表达呈负相关,与MMP-7表达呈正相关。结论 Cyclin D1、MMP-7和PRB2/p130蛋白的异常表达在涎腺腺样囊性癌发生、发展和复发中起重要作用,联合检测多个指标可能对患者预后的判断有指导意义。     

特异性下调葡萄糖调节蛋白78降低人涎腺腺样囊性癌侵袭和转移的能力

目的 探讨特异性下调葡萄糖调节蛋白78（GRP78）对涎腺腺样囊性癌（ACC）侵袭和转移能力的影响; 方法 应用siRNA干扰技术,特异性下调ACC-2细胞内GRP78的表达,通过划痕实验、Transwell实验、四甲基偶氮唑盐（MTT）实验和细胞骨架染色,观察下调GRP78表达后细胞的变化,并应用免疫印迹法检测下调GRP78表达对基质金属蛋白酶（MMP）-2、MMP-9、基质金属蛋白酶抑制因子(TIMP）-1和TIMP-2的影响。 结果 划痕实验显示,下调GRP78的表达能够抑制划痕愈合;Transwell实验发现,下调GRP78在ACC-2的表达明显降低穿过的细胞数;下调GRP78表达后细胞骨架染色发现,骨架纤维明显增粗;MTT实验显示,下调GRP78后细胞的增殖能力降低1/3;免疫印迹法检测结果发现,下调GRP78表达后ACC-2细胞的MMP-2、MMP-9的表达降低,TIMP-1、TIMP-2的表达增高。 结论 特异性下调GRP78的表达能够降低ACC的侵袭和转移能力,这与GRP78参与调节MMP和TIMP的表达有关。     

限制片段差异显示聚合酶链反应建立人类疾病表达谱的研究

目的 用限制片段差异显示聚合酶链反应(restriclion fragment differential display-polymerase chain reaction，RFDD-PCR)技术建立基因表达谱，分析比较人类疾病表达谱差异的可行性。方法 采集乳腺癌根治术患者的乳腺癌组织、乳腺癌转移淋巴结及正常乳腺组织，用RFDD-PCR技术平行操作，获得3种组织全部转录物组片段。然后以7％尿素变性聚丙烯酰胺凝胶电泳进行表达差异基因片段的分离、显示，结合http：//www．Qbiogene．com／display／数据库资料，进行生物信息学分析，初步筛选各组织问有表达差异的基因。结果 建立了乳腺癌组织、乳腺癌转移淋巴结及正常乳腺组织的基因表达谱，获得基因片段5407个，其中在癌组织和正常组织问有表达差异的片段为1491个，癌组织与转移淋巴结问的差异片段有1176个，而转移淋巴结与正常组织问的差异片段为1462个，分别占表达数的40．9％，33．9％，39．6％。这些差异片段涉及细胞增殖分化，信号转导，炎性反应，肿瘤转移等多方面功能。结论RFDD-PCR技术可以检测出大量表达基因，并同时比较3种或3种以上样本的表达差异，适用于人类疾病差异表达谱分析，筛选出疾病的候选基因。同时，还有可能找到新基因或新表达序列标签。     

涎腺腺样囊性癌66例临床分析

目的探讨涎腺腺样囊性癌部位、分期、治疗方法与复发及转移关系。方法对66例涎腺腺样囊性癌患者的病历资料作临床分析。结果腮腺23例,舌下腺14例,颌下腺6例,腭部12例,颊部8例,舌根3例。治疗方法,腮腺腺腺样囊性癌行全腮腺摘除13例,腮腺浅叶+部分深叶摘除9例,腮腺浅叶摘除1例;保留面神经18例,面神经切除5例;行肩胛舌骨上淋巴清扫15例。颌下腺、舌下腺、颊部、舌根腺样囊性癌均行腺体及局部扩大切除+同侧肩胛舌骨上淋巴清扫术;腭部腺腺样囊性癌行局部扩大切除+同侧肩胛舌骨上淋巴清扫术6例,单纯行局部扩大切除2例;术后放疗60例,同时行术后放化疗3例。术后复查3~10年,局部复发8例,远处转移9例。临床分期,Ⅰ期10例、Ⅱ42期例、Ⅲ期8例、Ⅳ期6例。结论颌下腺、舌下腺、颊部、舌根、腭部腺样囊性癌应同期行局部扩大切除+同侧肩胛舌骨上淋巴清扫术;腮腺腺腺样囊性癌如无神经受损症状及术中肿物与神经无明显粘连可保留面神经,术前检查如淋巴结无明显肿大可不作颈淋巴清扫术。术后均应行放疗。术后化疗效果如何本组病例不明确。Ⅲ、Ⅳ期较Ⅰ、Ⅱ期复发、转移为高。     

生物治疗涎腺腺样囊性癌的研究进展

涎腺腺样囊性癌是目前最为常见的涎腺恶性肿瘤之一,该肿瘤具有显著的两大生物学特性：嗜神经侵袭性和肺高转移性,而这两大生物学特性也直接影响着涎腺腺样囊性癌的治疗和预后.该肿瘤对手术、放射治疗及化学药物治疗效果均不十分理想,因此,生物治疗引起了国内外学者的广泛关注研究,并取得了一定的成果.     

SIKVAV, a laminin alpha1-derived peptide, interacts with integrins and increases protease activity of a human salivary gland adenoid cystic carcinoma cell line through the ERK 1/2 signaling pathway

Adenoid cystic carcinoma is a frequently occurring malignant salivary gland neoplasm. We studied the induction of protease activity by the laminin-derived peptide, SIKVAV, in cells (CAC2) derived from this neoplasm. Laminin alpha1 and matrix metalloproteinases (MMPs) 2 and 9 were immunolocalized in adenoid cystic carcinoma cells in vivo and in vitro. CAC2 cells cultured on SIKVAV showed a dose-dependent increase of MMP9 as detected by zymography and colocalization of alpha3 and alpha6 integrins. Small interfering RNA (siRNA) knockdown of integrin expression in CAC2 cells resulted in decreased adhesion to the peptide. SIKVAV affinity chromatography and immunoblot analysis showed that alpha3, alpha6, and beta1 integrins were eluted from the SIKVAV column, which was confirmed by mass spectrometry and a solid-phase binding assay. Small interfering RNA experiments also showed that these integrins, through extracellular signal-regulated kinase (ERK) 1/2 signaling, regulate MMP secretion induced by SIKVAV in CAC2 cells. We propose that SIKVAV increases protease activity of a human salivary gland adenoid cystic carcinoma cell line through alpha3beta1 and alpha6beta1 integrins and the ERK 1/2 signaling pathway.     

Metastatic and non-metastatic colorectal cancer (CRC) cells induce host metalloproteinase production in vivo

Numerous studies have demonstrated the persistent localization of matrix metalloproteinase (MMP) expression to the interface between invading human colorectal cancer (CRC) cells and surrounding stroma supporting a role for MMPs in CRC invasion and metastasis. The present study sought to determine whether CRC cells of varying metastatic potential would have differential effects on host MMP release. Subcutaneous CRC tumors were generated in BALB/c nude mice using three CRC cell lines: SW480, SW620, and the highly metastatic SW620S5 clone. Representative samples from the subcutaneous CRC were then orthotopically implanted on the cecum of recipient nude mice. Subcutaneous and cecal tumors were analyzed for MMP expression via zymography, western blot, and RT-PCR. In vitro, none of the three cell lines expressed MMP-2 nor MMP-9. In contradistinction, the subcutaneous tumors expressed limited amounts of MMP-2 and MMP-9 while the cecal tumors expressed significant amounts of MMP-2 and MMP-9 as well as other smaller members of the MMP family. MMP-9 mRNA and protein was confirmed as host in origin by RT-PCR with mouse specific primers and a mouse MMP-9 molecular weight of 105 kDa as determined by zymography and western blot analysis. In situ hybridization also localized the mRNA for MMP-9 to the host stromal cells. In conclusion, CRC cells appear incapable of producing MMP-2 and MMP-9 in vitro but are capable of up-regulating host MMP production in vivo. Enhanced host MMP-9 production in metastatic CRC cell-derived subcutaneous and cecal tumors suggests that metastatic colon cells may acquire the expression of important MMP regulating factor(s) in vivo.     

Dedifferentiated adenoid cystic carcinoma: a clinicopathologic study of 6 cases.

Dedifferentiated adenoid cystic carcinomas are a recently defined, rare variant of adenoid cystic carcinomas characterized histologically by two components: conventional low-grade adenoid cystic carcinoma and high-grade "dedifferentiated" carcinoma. We examined six cases and analyzed their clinicopathologic profiles, including immunohistochemical features and p53 gene alterations. The 6 patients (3 men and 3 women) had a mean age of 46.8 years (range, 34-70 y). The mean size of the tumors was 3.5 cm (range, 1.7-6 cm). The submandibular gland, maxillary sinus, and nasal cavity were involved in 2 cases each. Postoperatively, 5 patients had local recurrence and 5 developed metastatic disease. Five patients died of disease at a mean of 33.7 months after diagnosis (range, 6-69 mo), and one other was alive with disease at 60 months. Histologically, the conventional low-grade adenoid cystic carcinoma component of the tumors consisted of a mixture of cribriform and tubular patterns with scant solid areas. The high-grade dedifferentiated carcinoma component was either a poorly differentiated adenocarcinoma (4 cases) or undifferentiated carcinoma (2 cases). Three tumors were studied immunohistochemically. Myoepithelial markers were expressed in low-grade adenoid cystic carcinoma but not in the dedifferentiated component. In 2 cases, diffusely positive p53 immunoreactivity together with HER-2/neu overexpression was restricted to the dedifferentiated component. Loss of pRb expression was demonstrated only in the dedifferentiated component of the 1 other case. The Ki-67-labeling index was higher in the dedifferentiated component than in the low-grade adenoid cystic carcinoma component. Furthermore, molecular analysis of 2 cases demonstrated the loss of heterozygosity at p53 microsatellite loci, accompanied by p53 gene point mutation, only in the dedifferentiated carcinoma component of 1 case, which was positive for p53 immunostaining. These results indicate that dedifferentiated adenoid cystic carcinoma is a highly aggressive tumor. Because of frequent recurrence and metastasis, the clinical course is short, similar to that of adenoid cystic carcinomas with a predominant solid growth pattern. Limited evidence suggests that p53 abnormalities in combination with HER-2/neu overexpression or loss of pRb expression may have a role in dedifferentiation of adenoid cystic carcinoma.