肺巨细胞癌高低转移株转移能力差异相关分子的研究

目的 鉴定在肺巨细胞癌高、低转移株差异表达的转移相关分子,探讨肺巨细胞癌的转移机制。方法 以肺巨细胞癌高、低转移株(即PLA901D、PLA801C)为转移模型,通过细胞体外迁移和侵袭实验,验证高、低转移株间细胞体外迁移和侵袭能力的差异。以明胶酶谱实验,分析肿瘤细胞分泌基质金属蛋白酶(MMP-2和MMP-9)的活性;以Western blot技术,检测肿瘤细胞分泌的MMP-2、MMP-9、组织基质金属蛋白酶抑制剂(TIMP-1和TIMP-2)的蛋白水平,以及p53、细胞增殖核抗原(PCNA)、p16、CD44(V6)异构体、细胞骨架蛋白(CKl8)、上皮细胞间黏附分子(E-cadherin)和肿瘤转移抑制蛋白(nm23-H1)的蛋白水平;以半定量RT-PCR技术,检测细胞内MMP-2、MMP-9、TIMP-1、TIMP-2和血管内皮生长因子(VEGF)的mRNA水平。结果 肺巨细胞癌高转移株PLA801D的细胞体外侵袭能力显著高于低转移株PLA801C,大约为后者的4倍。PLA801D细胞株分泌的MMP-2活性高于PLA801C细胞株,其原因主要是由于分泌的MMP-2蛋白以及细胞内MMP-2 mRNA水平显著增高所致。在PLA801D细胞株内,p53、PCNA、CKl8蛋白和VEGF mRNA为显著高表达,p16、E-cadherin和nm23-H1蛋白为显著低表达,而MMP-9、TIMP-1、TIMP-2和CD44(V6)蛋白的表达在高、低转移株间的差异无显著性。结论 肺巨细胞癌高、低转移株间存在转移能力和转移表型的显著差异,经鉴定的差异表达分子可能在促进或抑制肺巨细胞癌细胞的转移过程中发挥重要作用。     

高转移人肺巨细胞癌细胞67—KD层粘连蛋白受体基因表达

在逆转录ＰＣＲ特异扩增６７－ＫＤ层粘连蛋白受体（简称ＬＮ－Ｒ）ｃＤＮＡ片段并制备成探针的基础上，观察了高转移人肺巨细胞癌（简称ＰＧ）细胞和低转移人肺腺癌（简称ＰＡａ）细胞６７－ＫＤＬＮ－Ｒ ｍＲＮＡ表达水平。结果表明，两种细胞存在大小相同的特异转录体，约１．７ｋｂ左右；高转移ＰＧ瘤细胞比低转移ＰＡａ瘤细胞表达较高水平的６７－ＫＤＬＮ－Ｒ ｍＲＮＡ，同时ＰＧ瘤细胞存在明显的该基因扩增；不同剂量６７－     

不同转移潜能人大细胞肺癌细胞株转移相关microRNAs的筛选研究

背景与目的微小RNA(microRNAs,miRNAs)参与调节肿瘤发生发展的多个过程,包括细胞的分裂增殖、细胞周期、凋亡、血管形成、侵袭和转移等。本研究应用miRNA芯片检测具有高低不同转移潜能人大细胞肺癌细胞株L9981和NL9980的miRNA表达谱,从中筛选出与大细胞肺癌转移相关的miRNAs。方法收集L9981和NL9980细胞,抽提总RNA进行CY3标记,将标记RNA在miRNA芯片上进行杂交反应。通过数据统计分析,筛选出表达明显差异的miRNAs。应用Real-timePCR验证芯片结果,并应用生物信息学方法预测靶基因。结果在不同转移潜能人大细胞肺癌L9981和NL9980细胞株中共筛选到22个表达明显差异的miRNAs。与NL9980相比,在L9981中有13个miRNAs表达上调,9个表达下调。Real-timePCR验证miR-125a-3p在细胞中的表达水平与芯片结果趋势一致,预测其靶基因可能为胰岛素样生长因子2。结论筛选得到与大细胞肺癌转移相关的miRNA表达谱。     

乳腺癌转移相关microRNA-200c调控的基因网络分析

目的:结合生物信息学分析手段,筛选乳腺癌转移相关微RNA (microRNA,miRNA)-200c调控的基因网络.方法:采用Affymetrix(R) miRNA生物芯片分析12株乳腺细胞中差异表达的miRNA;应用脂质体转染法将miR-200c模拟物(mimic)转入4株高转移性的乳腺癌细胞株(BT549、HS578T、MDA-MB-231和SUM159PT)中,再用Affymetrix(R) mRNA生物芯片检测转染miR-200c mimic后高转移性乳腺癌细胞株中差异表达的基因.采用生物分子功能注释系统(CapitalBio Molecule Annotation System,MAS),筛选miR-200c调控的信号通路与基因网络.结果:12个乳腺细胞株中筛选出9个差异表达的miRNA (P＜0.01,倍数值≥20或≤-20),其中以miR-200c在高转移性乳腺癌细胞株中下调幅度最显著.4个转染miR-200c mimic的乳腺癌细胞株中共有33个共上调基因及13个共下调基因.实时荧光定量-PCR与蛋白质印迹法验证结果显示,共调基因锌指E框结合同源框1(zinc finger E-box binding homeobox1,ZEB1)mRNA和蛋白在4个转染细胞株中均下调.MAS生物信息学分析结果显示,转染miR-200c mimic的乳腺癌细胞株中共有的差异表达基因相关信号通路包括嗅觉传导(olfactory transduction)通路、细胞因子-细胞因子受体关联(cytokine-cytokine receptor interaction)通路和细胞黏附分子(cell adhesion molecules,CAMs)通路等,不同的信号通路可以通过共调基因相互联系,在特定的信号通路中,共调基因间也存在密切的相互联系.结论:以高通量生物芯片检测为基础,运用生物信息学分析手段,多元化筛选获得miR-200c 调控的基因网络,为后续展开miR-200c作用机制的研究提供了明确的方向.     

人肺巨细胞癌高和低转移株中相关转录因子分析

目的：为了阐明人肺巨细胞癌高转移株(95D)细胞高表达uPA、低转移株(95C)细胞低表达uPA的原因，分析两株细胞中有关的转录因子的差异。方法：uPA基因启动子不同片段与Luciferase报告基因的融合基因真核表达质粒分别平行转染95C、95D细胞，比较活性；用EMSA分析95D、95C细胞中与寡核苷酸探针特异结合的转录因子差异；用RT-PCR分析两株细胞中相应转录因子mRNA相对表达水平。结果：三个不同长度的uPA基因启动子在95D细胞中的活性均相应高于95C细胞中的活性，分别是95C细胞的1．32、1．66和1．61倍，EMSA表明95D细胞中与AP1、AF2、NFKB及SP1探针结合的转录因子量均高于95C细胞中；RT-PCR表明AP2、NKkB及SP1转录因子mRNA表达水平分别是95C细胞的1．57、1．77和1．28倍。结论：转录因子AP1、AP2、NKkB及SP1在95D细胞中含量高于95C细胞可能是uPA在95D与95C细胞中表达差别的原因之一．     

胃癌转移相关miRNA的差异表达谱及miR-218的生物学分析

目的 探讨胃癌转移相关微小RNA（miRNA）的差异表达情况并进行miR-218的生物学分析。方法 采用实时荧光定量PCR（qPCR）及miRNA芯片法检测低转移潜能的胃癌细胞亚系（SGC7901-NM、MKN28-NM）与高转移潜能的胃癌细胞亚系（SGC7901-M、MKN28-M）间miRNA的差异表达。提取不同转移潜能胃癌细胞系和10例胃癌冰冻组织及相应的转移淋巴结中的总RNA,利用qPCR检测miR-218在不同细胞及组织中的表达情况。结果 对不同转移潜能的胃癌细胞亚系进行芯片检测发现,与SGC7901-NM细胞比较,SGC7901-M 细胞有47个分子表达下调,15个分子表达上调。与MKN28-NM细胞比较,MKN28-M细胞有41个分子表达下调,83个分子表达上调。在SGC7901-M及MKN28-M细胞中,34个分子表达均出现下降,11个分子表达均出现上升。对不同转移潜能的胃癌细胞亚系以及人永生化正常胃黏膜细胞系GES进行检测可以发现,4种不同转移潜能的胃癌细胞亚系中miR-218的表达均低于正常胃黏膜细胞系GES,差异有统计学意义（P＜0.05）,且在高转移潜能胃癌细胞亚系中miR-218的表达均低于低转移潜能胃癌细胞系,差异有统计学意义（P＜0.05）。胃癌转移淋巴结中miR-218的表达水平为0.23±0.02,低于胃癌原发灶的1.09±0.05,差异有统计学意义（P＜0.05）。结论 胃癌转移相关miRNA会出现差异表达情况,高转移潜能胃癌细胞中的miR-218表达水平上调可能与胃癌转移存在一定关系。     

RAB5A对人肺腺癌细胞系侵袭转移作用的研究

目的：探讨RAB5A基因在人肺腺癌细胞系侵袭和转移中的作用。方法：采用体外重建基底膜侵袭实验和癌细胞粘附能力；癌细胞趋化性运动能力、癌细胞分泌明胶酶的能力及活性的测定，分析RAB5A正义真核表达载体转染后的AGZY83－a和反义RNA转染后的Anip973细胞的侵袭、转移能力的改变。结果：RAB5A正义表达载体PcDNA3.1-RAB5A转染的AGZY83－a重建基底膜侵袭能力明显增强，统计学意义显著（P＜0．0005），细胞趋化性运动能力显著增高（P＜0．0005），对基底膜成分的粘附能力增大（P＜0．05），以及明胶酶分泌及活性增强等一系列趋向Anip973的变化。PcDNA3-AntiRAB5A反义RNA表达载体对Anip973重建基底膜侵袭力明显下降（P＜0．0025），趋化性运动能力显著降低（P＜0．005），对基底膜成分粘附能力降低（P＜0．05），以及明胶酶分泌减少等一系列趋向AGZY83－a的变化。结论：RAB5A在肺腺癌侵袭轩移表型形成中发挥重要的作用。反义RNA可阻断RAB5A基因的翻译过程。     

肺转移瘤的放射治疗

肺转移瘤是最常见的转移性肿瘤之一,手术仅适用于局限性肺转移的治疗,并且大部分患者不适合行手术治疗。化疗是主要治疗方法,但是疗效欠佳。研究证实,随着三维适形放疗、体部立体定向放射治疗等精确放疗的不断发展,放疗对于肺转移患者有效,尤其对于局限性转移,体部立体定向放射治疗可以取得较好的生存期,但仍需大规模前瞻性研究。     

肺转移瘤的放射治疗

肺转移瘤是最常见的转移性肿瘤之一,手术仅适用于局限性肺转移的治疗,并且大部分患者不适合行手术治疗.化疗是主要治疗方法,但是疗效欠佳.研究证实,随着三维适形放疗、体部立体定向放射治疗等精确放疗的不断发展,放疗对于肺转移患者有效,尤其对于局限性转移,体部立体定向放射治疗可以取得较好的生存期,但仍需大规模前瞻性研究.     

肺鳞癌患者淋巴转移相关基因的筛选

背景与目的筛选肺鳞癌患者淋巴转移差异表达基因群。方法原发癌组织及区域淋巴结取自10例接受根治性手术的肺鳞癌患者。根据组织来源将标本分为三组:不伴     

Spontaneous metastasis of clonal cell subpopulations of human lung giant cell carcinoma after subcutaneous inoculation in nude mice

Four sub-lines (strain A, C, D, E) were isolated from a human lung giant cell carcinoma cell line (PLA-801) by the single cell cloning technique. The incidence of spontaneous metastasis was higher in strain D, moderate in strains A, E and lower in strain C after they were inoculated subcutaneously into the nude mice. The lung and lymph-nodes were the major target organs of metastasis. Morphological studies (light microscope, electron microscope and immunohistochemistry) showed that the cytoplasm of metastatic tumor cells was rich in microfilament and positive for Vimentin. The results strongly support the theory on tumor cell heterogenicity. In addition, these clones, especially strains D and C, could be an ideal model for the study of tumor metastasis.     

Identification of liver metastasis-related genes in a novel human pancreatic carcinoma cell model by microarray analysis

Pancreatic cancer with liver metastases has a poor prognosis and the molecular mechanisms remain unclear. In this study, SW1990HM, a highly metastatic human pancreatic carcinoma line was subcloned from SW1990 by intrasplenic injection. In vivo and in vitro tumorigenicity, metastatic potential, in vitro invasion, cell growth curves, plate efficiency and S-phase cell numbers were higher in SW1990HM cells. Gene expression profiles of SW1990HM and SW1990 cells showed 40 metastasis-related genes expressed with a 3-fold difference. Thirteen of these 32.5% (13/40) were adhesion and extracellular-matrix related and twelve 30% (12/40) were cell growth and proliferation related, such as MMP10, MMP9, MMP7, CDH1, MGAT5, CTNNA1, IGF1, IL8RB, ITGA7, MDM2, MET, SSTR2 and VEGF, which were related to the onset and progression of tumor metastasis. Thus, SW1990HM is an attractive model to study metastasis and identify potential therapeutic targets.     

Neurotensin in human small cell lung carcinoma

High levels of neurotensin-like immunoreactivity were found in human small cell lung carcinoma lines. No immunoreactivity was present in non-small cell carcinoma lines and only low amounts in postmortem human lung tissue. The immunoreactive material co-eluted with synthetic neurotensin on two different chromatographic systems. No evidence was obtained for the presence of specific neurotensin binding sites in any of the small cell carcinoma lines examined. The results suggest that small lung cell carcinoma lines may be useful for studying the biosynthesis of human neurotensin.     

Detection of human lung epithelial cell growth factors produced by a lung carcinoma cell line: use in culture of primary solid lung tumors

Serum-free medium conditioned for 72 h by a human bronchioloalveolar carcinoma of lung, A549-1, stimulated the colony formation of normal human bronchial epithelial cells, newly cultured cells from human solid lung tumors, and established human lung tumor cell lines, including A549-1 cells themselves. This activity was concentration dependent and was stable to acid. Growth factors in A549-1 conditioned medium (CM) supported culture of solid lung tumors; primary cell cultures were obtained from nine of 10 solid lung tumors of non-small cell origin and from one small cell tumor using A549-1 CM. In addition, three cell lines have been established to date from these primary cultures. Gel filtration of concentrated A549-1 CM on Biogel P-10 separated the growth promoting activity into four regions of apparent Mr 70,000, 12,000, 8,000, and 6,000, and two broad regions of apparent Mr 3000-5000. All but the 12,000 Mr fraction contained activity which competed for specific binding of epidermal growth factor (EGF) to A431 cell membranes. CM was superior to both EGF and TGF alpha in stimulating growth of normal and neoplastic lung cells. EGF also was inhibitory to tumor cells while TGF alpha stimulated both normal and tumor cell growth. TGF beta was also found in CM but inhibited normal and neoplastic lung epithelial cell growth. Of other substances tested, ILGF-I stimulated colony formation. The results suggest that autocrine factors may be important in non-small cell lung tumor cell growth and that differences in response to EGF and TGF alpha may provide the basis for selective culturing of normal and neoplastic lung epithelial cells.     

Identificating 14-3-3 sigma as a lymph node metastasis-related protein in human lung squamous carcinoma

In this study, we aim to screen metastasis-related proteins in human lung squamous carcinoma (LSC) using laser capture microdissection and a proteomic approach. Twenty two differential proteins were identified from pooled microdissected primary LSC and matched lymph node (LN) metastatic tissues. Expression of the differential protein 14-3-3 sigma was determined by Western blotting and immunohistochemistry. In cell invasion assay, down-regulated 14-3-3 sigma by siRNA increased in vitro invasive ability of HTB-182 and A549 cells, up-regulation of 14-3-3 sigma by pcDNA3.0/14-3-3 sigma decreased in vitro invasive ability of HTB-182 and A549 cells. The data suggest that 14-3-3 sigma is a potential LN metastasis-related protein in LSC, and its dysregulation might play an important role in the LN metastatic process of LSC.     

A three-dimensional cell biology model of human hepatocellular carcinoma in vitro

We established an in vitro 3-D model of metastatic hepatocellular carcinoma (HCC) by culturing MHCC97H cells on molecular scaffolds within a rotating wall vessel bioreactor. Morphological and biochemical analyses revealed that the 3-D HCC model mirrored many clinical pathological features of HCC in vivo, including cancer cell morphology, tissue ultrastructure, protein production and secretion, glucose metabolism, tissue-specific gene expression, and apoptosis. Xenografts into livers of nude mice resulted in tumorigenesis and distant metastasis. This 3-D HCC spheroid is a promising model for HCC tumor biology, anticancer drug screening, and for the establishment of HCC animal models.     

喉鳞状细胞癌组织miRNA表达的初步研究

目的:探讨喉鳞状细胞癌组织miRNA表达.方法:对我院6例手术并经病理确诊的喉鳞状细胞癌患者,及6例良性病变惠者,提取喉部组织,应用miRNA基因芯片检测两组之间不同miRNA水平的表达情况.结果:与对照组相比,喉鳞状细胞癌组织中表达升高的有,miR-200b、miR-21、let-7i、let-7c、miR-93、miR19a、miR-106b和miR19b-2;下调的有,miR-543、miR-520h、miR-582-3p、miR-1305和miR-203.未见或鲜见报道的有miR-1305、miR-203、miR19a和miR19b-2.结论:喉鳞状细胞癌中存在miRNA的表达失调,为进一步的功能学研究和扩大实验提供了线索.     

Detection in colorectal carcinoma patients of antibody cytotoxic to established cell strains derived from carcinoma of the human colon and rectum.

Sera from eight of 15 patients with colonic carcinoma exhibited demonstrable cytotoxicity against an established cell strain derived from adenocarcinoma of the ileocecum, HCT-8. Sera from 12 of 16 patients with rectal carcinoma were cytotoxic for an established cell strain derived from an adenocarcinoma of the rectum, HRT-18. Patients with colonic carcinoma exhibited serum cytotoxicity against only the colonic target cells, whereas patients with rectal carcinoma gave significant cytotoxicity against both cell strains. This cytotoxicity was shown to be complement-dependent and appeared to be specific for colonic and/or rectal carcinoma cells. Although the cells produced carcinoembryonic antigen (CEA) in vitro, the cytotoxic antibody response in these patients did not appear to be directed against CEA. Serum cytotoxicity was not demonstrated against two other cell strains, HCT-48 and HT-29, derived from adenocarcinomas of the human colon, except for a reaction against a blood-group-related antigen. These cell strains had comparable levels of cell-associated CEA. The routine titration of cytotoxic antibody against these established cell cultures may provide meaningful information on the host's immune response to colorectal neoplasms.     

Extraosseous giant cell tumour of lung

A rare case of Extraosseous Osteoclastoma like giant cell tumour of the lung is presented.