树突状细胞与肿瘤细胞融合后对肿瘤发生和生长的抑制作用

目的研究树突状细胞（ＤＣ）与肿瘤细胞（ＳＰ２／０）融合后在体内的抗肿瘤效应。方法体外分离诱导扩增ＢＡＬＢ／Ｃ小鼠骨髓ＤＣ,然后与ＳＰ２／０细胞进行融合,将融合细胞接种于同基因小鼠皮下,观察肿瘤的生长及组织病理学特征。结果ＤＣ与ＳＰ２／０细胞融融后接种于小鼠能抑制肿瘤的生长,肿瘤的发生、生长速度及病理组织学改变的显示出ＤＣ－ＳＰ２／０融合细胞的抑制效应。结论ＤＣ与肿瘤细胞融合后在体内可产生抗肿瘤免疫     

流感疫苗对慢性粒细胞白血病源性树突状细胞功能影响的研究

 目的 探讨流感疫苗对白血病源性树突状细胞（DC）功能的影响。方法 分离慢性粒细胞白血病（CML）患者骨髓单个核细胞,加入GM-CSF和IL-4诱导培养7 d,将所得细胞分为4组：完整失活流感疫苗（WIV）组;裂解流感疫苗（SIV）组;TNF-α组;对照组,用不同培养方法再培育24 h后,流式细胞仪分析DC免疫表型,ELISA法检测上清液中IL-12浓度。结果 CML源性DC细胞表面分子表达、上清液中IL-12浓度在WIV及SIV组较TNF-α和对照组显著增高,差异有统计学意义（P＜0.05）;其中WIV组高于SIV组（P＜0.05）,而TNF-α组与对照组差异无统计学意义（P＞0.05）。结论 流感疫苗具有刺激CML源DC成熟的功能。     

肝脂素（YS2H）对K562细胞超微结构的影响

目的观察肝脂素（ＹＳ２Ｈ）对Ｋ５６２白血病细胞超微结构的影响。方法应用光镜及电镜观察经不同浓度肝脂素作用一定时间后的Ｋ５６２细胞形态及超微结构的变化。结果光镜下,肝脂素可使Ｋ５６２细胞出现细胞大小不均,形态不规则,细胞核染色质略浓密,核仁模糊,胞浆出现空泡,部分胞浆有灰蓝色或灰红色区域等变化;电镜下,细胞内出现空泡、脂滴、粗面内质网扩张、脱颗粒、线粒体肿胀、嵴减少等变化。结论肝脂素具有抗白血病细胞     

IFN-α对CML树突状细胞表型及功能的影响

临床观察表明IFN-α治疗早期慢性期CML患者有效,并且IFN-α治疗CML达到部分细胞遗传学缓解以上的患者较之IFN-α治疗无效者其生存期明显延长.IFN-α治疗CML有效的机制尚不清楚,可能与其增强DC的表型及功能有关.DC是体内唯一能激活初始型T细胞的抗原递呈细胞,在机体免疫系统中处于中心地位.我们采用CML患者骨髓单个核细胞,体外有血清培养体系中观察IFN-α对CML-DCs的分化及其功能的影响.     

DC-CIK对结直肠癌根治术后肝转移患者疗效和循环肿瘤细胞的影响

目的: 探讨树突状细胞-细胞因子诱导的杀伤细胞（dendritic cell-cytokine induced killer cell, DC-CIK）对结直肠癌（colorectal carcinoma, CRC）根治术后肝转移患者循环肿瘤细胞（circulating tumor cell, CTC）数量、疗效和预后的影响。方法: 回顾性分析2009 年7 月至2015 年12 月在解放军第309 医院普通外科采用DC-CIK+常规疗法治疗的CRC根治术后肝转移患者62例（DC-CIK组）和同期未接受DC-CIK治疗的70 例患者（常规组）的临床资料,同时抽取部分患者（DC-CIK组21 例,常规组24 例）的外周静脉血,用CellSearch R 免疫磁珠技术检测血中CTC的数量,分析比较两组患者的疗效和预后。结果：DC-CIK 组治疗后患者CTC数量明显低于治疗前[ （1.0±1.1）vs（2.7±2.0）个,P<0.01],而常规组CTC变化不明显（P>0.05）;DC-CIK组肝转移灶手术切除率、治疗客观有效率、患者无进展生存和总生存均显著优于常规组（P<0.05 或P<0.01）。结论：DC-CIK可减少CRC根治术后肝转移患者CTC数量并延长患者生存时间,在规范化实施的前提下具有可靠的抗肿瘤效果。     

透毒复方对急性髓系白血病完全缓解患者CD+34细胞源树突状细胞生物学效应的影响

 目的　研究透毒复方青蒿鳖甲汤对急性髓系白血病完全缓解期（AML-CR）患者骨髓CD+34 细胞来源树突状细胞（DC）生物学效应的影响。方法　分离纯化骨髓CD+34 细胞,体外不同浓度透毒中药含药血清与细胞因子联合诱导分化为DC,观察DC形态特征,流式细胞术检测DC表面分子CD80、CD83、CD86的表达,将DC分别与自体、异体外周血T细胞共同培养,四甲基偶氮唑蓝（MTT）比色法检测激活的T细胞不同效靶比对人类白血病细胞株（K562细胞）的杀伤效应。结果　青蒿鳖甲汤联合细胞因子能促进AML-CR患者骨髓CD+34 分化为形态典型DC,不同剂量含药血清均能上调DC表面特征性分子及共刺激分子CD80、CD83、CD86比例,较单纯细胞因子组比较差异有统计学意义（P＜0.01）,且含药血清组较单纯细胞因子更能激发外周血T淋巴细胞对K562细胞的杀伤作用,两者间比较差异有统计学意义（P＜0.01）。结论　青蒿鳖甲汤能促进髓系微小残留白血病患者CD+34 细胞向DC转化,提高DC的生物学效应。     

磷酸钙纳米颗粒修饰的甲胎蛋白和树突状细胞疫苗对小鼠皮下移植瘤的抑制作用

目的 探讨转染小鼠带有信号肽的AFP_1 cDNA和去掉信号肽的AFP_2 cDNA树突状细胞(DC)在体外的免疫活性,以及其对Balb/c小鼠皮下移植瘤的抑制作用.方法 应用磷酸钙纳米颗粒将带有信号肽的AFP_1 cDNA和去掉信号肽的AFP_2 cDNA 真核表达载体pcDNA3.1转染DC,将DC疫苗与同源小鼠脾淋巴细胞混合培养,采用酶联免疫吸附(ELISA)法,检测上清液中干扰素γ(IFN-γ)的表达情况.采用四甲基偶氮唑蓝(MTT)法,检测AFP_1/DC和AFP_2/DC刺激同基因小鼠脾淋巴细胞增殖能力及诱导特异性细胞毒性T淋巴细胞(CTL)的杀伤能力.观察AFP_1/DC和AFP_2/DC对Balb/c小鼠皮下移植瘤生长的抑制作用.结果 AFP_2/DC能明显促进脾淋巴细胞增殖并提高CTL的特异性杀伤作用.AFP_1/DC和AFP_2/DC瘤内注射均可显著抑制肝癌移植瘤的生长,但AFP_2/DC的抑制作用更明显,治疗2周后,AFP_2/DC组小鼠肿瘤体积为(726.7±298.2)mm3,明显小于AFP_1/DC组[(1486.2±457.2)mm~3]和空质粒对照组[(2137.2±547.2)mm~3,P<0.05].AFP_2/DC组和AFP_1/DC组的抑瘤率分别达79.2%和39.7%,而空质粒对照组和空白对照组的抑瘤率为0.AFP_2/DC组和AFP_1/DC 组小鼠的生存时间分别为(58.5±4.2)d和(45.2±4.8)d,较空质粒对照组[(30.6±6.2)d]显著延长(P<0.05).结论 AFP_2/DC 疫苗在体外能够诱导出高效而特异的抗肿瘤免疫效应,在体内具有抑制Balb/c小鼠皮下移植瘤生长的作用.     

热休克肿瘤细胞抗原负载的树突状细胞对结肠癌小鼠的治疗作用

背景与目的:目前通过树突状细胞诱导机体抗肿瘤免疫已经成为肿瘤免疫治疗的一种重要途径,热休克处理肿瘤细胞可提高肿瘤细胞抗原负载的树突状细胞分化成熟,增强其体内抗肿瘤作用.本实验研究热休克肿瘤细胞抗原负载的骨髓来源的树突状细胞(dendritic cell,DC)对结肠癌小鼠的治疗作用.方法:20只小鼠随分为4组,将小鼠结肠癌细胞CT26热休克处理后超声破膜,以其细胞裂解液负载BALB/c小鼠骨髓来源的DC,观察DC诱导肿瘤特异性细胞毒性T淋巴细胞(cytotoxic T lymphocyte,CTL)杀伤活性;并将DC接种于荷瘤小鼠皮下,观察其对肿瘤生长的抑制作用及对荷瘤小鼠生存期的影响.结果:与冻融抗原-DC组、DC组、PBS组相比,热休克抗原-DC诱导的CTL对CT26肿瘤细胞具有显著的杀伤作用,相同效靶比(P=0.00)下其肿瘤特异性细胞毒活性强于前者{(0.99±0.19)g vs.[(1.27±0.28)g、(2.19±0.35)g、(2.14±0.27)g],P均=0.00};用其进行免疫后对小鼠肿瘤的生长具有显著的抑制作用[(128±18)mm3 vs. (313±52)mm3,P=0.04],并能显著延长荷瘤小鼠的生存时间{(57±7)d vs. [(40±3)d、(25±3)d、(24±3)d],P均=0.00}.结论:热休克肿瘤细胞抗原负载的树突状细胞对结肠癌小鼠具有显著的治疗效果.     

IL-27基因转染对树突状细胞表型和功能的影响

目的 探讨转染IL-27基因体外对人外周血单个核细胞来源树突状细胞表型和功能的影响。方法 采集健康成人外周血,密度梯度离心法获得外周血单个核细胞,用GM-CSF、IL-4诱导树突状细胞（Dendritic cell, DC）,第5天后将DC分为3组：IL-27基因转染组、TNF-α组和阴性对照组。倒置显微镜观察DC形态;流式细胞术检测DC表面分子CD1a、CD80、CD83和CD86的表达情况;MTT法检测DC刺激T细胞增殖的能力;ELISA法检测细胞培养上清中IL-12和IFN-γ的含量。结果 转染IL-27基因后外周血单个核细胞来源DC呈现典型的成熟DC形态学特征。IL 27基因转染组DC表面CD1a、CD80、CD83和CD86表达水平较阴性对照组均明显上调（P<0.05）。IL-27基因转染组DC诱导T细胞增殖的能力较对照组DC明显增高（P<0.05）。IL-27基因转染组DC培养上清中IL-12和IFN-γ的含量均明显高于对照组DC（P<0.01）。结论 转染IL-27基因可以上调成熟DC的细胞表型,增强DC的免疫学活性。     

急性髓系白血病细胞对树突状细胞分化、成熟和调节性T细胞生成的影响

Objective To investigate the effects of soluble factors secreted by acute myeloid leukemia (AML) cells on the phenotypical and functional properties of DCs derived from normal mononuclear cells. Methods Mononuclear cells were cultured with interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF), in the presence or absence of 24 h culture supernatants from fresh primary AML cells, to generate immature DCs. The maturation of DCs was induced by cytokines IL-1beta, IL-6, tumor necrosis factor-alpha (TNF-alpha), and prostaglandin-2 (PGE-2). The phenotypic alterations of DCs and DCs-primed CD4 T cells were evaluated using flow cytometry. Precursor frequency (PF) was calculated to monitor the allostimulatory effects of DCs on CD4 and CD8 T cells. ResultsAML cell supernatant-treated DCs showed significantly lower expression of co-stimulatory molecules CD80 and CD86, and reduced response to cytokines IL-1beta, IL-6, TNF-alpha,and PGE-2. The allostimulatory effects of AML cell supernatant-treated DCs on CD4 and CD8 T cells were significantly lower than those of normal mature DCs [PF (1.8±0.5)% vs. (5.2±1.6)% for CD4 T cells, (2.1±0.6)% vs. (6.5±2.0)%for CD8 T cells, P＜0.01]. These AML supematant-induced DCs could also induce allogeneic CD4 T cells to differentiate into CD4 CD25high T cells, which had immunophenotyping characteristics of regulatory T cells, i.e. they expressed Foxp3 but not active maker CD69. Conclusion This study demonstrates that soluble factors secreted by AML cells can inhibit development and functions of DCs. In addition, AML supematant-induced DCs can induce the generation of CD4 CD25high T cells from CD4 T cells, which may be a mechanism of increased prevalence of CD4 CD25high regulatory T cells and immune dysfunction in AML patients.     

肝癌特异性靶标致敏DC诱导CIK细胞对肝癌HuH-7细胞及移植瘤的抑制

目的：观察负载肝癌特异性DC靶标的树突状细胞（dendritic cell,DC）与细胞因子诱导的杀伤（cytokine-induced killer cell,CIK）细胞共培养后对肝癌细胞HuH-7的杀伤作用以及对裸鼠肝癌移植瘤的治疗效果。 方法： 外周血单个核细胞经不同细胞因子作用后培养成DC和CIK细胞。分别利用肝癌特异性DC靶标和肝癌HuH-7细胞冻融抗原刺激DC,ELISA法测定其对DC IL-12分泌的影响。DC HuH-7、DC target分别与CIK细胞共培养后,ELISA法检测其对CIK细胞IFN-γ分泌的影响,CCK-8法检测效靶比为10〖DK〗∶1、20〖DK〗∶1、40〖DK〗∶1、100〖DK〗∶1时DCHuH-7-CIK和DC target-CIK细胞对HuH-7细胞的杀伤作用。构建HuH-7细胞裸鼠皮下移植瘤模型,随机分为对照组、CIK组、DC HuH-7-CIK组和DC target-CIK组,尾静脉注射细胞悬液,观察效应细胞的抑瘤效果。 结果： DC HuH-7与DC target的IL-12 p70分泌水平较DC显著升高\[（179.33±14.04）、（173.33±6.66） vs （59.33±1184）pg/ml,均 P <0.01\];与CIK细胞共培养后,DC HuH-7-CIK与DC target-CIK细胞分泌IFN-γ的能力也显著高于DC-CIK细胞（均 P <0.01）,且DC HuH-7-CIK与DC target-CIK细胞之间无显著差异（ P >0.05）。在相同效靶比时,DC HuH-7-CIK细胞以及DC target-CIK细胞对HuH-7细胞的杀伤活性明显高于单纯CIK细胞（ P <0.05）,且DC HuH-7-CIK与DC target-CIK组之间无显著差异（ P >0.05）。DC target-CIK细胞对裸鼠肝癌移植瘤的抑制作用与DCHuH-7-CIK细胞相仿,且均显著高于单纯CIK细胞\[(78.48±1458)%、(85.78±15.69)% vs (54.69±28.07)%,均 P <0.05\],无明显不良反应。 结论： 肝癌特异性DC靶标能够显著提高CIK细胞对HuH-7细胞及其裸鼠移植瘤的杀伤活性,可替代肝癌组织抗原负载DC。     

Influence of drug-induced apoptotic death on processing and presentation of tumor antigens by dendritic cells

Here we have studied the effects of apoptotic cell death induced by chemotherapic agents on tumor phagocytosis by dendritic cells (DC) and presentation of the relevant antigen to T lymphocytes. Annexin-V-FITC (Ann-V) and propidium iodide (PI) staining was used to assess early apoptotic (Ann-V(+)/PI(-)) vs. late apoptotic/secondary necrotic (Ann-V(+)/PI(+)) death after a 24 hr observation of untreated and drug-treated gastric carcinoma cells. After treatments, the HLA-A*0201(+) tumor cell line KATO III was exposed for 24 hr to allogeneic, HLA-related GM-CSF, IL-4-driven immature (i) DC. Tumor-loaded iDC were tested for IL-12 release in an ELISA assay, incubated with the DC-maturating factor TNF-alpha and used as stimulators for autologous T lymphocytes. Generation of antitumor T response against KATO cells was evaluated in an anti-MHC class I MAb-blocked Interferon-gamma ELISPOT assay. After treatment with Cis-platin (cis), all dying cells were in early apoptosis, whereas secondary necrosis was the prevalent death pattern observed after epirubicin (epi) and doxorubicin (doxo). Doxo and epi increased tumor expression of heat shock protein (hsp) 70 and uptake of tumor cell components by DC, whereas cis treatment had no effect on hsp70 and was associated with poor tumor uptake by DC. Significant upmodulation of IL-12 was observed by DC that had taken up the doxo- and epi-treated tumors (p< 0.005 and p< 0.01, respectively). Increased IFN-gamma release was also observed after stimulation of T lymphocytes with DC loaded with doxo- and epi-treated (p< 0.02 and p< 0.005, respectively) but not with cis-treated DC. These data show that the products of early apoptosis cannot efficiently cross-activate MHC class I-restricted anti-tumor lymphocytes even in the presence of DC maturating factors, whereas secondary necrosis is associated with robust T cell response.     

IL-27促进人单个核细胞来源树突状细胞的分化成熟及其作用机制

目的:探讨IL-27对人外周血单个核细胞来源的树突状细胞(dendritic cell,DC)形态和功能的影响及其作用机制。方法:从正常健康人外周血中分离出单个核细胞,将其用GM-CSF、IL-4体外培养7 d,并于培养的第5天加入不同刺激因子,并将细胞分为4组:阴性对照组、阳性对照组(20 ng/ml TNF-α)、IL-27(20 ng/ml)组、IL-27+TNF-α组(即双细胞因子组,10ng/ml TNF-α+10 ng/ml IL-27)。用倒置显微镜观察培养7 d的DC形态,用流式细胞术检测DC表面共刺激分子CD1a/CD83和CD80/CD86的水平,RT-PCR检测DC表面趋化因子受体CCR5、CCR7 mRNA的表达,混合淋巴细胞实验检测DC刺激同种异体T淋巴细胞增殖的能力,Western blotting检测DC信号通路蛋白P-STAT1/STAT3的含量。结果:IL-27组和双细胞因子组诱导7 d时DC呈现典型的成熟形态学特征;DC表面CD1a和CD83双阳性表达[(35.75±4.10)%、(52.49±2.65)%vs(23.29±4.49)%,P<0.05]、CD80和CD86双阳性表达[(39.06±1.61)%、(54.10±0.46)%vs(22.66±3.20)%,P<0.05]、趋化因子受体CCR7 mRNA[3.98±0.09、4.75±0.11 vs 3.09±0.18,P<0.05]和转录因子蛋白P-STAT1/STAT3水平均较阴性对照组明显上调,而CCR5 mRNA[0.99±0.03、0.61±0.02 vs 1.23±0.26,P<0.05]表达含量则明显下降;IL-27组和双细胞因子组DC均可明显刺激T细胞增殖,且随DC与T细胞比例增加而增强,以双细胞因子组的刺激作用更为明显。结论:细胞因子IL-27可以直接或者协同TNF-α诱导人DC分化成熟,并增强DCs的抗原提呈功能,其机制可能与活化P-STAT1/STAT3信号通路有关。     

胶质瘤细胞miR-153上调对树突状细胞分化成熟及Nrf2表达的影响

目的:探讨胶质瘤细胞miR-153上调对树突状细胞分化成熟及Nrf2表达的影响。方法:体外培养小鼠胶质瘤细胞系GL261,随机分为对照组、miR-153 mimics阴性对照组、miR-153 mimics组,以miR-153 mimics阴性对照、miR-153 mimics分别处理GL261,以实时荧光定量PCR（qRT-PCR）检测各组GL261细胞miR-153、VEGF-A及IL-10 mRNA水平;以蛋白免疫印迹法检测各组GL261细胞VEGF-A、IL-10、Nrf2蛋白表达;以流式细胞仪检测DC2.4细胞表面共刺激分子MHC-II、CD80、CD86、CD40表达水平;将小鼠T淋巴细胞系与上述各组DC2.4细胞共培养,以CCK-8检测T细胞增殖情况。结果:与对照组相比,miR-153 mimics组GL261细胞miR-153、VEGF-A及IL-10 mRNA水平,VEGF-A及IL-10蛋白表达明显降低（P＜0.05）。DC2.4表面共刺激分子MHC-II、CD80、CD86及CD40表达水平,DC2.4细胞Nrf2蛋白表达,T细胞活力明显升高（P＜0.05）;miR-153 mimics阴性对照组细胞各指标无明显变化（P＞0.05）。结论:上调miR-153可抑制胶质瘤细胞分泌免疫抑制性因子VEGF-A、IL-10,促进树突状细胞分化成熟并上调其Nrf2蛋白表达。     

急性髓细胞性白血病细胞培养上清对树突细胞分化、成熟、凋亡及功能的影响

背景与目的:树突细胞(dendritic cell,DC)存机体抗瘤免疫中起重要作用.研究已表明肿瘤患者和荷瘤动物体内DC功能受损并伴有DC数量的降低.最近的研究提示这种DC功能的受损可能是肿瘤细胞分泌的可溶性因子介导的免疫抑制所致.本研究拟观察急性髓系白血病(acute myeloid leukemia,AML)细胞分泌的可溶性因子对单核细胞来源DC的分化、成熟、凋亡及其功能的影响.方法:原代AML细胞培养24 h后收集上清.在含有或不含有AML细胞培养上清的培养液中,利用IL-4和GM-CSF刺激单核细胞分化成不成熟DC(immature DC,iDC),IL-1β、IL-6、TNF-α和PGE-2促进DC成熟.流式细胞仪检测DC表型和凋亡率的变化.计算前体细胞频率(precursorfrequency,PF),观察DC对异基因CD4 T细胞和CD8 T细胞的刺激功能.结果:AML细胞培养上清可显著抑制DC表面协同刺激分子CD80和CD86的表达,并可降低促成熟细胞因子对DC的促成熟作用.同时,与对照组相比,AML细胞培养上清可显著诱导DC的凋亡,凋亡率分别为(15.1±4.2)%和(29.4±9.5)%(P＜0.01).相对于正常成熟DC,AML细胞培养上清诱生的DC对异基因CD4 T细胞和CD8 T细胞的刺激功能显著降低(P＜0.01),其中CD4 T细胞的前体细胞频率分别为(5.2±1.6)%和(1.8±0.5)%,CD8 T细胞的前体细胞频率分别为(6.5±2.0)%和(2.1±0.6)%.结论:AML细胞分泌的可溶性因子可抑制DC的发育和功能.     

Silibinin inhibits ultraviolet B radiation‐induced mast cells recruitment and bone morphogenetic protein 2 expression in the skin at early stages in Ptch(+/−) mouse model of basal cell carcinoma

Around 80% of nonmelanoma skin cancers (NMSCs) are basal cell carcinoma (BCC), still studies evaluating the efficacy of chemopreventive agents during early stage/s of BCC development are lacking. Accordingly, utilizing the well‐established patched (Ptch)+/? mouse model of ultraviolet B (UVB) radiation‐induced BCC formation, we excised skin samples from UVB exposed Ptch+/? and Ptch+/+ mice before tumor formation to study the promotion/progression of BCC and to determine the efficacy and target/s of silibinin, a well‐known skin cancer chemopreventive agent. UVB exposure for 1 month increased the number of mast cells in Ptch+/? mice by ~48% (P < 0.05), which was completely inhibited by silibinin. Polymerase chain reaction profiler array analysis of skin samples showed strong molecular differences between Ptch+/+ and Ptch+/? mice which were either unexposed or UVB irradiated+/? silibinin treatment. Most notably, silibinin treatment significant decreased the expression of BMP‐2, Bbc3, PUMA, and Ccnd1 in Ptch+/? mice irradiated with silibinin + UVB. Additional studies showed that silibinin targets UVB‐induced expression of bone morphogenetic protein 2 (BMP‐2) in Ptch+/? mouse skin. Last, our studies found that silibinin strongly attenuates UVB‐induced BMP‐2 expression and DNA damage in Ptch+/? mouse skin ex vivo only after single UVB exposure. Together, our results suggest a possible role of mast cell recruitment and BMP‐2 activation in the early stages of BCC development; these are strongly inhibited by silibinin suggesting its possible chemopreventive efficacy against BCC formation in long‐term UVB exposure regimen.     

Organ specificity of tumor metastasis: role of preferential adhesion,invasion and growth of malignant cells at specific secondary sites

The locations of distant secondary tumors in many clinical cancers and animal tumors are nonrandom, and their distributions cannot be explained by simple anatomical or mechanical hypotheses based on the simple lodgment or trapping of tumor cell emboli in the first capillary bed encountered. Evidence from certain experimental tumor systems supports Paget's seed and soil hypothesis on the nonrandom distributions of metastases, in which the unique properties of particular tumor cells (seeds) and the different characteristics of each organ microenvironment (soil) collectively determine the organ preference of metastasis. Experimentally, differential tumor cell adhesion to organ-derived microvessel endothelial cells and organ parenchymal cells, differential invasion of basement membranes and organ tissues, and differential responses to organ-derived growth-stimulatory and-inhibitory factors all appear to be important determinants in explaining the organ preference of metastasis. Each tumor system may achieve organ specificity because of its own unique set of multiple metastasis-associated properties and responses to host microenvironments. As neoplasms progress to more highly malignant states multisite metastases are more likely and organ-specific metastases may be masked or circumvented owing to stochastic events, tumor cell diversification, host selection processes, and increased production of tumor autocrine molecules that may modulate adhesion, invasion, growth, and other properties important in metastasis. The importance of each of these properties, however, appears to vary considerably among different metastatic tumor systems. These and other tumor cell and host properties may eventually be used to predict and explain the unique metastastic distributions to certain human malignancies.     

p21(Waf1/Cip1) and p53 are downstream effectors of protein kinase C delta in tumor suppression and differentiation in human colon cancer cells

We have previously demonstrated that the delta isoform of protein kinase C (PKCdelta) is importantly involved in cell growth inhibition and tumor suppression in colon cancer cells. To investigate further the activity and mechanism of action of PKCdelta, we have retrovirally transduced a PKCdelta cDNA in HCT116 human colon cancer cells. PKCdelta-overexpressing cells (HCT116/PKCdelta) were growth-inhibited, showed marked morphologic changes and underwent multinucleation and phenotypic changes characteristic of mitotic catastrophe. Compared to controls, HCT116/PKCdelta cells showed a highly attenuated tumorigenic profile and poor anchorage-independent growth. In addition, transfected cells established junction-coordinated intercellular communications, expressed cell surface microvilli and overexpressed the colon differentiation marker alkaline phosphatase. HCT116/PKCdelta cells also produced the 89 kDa, carboxy-terminal catalytic domain of PARP. In HCT116/PKCdelta cells, p21(Waf1/Cip1) and p53 were transiently upregulated for 48 hr after PKCdelta transduction. In a p21 null subline of HCT116 cells (HCT116/p21null), overexpression of PKCdelta did not affect tumorigenicity or differentiation, indicating that p21 is essential for the antitumorigenic activity of PKCdelta. Similarly, overexpression of PKCdelta caused no significant phenotypic changes in HCT116/E6 cells, an HCT116 subline in which the p53 protein is downregulated by the human papillomavirus E6 gene product. We conclude that overexpression of PKCdelta in human colon cancer cells induces multiple antineoplastic effects that depend on the activities of p21(Waf1/Cip1) and p53.     

叉头盒蛋白A1过表达对SW480细胞增殖和凋亡及EMT形成影响

目的探讨叉头盒蛋白A1(fork-head box protein A1,FOXA1)过表达对结肠癌SW480细胞增殖、凋亡及上皮间质转化(epithelial-mesenchymal transition,EMT)形成的影响,为结肠癌发生发展机制研究提供一定参考。方法收集南京医科大学第一附属医院2014-03-01-2016-10-31结肠癌手术切除癌组织及其配对正常组织标本各50例。体外培养结肠癌细胞SW480、T84、DIFI及人正常结肠上皮细胞NCM460,以随机数字表法分成空白对照组(CK组)、转染含慢病毒载体对照液的阴性对照组(NC组)和转染含FOXA1过表达慢病毒载体的FOXA1过表达(FOXA1过表达组)3组。采用qRT-PCR法检测细胞中FOXA1mRNA表达水平,采用MTT法检测细胞增殖,采用AnnexinⅤ/PI双染法检测细胞凋亡,采用流式细胞术法检测细胞周期,采用蛋白质印迹法检测细胞中FOXA1蛋白及EMT过程相关蛋白的表达。结果结肠癌组织FOXA1mRNA及蛋白表达水平分别为0.42±0.12、0.28±0.07,低于正常组织的1.11±0.24、0.98±0.21,t值分别为18.183、22.361,均P<0.05。结肠癌SW480、T84、DIFI细胞中FOXA1mRNA分别为0.11±0.02、0.14±0.03和0.15±0.03,蛋白表达水平分别为0.34±0.07、0.38±0.09和0.37±0.08,均低于正常上皮细胞的0.27±0.06、0.67±0.09,F值分别为10.259、10.385,P值分别为0.004、0.004;与CK、NC组比较,FOXA1过表达组结肠癌SW480细胞凋亡率(F=32.511)、G0/G1期细胞比例(F=8.860)、FOXA1mRNA(F=325.500)及其蛋白(F=39.670)、E-cadherin(F=37.507)、Cytokeratin蛋白水平(F=14.957)均升高,均P<0.05;24(F=22.407)、48(F=65.290)和72h(F=46.778)的A值、S期细胞(F=11.042)、G2/M期细胞比(F=6.139)、N-cadherin(F=20.746)、Vimentin(F=26.493)、Twist(F=11.024)、β-catenin蛋白(F=29.359)表达水平均降低,均P<0.05。结论过表达FOXA1能够抑制结肠癌SW480细胞增殖,诱导其凋亡,可能抑制结肠癌细胞EMT过程。