贝利司他抑制骨肉瘤细胞活力的分子机制

目的:观察组蛋白脱乙酰酶抑制剂贝利司他对骨肉瘤细胞活力的影响,并初步研究其作用机制。方法:用不同浓度的贝利司他处理体外培养的骨肉瘤细胞系SAOS-2和U2OS后,采用MTT法检测细胞的活力,荧光探针和ELISA分别检测caspase-3/-7的酶活性以及DNA片段化以观察贝利司他对骨肉瘤细胞凋亡的影响,并采用Western blot检测组蛋白乙酰化水平、caspase-3、Bcl-xL和PTEN的表达,以及Akt和糖原合成酶激酶3β(GSK-3β)的磷酸化水平。用不同浓度的贝利司他和多柔比星(又称阿霉素)共同孵育U2OS和SAOS-2细胞,MTT观察其对细胞活力的影响,并计算其联合指数(CI)。结果:0.5、1、2.5和5μmol/L贝利司他处理U2OS和SAOS-2细胞48 h后,均可呈剂量依赖性方式抑制其活力,诱导DNA片段化,增强caspase-3/-7的活性,上调cleaved caspase-3水平,减少Bcl-xL的表达,促进细胞内组蛋白H3和H4乙酰化。Western blot结果显示,贝利司他处理后,U2OS和SAOS-2细胞中磷酸化Akt和GSK-3β水平显著降低(P...     

阿霉素联合塞来昔布诱导胃癌细胞凋亡的作用研究

目的:研究环氧化酶-2 (COX-2) 抑制剂塞来昔布联合阿霉素对胃癌MGC-803细胞株的凋亡诱导作用，并探讨其相互作用的可能的分子机制。方法:用MTT法检测MGC-803的增殖情况。用荧光显微镜、流式细胞术和DNA梯度电泳检测肿瘤细胞凋亡的情况。结果:随着阿霉素剂量的增加，MGC-803细胞的数量明显减少。细胞大部分静止于G₀/G₁期，S期细胞明显减少。 阿霉素（5 mg/L）联合塞来昔布（25 μmol/L）明显抑制MGC-803细胞的生长。肿瘤细胞经阿霉素或塞来昔布处理后荧光显微镜下可观察到典型的细胞凋亡形态变化。与两者单独用药相比，联合用药后的DNA梯度变化更为明显。联合用药 48 h 后MGC-803细胞堆积在G₀/G₁，而S期减少的细胞数量比两者分别用药时更为显著。结论：塞来昔布和阿霉素具有协调的诱导凋亡的作用，这对于将COX-2抑制剂用于肿瘤的临床辅助化疗具有重要意义。     

Notch1信号抑制对成骨肉瘤细胞化疗药物敏感性的影响

目的研究抑制Notch1信号对成骨肉瘤细胞化疗药物敏感性的影响及部分机制。方法将人成骨肉瘤细胞系OS-732按γ-分泌酶抑制剂（GSI）作用与否分为GSI组和对照组;脂质体法转染真核细胞表达质粒;Realtime PCR检测NICD、Hes1mRNA表达;流式细胞仪（FACS）检测Annexin V-PE/7-AAD双标的细胞凋亡;MTT法检测化疗药物对细胞的抑制率。结果 GSI组NICD和Hes1 mRNA均较对照组明显降低（P〈0.01）。GSI组化疗药物导致的瘤细胞抑制率和凋亡率均明显高于对照组。过表达Hes1可减弱GSI诱导的瘤细胞化疗药物敏感性增高。结论 GSI通过抑制Notch1受体激活和下游靶基因Hes1表达促进人成骨肉瘤细胞凋亡,从而增加人成骨肉瘤细胞对化疗药物的敏感性。     

阿霉素诱导人胆囊癌细胞耐药过程中糖基化神经酰胺合成酶及caspase 3的动态变化

目的：通过检测阿霉素诱导人胆囊癌细胞多药耐药过程中糖基化神经酰胺合成酶(GCSmRNA)、蛋白及caspase 3的变化情况，探讨神经酰胺（ceramide）代谢中起关键作用的酶GCS在人胆囊癌多药耐药产生过程中的作用。 方法： 阿霉素200 μg/L连续作用人胆囊癌细胞株GBC－SD 12周，采用MTT法、RT－PCR、Western blotting分别检测GBC－SD、GBC－SD（4周）、GBC－SD （12周）时的细胞毒性、GCSmRNA、蛋白的变化情况，荧光分光光度法检测此过程中caspase 3的变化。 结果： 诱导12周时，细胞耐药倍数增加3.8倍，GCSmRNA、蛋白表达明显上调(P<0.01)。3种细胞的增殖同阿霉素浓度呈负相关，GBC－SD、GBC－SD（4周）增殖同作用时间呈负相关，而GBC－SD （12周）细胞同作用时间无关。GCS的表达同caspase 3的活性呈负相关。 结论： 阿霉素通过诱导细胞凋亡抑制人胆囊癌细胞增殖，人胆囊癌细胞多药耐药的产生同GCS mRNA、蛋白表达的上调相关，GCS的表达上调抑制了caspase 3的激活，GCS可能是诱导胆囊癌多药耐药产生的机制之一。     

骨肉瘤瘤体周围炎症细胞浸润在假包膜形成中的作用

采用病理形态学与免疫组织化学方法，对６例无假包膜、１２例有假包膜和１６例术前化疗后有假包膜的骨肉瘤瘤体周围假包膜结构，进行形态学观察及对浸润的炎症细胞进行免疫学分类计数和统计学分析，发现假包膜组和化疗组瘤体周围浸润的巨噬细胞和Ｔ淋巴细胞显著高于无包膜组（Ｐ＜０．０１）。认为巨噬细胞和Ｔ淋巴细胞浸润在瘤体周围假包膜形成中起着重要作用。化疗有促进假包膜变成真性包膜的作用。     

骨肉瘤细胞凋亡和增殖与预后的关系

目的：探讨细胞凋亡指数与骨肉瘤病理、细胞增殖及预后的关系。方法：应用ＴＵＮＥＬ方法检测８０例骨肉瘤凋亡细胞,ＰＣＮＡ单克隆抗体ＰＣ１０免疫组化检测肿瘤组织增生,以１００个肿瘤细胞中凋亡细胞和增生细胞分别作为凋亡指数ＡＩ（％）和增生指数ＰＩ（％）。结果：８０例骨肉瘤中ＡＩ范围为０．６３％～１８．８％,其与骨肉瘤ＷＨＯ新分型及ＰＩ值密切相关（Ｐ〈０．０５）。而且ＡＩ〉７．２１的骨肉瘤比ＡＩ≥７．２１的     

抑制Treg联合瘤内转染Ad.GM-CSF增强阿霉素治疗肝癌效果的实验研究

目的:探讨抑制调节性T细胞(Treg)联合瘤内转染Ad.GM-CSF增强阿霉素治疗肝癌效果的可行性,并对其机制进行探讨。方法:建立C57BL/6J小鼠皮下肝癌模型,待肿瘤长至100-150mm3时随机分为6组,每组12只:(1)对照组(PBS);(2)环磷酰胺组(CTX);(3)阿霉素组(DOX);(4)Ad.GM-CSF组;(5)CTX+DOX组;(6)CTX+DOX+Ad.GM-CSF组。末次治疗后第5d每组处死其中4只小鼠,取脾脏测定CTL活性;取肿瘤组织行病理检查;每组另外的8只小鼠用于观察皮下肿瘤生长和小鼠生存期。结果:CTX可以显著增强DOX对肿瘤生长的抑制作用(P0.05),延长小鼠生存期[(62.13±4.21)dvs(79.88±9.00)d,P0.05],而联合应用Ad.GM-CSF可显著增强该效果[(79.88±9.00)dvs(106.13±5.23)d,P0.01]。同其它组相比,CTX+DOX+Ad.GM-CSF组小鼠瘤体内可见大量肿瘤细胞坏死,CD8+T细胞浸润显著增加,而小鼠脾脏CTL杀瘤活性显著增强(P0.05)。结论:以阿霉素为治疗基础的化疗联合抑制Treg并瘤内转染Ad.GM-CSF的免疫治疗具有协同抗肝癌作用,免疫化疗治疗晚期肝癌具有一定的应用前景。     

多柔比星通过Stat3-Oct-4/CD44途径诱导三阴性乳腺癌4T1细胞干性的产生

目的:观察多柔比星对三阴性乳腺癌细胞干性的诱导作用并研究其作用机制。方法:用不同终浓度(0、0.05、0.1和0.5μmol/L)多柔比星培养细胞,观察细胞形态及存活情况,筛选出最适多柔比星浓度(0.1μmol/L)用于后续实验。小鼠乳腺癌4T1细胞和人乳腺癌MDA-MB-468细胞经多柔比星(0.1μmol/L)持续刺激4周后分别命名为4T1-DOX和MDA-MB-468-DOX细胞。通过成球实验观察细胞的干性,免疫荧光检测CD133的表达,流式细胞术检测CD44的表达,Western blot检测Stat3、p-Stat3和Oct-4的表达。结果:成球实验发现4T1-DOX细胞成球能力较4T1细胞显著增强,MDA-MB-468-DOX细胞成球能力较MDA-MB-468细胞也显著增强(P 0.05);免疫荧光结果显示CD133在4T1-DOX细胞的表达比4T1细胞明显增高(P 0.05);流式细胞术结果表明CD44在4T1-DOX中表达较4T1细胞显著增高(P 0.05);Western blot结果显示Oct-4及p-Stat3在4T1-DOX细胞中的蛋白水平增高(P 0.05),而总Stat3的表达量未见显著改变。以上结果表明在0.1μmol/L多柔比星持续诱导下4T1细胞干性特征明显增强。加入Stat3抑制剂WP1066后,CD44、Oct-4和p-Stat3的蛋白水平均下降(P 0.05)。结论:多柔比星通过激活Stat3-Oct-4信号通路,使三阴性乳腺癌细胞成球能力增强,4T1细胞干性标志物CD133及CD44表达增多,诱导乳腺癌细胞干性的产生。因此,Stat3-Oct-4通路可能成为三阴性乳腺癌治疗的新靶标。     

过氧化氢诱导人皮肤成纤维细胞衰老模型的建立

目的应用过氧化氢(H_2O_2)诱导人皮肤成纤维细胞衰老,建立体外细胞衰老模型。方法组织块贴壁法分离培养人皮肤成纤维细胞,免疫荧光法对获得的细胞进行鉴定,采用50μmol/L、100μmol/L、200μmol/L、400μmol/L的H_2O_2处理细胞1h、2h两个时间点,然后正常培养4d,应用细胞增殖实验,衰老相关的β-半乳糖苷酶(SA-β-gal)染色实验,确定H_2O_2氧化应激下的损伤力和细胞衰老率。结果镜下可见细胞呈梭形或多角形,结合免疫荧光结果,分离获得的细胞即为成纤维细胞。用50μmol/L、100μmol/L H_2O_2处理1h、2h后虽然活细胞较多,但是SA-β-gal染色阳性率较低。200μmol/L、400μmol/L H_2O_2处理2h组SA-β-gal染色阳性率较高,但细胞量较少给以后实验带来较多困难。200μmol/L、400μmol/L H_2O_2处理1h组细胞数量,SA-β-gal染色阳性率最高且二者间无明显差异。结论选取200μmol/L H_2O_2处理1h作为建立衰老细胞模型。     

欧前胡素增强多柔比星对HeLa细胞的抗肿瘤效应

目的:研究欧前胡素是否能提高宫颈癌HeLa细胞株对多柔比星的敏感性。方法:MTT法检测HeLa细胞用欧前胡素和多柔比星处理后的活力。Western blot检测HeLa细胞用欧前胡素和多柔比星处理后Bcl-2蛋白家族成员(Mcl-1、Bcl-2、Bcl-x L、Bad和Bax)的表达水平。流式细胞术检测HeLa细胞用欧前胡素和多柔比星处理后的凋亡水平和线粒体膜电位的变化情况。构建Mcl-1真核表达载体,MTT法检测Mcl-1表达载体转染对欧前胡素联合多柔比星治疗宫颈癌效果的影响。结果:欧前胡素在体外可显著提高多柔比星对宫颈癌细胞系HeLa的杀伤活性。欧前胡素可显著降低HeLa细胞Mcl-1的表达,而多柔比星对Mcl-1的表达水平无影响。相比于欧前胡素或多柔比星单治疗组,两者联合可显著诱导HeLa细胞发生凋亡并降低其线粒体膜电位。体外转染Mcl-1真核表达载体显著降低多柔比星联合欧前胡素对HeLa细胞的杀伤活性。结论:欧前胡素通过靶向于Mcl-1增强多柔比星对宫颈癌细胞的杀伤活性。     

ATR signaling can drive cells into senescence in the absence of DNA breaks

The ATR kinase is a key transducer of "replicative stress," the type of genomic damage that has been postulated to be induced by oncogenes. Here we describe a cellular system in which we can unleash ATR activity at will, in the absence of any actual damage or additional signaling pathways triggered by DNA breaks. We demonstrate that activating ATR is sufficient to promote cell cycle arrest and, if persistent, triggers p53-dependent but Ink4a/ARF-independent senescence. Moreover, we show that an ectopic activation of ATR leads to a G1/S arrest in ATM-/- cells, providing the first evidence of functional complementation of ATM deficiency by ATR. Our system provides a novel platform for the study of the specific functions of ATR signaling and adds evidence for the tumor-suppressive potential of the DNA damage response.     

Artificial senescence of bovine retinal pigment epithelial cells induced by near-ultraviolet in vitro

RPE cells irradiated by near-ultraviolet (NUV) were characterized at cellular, biochemical and molecular levels in order to determine whether light-induced RPE changes contribute to the senescence of RPE cells in vitro. Biochemical and molecular parameters of cellular senescence were studied by using both bovine RPE cells at confluence repeatedly irradiated by NUV (peaking at 365 nm) and RPE cells at different levels of population doubling (PDL). After repeated NUV irradiation, RPE proliferation was markedly suppressed. In parallel, the BrdU index significantly reduced to a minimum level, similar to RPE cells undergoing multiple population doublings. NUV irradiation resulted in a decrease in cellular alkali-soluble melanin and an increase in lipofuscin-like fluorophores. The lipofuscin-like fluorophores, isolated from RPE cells exposing repeated NUV irradiation, represented a gradual hyperchromic change and red-shift, reaching the wavelength maxima (560–572 nm), at excitation wavelength of 365 nm, a typical range of ‘age pigment’. These phenomena were substantially eliminated in oxygen-free conditions. Both the NUV-irradiated RPE cells and RPE cells at 20 Pd expressed 4 to 8-fold and 2 to 4-fold less PEDF and TIMP-3 genes, respectively. As result of experiments using chronic photochemical treatment, RPE cells represented several characteristics of cellular senescence. In addition to alterations of the melanin/lipofuscin system, DNA synthesis was greatly suppressed in NUV-irradiated RPE cells, indicating replicative senescence. The phenomena of downregulation of the possible senescence markers imply that photochemical reactions of RPE cells accelerate the process of RPE senescence.     

Specific antitumor effects of tumor vaccine produced by electrofusion between osteosarcoma cell and dendritic cell in rats

Dendritic cells (DCs) are potent antigen-presenting cells capable of inducing primary T-cell responses.Severalimmunotherapy treatment strategies involve manipulation of DCs,both in vivo and ex vivo,to promote theimmunogenic presentation of tumor-associated antigens.In this study,an electrofusion protocol was developedto induce fusion between osteosarcoma cells and aliogeneic bone marrow-derived DCs.Preimmunization withirradiated electrofusion products was found to provide partial or complete protection from tumor challenge inthe UMR106 tumor model.Vaccinated survivors developed long immunological memory.The therapeuticpotential of this type of approach was suggested by the ability of UMR106-DC electrofusion products whichcould induce tumor rejection in a substantial percentage (60%) of hosts hearing pre-established tumor cells.These results tended to indicate that treatment with electrofused tumor cells and allogeneic DCs might becapable of inducing a potent antitumor response and could conceivably be applied to a wide range of cancerindications for which tumor-associated antigens have not been identified.Cellular & Molecular Immunology.2004;1(6):454-460.     

Giant cell rich osteosarcoma of the mandible with abundant spindle cells and osteoclast-like giant cells mimicking malignancy in giant cell tumor

Giant cell rich osteosarcoma is a relatively unusual histological form of osteosarcoma, common lesion usually presenting in the long bones of the appendicular skeleton. The occurrence in the mandible is exceptional rare. Histologically, this tumor tends to be a highly anaplastic, pleomorphic tumor in which the tumor cells may be: plasmacytoid, fusiform, ovoid, small round cells, clear cells, mono-or multinucleated giant cells, or, spindle cells. Herein, we present a case with the sternum and first thoracic vertebra metastasis from primary giant cell rich osteosarcoma of the mandible in a 28 year-old Chinese female. The tumor was predominantly composed of abundant spindle cells with marked atypia and numerous osteoclast-like giant cells reminiscent of malignancy in giant cell tumor. The unusual histological appearance can pose a great diagnostic challenge. It may be easily misdiagnosed, especially if the specimen is limited or from fine-needle aspiration.     

辣椒素诱导人骨肉瘤细胞MG-63发生免疫原性细胞死亡

目的探讨辣椒素与顺铂能否抑制人骨肉瘤细胞MG-63增殖并诱导其免疫原性死亡(ICD)。方法 MTT检测细胞的增殖;线粒体膜电位分析细胞凋亡;流式细胞计量术检测细胞膜上钙网蛋白(CRT)表达量;荧光素酶法检测细胞外ATP的释放;ELISA检测细胞上清中高迁移率族蛋白1(HMGB1)的分泌。结果辣椒素及顺铂作用均能抑制细胞MG-63增殖(P0.01),且呈剂量依赖性;辣椒素和顺铂均可诱导MG-63细胞凋亡(P0.01),但只有辣椒素能诱导MG-63细胞内质网中CRT转移到细胞膜表面,且促进ATP及HMGB1的释放(P0.01)。结论辣椒素可诱导人骨肉瘤细胞凋亡及免疫原性死亡。     

Correlation between the number of viable tumor cells and immune cells in the tumor microenvironment in non-small cell lung cancer after induction therapy

This study aims to determine the correlation between the percent viable tumor cells (%VTC) and the tumor microenvironment in resected non-small cell lung cancer after induction therapy. We enrolled 72 patients with non-small cell lung cancer (NSCLC) who received chemoradiotherapy (CRT) or chemotherapy (CT) prior to surgery. The ratio of the area of viable tumor cells to the total tumor area was calculated to obtain the %VTC. We also examined the number of CD4 (+), CD8 (+), CD20 (+) and FOXP3 (+) tumor-infiltrating lymphocytes (TILs), podoplanin (PDPN) (+) cancer-associated fibroblasts (CAFs), and CD204 (+) tumor-associated macrophages (TAMs) by immunohistochemistry (IHC). In the CRT group (n = 37), the tumors had significantly lower %VTC than the CT group (n = 35) (P < 0.001). In both of the CT group and CRT group, the %VTC showed a significant positive correlation with the number of CD204 (+)-TAMs (P = 0.014 and 0.005, respectively). Only in the CRT group, a higher number of CD204 (+) TAMs was associated with a shorter overall survival (OS) (P = 0.007) and recurrence-free survival (RFS) (P = 0.015). In the CRT group, the number of CD204 (+) TAMs is associated with %VTC and prognosis, suggesting that these cells may have tumor-promoting effects on the residual lung cancer in specific microenvironments after CRT.