rhTRAIL对耐顺铂人肺腺癌细胞凋亡的影响

背景与目的TRAIL和TNF、Fas一样均属TNF蛋白超家族成员。研究表明,TNFα可用于克服某些肿瘤细胞的化疗耐药性,而一些化疗药物可上调死亡受体DR的表达,从而促进TRAIL诱导的细胞凋亡。本研究旨在探讨重组人可溶性肿瘤坏死因子相关凋亡诱导配体蛋白rhTRAIL对耐顺铂人肺腺癌细胞凋亡的影响。方法常规体外培养耐顺铂人肺腺癌细胞株A549/DDP,应用MTT比色法、DPA法、流式细胞术和激酶法,检测rhTRAIL对耐顺铂人肺腺癌细胞株A549/DDP细胞增殖的抑制活性及其诱导细胞凋亡的效应。结果单用rhTRAIL时,低剂量rhTRAIL(6.25、12.5、25.0μg/L)对A549/DDP细胞的生长抑制率和各项凋亡指数(细胞凋亡率、DNA片段化比率和Caspase3活性)均无明显影响,P>0.05,但当rhTRAIL浓度在50μg/L时,细胞生长抑制率、细胞凋亡指数、DNA片段化比率和Caspase3活性均明显增高,各达68.6%、(27.13±0.66)%、(37.4±2.0)%和0.117±0.011(P<0.05)。当3mg/LDDP和rhTRAIL联用时,12.5μg/L的rhTRAIL即可使A549/DDP细胞生长抑制率、细胞凋亡指数、DNA片段化比率和Caspase3活性明显增高,各达30.4%、(19.39±0.54)%、(17.3±4.1)%和0.138±0.009(P<0.05)。结论单用较高剂量的rhTRAIL可以诱导耐顺铂人肺腺癌细胞A549/DDP细胞凋亡。低剂量顺铂可以增强rhTRAIL诱导A549/DDP细胞凋亡的效应。rhTRAIL可望成为耐药性肺癌治疗的一种重要的生物制剂。     

Targeted disruption of GAP-43 in P19 embryonal carcinoma cells inhibits neuronal differentiation. As well as acquisition of the morphological phenotype

GAP-43 is expressed in proliferating neuroblasts in vivo and in vitro, but its role during early neurogenesis has not been investigated. Here we show that neuroectodermal differentiation stimulated by retinoic acid (RA) in the embryonal carcinoma (EC) line P19 is accompanied by upregulation of GAP-43 expression in neuroepithelial precursor cells. In contrast, when upregulation of GAP-43 expression was prevented in 3 independent P19 lines because of a targeted insertion into the gene, generation of neuroepithelial precursors was inhibited. Consequently, neuronal number was significantly decreased, neuronal morphology was abnormal and fewer than 20% of all neurons were able to initiate neuritogenesis. Extracellular matrix (ECM) was unable to rescue initiation of neuritogenesis in the mutant cells, however those neurites that were extended responded normally to ECM-stimulated neurite outgrowth-promoting signals. These data suggest that GAP-43 function is required for commitment to a neuronal phenotype as well as initiation of neurite extension. However, stimulation of neurite outgrowth by ECM in P19s occurs independently of GAP-43.     

树突状细胞与卵巢癌细胞的融合及体外抗肿瘤作用的研究

目的 :比较脐血及以卵巢癌患者外周血来源的树突状细胞 (DC)的特点 ,研究DC 卵巢癌融合瘤苗的体外免疫应答效果。方法 :从脐血及卵巢癌患者外周血中诱导扩增DC ,从数量、形态、细胞表面标志及刺激增殖活性方面进行比较。体外培养卵巢癌患者癌细胞 ,将其与脐血DC在聚乙二醇 (PEG)介导下融合 ,活化自体T细胞 ,MTT法检测杀伤效果。结果 :体外诱导卵巢癌患者外周血DC ,每 2 0ml血能够获得 (0 .6～ 1.6 )× 10 6 个细胞 ;体外诱导脐血DC ,每 2 0ml血能够获得 (1.8～ 3.5 )× 10 6 个细胞 ,二者有明显差异 (P<0 .0 5 )。卵巢癌患者外周血DC在混和淋巴细胞反应 (mixedlymphocytereaction ,MLR)中显示了更为显著的刺激增殖活性。两者表达高水平的MHCII类分子和共刺激分子。经脐血DC 自体卵巢癌融合细胞活化的T细胞 ,对卵巢癌细胞株细胞的杀伤没有增加 ,而增强了对自体卵巢癌细胞的杀伤力。结论 :由脐血诱导可以获得更多数量的DC。脐血DC与自体卵巢癌细胞融合 ,能使肿瘤相关抗原有效提呈 ,激活T细胞 ,使它成为抗原特异性CTL ,有效杀伤自体癌细胞     

大鼠卵黄囊的分化及其肿瘤性转化研究

目的研究12d大鼠胚胎脏层卵黄囊(VYS)向多胚层组织分化的潜能和在逆转录病毒感染下的肿瘤性转化特征。方法在不同培养条件、移植位点的条件下,观察VYS体内外分化的改变;另外利用逆转录病毒载体将荧光蛋白基因(GFP)转染12d卵黄囊细胞,对GFP标记的转化细胞进行体内外研究。结果在不同培养条件下,均对体外培养的或体内移植的大鼠卵黄囊向三个胚层分化的进程无特异的导向性。将荧光蛋白标记卵黄囊克隆细胞接种在裸鼠皮下长出了未分化的间质细胞肉瘤。结论12d大鼠胚胎脏层卵黄囊具有向三胚层分化的潜能;逆转录病毒感染导致卵黄囊间质细胞发生肿瘤性转化。     

重组人粒细胞集落刺激因子对辐射诱发细胞恶性转化的影响

目的研究重组人粒细胞集落刺激因子(granulocyte colony-stimulatingfactor,G-CSF)对60Coγ射线诱发Wistar大鼠肺成纤维细胞恶性转化过程的影响。方法采用噻唑蓝(MTT)比色法、软琼脂克隆培养、流式细胞术(FCM)同时观察正常Wistar大鼠肺细胞、60Coγ射线照射细胞、经G-CSF预处理后60Coγ射线照射细胞、60Coγ射线照射后G-CSF持续处理细胞的增殖活性,克隆形成率和细胞周期的变化。结果G-CSF有抑制单纯照射细胞增殖的作用,使软琼脂克隆形成延迟,使进入细胞周期的细胞减少,细胞周期进程减慢。结论G-CSF能减缓细胞的恶性转化过程。     

A simple procedure for quantification of neurite outgrowth based on stereological principles

The molecular mechanisms controlling formation and remodelling of neuronal extensions are of considerable interest for the understanding of neuronal development and plasticity. Determination of neurite outgrowth in cell culture is a widely used approach to investigate these phenomena. This is generally done by a time consuming tracing of individual neurites and their branches. We have used stereological principles to determine the length of neurites. The total neuritic length per cell was estimated by counting the number of intersections between neurites and test lines of an unbiased counting frame superimposed on images of cell cultures obtained by conventional computer-assisted microscopy. The absolute length, L, of neurites per cell was subsequently estimated from the number of neurite intersections, I, per cell by means of the equation L=(πd/2)I describing the relationship between the number of neurite intersections and the vertical distance, d, between the test lines used. When measuring neurite outgrowth from PC12 cells and primary hippocampal neurons, data obtained by counting neuritic intersections correlated statistically significantly with data obtained using a conventional tracing technique. However, information was acquired more efficiently using the stereological approach. Thus, using the described set-up, the stereological procedure was approximately five times less time consuming than the conventional method based on neurite tracing. The study shows that stereological estimation of neuritic length provides a precise and efficient method for the study of neurite outgrowth in cultures of primary neurons and cell lines.     

A ventral approach to stereotaxy of the guinea pig brain

For skeletal muscles, a well-known match exists between the properties of motoneurones and those of their muscle fibres. Hence, the intramuscular distribution of different kinds of motoneuronal nerve endings (e.g. ‘slow’ versus ‘fast’) can be mapped by determining the distribution of the corresponding types of muscle fibre. As a background for further studies of motoneuronal plasticity, we needed precise measures of such distributions. Simple quantitative methods were developed for defining the position and extent of sub-populations of cells within a structure (e.g. the regional distribution of slow versus fast muscle fibres within a muscle cross-section): (a) The ‘mass vector method’ defined the relative position of the target cell cloud. A line was drawn between the calculated centre of mass for the target cells and that for the whole structure. The direction (a1) and length (a2) of this line gave a measure of the direction and degree of target cell eccentricity within the structure. (b) The ‘sector method’ delineated the region containing the target fibres. A circle around the centre of mass for the target fibres was subdivided into a number of equal sectors (standard setting: 20). The most remote point was found within each sector and a line joining these points defined the region of the target fibres. When applied to the ‘slow’ type I fibres of cross-sections from rat hindlimb muscles, the regional area estimates obtained by the sector method were highly correlated with, but 10% lower than those achieved by the well-established ‘convex hull’ method. Highly significant inter-muscular differences were observed for each one of the three new parameters described in this paper (a1, a2, b).     

CD44分子对胃癌细胞腹膜种植影响的体外实验研究

目的体外研究CD44分子对胃癌细胞(MKN45)腹膜种植转移的影响.方法在体外预先将抗CD44S抗体与MKN45细胞在细胞培养箱中作用45 min,然后在24孔培养板或Boyden小室中与生长良好的间皮细胞共同培养不同时间,在显微镜下直接计数与间皮细胞粘附的MKN45细胞,而用MTT法评估胃癌细胞在间皮细胞间的迁移和侵袭情况.结果在体外,抗CD44S抗体对MKN45细胞与间皮细胞间的粘附有明显的抑制作用(P＜0.01),在高浓度时对迁移和侵袭也有一定的抑制作用.结论CD44分子参与了胃癌细胞腹膜种植转移过程,抗CD44S抗体对胃癌细胞腹膜种植转移有一定的抑制作用.     

STAT1对人肾透明细胞癌放射敏感性的影响

目的 研究人肾透明细胞癌信号传递和转录活化因子1(STAT1)表达情况及抑制STAT1对该肿瘤细胞放射敏感性的影响.方法 采用免疫组织化学染色技术对比检测34例人肾透明细胞癌标本和12例正常肾组织标本的STAT1表达.用Western blotting法检测离体培养人肾透明细胞癌细胞(CRL-1932)、纤维母细胞(CCL-116)和wilm's瘤细胞(CRL-1441)的STAT1表达.用氟达拉滨和siRNA抑制CRL-1932细胞STAT1表达,并通过成克隆法和台盼蓝染色计数法研究抑制STAT1对CRL-1932细胞放射敏感性的影响.结果 人肾透明细胞癌标本的总STAT1和磷酸化STAT1表达均明显高于正常肾组织.Western blotting法显示CRL-1932细胞STAT1表达比CRL-1441、CCL-116细胞明显增高;药物氟达拉滨能显著抑制CRL-1932细胞磷酸化STAT1的表达,并显著增加CRL-1932细胞的放射敏感性,且放射增敏程度与药物浓度呈正相关.经STAT1 siRNA处理后CRL-1932细胞总STAT1和磷酸化STAT1的表达均被有效抑制,且细胞在照射后的存活分数显著下降.结论 肾透明细胞癌STAT1呈高表达,抑制STAT1对该细胞有放射增敏作用.     

血管内皮生长因子、微血管密度及Ki-67在宫颈鳞癌中的表达及意义

目的探讨血管内皮生长因子(VEGF)、微血管密度(MVD)及Ki-67在宫颈鳞状细胞癌中的表达及意义。方法采用免疫组织化学S-P法测定45例宫颈鳞癌组织和10例正常宫颈组织中VEGF、Ki-67和F8的表达。结果宫颈鳞癌中VEGF的表达阳性率为77.78%,Ki-67标记指数(Ki-67LI)和MVD分别为12.7±4.1和37±10,VEGF表达水平与肿瘤恶性程度、Ki-67 LI和MVD计数呈正相关。结论VEGF有促进肿瘤细胞增殖及血管生成的作用,联合检测VEGF、Ki-67和F8可作为病理诊断的补充,对患者预后判断和治疗方案的选择有重要的参考价值。