人骨髓基质细胞输注裸鼠后的体内分布

采用密度梯度离心和差速贴壁法分离培养人骨髓基质细胞.对细胞进行形态观察、表型分析、细胞生长曲线和成骨分化潜能测定.然后将第4代细胞通过尾静脉输注到未经任何处理的裸鼠体内,移植1周和4周后,利用RT-PCR检测人Alu基因在裸鼠不同脏器组织中的表达以判断人源性供体细胞的植入情况.结果表明培养的人骨髓基质细胞为CD34 -CD45-成纤维样细胞,并具有向成骨分化的能力,但第1至第5代人骨髓基质细胞增殖速度无显著性差异(P＞0.05).经尾静脉输注后,人源性供体细胞可在肺、肝、脾、肾、肌肉、心、肠、脑等多种脏器或组织中均有不同程度的分布.     

人肝癌细胞与肝星形细胞裸鼠皮下共培养体系的建立

目的:建立裸鼠皮下共培养人肝癌细胞与肝星形细胞模型,观察人肝癌细胞与肝星形细胞间相互作用后超微结构的改变.方法:将16只裸鼠分为两组,肝癌细胞单独培养组和癌细胞与肝星形细胞共培养组,40天后将荷瘤组织切片行光镜及透射电镜观察.结果:肝癌细胞单独培养组中可观察到肝癌细胞的胞质液化及早期细胞凋亡现象,而共培养组中可见肝星形细胞时肝癌细胞的趋化现象,可观察到肝癌细胞结构完整且有增殖趋势.结论:裸鼠皮下荷瘤三维立体模型建立成功,该模型能够模拟肝癌微环境中肝癌细胞与肝星形细胞问的作用,为进一步研究肝癌细胞与肝星形细胞间的相互作用奠定了基础.     

罗格列酮对裸鼠移植性人肝癌细胞生长的抑制作用

目的:研究罗格列酮(rosiiglitazone,ROZ)对裸鼠体内人肝癌SMMC7721细胞增殖的影响.方法:建立人肝癌裸鼠移植瘤模型,实验动物分为罗格列酮治疗组及对照组.观察罗格列酮对移植瘤体积和重量的变化,采用光镜观察移植瘤细胞形态学变化,流式细胞术检测移植瘤细胞周期分布,免疫组织化学和蛋白印迹检测瘤组织内增殖细胞核抗原(proliferating cellnuclear antigen,PCNA)蛋白表达变化.结果:罗格列酮在体内能明显抑制移植瘤的生长,抑瘤率为50.81%;组织学显示治疗组瘤细胞异型性降低,核质比下降;流式细胞术分析显示,对照组移植瘤细胞处于G2/M期细胞为13.1%,与对照组比较,治疗组处于G2/M期细胞显著增多(29.1%).免疫组织化学和蛋白印迹检测均显示检测显示,经罗格列酮处理后,裸鼠移植瘤组织PCNA蛋白表达明显下调(P＜0.05).结论:罗格列酮可体内抑制人肝癌SMMC7721细胞增殖,并使癌细胞阻滞于G2/M期.     

7-二氟甲氧基-5,4'-二正辛烷氧基金雀异黄素对人卵巢癌裸鼠移植瘤生长的影响

目的:研究7-二氟甲氧基-5,4'-二正辛烷氧基金雀异黄素(7-difluoromethoxyl-5,4'-di-n-octylygenistein,DFOG)对人卵巢癌裸鼠移植瘤生长的影响。方法:建立人卵巢癌HO-8910细胞裸鼠皮下移植瘤,随机分为5组,每组5只,即生理盐水组(Saline),紫杉醇组(TAX),DFOG此处为3组(5、10、20 mg/kg)组,观察各组移植瘤的体积和重量变化,观察各组荷瘤裸鼠重量及其血清乳酸脱氢酶(LDH)、谷丙转氨酶(ALT)、肌酐值(Cr)、外周血白细胞计数(WBC)的变化。FCM检测移植瘤组织细胞的凋亡率,Western blot检测移植瘤组织细胞cortactin蛋白的表达变化。结果:DFOG各组显著抑制人卵巢癌HO-8910细胞裸鼠移植瘤生长,与Saline组比较,移植瘤体积明显减小(P均0.05),其中DFOG10mg/kg组对移植瘤抑制率与TAX组相当(P0.05);与Saline组比较,DFOG各组移植瘤重量明显减轻(P均0.05),其中DFOG10mg/kg组对移植瘤重量减少率与TAX组相当(P0.05)。与Saline组及DFOG各组比较,TAX组荷瘤裸鼠重量显著降低(P均0.05),而Saline组和DFOG各组之间荷瘤裸鼠重量比较差异无统计学意义(P0.05);与Saline组及DFOG各组比较,TAX组荷瘤裸鼠血清LDH、ALT、Cr值和外周血WBC计数显著降低(P均0.05),而Saline组与DFOG各组比较,荷瘤裸鼠血清LDH、ALT、Cr值和外周血WBC计数无明显差异(P0.05)。DFOG各组作用HO-8910细胞移植瘤16 d后,HO-8910细胞发生凋亡,与Saline组比较(P均0.05),其中DFOG10 mg/kg组凋亡率与TAX组相当(P0.05);同时cortactin蛋白表达降低,与Saline组比较(P均0.05),其中DFOG10 mg/kg组凋亡率与TAX组相当(P0.05)。结论:DFOG能抑制卵巢癌HO-8910细胞裸鼠移植瘤生长,下调cortactin蛋白表达和诱导HO-8910细胞凋亡。     

人IL-17F对裸鼠人肝癌移植瘤生长的影响

利用RT-PCR方法从PHA活化的人外周血单个核细胞(PBMCs)中克隆hIL-17F基因,亚克隆至逆转录病毒载体pSIV-1,与辅助病毒载体pHIT456和pHIT60脂质体法共转染293T包装细胞,获得的成熟重组逆转录病毒(RV-hIL-17F)再感染SMMC-7721人肝癌细胞,并经G418筛选建立hIL-17F转基因肝癌细胞。PCR、RT-PCR和Westernblot结果表明hIL-17F基因在肝癌细胞中能成功整合、转录和表达。MTT和FCM结果表明hIL-17F不能改变SMMC-7721肝癌细胞的增殖活力和细胞周期,但ELISA结果表明其能明显下调肝癌细胞IL-6、IL-8和VEGF的表达。转基因肝癌细胞rhIL-17F表达上清具有抑制ECV304人脐静脉内皮细胞生长的作用。裸鼠皮下成瘤试验结果表明hIL-17F转基因肝癌细胞裸鼠致瘤能力明显减弱,VEGF和CD34表达降低,血管形成显著减少。hIL-17F可通过减少肿瘤血管形成显著抑制裸鼠人肝癌移植瘤的生长,为其进一步开展肿瘤血管靶向基因治疗和开发抗血管新药提供了一定的实验依据。     

癌基因及癌发育基因在裸小鼠人肝癌移植瘤中表达

本文采用Northern blot,斑点杂交,western Immunoblot,凝集素亲和电泳及聚丙烯酰胺梯度电泳方法分别检测裸小鼠肝癌移植瘤中C-ras基因簇(N-ras,H-ras及K-ras),甲种胎儿蛋白以及γ谷氨酰转肽酶(GGT)基因及其产物的表达。结果提示C-ras基因簇均有不同程度的表达,其中N-ras基因表达水平要比H-ras高8倍,比K-ras高20倍左右,同样AFP及GGT基因均有较高水平的表达。经ras-p21单克隆抗体检测,发现肝癌组织抽提液中p21呈现阳性反应;AFP亚型分析提示AFP分子变异体属肝癌特异性分子亚型;GGT同Ⅰ酶酶谱分析表明组织中仅合成肝癌特异性GGT同Ⅰ酶酶谱(Ⅰ’,Ⅱ’)。上述结果充分表明癌基因及癌发育基因的高表达可能与癌细胞去分化性,分裂旺盛程度及细胞恶性度相关。最后文内还对裸小鼠人肝癌移植瘤中二类基因的相关性作一定的讨论。     

Cutting Edge: Suppression of GM-CSF Expression in Murine and Human T Cells by IL-27

GM-CSF is a potent proinflammatory cytokine that plays a pathogenic role in the CNS inflammatory disease experimental autoimmune encephalomyelitis. As IL-27 alleviates experimental autoimmune encephalomyelitis, we hypothesized that IL-27 suppresses GM-CSF expression by T cells. We found that IL-27 suppressed GM-CSF expression in CD4(+) and CD8(+) T cells in splenocyte and purified T cell cultures. IL-27 suppressed GM-CSF in Th1, but not Th17, cells. IL-27 also suppressed GM-CSF expression by human T cells in nonpolarized and Th1- but not Th17-polarized PBMC cultures. In vivo, IL-27p28 deficiency resulted in increased GM-CSF expression by CNS-infiltrating T cells during Toxoplasma gondii infection. Although in vitro suppression of GM-CSF by IL-27 was independent of IL-2 suppression, IL-10 upregulation, or SOCS3 signaling, we observed that IL-27-driven suppression of GM-CSF was STAT1 dependent. Our findings demonstrate that IL-27 is a robust negative regulator of GM-CSF expression in T cells, which likely inhibits T cell pathogenicity in CNS inflammation.     

L1 Cell Adhesion Molecule-Specific Chimeric Antigen Receptor-Redirected Human T Cells Exhibit Specific and Efficient Antitumor Activity against Human Ovarian Cancer in Mice

New therapeutic modalities are needed for ovarian cancer, the most lethal gynecologic malignancy. Recent clinical trials have demonstrated the impressive therapeutic potential of adoptive therapy using chimeric antigen receptor (CAR)-redirected T cells to target hematological cancers, and emerging studies suggest a similar impact may be achieved for solid cancers. We sought determine whether genetically-modified T cells targeting the CE7-epitope of L1-CAM, a cell adhesion molecule aberrantly expressed in several cancers, have promise as an immunotherapy for ovarian cancer, first demonstrating that L1-CAM was highly over-expressed on a panel of ovarian cancer cell lines, primary ovarian tumor tissue specimens, and ascites-derived primary cancer cells. Human central memory derived T cells (T_CM) were then genetically modified to express an anti-L1-CAM CAR (CE7R), which directed effector function upon tumor antigen stimulation as assessed by in vitro cytokine secretion and cytotoxicity assays. We also found that CE7R⁺ T cells were able to target primary ovarian cancer cells. Intraperitoneal (i.p.) administration of CE7R⁺ T_CM induced a significant regression of i.p. established SK-OV-3 xenograft tumors in mice, inhibited ascites formation, and conferred a significant survival advantage compared with control-treated animals. Taken together, these studies indicate that adoptive transfer of L1-CAM-specific CE7R⁺ T cells may offer a novel and effective immunotherapy strategy for advanced ovarian cancer.     

格列卫与多西紫杉醇联合对人乳腺癌细胞株MCF-7及其裸鼠移植瘤的作用

目的：探讨分子靶向药物格列卫与多西紫杉醇联合对人乳腺癌细胞株MCF-7的凋亡及其裸鼠皮下移植瘤生长的影响。方法：采用流式细胞检测仪检测MCF-7细胞在格列卫与多西紫杉醇单独处理及共同处理条件下的凋亡率;建立人乳腺癌细胞株MCF-7裸鼠皮下移植瘤模型,观察格列卫与多西紫杉醇单独治疗组及联合治疗组移植瘤的生长,计算抑瘤率。结果：MCF-7细胞在格列卫与多西紫杉醇共同处理条件下的凋亡率（50.86%）远高于多西紫杉醇单独处理（22.06%）及同剂量格列卫组单独处理（8.13%）条件下的凋亡率;高剂量多西紫杉醇与格列卫联合组肿瘤质量与单独多西紫杉醇组比较,差异虽然没有显著性,但联合组抑瘤率高达99.55%,高于多西紫杉醇组（97.43%）,q值为1.014;中剂量多西紫杉醇与格列卫联合组肿瘤质量与单独多西紫杉醇组比较,差异有高度显著性,联合组抑瘤率达96.53%,高于多西紫杉醇组（92.01%）,q值为1.02;低剂量多西紫杉醇与格列卫联合组肿瘤质量与单独多西紫杉醇组比较,差异有高度显著性,联合组抑瘤率达68.20%,高于多西紫杉醇组（58.40%）,q值为1.004。结论：格列卫与凋亡诱导剂多西紫杉醇共同处理MCF—7细胞能达到协同诱导凋亡的效果;高、中、低剂量的多西紫杉醇与格列卫联合对人乳腺癌细胞株MCF-7裸鼠移植瘤增殖的抑制具有相加作用。