具有缺氧反应性的Notch信号抑制蛋白调节鼠结肠癌细胞的体内生长

目的:研究缺氧细胞中的Notch信号对小鼠结肠癌细胞体内生长的影响.方法:将缺氧诱导因子 (hypoxia-inducible factor,HIF)- 1α中的氧依赖性蛋白降解结构域(oxygen-dependent degradation domain,ODD)、抑制Notch信号的mastermind样(mastermind-like,MAML)突变体蛋白1和绿色荧光蛋白(green fluorescent protein, GFP)重组构建融合蛋白表达载体.通过在免疫荧光显微镜下观察荧光强度,应用Western 印迹和实时荧光定量PCR(real-time fluorogentic quantitative-PCR,RFQ-PCR)检测融合蛋白对缺氧的反应性和抑制Notch信号的能力.通过失巢凋亡实验和小鼠体内肿瘤形成实验,观察融合蛋白DNMAML1-ODD-GFP(DOG)对CT26结肠癌细胞体外失巢凋亡和小鼠体内肿瘤生长的影响.结果:DOG融合蛋白在细胞中的稳定表达依赖于缺氧条件,该融合蛋白能够靶向抑制缺氧CT26细胞中的Notch信号靶基因Hes1 mRNA的表达(P<0.01).体外失巢凋亡实验表明,DOG融合蛋白可显著促进缺氧CT26细胞的失巢凋亡(P<0.01).小鼠体内成瘤实验显示,DOG融合蛋白的表达可抑制结肠癌细胞CT26在小鼠体内的生长(P<0.01).结论:成功构建了靶向阻断缺氧细胞中Notch信号的融合蛋白表达载体.缺氧结肠癌细胞中的Notch信号对于肿瘤生长起重要作用.     

间隙连接蛋白Connexin32对肝癌侵袭和转移能力的影响

目的:探讨肝癌细胞中间隙连接蛋白Connexin32对肝癌细胞体内侵袭和转移能力的影响.方法:利用可以通过增减细胞外环境中Doxycycline调控Connexin32蛋白表达的肝癌细胞HuH7 Tet-off Cx32亚克隆,将之原位移植到重度联合免疫缺陷鼠肝脏浆膜下,通过饮用水中添加(去除)Doxycycline调控动物体内Connexin32的表达,8周后处死小鼠,观察肿瘤形成及转移情况,蛋白质印迹法及免疫组织化学方法检测其与对照组的Connexin32蛋白表达情况.结果:移植HuH7 Tet-off Cx32细胞的小鼠,在饮用水中去除Doxycycline诱发Connexin32高表达条件下,形成明显的肝内转移灶和腹膜转移灶;而饮用水中添加了Doxycycline的小鼠没有发现明显的转移灶形成.免疫组化证实,高表达的Connexin32蛋白定位于细胞质.蛋白质印迹法结果显示,Doxycycline在小鼠体内呈现较好的诱导性.结论:细胞质内异位蓄积的Connexin32蛋白明显增强肝癌细胞的体内侵袭和转移能力.     

mSDF-1γ/GM-CS融合蛋白的制备及其对辐射小鼠造血损伤恢复的促进

目的: 利用毕赤酵母表达系统表达mSDF-1γ/GM-CSF融合蛋白,研究该融合蛋白对辐射小鼠的促造血增殖作用和免疫增强作用.方法:化学合成mSDF-1γ/GM-CS基因,构建携带该基因的表达载体,转化酵母后分泌表达重组融合蛋白,离子交换柱纯化,SDS-PAGE和Western blotting分析鉴定.60Co γ射线照射制备小鼠辐射模型,以融合蛋白皮下注射进行治疗,观察辐射小鼠骨髓造血细胞的增殖活性及免疫细胞的趋化活性.结果:成功构建表达载体pPIC9K- SDF1-rh-GM-CSF1,转化毕赤酵母菌株GS115后表达融合蛋白mSDF-1γ/GM-CS,其相对分子质量为32 000,含量为78 ng/ml.融合蛋白治疗后,辐射小鼠骨髓单个核细胞增殖活性、GM-CFU形成能力明显提高(均P<0.01)、骨髓细胞凋亡率非常明显地降低(P<0.05或P<0.01),小鼠外周血CD3+CD4+T细胞数显著高于辐射组和GM-CSF治疗组(均P<0.05).结论: mSDF-1γ/GM-CSF融合蛋白具有促进造血和免疫细胞增殖的双重活性,有望在抗肿瘤免疫和造血调控中开发成为有应用前景的新型细胞因子.     

cEGFR-rFc融合DNA疫苗抗小鼠黑素移植瘤的效果

目的:构建cEGFR-rFc融合DNA疫苗, 观察其对小鼠黑素移植瘤的抑制作用.方法:以异种同源鸡EGFR(chicken EGFR,cEGFR)膜外部分与兔疫球蛋白IgG的Fc段(rabbit IgG Fc,rFc)为基础构建真核表达载体pVAX1/cEGFR-rFc,脂质体法转染黑素瘤B16 细胞,免疫荧光法检测融合蛋白在B16细胞中的表达;以融合DNA疫苗免疫C57BL/6J小鼠,Western blotting法检测融合蛋白在小鼠体内的稳定表达.疫苗免疫小鼠接种B16黑素瘤细胞,14 d后处死部分小鼠,取出瘤体称重,计算抑瘤率;同时观察小鼠荷瘤后的生存率;ELASIA法检测DNA疫苗免疫小鼠血清抗EGFR抗体滴度,流式细胞仪测定小鼠脾细胞淋巴细胞亚群.结果:成功构建融合DNA疫苗pVAX1/ cEGFR-rFc,重组载体转染B16细胞后能检测到融合蛋白显著表达,疫苗免疫小鼠后能检测融合蛋白体内稳定表达.融合DNA疫苗能够延缓小鼠黑素移植瘤的生长,抑瘤率为54%(P<0.01);能延长荷瘤小鼠的生存期(疫苗组小鼠接种瘤细胞后30 d的生存率为40%);融合DNA疫苗诱发小鼠产生高滴度抗EGFR抗体(效价1∶1 000)和T细胞免疫(疫苗组小鼠脾脏CD8 T淋巴细胞数目显著增加, P<0.01).结论:cEGFR-rFc融合DNA疫苗能产生有效对抗黑素瘤B16细胞的免疫效应.     

人肺癌裸小鼠模型活体成像的动态观察

目的:建立稳定表达绿色荧光蛋白的人肺癌细胞系,并探讨小动物活体荧光成像系统在肺癌皮下移植瘤模型中的应用.方法:用慢病毒转染的方法建立表达绿色荧光蛋白的人肺癌细胞系NCI-H460-GFP,接种至裸小鼠体内建立皮下移植瘤模型,通过小动物活体成像系统连续5周观察肿瘤在小鼠皮下的动态生长情况.结果:建立了转染率接近100%的人肺癌NCI-H460-GFP细胞系,在体外及裸小鼠体内均能够长期稳定表达绿色荧光蛋白.活体荧光成像观察发现,1～4周随着肿瘤体积逐渐增大,平均荧光光子数逐渐增加;5周时随着肿瘤出现明显坏死,平均荧光光子数呈现下降趋势.结论:稳定表达绿色荧光蛋白的NCI-H460-GFP细胞系及其动物模型可以为肺癌研究提供理想的实验材料,应用小动物活体成像系统能够客观定量评价肿瘤在动物体内的生长情况.     

肝癌相关基因pp1158的表达及功能研究

目的 对pp1158基因的蛋白表达和体内外功能进行研究。方法 用pET-32a融合表达载体在大肠杆菌BL21（DE3）对该基因进行表达,制备兔源性多克隆抗体;用体外细胞集落形成试验、裸鼠体内成瘤实验、免疫组化及蛋白印迹,检测该基因对肿瘤细胞生长的影响及表达差异;用Northern blot检测pp1158在组织中的表达差异。结果 pp1158对BEL 7402肝癌细胞系的体外集落形成有明显抑制作用,抑制率为58．3％（P＜0．01）;对BEL 7402细胞的成瘤有明显抑制作用（P＜0．05）。蛋白印迹结果显示,转染pCMV-Script-pp1158的BEL 7402细胞表达pp1158蛋白。免疫组化结果显示,pp1158基因在人组织中的表达为：正常肝组织≥癌旁肝组织＞肝癌组织。Northern blot结果显示,pp1158在人胎盘组织中高表达,在心、肝、骨骼肌、胰腺、肺等脏器中度表达,而在脑和肾组织中表达很低。结论 pp1158为肝细胞性肝癌的侯选抑瘤基因。     

川楝素对H22肝癌荷瘤小鼠的抑瘤作用

目的:建立荷瘤小鼠模型,探讨川楝素对肝癌的抑瘤作用及其机制.方法:建立H22肝癌移植瘤小鼠模型,随机分为0.9%氯化钠溶液对照组、环磷酰胺(cyclophosphamide,CTX)组(20 mg/kg)、川楝素低剂量组(0.173 mg/kg)和川楝素高剂量组(0.690 mg/kg)共4组.各组药物处理后,测量小鼠体内肿瘤大小,观察肿瘤的生长曲线;剥瘤后称重,计算小鼠的肿瘤抑制率;行肿瘤组织病理形态学及HE染色观察,透射电子显微镜观察其超微结构改变;同时,观察川楝素对荷瘤小鼠心、肝、脾、肾、胸腺及睾丸组织的影响;免疫组织化学法检测肿瘤组织内Bcl-2、Bax和Fas蛋白的表达.结果:川楝素可显著抑制小鼠体内肿瘤的生长,川楝素低剂量组(0.173 mg/kg)和高剂量组(0.690 mg/kg)的抑瘤率分别为66.23%和87.01%(P＜0.05);透射电子显微镜观察可见肿瘤组织超微结构中出现凋亡小体;HE染色显示小鼠心、肝、脾、肾及睾丸脏器形态正常,而胸腺组织中可见胸腺小叶的数量及面积减少甚至消失;免疫组织化学检测证实,小鼠肿瘤组织内Bcl-2蛋白表达下调,Bax和Fas蛋白表达上调(P＜0.05).结论:川楝素能够明显抑制小鼠移植瘤的生长,此作用可能与抑制肿瘤细胞增殖及促进肿瘤细胞凋亡有关.     

Tat-p53融合蛋白在乳腺癌细胞的表达、纯化及其转导活性

目的:研究Tat-p53融合蛋白在乳腺癌细胞的表达、纯化及其转导活性,探讨蛋白转导方法在乳腺癌治疗中应用的可能性。方法:利用RT-PCR方法从A549细胞系中分离野生型p53基因,将该基因克隆入pTAT-HA原核表达载体,在大肠杆菌BL21(DE3)LysS内诱导表达并进行纯化。将Tat-p53融合蛋白加入MCF-7人乳腺癌细胞培养上清,检测Tat-p53融合蛋白导入MCF-7人乳腺癌细胞的情况。结果:成功地构建了含有野生型p53基因的原核表达载体,表达纯化了Tat-p53融合蛋白,证实Tat-p53可以高效转入MCF-7人乳腺癌细胞内。结论:TatPTD可以发挥高效的蛋白转导特性将p53导入人乳腺癌细胞,p53在细胞核内可以发挥生物学活性,抑制肿瘤细胞生长并诱导其发生凋亡。     

癌基因LMO3的原核表达及多克隆抗体的制备及应用

目的:制备抗神经特异性表达的癌基因LMO3的多克隆抗体,进行LMO3分子检测和功能研究.方法:采用RT-PCR方法扩增LMO3的基因序列,构建其原核表达载体pET32a-LMO3.在大肠埃希菌中诱导融合蛋白表达,SDS-PAGE分离纯化原核表达的融合蛋白,PBS溶解聚丙烯酰胺凝胶颗粒中的融合蛋白,乳化后免疫新西兰兔制备多克隆抗体.采用免疫印迹、免疫组化等对该抗体的特异性进行鉴定.结果:构建的重组质粒经酶切鉴定和测序证实序列正确.经诱导表达在相对分子质量为37×103处有一条明显的蛋白条带,与预期值一致;免疫动物所得抗血清效价为1∶16 000,该抗体能够识别原核表达的LMO3蛋白及细胞内源性表达的LMO3蛋白;免疫组化显示,LMO3在SHG44胶质瘤细胞株及脑胶质瘤组织中均有表达. 结论:制备了能识别天然LMO3分子的抗LMO3的多克隆抗体,为检测LMO3分子的表达和进一步研究其功能提供了有力的工具.     

癌蛋白iASPP对小鼠骨髓细胞增生促进作用研究

 目的　研究iASPP（一个新发现的p53家族抑制性成员）在小鼠骨髓细胞中过表达对细胞增生作用的影响。方法　构建反转录病毒载体,通过基因转导方法,使小鼠骨髓细胞过表达人类iASPP或（和）突变的N-Ras,采用集落形成实验检测细胞体外增生能力的变化。结果　转导iASPP的小鼠骨髓细胞与转导空载体对照组相比集落形成能力显著增强（P＜0.01）,转导突变的N-Ras也能提高小鼠骨髓细胞的体外增生能力,二者差异无统计学意义（P＞0.05）,但iASPP联合突变的N-Ras可使集落的数量显著多于单基因组（P＜0.05）及空载体对照组（P＜0.01）,而且集落体积增大。结论　癌蛋白iASPP可以显著提高小鼠骨髓细胞的体外增生能力,并且与突变的N-Ras具有协同作用,因而iASPP有望成为研究肿瘤及白血病治疗的新靶点。     

Irinotecan and radiation in vitro and in vivo

Loss of chromosome sequences at 13q14 (Rbl) and 17p13 (p53) associated with squamous cell carcinoma of head and neck (SCCHN) was evaluated in 12 recurrent tumors and 51 primary tumors from 63 patients. The incidence of LOH at 17p13 was 19 of 50 (38%) tumors, and at 13q14 was 21 of 57 (37%). LOH affecting Rbl and/or p53 was observed in 30 of 63 (48%) SCCHN. Coincident LOH at Rbl and p53 was detected in 10 of 46 (22%) tumors. There were nine cases in which primary and metastatic tumors were obtained from the same patient. Of these, seven were informative and five of these (71%) manifested LOH at p53 in both primary and metastatic sites. Examination of Rbl in these same tumors showed LOH in six of the nine metastases, and of these six, only three revealed LOH in the primary tumor. LOH at p53 or Rbl alone showed no correlation with clinical outcome. However, tumors that manifested LOH at both loci were associated with poorer patient outcome and poorer histological differentiation.     

TCP and NTCP in preclinical and clinical research in Europe

European research in radiation oncology has a long and successful tradition. The aim of this research is to increase the therapeutic window of radiotherapy by increasing the tumor control probability (TCP) and/or by decreasing the normal tissue complication probability (NTCP). This paper summarizes the basic radiobiological concept underlying treatment optimization by TCP-NTCP data and discusses some of the limitations of currently used models. These are controversial in many aspects and cannot be recommended for clinical routine practice but should rather be considered as a research tool.     

Advances in molecular diagnostics and therapeutics in head and neck cancer

Opinion statement Extensive treatment-related morbidities and stagnant survival rates over the past few decades for patients with squamous cell cancer of the head and neck (SCCHN) emphasize the need for novel diagnostics and therapeutics based on the molecular characteristics of the tumor. The development of an early detection test remains largely preliminary. Much attention has recently been given to saliva-based early detection assays that use accepted tumor markers such as p53 and DNA methylation. Most of these studies have focused on feasibility as opposed to prospective clinical trials. To date, early detection saliva assays have failed to yield a high enough sensitivity and specificity for broad population-based screening. The use of saliva as a noninvasive, inexpensive, and accessible diagnostic substrate remains desirable. Unlike SCCHN diagnostics, molecular-targeted therapies for SCCHN will soon be a reality, with many more compounds in the pipeline. The most promising of these drugs target the epidermal growth factor receptor (EGFR), which is known to be overexpressed in squamous cell carcinomas. Cetuximab, a monoclonal EGFR antibody, has shown efficacy in combination with radiotherapy in advanced SCCHN in a recent phase III trial and is currently being petitioned for US Food and Drug Administration approval. Likewise, erlotinib, an EGFR tyrosine kinase inhibitor, has shown favorable results in phase II trials as monotherapy and in combination with chemotherapy. Gefitinib, another EGFR tyrosine kinase inhibitor, has shown efficacy as monotherapy, in combination with chemotherapy, and with chemoradiotherapy. At least two phase III trials of gefitinib in patients with advanced SCCHN are ongoing. Such low-toxicity, tumor-specific targeting strategies will soon be available for patients with head and neck cancer. The challenge is to establish assays to determine which patients are most likely to benefit from these agents.     

Daunorubicin and daunorubicinol pharmacokinetics in plasma and tissues in the rat

Recent evidence suggests that 13-hydroxy metabolites of anthracyclines may contribute to cardiotoxicity. This study was designed to determine the pharmacokinetics of daunorubicin and the 13-hydroxy metabolite daunorubicinol in plasma and tissues, including the heart. Fisher 344 rats received 5 mg kg^–1 daunorubicin i.v. by bolus injection. Rats were killed at selected intervals for up to 1 week after daunorubicin administration for determination of concentrations of daunorubicin and daunorubicinol in the plasma, heart, liver, kidney, lung, and skeletal muscle. Peak concentrations of daunorubicin were higher than those of daunorubicinol in the plasma (133±7 versus 36±2 ng ml^–1;P<0.05), heart (15.2±1.4 versus 3.4±0.4 g g^–1;P<0.05), and other tissues. However, the apparent elimination half-life of daunorubicinol was longer than that of daunorubicin in most tissues, including the plasma (23.1 versus 14.5 h) and heart (38.5 versus 19.3 h). In addition, areas under the concentration/time curves (AUC) obtained for daunorubicinol exceeded those found for daunorubicin in almost all tissues, with the ratios being 1.9 in plasma and 1.7 in the heart. The ratio of daunorubicinol to daunorubicin concentrations increased dramatically with time from <1 at up to 1 h to 87 at 168 h in cardiac tissue. Thus, following daunorubicin injection, cumulative exposure (AUC) to daunorubicinol was greater than that to daunorubicin in the plasma and heart. If daunorubicinol has equivalent or greater potency than daunorubicin in causing impairment of myocardial function, it may make an important contribution to the pathogenesis of cardiotoxicity.     

Estrogen and Progesterone in Normal Mammary Gland Development and in Cancer

There is emerging evidence that the mammary epithelium in both mice and humans is arranged as a hierarchy that spans from stem cells to differentiated hormone-sensing, milk-producing and myoepithelial cells. It is well established that estrogen is an important mediator of mammary gland morphogenesis and exposure to this hormone is associated with increased breast cancer risk. Yet surprisingly, the primitive cells of the mammary epithelium do not express the estrogen receptor-α (ERα) or the progesterone receptor. This article will review the mammary epithelial cell hierarchy, possible cells of origin of different types of breast tumors, and the potential mechanisms on how estrogen and progesterone may influence the different subcomponents in normal development and in cancer. Also presented are some hypothetical scenarios on how this underlying biology may be reflected in the behavior of ERα(+) and ERα(-) breast tumors.     

Catalysis of nitrosation in vitro and in vivo in rats by catechin and resorcinol and inhibition by chlorogenic acid

Measurements were made of the effects of phenolic compounds,some of which are present in the human diet, on the nitrosationof proline by nitrite to give N-nitrosoproline (NPRO). In vitro,resorcinol, catechin, p-nitrosophenol and phenol were catalystsand chlorogenic acid an inhibitor; guaiacol showed a marginalcatalytic effect. Both the catalytic and the inhibiting effectswere dependent on pH and on the concentration of phenolic compounds;catalysis by resorcinol and catechin was increased at optimalratios of [nitrite]: [phenolic compound]. Endogenous nitrosationwas examined in vivo by co-administration of nitrite, prolineand a phenolic compound to rats and by monitoring the amountof NPRO excreted in the urine. Under similar experimental conditions,the catalytic effects observed in vivo decreased in the sameorder as those observed in vitro: resorcinol > p-nitrosophenol> catechin > phenol guaiacol; chlorogenic acid acted asan inhibitor. Catalysis and inhibition of N-nitrosation in ratsin vivo appears to occur via mechanisms similar to those invitro, although the effects in vivo were smaller. The implicationsof our findings for the endogenous formation of N-nitroso compoundsand for variations in exposure due to different dietary constituentsin humans are discussed.