Dual-color imaging of angiogenesis and its inhibition in bone and soft tissue sarcoma

BACKGROUND: Angiogenesis is a critical step in tumor growth, progression, and metastasis. Soft tissue and bone sarcoma are resistant to most therapeutic approaches. Angiogenesis of these tumors may be an effective target. We hypothesized that we could inhibit tumor growth by targeting angiogenesis in a mouse model of sarcoma. We demonstrate in this report, using powerful color-coded fluorescent imageable tumor-host models, the onset of angiogenesis of these sarcomas and its inhibition. MATERIALS AND METHODS: Transgenic mice were used as the host in which green fluorescent protein (GFP) is driven by a regulatory element of the stem cell marker nestin (ND-GFP). Nascent blood vessels express ND-GFP in this model. We visualized, by dual-color fluorescence imaging, angiogenesis of sarcoma formed by the HT-1080 human fibrosarcoma cell line expressing red fluorescent protein (RFP) in the ND-GFP mice. Tumor cells were injected into either the muscle or the bone. RESULTS: Nestin was highly expressed in proliferating endothelial cells and nascent blood vessels in the growing tumors, including the surrounding tissues. Immunohistochemical staining showed that CD31 colocalized in ND-GFP-expressing nascent blood vessels. The density of nascent blood vessels in the tumor was readily quantitated. The mice were given daily i.p. injections of 5 mg/kg of doxorubicin after implantation of tumor cells. Doxorubicin significantly decreased the mean nascent blood vessel density in the tumors as well as decreased tumor volume. CONCLUSION: The dual-color model of the ND-GFP nude mouse and RFP sarcoma cells is useful for the visualization and quantitation of bone and soft tissue tumor angiogenesis and evaluation of angiogenic inhibitors for such tumors. These data suggest targeting angiogenesis of sarcomas as a promising clinical approach.     

σ因子在四种结核分枝杆菌临床株中的表达分析

目的 探索在体外培养的环境下,结核分枝杆菌13种σ因子在临床分离的敏感菌株、单耐异烟肼(INH)、单耐利福平(RFP))、耐多药(同时耐INH和RFP)菌株中的表达情况。方法 选取2018年1—12月天津市结核病控制中心门诊部确诊的结核病患者,对其痰标本快培阳性的实验菌株进行比例法药敏实验,随机分别选择10株全敏菌株、单耐异烟肼、单耐利福平、同时耐利福平和异烟肼的菌株进行Hain实验,随机分别选择10株katG 突变的单耐异烟肼菌株、rpoB 突变的单耐利福平菌株、katG 和rpoB 同时突变的耐多药临床分离株。以传统的Trizol方法提取全菌RNA,设计σ因子的特异性引物,以荧光定量PCR的方法,检测σ因子在转录水平上RNA的表达情况。以核糖体16s为参照基因,利用2^-ΔCt分析σ因子的转录倍数。将稳定表达的sigA作为统计分析的底物,单因素方差(ANOVA)分析其余σ因子的表达差异。结果 σ因子在四种临床分离株中的表达情况与H37Rv相比,虽然所有的σ因子转录水平都提高,但是差异无统计学意义(P >0.05)。三种耐药表型菌株和敏感菌相比,σ因子表达量也都上调,在单耐异烟肼的菌株中,sigC、sigG、sigI、sigJ、sigK表达倍数最高,单因素方差分析差异有统计学意义(P <0.05)。在单耐利福平表型中,只有sigC、sigG表达倍数最高,但差异无统计学意义(P >0.05)。在耐多药菌株中,sigC表达倍数最高,表达倍数>10(P <0.05)。结论 耐药基因的突变能够上调σ 因子的转录水平,σ 因子中sigB、sigC、sigG、sigI、sigJ、sigK可能会成为研究耐药新药方面的新的靶标。     

RFP—BSA免疫家兔及RFP特异IgG的检测

用利福平(RFP)与牛血清白蛋白(BSA)在碱性条件下反应生成稳定的偶联物。偶联比约为RFP:BSA=3.1:1。用此偶联物免疫家兔获得抗血清经ELISA竞争抑制实验证明了RFP特异IgG的存在。其效价约为1:4000。说明RFP具有免疫原性,在一定条件下可以使动物致敏。初步建立了田ELISA检测RFP特异IgG的方法。     

HPLC对30例肺结核患者口服利福平胶囊的血药浓度监测

目的建立监测肺结核患者口服利福平后的血药浓度,为临床合理用药提供可靠依据。方法高效液相法色谱条件:NOVAPAKC18柱,(4μm,3.9mm×150mm);流动相:甲醇0.02mol·L-1醋酸钠溶液(62∶40);流速1.0ml/min,检测波长为334nm;柱温20℃。结果血清中利福平在0.5～16.0μg·ml-1范围内浓度与峰面积线性关系良好,r=0.9992,平均回收率为99.43%。30例肺结核患者一次口服利福平600mg后,28例血药浓度检测结果在0.51～7.81μg·ml-1之间,2例血药浓度小于0.5μg·ml-1。结论本法准确、灵敏,用血量少,适用于治疗药物监测,可为临床合理用药提供数据参考。