三种中药制剂Ams-11、Fw-13、Tul-17逆转肿瘤细胞多药耐药性的研究

为寻找能有效逆转肿瘤细胞多药耐药性的药物,通过体外细胞实验对Ams-11、Fw-13、Tul-17三种中药制剂逆转肿瘤细胞多药耐药性的作用进行了分析。并用流式细胞仪测定了Tul-17处理细胞后药物累积程度的变化及细胞P糖蛋白表达情况。为进一步研究体外细胞实验筛选出的多药耐药逆转剂在体内的药效学,将其中Fw13用于人白血病K562/ADR裸鼠移植瘤逆转试验。结果:在无细胞毒性的剂量范围内,该三种中药制剂均能明显增强多药耐药细胞对抗癌药物的敏感性,而且其逆转作用呈剂量依赖关系。Tu-17处理后,K562耐药细胞表达的P糖蛋白较对照降低1.5倍,对罗丹明123的累积量是对照的2.5倍。用Fw13治疗人白血病K562/ADR裸鼠移植瘤,可将硫酸长春新碱(VCR)对K562/ADR的抑瘤率从19.79%提高到86.59%,与单独VCR治疗疗效有显著性差异(P<0.05)。结果表明,这三种中药制剂可望成为肿瘤多药耐药逆转剂,在肿瘤化疗中发挥作用。     

Inhibition of telomerase in tumor cells by ribozyme targeting telomerase RNA component

Telomerase plays an important role in cell proliferation and carcinogenesis and is believed to be a good target for anti-cancer drugs. Elimination of template function of telomerase RNA may repress the telomerase activity. A hammer-headed ribozyme (telomerase ribozyme, te-loRZ) directed against the RNA component of human telomerase (hTR) was designed and synthesized. TeloRZ showed a specific cleavage activity against the hTR. The cleavage efficacy reached 60%. A eukaryotic expression plasmid containing teloRZ gene was inducted into HeLa cells by lipofectamine, the telomerase activity in HeLa cells expressing teloRZ decreased to one eighth of that in the control cells. The doubling time increased significantly and the apoptosis ratio was elevated with increasing population doublings (PDS). After 19-20 PDS 95% cells were apoptotic. To further investigate the effect of teloRZ on tumor growth, the eukaryotic expression plasmid containing teloRZ was injected into transplanted tumor of nude mouse. The teloRZ e     

Ribozyme抑制端粒酶活性的肿瘤基因治疗

针对端粒酶RNA组分模板区设计合成锤头状端粒酶核酶(teloRZ),构建了teloRZ基因体外转录质粒P^SPT19-teloRZ和真核表达质粒P^{XJ-neo-teloRZ}.用T₇/SP₆体外转录系统检测了teloRZ对端粒酶RNA组分的体外切割效率,达60%左右.用脂质体介导法, 将P^{XJ-neo-teloRZ}导入HeLa细胞和裸鼠移植瘤,结果使HeLa细胞端粒酶活性降至对照细胞的11%～12.5%, 细胞生长速度明显变慢,传至19～20代时出现95%凋亡.裸鼠移植瘤注射P^{XJ-neo-teloRZ} 14 d后,端粒酶活性下降69%,抑瘤率达62%.实验表明,该核酶可望成为有效的端粒酶抑制剂,在肿瘤治疗中发挥作用.     

牙龈卟啉菌特异性克隆探针的筛选

目的 从牙龈卟啉菌47A－1的基因文库中筛选出特异性片段,制备成特异性克隆探针,方法 将牙龈卟啉菌47A－1基因文库中的重组质粒大量扩增和纯化,采用地高辛标记法制备成探针,与口腔中14种常见细菌DNA进行杂交鉴定,检测其特异性,从中筛选出对牙龈叶卟啉菌具有特异性的克隆探针。结果 重组质粒pZJ1与牙龈卟啉菌47A－1杂交,而与其它细菌DNA均不杂交,包括牙龈卟啉菌ATCC33277和W83。结论 重组质粒pZJI可制备成高特异笥克隆探针。     

Inhibition of telomerase in tumor cells by ribozyme targeting telomerase RNA component

Telomerase plays an important role in cell proliferation and carcinogenesis and is believed to be a good target for anti-cancer drugs. Elimination of template function of telomerase RNA may repress the telomerase activity. A hammer-headed ribozyme (telomerase ribozyme, teloRZ) directed against the RNA component of human telomerase (hTR) was designed and synthesized. TeloRZ showed a specific cleavage activity against the hTR. The cleavage efficacy reached 60%. A eukaryotic expression plasmid containing teloRZ gene was inducted into HeLa cells by lipofectamine, the telomerase activity in HeLa cells expressing teloRZ decreased to one eighth of that in the control cells. The doubling time increased significantly and the apoptosis ratio was elevated with increasing population doublings (PDS). After 19–20 PDS 95% cells were apoptotic. To further investigate the effect of teloRZ on tumor growth, the eukaryotic expression plasmid containing teloRZ was injected into transplanted tumor of nude mouse. The teloRZ effectively inhibited the telomerase activity in transplanted tumor, promoted apoptosis of the transplanted tumor cells, and decreased the tumor size significantly. These results indicate that teloRZ can effectively inhibit telomerase activity and growth of tumor cells, and suggest the potential use of this ribozyme in anti-cancer therapy.     

牙龈卟啉菌基因文库的建立

目的 通过构建牙龈卟啉菌47A－1的基因文库,为进一步深入研究牙龈卟菌的基因结构鉴定基础。方法 选择Sau3AI对牙龈卟啉菌47A－1染色体DNA作酶切,获得的DNA片段与BamBI酶解的质粒pUC18作体外连续并转化大肠杆菌JM109,在含有IPTG、X-gal和氨苄青霉素的LB琼脂平板上,用蓝、白筛选出选出含重组质粒的大肠杆菌扩增并分析。结果 得到的重组质粒经酶切、电泳分析,证实包含牙龈卟啉菌47A－1的插入片段。结论 牙龈卟啉菌47A－1的基因文库构建成功。