目的:探讨插入甘氨酸(G ly)对H IV Tat-TK融合蛋白功能的影响。方法:利用基因重叠(gene sp lic ing by overlap ex-tension,gene SOE ing)PCR技术,将不同长度的甘氨酸密码子(0、2、4、6个)插入H IV Tat-TK融合基因,经转染、鉴定证实后...目的:探讨插入甘氨酸(G ly)对H IV Tat-TK融合蛋白功能的影响。方法:利用基因重叠(gene sp lic ing by overlap ex-tension,gene SOE ing)PCR技术,将不同长度的甘氨酸密码子(0、2、4、6个)插入H IV Tat-TK融合基因,经转染、鉴定证实后诱导表达,并经偶联Tat单克隆抗体的Sepharose CL-4B亲和层析柱纯化。4种H IV Tat-(G ly)n-TK系列融合蛋白(n=0、2、4、6)、H IV Tat蛋白、TK蛋白(1μg/m l)分别与HepG2细胞在普通培养基共培养24 h后,间接免疫荧光检测各自透过细胞膜效率;在加入更昔洛韦的培养基培养3 d后,锥虫蓝染色计算细胞死亡率,流式细胞仪检测细胞凋亡率。结果:精确克隆出H IV Tat-(G ly)n-TK系列融合基因,成功表达H IV Tat-(G ly)n-TK系列融合蛋白以及H IV Tat和TK蛋白。H IV Tat-(G ly)n-TK系列融合蛋白与H IV Tat蛋白透过细胞膜的效率相似,但单独TK蛋白无法进入细胞;在含有更昔洛韦的培养基中H IV Tat-(G ly)4-TK融合蛋白致HepG2细胞凋亡率最高(14.77%),其余依次为H IV Tat-(G ly)2-TK融合蛋白(12.69%)、H IV Tat-TK(8.31%)、H IV Tat-(G ly)6-TK(4.36%)和H IV Tat组(1.03%),组间均有显著性差异(P<0.05);细胞死亡率也发现类似的结果(分别为80.2%、65.4%、58.4%、56.7%、9.1%,组间有显著差异,P<0.05)。结论:插入2、4、6个甘氨酸对H IV Tat-TK融合蛋白上游Tat蛋白的细胞融合穿透功能不产生影响,而对融合蛋白下游TK蛋白介导的更昔洛韦的细胞毒作用干扰较大,其中插入4个甘氨酸对TK蛋白介导的更昔洛韦的细胞毒作用影响最小。展开更多
Objective To study the antineoplastic effect of the calcium channel blocker verapamil and 5-fluorouracil intraperitoneal chemotherapy onhepatocarcinoma-bearing rats, and examine the action between calcium channel bloc...Objective To study the antineoplastic effect of the calcium channel blocker verapamil and 5-fluorouracil intraperitoneal chemotherapy onhepatocarcinoma-bearing rats, and examine the action between calcium channel blockers and cytotoxic drugs.Methods We adopted the method of subcapsular implantation of carcinoma tissues of walker-256 in the left liver lobe as a model of livercarcinoma-bearing rats. All experimental animals were divided into four groups. On the sixth day post implantation, in group A (controlgroup) 6 ml of saline was injected intraperitoneally once a day for 3 days. In group B (single chemotherapy group) 6 ml of 5-Fu 75 mg/kg was injected intraperitoneally once a day for 3 days. In group C (combination of treatment group) both 5-Fu (75 mg/kg) and verapamil(25 mg/kg) were administered simultaneously as in A and B. In group D (simple verapamil group) only 6 ml of verapamil (25 mg/kg)was administered as above.Results Compared with groups A, B and D, The volume of cancer and the contents of liver cancer DNA and protein were significantlyreduced. The rates of inhibiting cancer (89.9% in group C and 35.4% in group B) were significantly increased in group C. Group C hadsignificantly long survival time compared to groups A, B and D ( P < 0.05) . By light microscopy, a number of focal necroses were foundin cancer tissue in group C.Conclusion Calcium channel blockers can enhance the antineoplastic effect of 5-Fu intraperitoneal chemotherapy to liver cancer ; Theuse of verapamil can not increase the toxicity of 5-Fu.展开更多
Endocytosis occurs at the cell surface and involves internalization of the plasma membrane （PM） along with its constituent membrane proteins and lipids. Endocytosis is involved in sampling of the extracellular milie...Endocytosis occurs at the cell surface and involves internalization of the plasma membrane （PM） along with its constituent membrane proteins and lipids. Endocytosis is involved in sampling of the extracellular milieu and also serves to regulate various processes initiated at the cell surface. These include nutrient uptake, signaling from cell- surface receptors, and many other processes essential for cell and tissue functioning in metazoans. It is also central to the maintenance of PM lipid and protein homeostasis. There are multiple means of internalization that operate concurrently, at the cell surface. With advancement in high-resolution visualization techniques, it is now possible to track multiple endocytic cargo at the same time, revealing a remarkable diversity of endocytic processes in a single cell. A combination of live cell imaging and efficient genetic manipulations has also aided in understanding the functional hierarchy of molecular players in these mechanisms of internalization. Here we provide an account of various endocytic routes, their mechanisms of operation and occurrence across phyla.展开更多
文摘目的:探讨插入甘氨酸(G ly)对H IV Tat-TK融合蛋白功能的影响。方法:利用基因重叠(gene sp lic ing by overlap ex-tension,gene SOE ing)PCR技术,将不同长度的甘氨酸密码子(0、2、4、6个)插入H IV Tat-TK融合基因,经转染、鉴定证实后诱导表达,并经偶联Tat单克隆抗体的Sepharose CL-4B亲和层析柱纯化。4种H IV Tat-(G ly)n-TK系列融合蛋白(n=0、2、4、6)、H IV Tat蛋白、TK蛋白(1μg/m l)分别与HepG2细胞在普通培养基共培养24 h后,间接免疫荧光检测各自透过细胞膜效率;在加入更昔洛韦的培养基培养3 d后,锥虫蓝染色计算细胞死亡率,流式细胞仪检测细胞凋亡率。结果:精确克隆出H IV Tat-(G ly)n-TK系列融合基因,成功表达H IV Tat-(G ly)n-TK系列融合蛋白以及H IV Tat和TK蛋白。H IV Tat-(G ly)n-TK系列融合蛋白与H IV Tat蛋白透过细胞膜的效率相似,但单独TK蛋白无法进入细胞;在含有更昔洛韦的培养基中H IV Tat-(G ly)4-TK融合蛋白致HepG2细胞凋亡率最高(14.77%),其余依次为H IV Tat-(G ly)2-TK融合蛋白(12.69%)、H IV Tat-TK(8.31%)、H IV Tat-(G ly)6-TK(4.36%)和H IV Tat组(1.03%),组间均有显著性差异(P<0.05);细胞死亡率也发现类似的结果(分别为80.2%、65.4%、58.4%、56.7%、9.1%,组间有显著差异,P<0.05)。结论:插入2、4、6个甘氨酸对H IV Tat-TK融合蛋白上游Tat蛋白的细胞融合穿透功能不产生影响,而对融合蛋白下游TK蛋白介导的更昔洛韦的细胞毒作用干扰较大,其中插入4个甘氨酸对TK蛋白介导的更昔洛韦的细胞毒作用影响最小。
文摘Objective To study the antineoplastic effect of the calcium channel blocker verapamil and 5-fluorouracil intraperitoneal chemotherapy onhepatocarcinoma-bearing rats, and examine the action between calcium channel blockers and cytotoxic drugs.Methods We adopted the method of subcapsular implantation of carcinoma tissues of walker-256 in the left liver lobe as a model of livercarcinoma-bearing rats. All experimental animals were divided into four groups. On the sixth day post implantation, in group A (controlgroup) 6 ml of saline was injected intraperitoneally once a day for 3 days. In group B (single chemotherapy group) 6 ml of 5-Fu 75 mg/kg was injected intraperitoneally once a day for 3 days. In group C (combination of treatment group) both 5-Fu (75 mg/kg) and verapamil(25 mg/kg) were administered simultaneously as in A and B. In group D (simple verapamil group) only 6 ml of verapamil (25 mg/kg)was administered as above.Results Compared with groups A, B and D, The volume of cancer and the contents of liver cancer DNA and protein were significantlyreduced. The rates of inhibiting cancer (89.9% in group C and 35.4% in group B) were significantly increased in group C. Group C hadsignificantly long survival time compared to groups A, B and D ( P < 0.05) . By light microscopy, a number of focal necroses were foundin cancer tissue in group C.Conclusion Calcium channel blockers can enhance the antineoplastic effect of 5-Fu intraperitoneal chemotherapy to liver cancer ; Theuse of verapamil can not increase the toxicity of 5-Fu.
文摘Endocytosis occurs at the cell surface and involves internalization of the plasma membrane （PM） along with its constituent membrane proteins and lipids. Endocytosis is involved in sampling of the extracellular milieu and also serves to regulate various processes initiated at the cell surface. These include nutrient uptake, signaling from cell- surface receptors, and many other processes essential for cell and tissue functioning in metazoans. It is also central to the maintenance of PM lipid and protein homeostasis. There are multiple means of internalization that operate concurrently, at the cell surface. With advancement in high-resolution visualization techniques, it is now possible to track multiple endocytic cargo at the same time, revealing a remarkable diversity of endocytic processes in a single cell. A combination of live cell imaging and efficient genetic manipulations has also aided in understanding the functional hierarchy of molecular players in these mechanisms of internalization. Here we provide an account of various endocytic routes, their mechanisms of operation and occurrence across phyla.
