论文部分内容阅读
目的针对复发转移结直肠癌细胞膜分子靶点,探讨可溶性Fas偶联蛋白激酶C(PKC)抑制剂对结直肠癌细胞的靶向杀伤作用。方法采用RT-PCR扩增、克隆Fas胞外区,构建真核表达载体pGEX-4T-1-sFas,采用GST融和蛋白纯化法纯化sFas。采用化学偶联法连接sFas与PKC抑制剂Calphoetin C。通过细胞抑制率测定法(MTT法)检测偶联物对FasL阳性结直肠癌细胞的杀伤作用。结果扩增、克隆Fas胞外区571 bp经限制性酶切和DNA序列测定证实无误。转化宿主菌BL21表达纯化,经Western blotting确认sFas蛋白产物。将sFas与PKC抑制剂Calphostin C经化学偶联得到偶联物sFas-Calphostin C。利用PKC酶检测系统检测偶联物具有抑制PKC酶活性作用。偶联物(40 mg/L)对FasL表达阳性结直肠癌细胞HR-8348癌细胞生长抑制率(39．04%)较FasL表达阴性HR-8348癌细胞(33．69%)明显提高(t=4．093,P＜0．05),对外周血单个核细胞杀伤作用不明显(23．08%),偶联物联合氟尿嘧啶对FasL阳性HR-8348细胞的细胞抑制率(68．38±5．07)%明显高于氟尿嘧啶对FasL阳性HR-8348细胞的细胞抑制率(36．02±1．50)%(t=14．05,P＜0．001)。结论重组可溶性Fas偶联PKC抑制剂对侵袭行为较强的结直肠癌细胞具有较强的靶向杀伤作用。
Objective To investigate the molecular target of the remittent metastatic colorectal cancer cell membrane and to investigate the target killing effect of soluble Fas coupled protein kinase C (PKC) inhibitor on colorectal cancer cells. Methods The extracellular region of Fas was cloned by RT-PCR and the eukaryotic expression vector pGEX-4T-1-sFas was constructed. The sFas was purified by GST fusion protein purification. Chemically coupled sFas and PKC inhibitor Calphoetin C were used. The cytotoxicity of the conjugates on FasL positive colorectal cancer cells was determined by MTT assay. The results of amplification, cloned Fas extracellular region 571 bp restriction endonuclease and DNA sequencing confirmed. The transformed host strain BL21 was expressed and purified, and the sFas protein product was confirmed by Western blotting. The conjugate sFas-Calphostin C was obtained by chemical coupling of sFas with the PKC inhibitor Calphostin C. The use of PKC enzyme detection system to detect conjugates inhibit PKC enzyme activity. Conjugate (40 mg / L) significantly inhibited the growth of FasL-positive HR-8348 cancer cells (39.04%) and FasL-negative HR-8348 cancer cells (33.69% = 4.093, P <0.05). The cytotoxicity of the conjugate combined with fluorouracil on FasL positive HR-8348 cells was not significantly different (23.08%) (68.38 ± 5.07)% was significantly higher than that of fluorouracil on FasL positive HR-8348 cells (36.02 ± 1.50)% (t = 14.05, P <0.001). Conclusion Recombinant soluble Fas-conjugated PKC inhibitors have a strong target-killing effect on colorectal cancer cells with strong invasiveness.