重组可溶性Fas偶联蛋白激酶C抑制剂对结直肠癌细胞靶向杀伤作用的研究

Target lethal effect of recombinant soluble Fas coupled with protein kinase C inhibitor on colorectal carcinoma cells

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 study the target killing effect of soluble Fas（sFas） coupled with protein kinase C（PKC） inhibitor on colorectal carcinoma cells. Methods The extracellular region of Fas protein was cloned and amplified by RT-PCR, and the expressing vector pGEX-4T-1-sFas was constructed. The sFas protein was purified by GST fusion protein purification system and coupled with Calphostin C（one kind of PKC inhibitor）. The killing effect of soluble Fas coupled with PKC inhibitor on FasL-positive colorectal carcinoma cells was detected. Results After amplifying and cloning, the extracellular region of Fas protein, a 571 bp fragment, was proved by limited enzyme cutting and DNA sequencing. The expressed and purified protein was identified by Western Blot after transformed into E. coil BL21. The coupled sFas-Calphostin C showed suppressant activity on PKC kinase by the PKC kinase activity assay kit. The growth suppression rate of FasL-positive celorectal carcinoma HR-8348 cells treated with sFas-Calphostin C was significantly higher than that of FasL-negative cells, but the killing effect of sFas-Calphostin C on normal h,Jrnan monocyte was not obvious. Compared with 5-Fu alone, the growth suppression rate of FasL-positive colorectal carcinoma HR-8348 cells was significantly raised by sFas-Calphostin C comhined with 5-Fu. Conclusion The recombinant of soluble Fas and PKC inhibitor shows target killing effect on colorectal carcinoma cells.