穿膜肽mCLOCK’s DNA-BIND作为药物载体的生物安全性初步评价

Preliminary Assessment of Biologic Security of a Membrane Penetrating Peptide mClock's DNA-BIND as a Drug-carrier

目的研究一种新的来源于节律蛋白mCLOCK's DNA结合域的穿膜肽mCLOCK's DNA-BIND(CDB)作为药物携带载体的生物安全性。方法化学合成穿膜肽CDB。体外采用Phorbol12-Myristate13-acetate(PMA)诱导NIH3T3细胞的近日节律。于诱导后不同时间点,CDB与小鼠成纤维细胞NIH3T3细胞共孵育,RT-PCR检测其对近日节律核心分子mPeriod1(mPer1)基因表达的影响。此外,培养的NIH3T3直接与CDB共孵育12h和24h后,MTT法检测其对细胞增殖的影响,流式细胞术检测其对细胞周期和凋亡的作用。结果CDB没有明显影响PMA诱导的mPer1节律基因表达。此外,CDB不干扰NIH3T3细胞增殖及生长周期,亦无明显的诱导凋亡作用。结论CDB对细胞近日节律、细胞增殖、生长周期及细胞凋亡均无明显毒副效应,可望作为一种安全有效药物载体,并具有广泛的临床应用前景。

Objective To investigate the biologic security of a novel membrane penetrating peptide （ MPP）-mCLOCK＇ s DNA-BIND （CDB） derived from circadian protein mCLOCK＇ s DNA-binding domain as a drug-carrier for intracellular treatment. Methods The CDB was synthesized by chemical method. The circadian gene expression in NIH3T3 fibroblasts was induced by PMA（ Phorbol 12-Myristate 13-acetate）. At the indicated times, the influence of CDB on the circadian gene expression of mPeriod1 was sequently detected by RT-PCR. In addition, NIH3T3 fibroblasts were incubated with CDB directly. After 12 h or 24 h incubation, cell proliferation was detected by MTT, cell cycle and apoptosis were detected by flow cytometer. Results Circadian oscillation of mPeriod1 gene induced by PMA in cultured NIH3T3 cells was not influenced by CDB, and influence of CDB on cell proliferation, cell cycle and apoptosis was not observed. Conclusion There is no toxic or adverse effect on circadian rhythm, cell proliferation, cell cycle and apoptosis in cultured cells treated with CDB. As a result, CDB can be used as a safe and efficient drug-carrier for intracellular treatment, and will have broad perspective in clinical applications.