枯草芽孢杆菌发酵产物CLPs抗肿瘤活性研究

Study on Antitumor Activity of CLPs from Bacillus Subtilis Fermentation

由枯草芽孢杆菌(Bacillus subtilis)发酵并分离纯化得到环脂肽(CLPs),研究其对肿瘤细胞MCF-7和正常细胞HELF的细胞毒作用,探索CLPs对LDH释放的影响,并用流式细胞仪检测CLPs对MCF-7细胞凋亡的影响。采用MTT法检测CLPs对肿瘤细胞和正常细胞的细胞毒性,LDH试剂盒检测对LDH释放的影响,AnnexinV/PI染色观察CLPs对肿瘤细胞凋亡的影响。CLPs对肿瘤细胞MCF-7有细胞毒性,CLPs作用24 h时抑制率可达88%,CLPs对正常HELF细胞无细胞毒性;CLPs导致MCF-7细胞LDH释放,CLPs质量浓度100μg/mL,作用24 h时,LDH释放率由5.10%增加到31.67%,CLPs对HELF细胞LDH没有影响;CLPs能导致MCF-7细胞发生凋亡。CLPs能选择性作用于MCF-7细胞,增加MCF-7细胞LDH释放,并导致MCF-7细胞发生凋亡。

Viral vectors and plasmid vectors are the most frequently used gene expression vectors and they may cause unwanted side effects including immunologic,inflammatory reaction, cytotoxicity and gene silence. Here a new non-viral gene expression mini vector based on AAV-ITR is developed, and the ITRs will protect the mini vector from the degradation by nucleases in the cell for its T-shaped structure. The GFP is selected as reported gene for AAV-ITR mini vector. Single-stranded DNA is prepared by thermal denaturation method,and then the plasmid with GFP gene, double-stranded DNA vector and AAV-ITR mini vector are transfected intc eukaryotic cells respectively. The results were tested by fluorescence microscope and flow cytometry. The results show that the AAV-ITR mini vector has a higher efficiency of transfection and expression. It can be preliminarily concluded that AAV-ITR mini vector could be a new non-viral gene therapy vector with single strand.