穿膜肽TAT和R9的生物学效应的体内体外研究

In vitro and in vivo Studies on the Biological Effects of Transmembrane Peptides TAT and R9

本文比较了两种常用穿膜肽TAT和R9的体外穿膜效率、对细胞影响以及小鼠活体内不同器官的穿膜效率。非特异性穿膜肽TAT和R9分别为11个氨基酸和9个氨基酸的短肽,能够有效地穿过细胞膜进入细胞。本研究纯化制备出融合蛋白TAT-EGFP及R9-EGFP多肽,用相同浓度的活性TAT-EGFP和R9-EGFP处理肺癌细胞,观察比较二者的穿膜活性及效率;设置不同浓度梯度检测两种穿膜肽是否对细胞产生影响;通过小鼠腹腔注射TAT-EGFP和R9-EGFP活性肽段,研究二者在活体能够穿膜到达的靶位置,并比较分析二者穿膜的效果器官分布。结果显示制备获得的两种融合蛋白TAT-EGFP和R9-EGFP均能有效穿过实现穿膜功能,两种穿膜肽对乳腺癌细胞影响较小。活体试验证明两种穿膜肽在心、肝、脾、肺、肾器官均有分布,R9主要富集在小鼠的肾和肝脏中,TAT在6种被研究的器官都有富集,两种细胞穿膜肽都能透过血脑屏障到达脑部。相对来说,TAT在小鼠活体中的穿膜效果更好,荧光富集多,R9整体穿膜效果较弱。本研究分析比较了两种穿膜肽的细胞安全性浓度和活体的穿膜能力,为TAT和R9两种穿膜肽在后续试验中的合理有效的选择和使用,提供了基础数据和指导性依据。

In this paper, the in vitro permeation efficiency of two commonly used transmembrane peptides TAT and R9, the effects on cells and the transmembrane efficiencies of different organs in mice were compared. The non-specific transmembrane peptides TAT and R9 are short peptides of 11 amino acids and 9 amino acids, respectively, which are able to efficiently enter cell through cell membrane. In this study, the fusion proteins TAT-EGFP and R9-EGFP were purified, and the lung cancer cells were treated with the same concentration of active TAT-EGFP and R9-EGFP. The transmembrane activity and efficiency of the two were observed and compared. To explore whether the transmembrane peptide has an effect on the cells, the TAT-EGFP and R9-EGFP active peptides were injected intraperitoneally into mice to study the target positions of the two in vivo , and the distribution of the effect organs of the two membranes was compared. The results showed that the two fusion proteins TAT- EGFP and R9-EGFP were able to effectively penetrate the membrane, and the two transmembrane peptides had less effect on breast cancer cells. In vivo experiments showed that the two transmembrane peptides were distributed in the heart, liver, spleen, lung and kidney organs. R9 was mainly enriched in the kidney and liver of mice, and TAT was enriched in the six organs studied. The two cell-penetrating peptides can pass through the blood-brain barrier to reach the brain. Relatively speaking, TAT has a better transmembrane effect in mice and gives more fluorescence enrichment, and R9 overall membrane penetration is weaker. Through the analysis of this study, the cell safety concentration of two transmembrane peptides and the transmembrane ability of living cells were compared, which provided basic data and a guidance for the rational and effective selection and use of TAT and R9 transmembrane peptides in subsequent experiments.