新型蛋白转导域PTD4的细胞内转导及其组织分布

Intracellular Translocation and Tissue Distribution of a New Protein Transduction Domain PTD4

目的:合成新型蛋白转导域PTD4,研究其细胞内穿膜效应及在体组织分布。方法:设计并采用固相合成法合成新型蛋白转导域PTD4,用FAM(羧基荧光素)进行标记,采用高效液相色谱仪(HPLC)和质谱仪测定其纯度与相对分子质量;采用荧光倒置显微镜和激光扫描共聚焦显微镜观察其在细胞内的穿膜情况;采用裸鼠活体成像观察穿膜肽在体内的组织分布及代谢特性,并切片观察各组织器官的分布情况。结果:合成了新型蛋白转导域PTD4,纯度为95.76%,相对分子质量为1776.5;PTD4在体外的穿膜效应有时间与浓度依赖性,约24 h代谢完毕,且共聚焦显微镜细胞层扫证实穿膜肽进入胞内;尾静脉注射的PTD4在裸鼠体内可迅速分布于各组织器官,但组织分布不均一,且有时间与浓度依赖性,6 h后荧光强度下降了大半,且腹腔注射方法不如尾静脉注射的穿膜效率高。结论:采用Fmoc固相肽合成法可成功合成蛋白转导域PTD4;PTD4能高效穿透细胞膜,穿膜效应呈时间与浓度依赖性;PTD4在体内能迅速分布于各组织细胞。

Objective： To synthesize a new protein transduction domain 4（PTD4） and evaluate its transductionefficiency in vitro and biodistribution in vivo. Methods： The peptides were synthesized by solid phase peptide syn-thesis using fluorenylmethyloxycarbonyl（Fmoc） as a protecting group alpha amino acids. FAM（carboxy fluoresceinfluorescence marker） was used to obtain PTD4- FAM. High performance liquid chromatography analyzer（HPLC）and mass spectrometry were applied to determinate the purity and molecular weight. Fluorescence microscopy andlaser scanning confocal were used to observe the effect of cell penetrating peptide PTD4 on the transmembrane ac-tivity and the trend with the time and the concentration in vitro. Small animal imaging system was used to ob-serve the dynamic distribution of PTD4 in nude mice. Results： The purity of the synthesized protein transductiondomain was 95.76% measured by HPLC analisis, and the mass spectra of relative molecular weight was 1776.5Da. Group A at each time point cannot detect green fluorescence signal. Group B/C/D at each time point could de-tect the signal, with the intensity decreased by the time and highest in 15 min and 1 h. The fluorescence intensi-ty decreased significantly after 12 h, only a little weak fluorescence after 24 h. Confocal on cell layer scan foundthat each layer of the expression of green fluorescence intensity was not the same. Nude mice injected saline cannot detect any fluorescence. The fluorescence intensity at different concentration reached the maximum 15 minpost injection, then the fluorescence intensity decreased along with time, fell by more than half after 10 h. Underthe same concentration, the detection of fluorescence intensity decreased successively. Conclusion： Using Fmoc sol-id-phase synthesis method, we synthesized the PTD4-FAM. PTD4 can penetrate the cell membrane, with the mem-brane penetrating effect in a time and dose dependent manner. PTD4 also can translocate into tissue cells, and rapidly distribute in vivo.