【摘要】 目的 筛选人源喉癌Hep-2细胞株特异结合的短肽，作为喉癌靶向治疗的载体。 方法 体外培养Hep-2细胞株作为靶细胞，人正常喉黏膜上皮细胞为吸附细胞；用噬菌体展示十二肽库进行3轮差减筛选，随机挑取10个噬菌体克隆进行测序；采用酶联免疫吸附（enzyme linked immunosorbent assay，ELISA）法鉴定噬菌体与Hep-2细胞的结合活性；通过免疫荧光鉴定喉癌细胞特异性结合肽（F2）噬菌体阳性克隆与喉癌细胞结合的特异性。 结果 经过3轮筛选后，噬菌体在靶细胞Hep-2上出现明显富集；ELISA分析鉴定显示5个阳性克隆能与Hep-2细胞特异结合，其中F2噬菌体克隆对喉癌细胞的结合靶向性明显高于对照细胞（Plt;0.05）； 免疫荧光显色显示，F2能特异性地与喉癌细胞结合。 结论 利用噬菌体展示肽库技术，可以成功筛选到F2，其可能成为喉癌靶向治疗的载体。【Abstract】 Objective To obtain the polypeptides specifically bound to laryngeal squamous cell carcinoma line (Hep-2) and use it as a potential therapeutic vector targeting laryngeal squamous cell carcinoma patients. Methods With the Hep-2 cells as the target cells and human normal laryngeal squamous epithelial cells (HNLE cells) as the absorber cells, 3 rounds of panning from a Ph.D.-12TM phage-display peptide library were carried out. Ten randomly selected phage clones were sent for sequence detection. The affinity of phage clones was detected by enzyme-linked immunosorbent assay (ELISA). The positive phage clones (F2) specifically bound to Hep-2 were identified by immunofluorescence detection. Results After 3 rounds of screening, 5 positive phage clones showed specific binding to Hep-2 cells and the affinity of positive phage clones (F2) was significantly higher than that of the control groups (Plt;0.05). The results of immunofluorescence detection indicated that F2 could be specifically bound to Hep-2. Conclusions Phage display peptide libraries technique can successfully screen the peptide specifically bound to Hep-2 cell line. Thus, it provides a potential vector for targeting therapy of laryngeal squamous cell carcinoma patients.
Objective To screen the possible regulatory proteins showing the ability for interaction with serum response factor ( SRF) in the progress of myofibroblast activation, and to see if the proteinprotein interaction is contributing to induce the expression of smooth muscle αactin ( α-SMA) . Methods Phage display cDNA libraries were constructed from the transdifferentiated airway epithelial cells and parental cells. Phage clones were then selectively amplified during the biopanning procedure by using SRF as a bait protein for the two cDNA libraries. Following four rounds of biopanning, recovered cDNAs were sequenced and the obtained sequences were aligned by BLAST tool to select the candidate gene. PAI-RBP1 of the candidate gene was cloned and sub-cloned into pcDNA3. 0 plasmid. Transient transfection and RT-PCR analysis were performed for investigation of the expression of α-SMA. Results Three candidate proteinbinding partners, PAI-RBP1, Nucleolin, and HF1OO, were identified. Among them, PAI-RBP1 pcDNA3. 0 plasmid was subjected to transient co-transfection with SRF, showing up-regulation of α-SMA expression. Conclusions Combined with phage display technique, through protein-protein interaction between core transcription factor and unknown proteins to find a newtranscriptional regulator may serve as an effective strategy. Three novel SRF binding proteins were found from transdifferentiated cells. This study indicates that PAI-RBP1 involves in the activation of myofibroblast by induction of α-SMA expression.