正電奈米粒子作為蛋白質輸送系統之載體以製造人類誘導多能幹細胞之研究

Abstract

誘導式多能性幹細胞 (iPSC) 在再生醫學領域中具備極佳的潛能，它可 應用在疾病的機制探討、新藥開發、幹細胞治療以及組織工程的研究之中。與胚胎幹細胞不同，誘導式多能性幹細胞避開了道德爭議的問題。然而第一代以病毒製備的誘導式多能性幹細胞面臨基因突變的可能性與致癌性，從而陸續有研究欲改善製備的方法。以蛋白質為基礎的方法相較於其他非DNA相關的方法具備較高的穩定性，但是針對細胞的蛋白質輸送現今依然面臨挑戰。 藥物輸送系統相較於傳統的藥物劑型具備許多優點:保護藥物、增加在血液循環的半衰期、控制釋放速率以及主動標靶等等。奈米粒子是常見的一種藥物輸送系統，蛋白質被奈米粒子攜帶而進入細胞之中可以大幅增加輸送的效率，並且保護蛋白質不受酵素破壞。 利用明膠聚丙烯亞胺奈米粒子攜帶以誘導細胞再程序化之功能蛋白質進入人類纖維細胞中，以製備絕對安全的誘導式多能性幹細胞。在研究中我首先證明了明膠聚丙烯亞胺奈米粒子蛋白傳送系統的可行性。我製備出的奈米粒子，顆粒直徑約為100 nm，表面界面電位為 +43.2。細胞毒性測試中顯示奈米粒子的毒性是可接受的。在BSA結合實驗中證明明膠聚丙烯亞胺奈米粒子，有能力吸附BSA在粒子表面，並且在重量比為1:1時有最大的結合效率。為了進行近一步蛋白質功能測試，我利用大腸桿菌系統表現並純化出His-eGFP的蛋白質。利用明膠聚丙烯亞胺奈米粒子投遞His-eGFP至細胞中的實驗顯示，確實蛋白質可以順利進入細胞內部還維持良好功能，順利表現綠色螢光。在建立誘導細胞再程序化之蛋白質實驗中，在BL21菌株中始終得不到理想的蛋白質表現量。再BL21 StarTM (DE3)的菌株中表現下，目標蛋白之表現量有顯著的上升。利用冷光素酶檢測法，確定設計標的Nanog啟動子的小RNA片段能夠引導在程序蛋白開啟基因表現。未來，需要進行再程序化之功能蛋白質的細胞遞送實驗進一步測試其功能性。最終期望以新方法生產安全的誘導式多能性幹細胞，並且在未來應用在臨床上。

Induced pluripotent stem cell (iPSC) possesses high potentials in regeneration medicine. It has been used in several applications such as disease mechanism study, drug development, stem cell therapy, and tissue engineering. Because of the problem of ethical controversy which hinders the development of embryonic stem cell research, iPSC is a better platform for the stem cell research. The first generation of iPSC is generated from somatic cells by virus infection. Therefore, the safety issue should be concerned because of the possibility of gene mutation and carcinogenesis. To solve this problem, there has been different ways to generate iPSC. One of them is the protein-based method. The research in protein delivery with efficiency and safety is still under development. Comparing to the traditional dosage form, drug delivery system has many advantages such as drug protection, the improvement of half-life, controlling release, and active targeting. The nanoparticle is one of drug delivery system, which is reported to enhance the efficiency in protein delivery. In this study, I try to force express Yamanaka factors in Hs68 by protein-based reprogramming proteins (RPs) delivery with Gelatin-PEI nanoparticle to generate the absolutely safe iPSC. First, Gelatin-PEI nanoparticles were produced. The average diameter of them was about 100 nm, and the zeta potential on the surface was +43.2. The MTT assay showed that Gelatin-PEI nanoparticles are safe with the acceptable cytotoxicity. In the experiment of BSA binding assay, Gelatin-PEI nanoparticles could bind with BSA, and the highest efficiency was achieved when the wt/wt equal to 1. For the functional test after delivery, eGFP was cloned and expressed in E.coli system, and then it was purified by Ni-NTA spin column. The last, in the experiment of eGFP delivery into Hs68 cell line, Gelatin-PEI nanoparticle was capable of carrying eGFP, internalizing to Hs68, and helping it escape from endosome. These data indicated Gelatin-PEI nanoparticle is a high potential Nanocarrier in protein delivery. Next, RPs was cloned and expressed in BL21. BL21 StarTM (DE3) strain was used to express RPs with relative high expression level. The small RNA sequence was produced and identified to guide RPs to activate the Nanog-luc gene expression. Further functional tests of RPs delivery system should be evaluated in the future. Overall, I expect to develop a new method to generate iPSC for clinical application.