提升上清液Wnt3a蛋白含量的L-Wnt3a细胞培养体系及其促进多能干细胞诱导黑素细胞分化的效应研究  

L-Wnt3a cell culture system with increased contents of Wnt3a in supernatant and its efficacy of inducing differentiation of pluripotent stem cells into melanocytes

作　　者：曲勃维 刘莉萍[1] 鲁浩 李遇梅[1] QU Bowei;LIU Liping;LU Hao;LI Yumei(Department of Dermatology,Affiliated Hospital of Jiangsu University,Zhenjiang Jiangsu 212001;Department of Dermatology,Affiliated Hospital of Inner Mongolia Medical University,Hohhot Inner Mongolia 010107,China)

机构地区：[1]江苏大学附属医院皮肤科,江苏镇江212001 [2]内蒙古医科大学附属医院皮肤科,内蒙古呼和浩特010107

出　　处：《江苏大学学报（医学版）》2022年第6期497-503,共7页Journal of Jiangsu University：Medicine Edition

基　　金：国家自然科学基金资助项目(81770621)。

摘　　要：目的:明确L-Wnt3a条件培养基的收集方案并标定Wnt3a蛋白浓度,验证其作为多能干细胞向黑素细胞分化体系中Wnt3a来源的作用。方法:通过观察L-Wnt3a细胞的生长特性及其分泌Wnt3a蛋白量特点,设定收集条件培养基的参数,使用ELISA法对收集到的条件培养基进行Wnt3a蛋白浓度的标定,配制出明确Wnt3a浓度的黑素细胞分化培养基,对胚胎干细胞株WA09及诱导多能干细胞株WTC-11细胞进行黑素细胞的悬浮诱导分化,对获得的诱导黑素细胞进行黑素相关基因表达检测及Masson-Fontana染色等相关验证。结果:优化后的L-Wnt3a条件培养基收集体系能够稳定高效获取Wnt3a蛋白,明确Wnt3a浓度的分化体系能够使WA09和WTC-11成功诱导黑素细胞,使用相应浓度的Wnt3a重组蛋白也能够达到相似的效果。结论:通过优化收集方案能够高效获得明确浓度的L-Wnt3a条件培养基,将其应用于明确Wnt3a浓度的培养体系能够成功诱导多能干细胞分化为功能性黑素细胞。Objective:To optimize the collection protocol and calibrate the Wnt3a protein concentration of L-Wnt3a conditioned medium(L-Wnt3a CM)and verify its role as a source of Wnt3a in pluripotent stem cell(PSC)to melanocytes differentiation system.Methods:The growth characteristics of L-Wnt3a cells and the characteristics of their secreted Wnt3a protein amount were observed to set the parameters of the collection method of L-Wnt3a conditioned medium,and the collected conditioned medium was calibrated for Wnt3a protein concentration using ELISA,and melanocyte differentiation medium with a defined Wnt3a concentration was formulated for the embryonic stem cell line WA09 and the induced pluripotent stem cell line WTC-11 cells were subjected to suspension-induced differentiation of melanocytes,and the obtained induced melanocytes were subjected to melanin gene expression assay and Masson-Fontana staining.Results:The new collection system was able to obtain Wnt3a protein stably and efficiently,and the differentiation system with a defined Wnt3a concentration was able to induce melanocytes successfully in WA09 and WTC-11,and the corresponding concentration of recombinant Wnt3a protein was able to achieve similar results.Conclusion:The precise concentration of L-Wnt3a CM could be efficiently obtained by optimizing the collection protocol and the use of a culture system with defined Wnt3a concentration also successfully induced of PSC differentiation into functional melanocytes.

关 键 词：黑素细胞 多能干细胞 Wnt3a蛋白 诱导多能干细胞 细胞分化 拟胚体 

分 类 号：R751.05[医药卫生—皮肤病学与性病学]