基于转谷氨酰胺酶交联明胶水凝胶的脂肪干细胞培养研究

THREE-DIMENSIONAL CULTURED ADIPOSE-DERIVED STEM CELLS BASED ON MICROBIAL TRANSGLUTAMINASE ENZYME CROSSLINKED GELATIN HYDROGEL

目的构建转谷氨酰胺酶（microbial transglutaminase，mTG）交联明胶水凝胶的脂肪干细胞（adipose—derived stemcells，ADSCs）三维培养体系，观察ADSCs在该水凝胶中生长情况。方法取SD大鼠腹股沟皮下脂肪组织，采用胶原酶消化及离心分离获取ADSCs，并培养传代。定量称取明胶及mTG，分别置于PBS中溶解后，按照一定比例混匀制备mTG交联明胶水凝胶。取P3～P4代ADSCs分别于水凝胶表面二维培养及包埋于水凝胶中进行三维培养。培养期间，采用倒置相差显微镜观察ADSCs的生长形态，并进行HE染色和Masson染色，观察细胞在材料中的分布形态；采用荧光显微镜和激光共聚焦显微镜观察ADSCs在材料中存活状况；扫描电镜观察ADSCs在水凝胶表面黏附性；采用Alamar—Blue法检测ADSCs在水凝胶中增殖情况，以培养皿中常规培养细胞作为对照。结果二维培养观察示，ADSCs在水凝胶表面生长良好、细胞功能正常，贴壁良好。三维培养观察示，ADSCs在mTG交联明胶水凝胶中生长良好，呈三维立体形态，细胞向各方向伸展；细胞死活染色显示凋亡细胞非常少。各时间点三维培养的细胞数量显著低于常规培养细胞，比较差异有统计学意义（P〈0．05）；但第8天开始，三维培养的细胞增殖明显加快，但常规培养细胞增殖开始减慢。结论基于mTG交联明胶水凝胶三维培养的ADSCs黏附性强，生长状况良好，成活率高。

Objective To study the growth of adipose-derived stem cells （ADSCs） planted in three-dimensional （3D） materials, a 3D cultured ADSCs system based on microbial transglutaminase （mTG） enzyme crosslinked gelatin hydrogel was constructed. Methods ADSCs were isolated from the subcutaneous adipose tissue of a Sprague Dawley rat by collagenase digestion and centrifugation, and were cultured for passage. The mTG enzyme crosslinked gelatin hydrogel was firstly synthesized by mixing gelatin and mTG, and then the ADSCs were encapsulated in situ （2D environment） and cultured in the 3D materials （3D environment）. The morphology and adhesion of cells were observed by inverted phase contrast microscope. In addition, HE staining and Masson staining were carried out to observe the distribution of cells in the material. Living and death situation of ADSCs in the materials was observed by fluorescence microscope and laser scanning confocal microscopy. Scanning electron microscopy was used to observe the adhesion of ADSCs on hydrogel surface. Alamar-Blue method was used to detect the proliferation of ADSCs in the hydrogel. Moreover, the results were compared between the cells cultured in 2D environment and those in 3D environment. Results The result of 2D culture showed that ADSCs grew well on the hydrogel surface with normal functioning and had good adhesion. The results of 3D culture showed that ADSCs grew well in 3D cultured mTG enzyme crosslinked gelatin hydrogel, and presented 3D shape. Cells obviously extended in all directions. The number of apoptotic cells was very small. The cells of 3D culture at each time point was significantly less than that of the conventional culture cells, difference was statistically significant （P〈0.05）. But after 8 days culture, the proliferation of the cells cultured in the mTG enzyme crosslinked gelatin hydrogel increased more quickly. Conclusion ADSCs can grow well with good adhesion and show high viability in 3D culture system constructed by mTG enzyme crosslinked gelatin hydrogel.