摘要
目的研究微生物转谷氨酰胺酶(microbial transglutaminase,mTG)交联明胶水凝胶对新生大鼠心肌细胞活力和增殖的影响,并探究该水凝胶在心肌组织工程应用中的适用性。方法采用微生物转谷氨酰胺酶交联明胶水凝胶作为基质材料,进行新生大鼠心肌细胞的体外培养。通过倒置光学显微镜观测心肌细胞生长状况,并选取培养一周内的心肌细胞进行细胞相容性评估:采用钙绿黄素-AM/碘化丙啶进行活/死染色,表征了在明胶水凝胶表面培养的心肌细胞的细胞活性;采用MTT法分析细胞在水凝胶表面的增殖情况。使用DAPI和心肌细胞标志蛋白cTnI、cTnT免疫荧光染色法,检测体外培养的心肌细胞在明胶水凝胶基底上的心肌源性和成熟状态。结果在37℃下加入mTG溶液的明胶快速凝胶,接种后的心肌细胞在4 h内完全粘附。明场下可清楚观测到心肌细胞的增殖和随时间逐渐同步的自发搏动行为。活/死染色和MTT测定结果显示,mTG-GA水凝胶上心肌细胞具有良好的活性,并保持了相当的增殖潜力。cTnI和cTnT荧光染色结果说明,相较于培养板上传统培养的心肌细胞,mTG-GA水凝胶上生长的心肌细胞不仅保持了良好的细胞活力,而且具有相当的心肌细胞表型和更高的特征蛋白表达。结论mTG交联明胶水凝胶具有高细胞相容性,适合作为心肌细胞体外培养材料,用于工程化心肌组织构建和其他心肌组织工程应用。
Objective This study aimed to investigate the effect of microbial transglutaminase(mTG)cross-linked gelatin hydrogel on the viability and proliferation of neonatal rat cardiomyocyte(NRCM)and to explore its applicability in myocardial tissue engineering.Methods Gelatin hydrogels crosslinked with microbial transglutaminase were utilized as the substrate for in vitro culture of NRCMs.The growth of cardiomyocytes was observed under a inverted optical phase contrast microscope,and compatibility assessment was performed on cardiomyocytes cultured within one week.Cell viability was evaluated by using live/dead staining(calcein-AM/propidium iodide)to characterize the cellular activity of cardiomyocytes cultured on the gelatin hydrogel surface.Cell proliferation on the hydrogel surface was assessed by using the MTT assay.Immunofluorescence staining with DAPI for cardiac-specific markers cTnI and cTnT was employed to investigate the cardiac origin and maturity of NRCMs cultured on the gelatin hydrogel substrate.Results Gelatin rapidly gelated at 37℃upon the addition of mTG solution.Proliferation of cardiomyocytes and synchronous spontaneous beating behavior were clearly observed under bright-field microscopy over time.Live/dead staining and MTT assay results demonstrated that cardiomyocytes on mTG-GA hydrogels exhibited robust viability and maintained considerable proliferative potential.Immunofluorescence staining for cTnI and cTnT indicated that cardiomyocytes grown on mTG-GA hydrogels not only retained excellent cell viability but also displayed a comparable cardiac cell phenotype and higher expression of characteristic proteins compared to traditional culture on tissue culture plates.Conclusions Microbial transglutaminase-crosslinked gelatin hydrogel exhibits excellent cell compatibility and is a suitable material for cardiomyocyte cultivation,making it conducive to the construction of engineered cardiac tissues and other applications in myocardial tissue engineering.
作者
郭坤铭
叶静
贺子亮
邹佐凤
杨刚
GUO Kunming;YE Jing;HE Ziliang;ZOU Zuofeng;YANG Gang(College of Biomedical Engineering,Sichuan University,Chengdu 610065;State Key Laboratory of Biotherapy,West China Hospital,Sichuan University,Chengdu 610041)
出处
《北京生物医学工程》
2024年第2期137-144,共8页
Beijing Biomedical Engineering
基金
川大-达州校地合作项目(2020CDDZ-23)资助。
