CAJ | 학술논문

背景:纤维蛋白是一种天然的可生物降解、组织相容性好的高分子材料,其中血纤维蛋白稳定因子XIII已经证明有利于未分化间充质干细胞在高度交联的凝胶支架内迁移,并促进其增殖与分化.目的:探讨大鼠胚胎间充质干细胞在不同质量浓度纤维蛋白凝胶内的形态学变化,以及生长增殖和成骨分化情况.方法:无菌条件下取胎鼠四肢,组织块消化法体外分离培养胚胎间充质干细胞.取传至第3代细胞,分别接种于20,10,5 g/L纤维蛋白凝胶内,并设立对照组,接种于无凝胶的正常培养基中.用激光扫描共聚焦显微镜观察细胞在凝胶内的形态学变化,荧光分光光度计测定细胞增殖状态,酶标仪和Von Kossa染色分析细胞碱性磷酸酶活性和钙盐沉积情况.结果与结论:培养7 d,在纤维蛋白凝胶内的大鼠胚胎间充质干细胞具有较长突起,且连接成网,而对照组细胞呈纺锤形或立方形.与对照组比较,凝胶组细胞增殖活跃,至培养第14天达峰值,以5 g/L凝胶细胞组荧光最强;凝胶组细胞碱性磷酸酶活性从14 d开始增强,21 d达峰值.培养14 d后20 g/L凝胶组在凝胶区出现小矿化结节,随后逐渐融合,而对照组无矿化结节出现.证实纤维蛋白凝胶支架能够支持大鼠胚胎间充质干细胞的存活与生长,且可以促进细胞的增殖和分化.5 g/L低浓度凝胶有利于细胞形态的发生,20 g/L高浓度凝胶有利于细胞的成骨分化.
배경:섬유단백시일충천연적가생물강해、조직상용성호적고분자재료,기중혈섬유단백은정인자XIII이경증명유리우미분화간충질간세포재고도교련적응효지가내천이,병촉진기증식여분화.목적:탐토대서배태간충질간세포재불동질량농도섬유단백응효내적형태학변화,이급생장증식화성골분화정황.방법:무균조건하취태서사지,조직괴소화법체외분리배양배태간충질간세포.취전지제3대세포,분별접충우20,10,5 g/L섬유단백응효내,병설립대조조,접충우무응효적정상배양기중.용격광소묘공취초현미경관찰세포재응효내적형태학변화,형광분광광도계측정세포증식상태,매표의화Von Kossa염색분석세포감성린산매활성화개염침적정황.결과여결론:배양7 d,재섬유단백응효내적대서배태간충질간세포구유교장돌기,차련접성망,이대조조세포정방추형혹립방형.여대조조비교,응효조세포증식활약,지배양제14천체봉치,이5 g/L응효세포조형광최강;응효조세포감성린산매활성종14 d개시증강,21 d체봉치.배양14 d후20 g/L응효조재응효구출현소광화결절,수후축점융합,이대조조무광화결절출현.증실섬유단백응효지가능구지지대서배태간충질간세포적존활여생장,차가이촉진세포적증식화분화.5 g/L저농도응효유리우세포형태적발생,20 g/L고농도응효유리우세포적성골분화.
BACKGROUND: Fibrin is a kind of high polymer materials with biodegradation and good histocompatibility. Fibrin stabilizing factor XIII has been verified that it can contribute to the migration of undifferentiated mesenchymal stem cells (MSCs) in gel scaffold with high crosslinking, and promote its proliferation and differentiation.OBJECTIVE: To analyze morphological changes of rat embryonic MSCs in fibrin gel of different mass concentrations, and to observe the growth, proliferation and osteogenic differentiation .METHODS: To isolate and culture fetal limbs mesenehymal stem cells (flMSCs) under aseptic condition using tissue digesting method. The third passage of flMSCs were incubated in three different concentrations (20,10 and 5 g/L) of fibrin gels. A control group was set and incubated in normal medium without gel. The cell morphology in fibrin gels was observed by confocal laser scanning microscope. Cell proliferation was assessed by ftuorospectrophotometer. A microplate reader and Von Kossa staining were used to analyze alkaline phosphatase (ALP) activity and calcium salts mineralization.RESULTS AND CONCLUSION: FlMSCs had long processes which connected each other and formed network in fibrin gels, but fusiform or cube cells were observed in the control group at 7 days. Compared with the control group, cell proliferation was great in the fibrin gel group, and peaked at day 14, especially in the 5 g/L fibrin gel group. ALP activity was increased at day 14 in the fibrin gel group, and reached a peak at day 21. Following 14 days of incubation, mineralization was observed in 20 g/L fibrin gel group, and then gradually fused. However, no mineralization was detected in the control group. Results verified that fibrin gel scaffold can support the survival and growth of rat embryonic MSCs, and promote cell proliferation and differentiation. 5 g/L fibrin gel contributes to cell morphological changes, and 20 g/L fibrin gel contributes to osteogenic differentiation.