人肾小管上皮细胞壳聚糖/明胶三维模型的建立
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摘要
目的:观察人肾小管上皮细胞在壳聚糖/明胶三维支架培养系统中的生长特性,构建人肾小管上皮细胞三维培养生长模型。方法:将人肾近曲小管上皮细胞接种于壳聚糖/明胶三维支架中培养,电镜观察培养不同时间后的细胞形态。用Annexin V&PI双染检测三维培养的细胞凋亡率。结果:电镜观察显示人肾小管细胞在壳聚糖/明胶三维支架中紧贴支架生长。三维培养细胞早期自发凋亡细胞率为2.53%,与二维培养细胞的3.26%相比显著减少(P<0.05);三维培养和二维培养细胞晚期凋亡细胞率分别为1.81%和2.61%,但两者差异无统计学意义(P>0.05)。结论:结果说明壳聚糖/明胶三维支架能够保持人肾小管上皮细胞的细胞活力,降低细胞早期凋亡率是进行人肾小管上皮细胞体外培养的有效途径。
Objective: To investigate the growth characteristics of renal proximal tubule cells cultured in the three- dimensional chitosan /gelatin scaffold. To establish a three- dimensional chitosan / gelatin scaffold for renal proximal tubule cells culture. Method: Renal proximal tubule cells were cultured in the three- dimensional chitosan / gelatin scaffold. Scanning electron microscope was used for the observation of cell morphology. The cells were stained by Annexin V& PI and apoptosis ratios were detected by flowcytomerty. Result: Renal proximal tubule cells grew in monolayer in the two dimensional culture system and the adhesion and interconnection of cells in the scaffolds are formed after cells seeded. The proportions of early stage of apoptosis cells were significantly lower in the cells cultured in three- dimensional chitosan / gelatin scaffold( 2. 53%) than those in the two- dimensional culture system( 3. 26%)( P < 0. 05). Cells cultured in the three- dimensional chitosan / gelatin scaffold displayed lower ratios of late stage of apoptosis( 1. 81%) compared with that cultured in monolayer( 2. 61%),while there was no significant difference( P > 0. 05). Conclusion: Renal proximal tubule cells cultured in the three- dimensional chitosan / gelatin scaffold displayed significantly lower ratio of early stage of apoptosis cells. This cell culture system can be used as a biomaterial plants for the investigation of tubular cells proliferation after injury.
Objective: To investigate the growth characteristics of renal proximal tubule cells cultured in the three- dimensional chitosan /gelatin scaffold. To establish a three- dimensional chitosan / gelatin scaffold for renal proximal tubule cells culture. Method: Renal proximal tubule cells were cultured in the three- dimensional chitosan / gelatin scaffold. Scanning electron microscope was used for the observation of cell morphology. The cells were stained by Annexin V& PI and apoptosis ratios were detected by flowcytomerty. Result: Renal proximal tubule cells grew in monolayer in the two dimensional culture system and the adhesion and interconnection of cells in the scaffolds are formed after cells seeded. The proportions of early stage of apoptosis cells were significantly lower in the cells cultured in three- dimensional chitosan / gelatin scaffold( 2. 53%) than those in the two- dimensional culture system( 3. 26%)( P < 0. 05). Cells cultured in the three- dimensional chitosan / gelatin scaffold displayed lower ratios of late stage of apoptosis( 1. 81%) compared with that cultured in monolayer( 2. 61%),while there was no significant difference( P > 0. 05). Conclusion: Renal proximal tubule cells cultured in the three- dimensional chitosan / gelatin scaffold displayed significantly lower ratio of early stage of apoptosis cells. This cell culture system can be used as a biomaterial plants for the investigation of tubular cells proliferation after injury.
引文
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[14]Tanaka H,Murphy CL,Murphy C.Chondrogenic differentiation of murine embryonic stem cells:effects of culture conditions and dexamethasone[J].Journal of Cellular Biochemistry,2004 Oct 15,93(3):454-462.
[2]Di Martino A,Sittinger M,Risbud MV.Chitosan:a versatile biopolymer for orthopaedic tissue-engineering[J].Biomaterials,2005 Oct,26(30):5983-5990.
[3]Breyner NM,Hell RC,Carvalho LR,et al.Effect of a three-dimensional chitosan porous scaffold on the differentiation of mesenchymal stem cells into chondrocytes[J].Cells Tissues Organs.,2010,191(2):119-128.doi:10.1159/000231472.Epub 2009 Jul 28.
[4]Ravindran S,Song Y,George A.Development of three-dimensional biomimetic scaffold to study epithelial-mesenchymal interactions[J].Tissue Engineering Part A,2010 Jan,16(1):327-342.doi:10.1089/ten.TEA.2009.0110.
[5]Gloria A,De Santis R,Ambrosio L.Polymer-based composite scaffolds for tissue engineering[J].Journal of Biomaterials Applications,2010May-Aug,8(2):57-67.
[6]Lima EG,Mauck RL,Han SH,et al.Functional tissue engineering of chondral and osteochondral constructs[J].Biorheology,2004,41(3-4):577-590.
[7]Schek RM,Taboas JM,Segvich SJ,et al.Engineered osteochondralgrafts using biphasic composite solid free-form fabricated scaffolds[J].Tissue Engineering,2004 Sep-Oct,10(9-10):1376-1385.
[8]Yamane S,Iwasaki N,Majima T,et al.Feasibility of chitosan-based hyaluronic acid hybrid biomaterial for a novel scaffold in cartilage tissue engineering[J].Biomaterials,2005 Feb,26(6):611-619.
[9]Mao J,Zhao L,De Yao K,et al.Study of novel chitosan-gelatin artificial skin in vitro[J].Journal of Biomedical Materials Research Part A,2003 Feb 1,64(2):301-308.
[10]Medrado GC,Machado CB,Valerio P,et al.The effect of a chitosangelatin matrix and dexamethasone on the behavior of rabbit mesenchymal stem cells[J].Biomedical Materials,2006 Sep,1(3):155-61.doi:10.1088/1748-6041/1/3/010.
[11]McMahon LA,Prendergast PJ,Campbell VA.A comparison of the involvement of p38,ERK1/2 and PI3K in growth factor-induced chondrogenic differentiation of mesenchymal stem cells[J].Biochemical Biophysical Research Communications,2008 Apr 18,368(4):990-995.
[12]Mehlhorn AT,Niemeyer P,Kaschte K,et al.Differential effects of BMP-2 and TGF-beta1 on chondrogenic differentiation of adipose derived stem cells[J].Cell Proliferation,2007 Dec,40(6):809-823.
[13]Huang CK,Huang W,Zuk P,et al.Genetic markers of osteogenesis and angiogenesis are altered in processed lipoaspirate cells when cultured on three-dimensional scaffolds[J].Plastic and Reconstructive Surgery,2008 Feb,121(2):411-423.
[14]Tanaka H,Murphy CL,Murphy C.Chondrogenic differentiation of murine embryonic stem cells:effects of culture conditions and dexamethasone[J].Journal of Cellular Biochemistry,2004 Oct 15,93(3):454-462.