Chinese Journal of Tissue Engineering Research ›› 2018, Vol. 22 ›› Issue (17): 2661-2668.doi: 10.3969/j.issn.2095-4344.0537
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Song Zhuo-yue, Wang Yang, Lian Xiao-lei, Ding Kang, Li Guang-heng
Revised:
2018-03-22
Online:
2018-06-18
Published:
2018-06-18
Contact:
Li Guang-heng, M.D., Professor, Doctoral supervisor, Department of Orthopedics, the First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan Province, China
About author:
Song Zhuo-yue, Master candidate, Department of Orthopedics, the First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan Province, China
Supported by:
the National Natural Science Foundation of China, No. 81472136
CLC Number:
Song Zhuo-yue, Wang Yang, Lian Xiao-lei, Ding Kang, Li Guang-heng. Human adipose-derived mesenchymal stem cells and synovial-derived mesenchymal stem cells synergistically inhibit the degeneration of inflammatory chondrocytes[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(17): 2661-2668.
2.1 原代细胞培养及细胞增殖情况 图1A为原代培养的3种细胞即ADMSCs、SDMSCs、炎性软骨细胞,均表现出类成纤维细胞的形态即细胞均呈现梭形,少数表现为多角形。软骨细胞呈现短梭形多分支,而另外2种细胞呈现长梭形。 图1B显示3种贴壁细胞在不同时间点细胞增殖情况,通过MTS细胞增殖实验发现第2天ADMSCs和SDMSCs的增殖速率开始大于炎性软骨细胞(ADMSCs 1.3±0.2,SDMSCs 1.1±0.1,软骨细胞0.7±0.1),差异有显著性意义(P < 0.05),但是前两者之间比较差异无显著性意义(P > 0.05)。从第4天开始ADMSCs的增殖速率开始大于SDMSCs(ADMSCs 1.65±0.1,SDMSCs 1.3±0.2,软骨细胞1.0±0.1),差异有显著性意义(P < 0.05),而SDMSCs和软骨细胞的增殖速率差异无显著性意义(P > 0.05)。 2.2 贴壁细胞成软骨分化能力的基因及蛋白水平差异 图2A及2B显示炎性软骨细胞的成软骨标志物Ⅱ型胶原蛋白及蛋白聚糖在mRNA水平的表达量要高于ADMSCs、SDMSCs,差异有非常显著性意义(P < 0.01),而ADMSCs与SDMSCs的成软骨能力在mRNA水平上差异无显著性意义。图2C为3种细胞Ⅱ型胶原免疫荧光染色的显微镜下形态,通过图2D的定量分析显示炎性软骨细胞在蛋白水平表达的Ⅱ型胶原明显高于ADMSCs、SDMSCs,差异有非常显著性意义(P < 0.01),SDMSCsⅡ型胶原蛋白水平高于ADMSCs,差异有显著性意义(P < 0.05)。 2.3 3D混合培养细胞团块成软骨分化的特殊染色及免疫组化染色 图3A是3D培养至14 d及21 d的3组细胞团块,可以看出培养至14,21 d的细胞团块大小依次为:A+S+C组>S+C组>A+C组,差异有显著性意义(P < 0.05)。图3B-D分别为培养至14 d及21 d的3组细胞团块石蜡切片进行阿尔新蓝染色、番红O染色及Ⅱ型胶原免疫组化染色在不同倍数(×40、×200)显微镜下的大体观,图3E为定量分析发现阿尔新蓝染色、番红O染色及Ⅱ型胶原免疫组化阳性区域面积大小依次为:A+S+C组>S+C组>A+C组,差异有显著性意义(P < 0.05)。 2.4 3D培养条件下不同时间点3组细胞团块蛋白水平、mRNA水平及细胞增殖情况的差异 为了进一步证明联合应用ADMSCs和SDMSCs对炎性软骨细胞退变的协同抑制作用,通过蛋白、基因及细胞增殖3方面进行解释。图4A-C分别显示在3个不同时间点S+C组分泌的促炎因子白细胞介素1β、白细胞介素6、肿瘤坏死因子α水平均较A+C组及A+S+C组高,差异有显著性意义(P < 0.05)。而A+C组和A+S+C组在第7,14,21天分泌的白细胞介素1β、白细胞介素6、肿瘤坏死因子α水平差异无显著性意义(P > 0.05)。图4D显示不同时间点A+C组和A+S+C组分泌的抗炎因子白细胞介素10水平明显高于S+C组,差异有非常显著性意义(P < 0.01)。图4E显示7,14,21 d成软骨分化的标志物蛋白聚糖在mRNA水平A+S+C组明显高于A+C组和S+C组,且第21天S+C组表达量高于A+C组,差异有显著性意义(P < 0.05)。图4F显示7,14,21 d软骨基质Ⅱ型胶原在mRNA水平A+S+C组明显高于A+C组和S+C组,差异有显著性意义(P < 0.05)。第14,21天 S+C组表达量高于A+C组,差异有显著性意义(P < 0.05)。图4G显示不同时间点3组细胞的增殖情况,在0,7 d 3组细胞个数差异无显著性意义(P > 0.05),而第14天及第21天,细胞总数:A+S+C组>S+C组>A+C组,差异有显著性意义(P < 0.05)。"
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