Human adipose-derived mesenchymal stem cells and synovial-derived mesenchymal stem cells synergistically inhibit the degeneration of inflammatory chondrocytes

doi:10.3969/j.issn.2095-4344.0537

Abstract

Abstract:

BACKGROUND: Intra-articular injection of cells targeting to change the microenvironment in lesions can act on early osteoarthritis of inflammatory chondrocytes. Implanted cells affect the progress of the disease by the cell characteristics.
OBJECTIVE: To explore the synergistic effect of mesenchymal stem cells from human knee adipose (ADMSCs) and synovial tissues (SDMSCs) to inhibit the degeneration of inflammatory chondrocytes.
METHODS: ADMSCs, SDMSCs and inflammatory chondrocytes were primary cultured. Under in vitro two-dimensional culture conditions, cell proliferation assay (MTS) was performed to detect the proliferation of three kinds of cells. Differences in chondrogenic markers at mRNA and protein levels between three kinds of adherent cells were detected by quantitative PCR and immunofluorescence. Under in vitro three-dimensional mixed culture conditions, three groups were set up: (1) ADMSCs+inflammatory chondrocytes (A+C group), (2) SDMSCs+inflammatory chondrocytes (S+C group), and (3) ADMSCs-SDMSCs+inflammatory chondrocytes (A+S+C group). Alcian blue staining, safranin O staining and type II collagen immunohistochemistry staining were performed on the mixed-cultured cell mass paraffin sections followed by quantitative analysis. Chondrogenic differentiation in each group was detected by quantitative PCR. Culture supernatants were collected to detect the secretion of pro-inflammatory and anti-inflammatory factors by enzyme-linked immunosorbent assay.
RESULTS AND CONCLUSION: Under the two-dimensional culture, the proliferative rate of ADMSCs was significantly higher than that of inflammatory chondrocytes and SDMSCs (P < 0.05). The expression of type II collagen mRNA and protein and proteoglycan protein in inflammatory chondrocytes was significantly higher than that in the other two kinds of cells (P < 0.01). Under the three-dimensional culture, the percentage of chondrogenic area per total area was significantly higher in the A+S+C group than the S+C and A+C groups (P < 0.05). The expression of type II collagen and proteoglycan was significantly higher in the A+S+C group than the S+C and A+C groups (P < 0.05). Compared with the other two groups, the S+C group showed higher levels of interleukin 1, interleukin 6, and tumor necrosis factor α, but lower level of interleukin 10 (P < 0.05). To conclude, the combined use of ADMSCs and SDMSCs synergistically inhibits the degeneration of inflammatory chondrocytes.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Adipose Tissue, Synovial Membrane, Mesenchymal Stem Cells, Chondrocytes, Coculture Techniques, Osteoarthritis, Tissue Engineering

CLC Number:

R394.2

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.

Figures/Tables 2

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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