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目的种子细胞来源是软骨组织工程的研究热点。探讨以RNA干扰(RNA interference,RNAi)技术干扰多聚蛋白聚糖酶(Aggrecanase)后的软骨细胞作为组织工程种子细胞的可行性。方法取成年SD大鼠肋软骨采用消化法分离培养软骨细胞。取第1代细胞分为空白阴性对照组(对照组)及慢病毒转染组(实验组)两组。将两组细胞分别与壳聚糖/明胶材料进行体外复合培养制备组织工程软骨,于培养后不同时间点通过HE、Masson染色,扫描电镜观察以及RT-PCR方法检测两组软骨中蛋白聚糖(Aggrecan)以及Aggrecanase-1、2的变化情况。结果组织学观察示培养2周时,对照组与实验组相比细胞数量及细胞外基质未见明显差别;随着培养时间延长,实验组较对照组细胞外基质分泌增多,细胞数目更多。RT-PCR结果显示培养4、8周,实验组细胞Aggrecan mRNA表达量明显高于对照组,Aggrecanase-1和Aggrecanase-2 mRNA表达量明显低于对照组,比较差异均有统计学意义(P<0.05)。扫描电镜显示实验组细胞数量明显多于对照组,且细胞间连接紧密。结论采用RNAi技术干扰Aggrecanase后的软骨细胞可以作为组织工程软骨种子细胞,其在材料上分泌Aggrecan的含量明显高于正常软骨细胞,其生物学活性需进一步研究。
The purpose of seed cells is the research focus of cartilage tissue engineering. To investigate the feasibility of using RNA interference (RNAi) technology to interfere with the chondrocytes of Aggrecanase as tissue-engineered seed cells. Methods Chondrocytes were isolated and cultured by the method of digestion in adult costal cartilage of adult SD rats. The first generation of cells were divided into blank negative control group (control group) and lentiviral transfection group (experimental group) two groups. Tissue engineered cartilage was prepared by in vitro culture of two groups of cells with chitosan / gelatin, respectively. HE and Masson staining, scanning electron microscopy and RT-PCR were used to detect the expression of proteoglycan Aggrecan) and Aggrecanase-1, 2 changes. Results Histological observation showed that there was no significant difference between the control group and the experimental group in cell number and extracellular matrix at 2 weeks of culture. With the prolongation of culture time, the extracellular matrix secretion and the number of cells in the experimental group increased more than those in the control group. The results of RT-PCR showed that the expression of Aggrecan mRNA in the experimental group was significantly higher than that in the control group after 4 and 8 weeks of culture, and the expression of Aggrecanase-1 and Aggrecanase-2 mRNA was significantly lower than that of the control group (P < 0.05). Scanning electron microscopy showed that the number of cells in the experimental group was significantly more than that of the control group, and the cell junctions were tight. Conclusions The chondrocytes that interfere with Aggrecanase by RNAi can be used as tissue-engineered cartilage seed cells. Aggrecan secreted on the material is significantly higher than that of normal chondrocytes, and its biological activity needs further study.