Chen Qi, Shi Fangfang, Yang Xi, Liu Limin, Song Yueming
Department of Orthopedics, Zhejiang Provincial People's Hospital, People's Hospital of Hangzhou Medical College, Hangzhou Zhejiang, 310014, P.R.China;Department of Orthopedics, West China Hospital, Sichuan University, Chengdu Sichuan, 610041, P.R.China.
Department of Hematology, Zhejiang Provincial People's Hospital, People's Hospital of Hangzhou Medical College, Hangzhou Zhejiang, 310014, P.R.China.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2018 Jun 15;32(6):660-667. doi: 10.7507/1002-1892.201707017.
To research the biological characteristics of different generations of rabbit nucleus pulposus cells (NPCs) that were cultured with natural culture and subculture method.
The thoracolumbar segments of New Zealand white rabbits (6-8 weeks old and weighing 1.5-2.5 kg) were obtained and nucleus pulposus were isolated from disc regions. And NPCs were harvested by enzymatic digestion from nucleus pulposus. Primary NPCs were counted as P0 generation. Then, NPCs were passaged by trypsin and counted as P1, P2, P3 with a totle of 4 generations. P0 to P3 generations NPCs were separately examined by observation of cell morphology and proliferation time, detection of apoptosis rates of cells by flow cytometry, and detection of hypoxia-inducible factor 1α (HIF-1α), matrix metalloproteinases 2 (MMP-2), Aggrecan, and collagen type Ⅱ proteins by immunofluorescence and Western blot.
The morphology of NPCs transformed from triangular or polygonal in P0 generation to spindle in P3 generation; the characteristic vacuolated cells gradually disappeared; and the cell volume and cell proliferation time increased. The cell apoptosis rates were 5.47%±0.91%, 13.77%±2.42%, 33.46%±1.82%, and 38.76%±1.50% from P0 to P3 generations, with the increase of culture time, and there were significant differences between 4 generations ( <0.05). Immunofluorescence staining showed that with the increase of cells generation, the fluorescence intensity of HIF-1α, collagen type Ⅱ, and Aggrecan decreased, and the fluorescence intensity of MMP-2 increased. Western blot results showed that the relative expression of HIF-1α protein was high in P0 generation, the P1 generation has a rising trend, and then gradually decreased; the differences between generations were significant ( <0.05). The relative expression of collagen type Ⅱ protein decreased from P0 to P3 generations and there were significant differences between generations ( <0.05). The relative expression of Aggrecan protein decreased from P0 to P2 generations and there were significant differences between generations ( <0.05); but no significant difference was found between P2 and P3 generations ( >0.05). The relative expression of MMP-2 protein increased significantly in P3 generation; except that the difference between P0 and P2 generations was not significant ( >0.05), the significant differences were found between the other generations ( <0.05).
Rabbit NPCs degeneration model was successfully established by the natural culture and subculture method. Transforming of NPCs morphology, increasing of cell apoptosis rates, decreasing of anabolism, and increasing of catabolism were presented in NPCs degeneration model.
研究采用自然培养和传代培养方法培养的不同代兔髓核细胞（NPCs）的生物学特性。
获取6 - 8周龄、体重1.5 - 2.5 kg的新西兰白兔的胸腰段，从椎间盘区域分离出髓核。通过酶消化法从髓核中获取NPCs。原代NPCs计为P0代。然后，用胰蛋白酶对NPCs进行传代，计为P1、P2、P3代，共4代。分别通过观察细胞形态和增殖时间、流式细胞术检测细胞凋亡率以及免疫荧光和蛋白质印迹法检测缺氧诱导因子1α（HIF - 1α）、基质金属蛋白酶2（MMP - 2）、聚集蛋白聚糖和Ⅱ型胶原蛋白，对P0至P3代NPCs进行检测。
NPCs的形态从P0代的三角形或多边形转变为P3代的梭形；特征性的空泡状细胞逐渐消失；细胞体积和细胞增殖时间增加。从P0代到P3代，细胞凋亡率分别为5.47%±0.91%、13.77%±2.42%、33.46%±1.82%和38.76%±1.50%，随培养时间增加，4代之间差异有统计学意义（<0.05）。免疫荧光染色显示，随着细胞代数增加，HIF - 1α、Ⅱ型胶原蛋白和聚集蛋白聚糖的荧光强度降低，MMP - 2的荧光强度增加。蛋白质印迹结果显示，HIF - 1α蛋白的相对表达在P0代较高，P1代有上升趋势，然后逐渐下降；代间差异有统计学意义（<0.05）。Ⅱ型胶原蛋白蛋白的相对表达从P0代到P3代降低，代间差异有统计学意义（<0.05）。聚集蛋白聚糖蛋白的相对表达从P0代到P2代降低，代间差异有统计学意义（<0.05）；但P2代和P3代之间差异无统计学意义（>0.05）。MMP - 2蛋白的相对表达在P3代显著增加；除P0代和P2代之间差异无统计学意义（>0.05）外，其他代间差异有统计学意义（<0.05）。
通过自然培养和传代培养方法成功建立了兔NPCs退变模型。在NPCs退变模型中呈现出NPCs形态改变、细胞凋亡率增加、合成代谢降低和分解代谢增加的现象。
