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背景:以往主要是针对肋软骨、关节软骨、鼻中隔软骨、耳软骨等透明软骨进行研究,而对人椎体终板软骨的体外培养尚不多见。目的:探讨Ⅰ型神经纤维瘤病伴萎缩性脊柱侧凸患者萎缩区椎体终板软骨细胞体外分离培养的可行性,并对其生物学特性进行观察。方法:对术中取得的7例Ⅰ型神经纤维瘤病伴萎缩性脊柱侧凸患者顶椎区椎体终板软骨采用两步酶消化结合组织块法进行体外分离培养。细胞传代后倒置相差显微镜下观察细胞形态;Ⅱ型胶原免疫组化染色对软骨细胞进行鉴定;MTT比色法测定细胞增殖情况,描绘第2代软骨细胞的生长曲线。结果与结论:软骨组织培养2周后周围开始有软骨细胞爬出,再经1周左右可以传代。随着传代次数的增加,软骨细胞逐渐由多角形、圆形、三角形和不规则形变为梭形,折光性下降。Ⅱ型胶原免疫组化染色可见细胞核周围被染成棕褐色。MTT比色法检测显示,第2代终板软骨细胞的生长曲线近似倒“S”形,在第4-7天进入快速增殖期,细胞呈对数生长;到第8-13天达平台期,细胞增殖停止;从第14天开始出现生长抑制。结果显示采用两步酶消化结合组织块法能够培养出纯度高,活性好的终板软骨细胞。第2代终板软骨细胞在第4-7天活力最好,增殖力旺盛,可以作为研究Ⅰ型神经纤维瘤病伴萎缩性脊柱侧凸发病机制的观察点。
Background: In the past, the studies focused on cartilage of cartilage, articular cartilage, cartilage of nasal septum, cartilage of ear cartilage and so on, but it was rare to culture human vertebral endplate cartilage in vitro. Objective: To investigate the feasibility of isolating and culturing vertebral end-plate chondrocytes in patients with type Ⅰ neurofibromatosis with atrophic scoliosis in vitro and to observe its biological characteristics. Methods: Seven cases of type Ⅰ neurofibromatosis with atrophic scoliosis in patients with vertebral endplate vertebral endplate cartilage using two-step enzyme digestion combined with tissue block method for in vitro isolation and culture. The cell morphology was observed under inverted phase contrast microscope. The type Ⅱ collagen immunohistochemistry was used to identify the chondrocytes. The proliferation of the second generation of chondrocytes was measured by MTT assay. RESULTS AND CONCLUSION: After 2 weeks of culture, cartilage tissue began to climb out of chondrocytes and passaged after about 1 week. With the increase of passage times, chondrocytes gradually changed from polygons, circles, triangles and irregularities to fusiform shapes, and the refractive index decreased. Type Ⅱ collagen immunohistochemical staining around the nucleus was stained tan. MTT colorimetric assay showed that the growth curve of the second generation of end-plate chondrocytes was approximately “S” -shaped, and entered a rapid proliferative phase on day 4-7 with a logarithmic growth; on day 8-13 During the plateau, cell proliferation ceased; growth inhibition began on the 14th day. The results showed that two-step enzyme digestion combined with tissue block method can produce high purity, good activity of end-plate chondrocytes. The second generation of end-plate chondrocytes had the highest viability and proliferative power at day 4-7, which could be used as a study point to investigate the pathogenesis of type I neurofibromatosis with atrophic scoliosis.