华西医学

华西医学

型胶原蛋白在不同损伤类型动物模型椎间盘组织内的含量变化规律

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目的 通过对损伤动物模型椎间盘软骨终板细胞内Ⅰ、Ⅱ型胶原 mRNA 的水平进行定量测定,明确椎间盘软骨细胞在椎间盘损伤后纤维化修复过程中的细胞学功能变化规律。 方法 建立兔椎间盘损伤模型,选用健康新西兰大白兔 40 只,随机分为纤维环穿刺组、上位软骨终板单次穿刺组、上下位软骨终板多次穿刺组、假手术对照组,分别于建模手术术后第 2 天及第 2、8、12、24 周在每组中随机选取 2 只实验动物获取椎间盘标本,逆转录聚合酶链反应测定椎间盘软骨终板细胞Ⅰ、Ⅱ型胶原 mRNA 水平。首先提取椎间盘软骨终板组织的 RNA,通过核酸测定仪测定 RNA 浓度及 RNA 浓度与蛋白浓度比值,采用 Revertaid M-Mulv 逆转录酶进行逆转录,设计Ⅰ、Ⅱ型胶原引物,建立聚合酶链反应体系,用 0.5×TBE 电泳缓冲液配制 2% 的琼脂糖凝胶电泳(120 V,40 min),紫外灯下观察扩增结果,测定不同电泳条带的灰度值。 结果 术后各手术组Ⅰ型胶原 mRNA 水平在术后 8 周后呈现进行性上升的趋势,上升的幅度与损伤程度相关。Ⅱ型胶原的 mRNA 水平在纤维环穿刺组及上位终板单次穿刺组中开始 2 周内出现一过性的上升,随后开始进行性下降;而在上下位软骨终板多次穿刺组则从术后开始一直呈现下降的趋势。 结论 损伤椎间盘髓核组织内软骨细胞Ⅰ型胶原的合成量随时间呈现持续升高的趋势,而Ⅱ型胶原的合成量在小幅升高后开始进行性下降。Ⅰ、Ⅱ型胶原在损伤椎间盘软骨细胞内的形成变化规律有别于自然退变。

ObjectiveTo quantitatively determine the levels of type Ⅰ and type Ⅱ collagen mRNA in the intervertebral disc cartilage endplate of injured animal model, and to clarify the cytological function of intervertebral disc chondrocytes during fibrosis repair after intervertebral disc injury.MethodsForty healthy New Zealand rabbits were randomly divided into fibrosus puncture group, upper cartilage endplate single puncture group, upper and lower cartilage endplate multiple puncture group, and sham group. Two experimental animals were randomly selected from each group on the 2nd day, and the 2nd, 8th, 12th, and 24th week after the animal modeling operation to obtain intervertebral disc specimens. The levels of type Ⅰ collagen and type Ⅱ collagen in cartilage endplate cells of the intervertebral disc were determined by reverse transcriptase polymerase chain reaction (PCR). RNA was extracted from the endplate of the intervertebral disc, and the RNA concentration and the ratio of RNA concentration to protein concentration were determined by nucleic acid analyzer. Reverse transcription was performed by Revertaid M-Mulv reverse transcriptase, type Ⅰ and type Ⅱ collagen primers were designed to establish a PCR reaction system, 2% agarose gel electrophoresis (120 V, 40 min) was prepared by using 0.5×TBE electrophoresis buffer. The amplification results were observed under ultraviolet light, and the gray values of different electrophoresis bands were determined.ResultsThe level of type Ⅰ collagen mRNA in each operation group showed a progressive increase after 8 weeks, and the magnitude of the increase was related to the degree of injury. The level of type Ⅱ collagen mRNA showed a transient increase in the fibrosis puncture group and the upper endplate single-puncture group in the first two weeks after the endplate punctures were completed, and then began to decline progressively; in the multiple puncture group, it showed a downward trend from the beginning of the operation. ConclusionsThe synthesis of type Ⅰ collagen in chondrocytes of the injured nucleus pulposus tissue continues to increase with time, while the synthesis of type Ⅱ collagen begin to decrease progressively after a small increase. The formation and change of type Ⅰ and type Ⅱ collagen in injured intervertebral disc chondrocytes are different from natural degeneration.

关键词: 损伤; 软骨终板; Ⅰ、Ⅱ型胶原; 逆转录聚合酶链反应

Key words: Injury; Cartilage endplate; Type Ⅰ/Ⅱ collagen; Reverse transcriptase polymerase chain reaction

引用本文: 赵小丹, 刘浩, 黄富国, 刘熹, 邓宇骁. 型胶原蛋白在不同损伤类型动物模型椎间盘组织内的含量变化规律 . 华西医学, 2018, 33(9): 1146-1152. doi: 10.7507/1002-0179.201807150 复制

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