摘要目的 探讨成人退变髓核细胞的体外微载体旋转立体培养模式及其对细胞外基质合成的影响.方法 标本取自2005年9月至2009年5月因椎间盘疾患而施行椎体间融合术的患者,对总共34个退变的髓核组织进行体外培养,将其随机分为单层培养组和微载体旋转立体培养组.对2种培养方式的对数生长期髓核细胞进行Ⅰ、Ⅱ型胶原的SP-ABC免疫组化法染色,并行Ⅰ、Ⅱ型胶原的Western blot定量检测；用35S标记放射免疫定量分别检测两组处于不同生长期细胞的蛋白多糖含量,数据采用两独立样本的t检验进行统计分析.结果 Ⅰ、Ⅱ型胶原的SP-ABC免疫组化法和Western blot定量检测均显示微载体培养组高于单层培养组.SP-ABC免疫组化法结果:Ⅰ型胶原:32.5±4.4比15.2±1.2,t=2.871,P＜0.01；Ⅱ型胶原:43.6±4.1比23.1 ±2.2,t=2.375,P＜0.05；Western blot定量检测结果:Ⅰ型胶原:0.62±0.08比0.50 ±0.06,t=3.327,P＜0.01;Ⅱ型胶原:1.46±0.08比0.86±0.04,t=2.453,P＜0.05.35S标记放射免疫显示两种生长期的髓核细胞,微载体培养组表达蛋白多糖的含量均高于单层培养组(稳定生长期:34 821±312比21 046±673,t=2.134,P＜0.05;对数生长期:45 134±175比32 193±713,t=2.801,P＜0.01).结论 微载体旋转立体培养法对成人退变髓核细胞基质内Ⅰ、Ⅱ型胶原和蛋白多糖的表达具有正向调控的作用,可以适用于成人退变髓核细胞的大量扩增.

abstractsObjective To evaluate the biological effect on the synthesis of the extracellular matrix (ECM) in the cultivation of adult degenerative nucleus pulposus cells using the stiring microcarrier system in vitro.Methods Thirty-four specimens were collected after intervertebral fusion operations of the patients with intervertebral disc herniation diseases from September 2005 to May 2009.The specimens were then randomly allocated into 2 groups for in vitro cultivation:monolayer culture group and microcarrier culture group.On the exponential phase,SP-ABC immunohistochemical staining and Western blot quantitative analysis were conducted in the two groups to detect the collagen type Ⅰ and Ⅱ.Proteoglycan contents of two groups in different growth phases were detected with 35S-sulfate incorporation assay.Results The expressions of collagen type Ⅰ and Ⅱ in microcarrier culture group were significantly higher than those in monolayer culture group:SP-ABC immunohistochemical staining (collagen type Ⅰ:32.5 ± 4.4 vs.15.2 ±1.2,t=2.871,P＜0.01; collagen typeⅡ:43.6±4.1 vs.23.1±2.2,t=2.375,P＜0.05); Western blot quantitative analysis (collagen type Ⅰ:0.62 ± 0.08 vs.0.50 ± 0.06,t =3.327,P ＜ 0.01 ; collagen type Ⅱ:1.46 ± 0.08 vs.0.86 ± 0.04,t =2.453,P ＜ 0.05).Nucleus pulposus cells cultivated in stiring microcarrier system showed significantly increased proteoglycan synthesis than monolayer culture group does on both exponential phase and stationary phase (exponential phase:34 821 ± 312 vs.21 046 ±673,t=2.134,P ＜ 0.05; stationary phase:45 134 ± 175 vs.32 193 ± 713,t =2.801,P ＜ 0.01).Conclusions The expression of collagen type Ⅰ,Ⅱ and proteoglycan of adalt degenerative nucleus pulposus cells are positive regulated by the stiring microcarrier system,which can be used in the mass amplification of the adult degenerative nucleus pulposus cells.