[搅拌式微载体培养系统体外培养成年退变髓核细胞细胞外基质的变化]

[The changes of extracellular matrix in adult degenerative nucleus pulposus cells with stiring microcarrier culture system in vitro].

作者信息

Ning Bin, Liu Hai-fei, Gong Wei-ming, Zhao Kai, DU Hong-xia, Liu Yong, Wang De-chun, Hu You-gu

机构信息

Department of Orthopaedic, Jinan Central Hospital Affiliated to Shandong University, Jinan 250013, China.

出版信息

Zhonghua Wai Ke Za Zhi. 2013 May 1;51(5):432-6.

PMID:23958167

Abstract

OBJECTIVE

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 (35)S-sulfate incorporation assay.

RESULT

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 type II: 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 adult 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.

摘要

目的

体外评价搅拌式微载体系统培养成人退变髓核细胞对细胞外基质（ECM）合成的生物学效应。

方法

收集2005年9月至2009年5月间因椎间盘突出症行椎间融合手术患者的34份标本，随机分为2组进行体外培养：单层培养组和微载体培养组。在指数生长期，对两组进行SP-ABC免疫组织化学染色和蛋白质印迹定量分析以检测Ⅰ型和Ⅱ型胶原。用（35）S-硫酸盐掺入法检测两组在不同生长阶段的蛋白聚糖含量。

结果

微载体培养组Ⅰ型和Ⅱ型胶原的表达明显高于单层培养组：SP-ABC免疫组织化学染色（Ⅰ型胶原：32.5±4.4对15.2±1.2，t=2.871，P<0.01；Ⅱ型胶原：43.6±4.1对23.1±2.2，t=2.375，P<0.05）；蛋白质印迹定量分析（Ⅰ型胶原：0.62±0.08对0.50±0.06，t=3.327，P<0.01；Ⅱ型胶原：1.46±0.08对0.86±0.04，t=2.453，P<0.05）。搅拌式微载体系统培养的髓核细胞在指数生长期和静止期的蛋白聚糖合成均明显高于单层培养组（指数生长期：34 821±312对21 046±673，t=2.134，P<0.05；静止期：45 134±175对32 193±713，t=2.801，P<0.01）。