白血病抑制因子对退变髓核细胞胞外基质的影响 及其机制研究

1. Department of Orthopedics，The First Affiliated Hospital of Chongqing Medical University； 2. Department of Orthopedics，Jiangxi Provincial People’s Hospital

摘要:

目的：探讨退变椎间盘髓核组织白血病抑制因子（leukemia inhibit factor，LIF）表达及其对胞外基质表达的影响。方法：40只新西兰兔随机分5组（1组为对照组，4组为实验组），每组8只。针刺法建立L4/5、L5/6椎间盘退变模型，术后0、2、4、8周对椎间盘进行MRI扫描及HE染色评价退变程度，免疫组化检测髓核组织LIF的表达。体外原代培养兔髓核细胞，不同浓度重组LIF蛋白刺激髓核细胞24、48、72 h，Western blot检测聚蛋白多糖（Aggrecan）、Ⅱ型胶原（COLLA2α1）的表达，免疫荧光检测最大浓度组和对照组Aggrecan的表达并行Western blot检测金属蛋白酶组织抑制物-1（tissue inhibitor of metalloproteinases，TIMP-1）和基质金属蛋白酶-3（matrixmetalloproteinase-3，MMP-3）的表达。结果：影像学和组织学评分显示椎间盘退变随造模时间延长而加重，各组间差异有统计学意义（1.000±0.000，2.000±0.535，2.750±0.463，3.875±0.354，P=0.000；4.000±0.000，7.375±0.518，9.375±1.302，11.750±0.463，P=0.000）。免疫组化显示2周组LIF表达最高，随着椎间盘退变加重，其表达降低，但各组均高于0周组，差异有统计学意义（559.608±68.689，16 135.613±577.329，8 149.739±457.189，2 018.254±211.870，P=0.000）。细胞学实验显示，加入不同浓度LIF刺激后，Aggrecan、COLLA2α1的蛋白表达与LIF刺激浓度呈正相关，各处理组与对照组差异有统计学意义（0.191±0.020，0.212±0.020，0.321±0.041，0.511±0.032，0.561±0.042，P=0.000），Aggrecan免疫荧光结果与之相符。处理组TIMP-1表达明显高于对照组（0.454±0.022，0.211±0.012，P=0.000），MMP-3明显低于对照组（0.243±0.013，0.362±0.021，P=0.000）。结论：LIF在退变髓核组织中表达上升，且具有促进胞外基质合成作用，其潜在机制与LIF能够上调TIMP-1并下调MMP-3作用相关。分为A组（AMD3100+NS动员组），B组（AMD3100+G-CSF动员组），C组（G-CSF+AMD3100动员组）和D组（NS空白对照组）；80只db/+小鼠为相应对照组，分组对应为A’组，B’组，C’组和D’组。每组在动员后第3 、7、10、14 天4个时间点于心脏部位取外周血，流式细胞仪检测CD34+/CD133+/Flk-1+细胞的比例。结果取平均值用均值±标准差（单位%）表示，统计学方法处理后进行比较和分析。结果：①D组和D’组在动员后第3、7、10、14 天4个时间点，CD34+/CD133+/Flk-1+细胞比例无差异（P >0.05），但D’组中CD34+/CD133+/Flk-1+细胞比例高于D组（P=0.003）。②A组、B组和C组db/db小鼠在动员后第3、7、10、14 天4个时间点，CD34+/CD133+/Flk-1+细胞比例逐渐上升，A组至第7 天达到峰值，随后逐渐下降；B组和C组至第10 天达到峰值，随后逐渐下降，且在达到峰值时CD34+/CD133+/Flk-1+细胞比例与其余3个时间点相比差异有意义（P=0.000）。在第10 天，B组和C组db/db小鼠外周血中CD34+/CD133+/Flk-1+细胞比例较A组和D组高（P=0.000），但B组和C组db/db小鼠外周血中CD34+/CD133+/Flk-1+细胞比例之间的差异无统计学意义（P >0.05）。③A’组、B’组和C’组db/+小鼠在动员后第3、7、10、14天4个时间点，CD34+/CD133+/Flk-1+细胞比例逐渐上升，至第7 天达到高峰，随后逐渐下降，且在第7天CD34+/CD133+/Flk-1+细胞比例与其余3个时间点相比差异有意义（P=0.000）。在达到峰值的第7天，C’组CD34+/CD133+/Flk-1+细胞比例较A’组、B’组和D’组高（P=0.000）。结论：AMD3100联合G-CSF动员骨髓EPCs的效果优于单一使用AMD3100的动员效果，在非糖尿病状态下，先使用G-CSF再联合AMD3100的动员作用更优，但在糖尿病状态下先使用AMD3100再联合G-CSF的动员作用更优。

Abstract:

Objective：To investigate the expression of leukemia inhibit factor（LIF） in degenerative disc nucleus pulposus and its role in degenerative nucleus pulposus extracellular matrix synthesis. Methods：Forty adult New Zealand white rabbits were randomly divid－ed into 5 groups（1 group as the control group，the remaining 4 groups as the experimental group），8 rabbits in each group. Interverte－bral disc degeneration model was established by puncturing the L4/5 and L5/6 intervertebral discs in the experimental group. MRI scoring was performed on the discs at 0，2，4，8 weeks immediately after the operation，and the discs were obtained and stained with hematoxylin and eosin to get histological scores and to determine the degree of degeneration. Immuno-histochemical staining was used to semi-quantitatively detect the expression of LIF in nucleus pulposus. Rabbit nucleus pulposus cells were harvested for primary culture；the expressions of Aggrecan，COLLA2α1 in human in－tervertebral disc nucleus pulposus cells were detected by Western blot at 24，48，72 h after treated with gradient concentration hu－man recombinant LIF protein. In maximal concentration group and control group，Aggrecan expression was again verified by immunofluorescence and the expressions of tissue inhibitor of metalloproteinases-1（TIMP-1） and matrixmetalloproteinase-3（MMP-3） were detected by Western blot. Results：The imaging scores and histological scores showed that the degeneration of intervertebral disc increased with the prolongation of modeling time，and there was a significant difference（1.000±0.000，2.000±0.535，2.750±0.463，3.875±0.354，P=0.000；4.000±0.000，7.375±0.518，9.375±1.302，11.750±0.463，P=0.000）. The LIF expression in puncture 2-week group was significantly higher，as disc degen－eration aggravated，the expression decreased gradually，and the expression level of each experimental group was higher than that of the 0 week group，the difference was statistically significant（559.608±68.689，16 135.613±577.329，8 149.739±457.189，2 018.254±211.870，P=0.000）. Cytology experiments showed that after stimulation of different LIF concentrations，Aggrecan，COLLA2α1 protein expression and LIF stimulus concentration was positively correlated，and there was a significant difference between each concentration group and the control group（0.191±0.020，0.212±0.020，0.321±0.041，0.511±0.032，0.561±0.042，P=0.000），and Aggrecan immunofluorescence results were consistent. TIMP-1 expression in the treatment group was significantly higher than that of control group（0.454±0.022，0.211±0.012，P=0.000）；MMP-3 expression in the treatment group was significantly lower than that in the control group（0.243±0.013，0.362±0.021，P=0.000）. Conclusion：LIF expression in degenerative nucleus pulposus is increased，and LIF can promote the synthesis of extracellular matrix，and its underlying mechanism is related to up-regulating TIMP-1 and down-regulating MMP-3.