白细胞介素1β对大鼠髓核间质干细胞生物学特性的影响.docx

白细胞介素1β对大鼠髓核间质干细胞生物学特性的影响
张  宁1，关晓明1，马  迅2，张  丽2，张  辉1，宋  亮1
1山西医科大学，山西省太原市  030001；2山西大医院骨科，山西省太原市  030032

Interleukin-1 beta affects the biological properties of rat nucleus pulposus-derived mesenchymal stem cells
Zhang Ning1, Guan Xiao-ming1, Ma Xun2, Zhang Li2, Zhang Hui1, Song Liang1
1 Shanxi Medical University, Taiyuan 030001, Shanxi Province, China; 2 Department of Orthopedics, Shanxi Dayi Hospital, Taiyuan 030032, Shanxi Province, China
摘要 
背景：内源性干细胞并未在椎间盘退变的进程发挥修复作用，作者推测可能与相关致病因素的异常作用有关，导致对髓核间质干细胞功能产生一定的抑制效应。
目的：观察炎症因子白细胞介素1β对大鼠髓核间质干细胞生物学特性的影响。
方法：取3月龄雄性SD大鼠脊柱腰段椎间盘髓核组织，用胶原酶，胰酶序贯消化法分离并培养髓核间质干细胞。采用流式细胞术检测细胞免疫表型CD24，CD34，CD45，CD90，CD105表达；利用RT-PCR检测干细胞基因SOX2，Nanog的表达。观察髓核间质干细胞成脂、成骨、成软骨分化能力。利用流式细胞术检测白细胞介素1β作用于髓核间质干细胞后的凋亡率，荧光定量PCR检测白细胞介素1β作用于髓核间质干细胞后SOX9，蛋白多糖，Ⅱ型胶原酶，caspase-3基因的表达变化。
结果与结论：体外分离培养出的细胞表现为纺锤样长梭形；CD90、CD105表达阳性，CD45、CD34、CD24表达阴性，表达干细胞基因SOX2，Nanog，具有成骨和成软骨分化能力，无成脂分化能力。CCK-8检测白细胞介素1β可以抑制髓核间质干细胞的增殖活性，与作用时间、作用浓度存在一定的依赖关系，最佳作用时间为48 h，最佳作用浓度为50 μg/L。流式细胞仪检测白细胞介素1β可以使髓核间质干细胞轻度凋亡；荧光定量PCR结果显示：SOX9，蛋白多糖，Ⅱ型胶原的mRNA表达下调，caspase-3表达上调。结果可见白细胞介素1β具有抑制髓核间质干细胞增殖活性并诱导其凋亡的作用，是导致椎间盘自身干细胞无法发挥修复作用的原因之一。

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

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窗体顶端

关键词 ： 干细胞； 骨髓干细胞； 髓核间质干细胞； 白细胞介素1β； 增殖活性； 凋亡； 大鼠； 山西省自然科学基金；   
Abstract：
BACKGROUND: Endogenous stem cells have no repair effects on the process of disc degeneration. Authors assumed that this maybe associate with abnormal effects of related etiological factor, resulting in an inhibitory effect on the function of nucleus pulposus-derived mesenchymal stem cells.
OBJECTIVE: To investigate the effects of inflammatory cytokine interleukin-1β on biological characteristics of nucleus pulposus-derived mesenchymal stem cells of rats.
METHODS: Lumbar spinal nucleus pulposus was obtained from 3-month-old male Sprague-Dawley rats. Nucleus pulposus-derived mesenchymal stem cells were isolated and cultured with collagenase and sequential trypsin digestion. The expression of CD24, CD34, CD45, CD90 and CD105 was detected using flow cytometry. Stem cell gene SOX2 and Nanog expression was measured using RT-PCR. Adipogenic, osteogenic and chondrogenic abilities of nucleus pulposus-derived mesenchymal stem cells were observed. The apoptotic rate of interleukin-1β-treated nucleus pulposus-derived mesenchymal stem cells was detected using flow cytometry. Fluorescent quantitative PCR was used to measure the expression of SOX9, proteoglycan, type II collagenase 
and caspase-3 gene after nucleus pulposus-derived mesenchymal stem cells were treated with interleukin-1β.
RESULTS AND CONCLUSION: Cells isolated and cultured in vitro showed spindle-like fusiform. Nucleus pulposus-derived mesenchymal stem cells were positive for CD90 and CD105, negative for CD45, CD34 and CD24. Nucleus pulposus-derived mesenchymal stem cells expressed SOX2 and Nanog, and were able to differentiate into osteocytes and chondrocytes, but not adipocytes. By the cell counting kit-8, interleukin-1β inhibited the proliferation capacity of nucleus pulposus-derived mesenchymal stem cells, showing a dependent relationship with action time and action concentration. The optimum reaction time was 48 hours, and the optimal concentration was 50 μg/L. Flow cytometry detected that interleukin-1β induced a slight apoptosis of nucleus pulposus-derived mesenchymal stem cells. Fluorescent quantitative PCR results revealed that mRNA expression of SOX9, proteoglycan, and type II collagenase was decreased, but caspase-3 was increased. Results indicated that interleukin-1β inhibited the proliferation of nucleus pulposus-derived mesenchymal stem cells and induced the apoptosis of nucleus pulposus-derived mesenchymal stem cells, and was a reason that intervertebral own stem cells could not exert repair effects.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

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Key words： intervertebral disk; mesenchymal stem cells; interleukin-1 beta; cell proliferation; apoptosis;
窗体底端

中图分类号: 
R394.2

基金资助: 山西省自然科学基金资助项目(2011011042-3)；太原市大学生创新创业专题(120164064)；山西省研究生优秀创新项目(20113069)
通讯作者: 马迅，硕士，教授，山西大医院骨科，山西省太原市 030032   
作者简介: 张宁，男，1987年生，山西省长治市长子县人，汉族，2014年山西医科大学毕业，硕士，主要从事脊柱退行性疾病的研究。

引用本文:   
张宁,关晓明,马迅等. 白细胞介素1β对大鼠髓核间质干细胞生物学特性的影响[J]. 中国组织工程研究, 2014, 18(28): 4437-4443.

Zhang Ning,Guan Xiao-ming,Ma Xun et al. Interleukin-1 beta affects the biological properties of rat nucleus pulposus-derived mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(28): 4437-4443.