成骨诱导因子Nell-1及Noggin短发卡RNA联合转染脂肪间充质干细胞的促成骨分化能力

doi:10.3969/j.issn.2095-4344.2116

中国组织工程研究 ›› 2020, Vol. 24 ›› Issue (31): 4966-4970.doi: 10.3969/j.issn.2095-4344.2116

• 脂肪干细胞 adipose-derived stem cells • 上一篇下一篇

成骨诱导因子Nell-1及Noggin短发卡RNA联合转染脂肪间充质干细胞的促成骨分化能力

刘浩1，刘军1，芮永军1，汤峰林2，陆淼2，丁涛2

1无锡市第九人民医院骨科，江苏省无锡市 214000；2南京医科大学附属无锡人民医院骨科，江苏省无锡市 214000

收稿日期:2019-11-25 修回日期:2019-11-29 接受日期:2020-01-02 出版日期:2020-11-08 发布日期:2020-09-03
通讯作者: 丁涛，博士，主任医师，硕士生导师，南京医科大学附属无锡人民医院，江苏省无锡市 214000
作者简介:刘浩，男，1990年生，硕士，主治医师，主要从事骨组织工程学研究。
基金资助:
南京医科大学科技发展基金重点项目(2016NJMUZD069)

Co-transfection by Nell-1 and Noggin-shRNA promotes osteoblast differentiation of adipose derived mesenchymal stem cells

Liu Hao1, Liu Jun1, Rui Yongjun1, Tang Fenglin2, Lu Miao2, Ding Tao2

1Department of Orthopedics, Wuxi 9th People’s Hospital, Wuxi 214000, Jiangsu Province, China; 2Department of Orthopedics, Wuxi People’s Hospital Affiliated to Nanjing Medical University, Wuxi 214000, Jiangsu Province, China

Received:2019-11-25 Revised:2019-11-29 Accepted:2020-01-02 Online:2020-11-08 Published:2020-09-03
Contact: Ding Tao, MD, Chief physician, Master’s supervisor, Department of Orthopedics, Wuxi People’s Hospital Affiliated to Nanjing Medical University, Wuxi 214000, Jiangsu Province, China
About author:Liu Hao, Master, Attending physician, Department of Orthopedics, Wuxi 9th People’s Hospital, Wuxi 214000, Jiangsu Province, China
Supported by:
the Major Project of Scientific Development Foundation of Nanjing Medical University, No. 2016NJMUZD069

摘要/Abstract

摘要：

文题释义：

Nell-1：是一种新近发现的促成骨诱导因子，在成骨分化和骨再生中起重要作用。研究表明，Nell-1甚至比经典的骨形态发生蛋白2、骨形态发生蛋白7具有更强的成骨效应。

shRNA：即短发卡RNA(short hairpin RNA)，应用于RNA干扰，其含有一段紧密发卡环的RNA序列，将其转染进入细胞，可以干扰靶基因表达。

背景：Nell-1在诱导成骨分化和新骨形成中的作用已有报道，但试图通过双基因联合转染起协同成骨作用的研究很少。

目的：体外环境下抑制细胞中Noggin基因的同时上调Nell-1基因，观察其对脂肪间充质干细胞成骨分化能力的影响。

方法：取健康成年大鼠脂肪组织获取脂肪间充质干细胞。将细胞分为3组，对照组转染空载体病毒Lv-EGFP(增强型绿色荧光蛋白)，Nell-1组单纯转染Lv-Nell-1，Nell-1+Noggin shRNA组同时转染Lv-Nell-1和Lv-Noggin shRNA。转染后3，7，14 d，应用实时荧光定量PCR检测Nell-1及成骨相关标志物(Col-Ⅰ、ALP、OCN)的表达，转染后14 d行茜素红染色。

结果与结论：①转染后3 d，对照组Nell-1 mRNA相对表达量与Nell-1组比较，差异无显著性意义(P > 0.05)。与Nell-1+Noggin shRNA组比较，对照组和Nell-1组Nell-1 mRNA表达量偏低，差异均有显著性意义(P < 0.05)。转染后7，14 d，Nell-1+Noggin shRNA组Nell-1 mRNA相对表达量仍高于对照组和Nell-1组，且Nell-1组高于对照组，两两比较差异均有显著性意义(P < 0.05)；②转染后3，7，14 d，Nell-1+Noggin shRNA组各成骨基因mRNA相对表达量均显著高于对照组和Nell-1组，且Nell-1组高于对照组，组间比较差异有显著性意义(P < 0.05)；③转染后14 d，Nell-1+Noggin shRNA组呈现较多细胞聚集形成的结节，Nell-1组钙结节数量少于Nell-1+Noggin shRNA组，对照组未见明显被染色的钙结节；④实验结果表明，Nell-1基因可以促进大鼠脂肪间充质干细胞成骨分化，而干扰Noggin基因会上调Nell-1基因的表达，形成显著的协同成骨分化作用。

ORCID: 0000-0002-4601-8391(丁涛)

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

关键词: Nell-1, Noggin, 共转染, 脂肪间充质干细胞, 成骨分化

Abstract:

BACKGROUND: Nell-1 can induce osteogenic differentiation and accelerate bone regeneration. However, little is reported about co-transfection by Nell-1 and Noggin-shRNA.

OBJECTIVE: To observe the effects of simultaneously down-regulating Noggin and up-regulating Nel-like molecule-1(Nell-1) on osteogenic differentiation of adipose derived mesenchymal stem cells (ADSCs) in vitro.

METHODS: ADSCs were isolated and cultured in vitro from adipose tissue of healthy rats, and divided into three groups. ADSCs in control group were transfected with lentiviral (Lv)-enhanced green fluorescent protein. In Nell-1 group, the cells were transfected with Lv-Nell-1. And in co-transfection group, the cells were co-transfected with Lv-Nell-1 and Lv-Noggin shRNA. At 3, 7 and 14 days after transfection, Nell-1 and osteogenesis-related genes (collagen type I, alkaline phosphatase and osteocalcin) were detected by real-time fluorescence quantitative PCR. At 14 days after transfection, ADSCs were detected by alizarin red staining.

RESULTS AND CONCLUSION: At 3 days after transfection, there was no significant difference in the Nell-1 mRNA expression between the control and Nell-1 groups (P > 0.05); and the Nell-1 mRNA expression in the control and Nell-1 groups was significantly lower than that in the co-transfection group (P < 0.05). At 7 and 14 days after transfection, the Nell-1 mRNA expression was still lower in the control and Nell-1 groups than in the co-transfection group, and moreover, the expression level in the Nell-1 group was significantly higher than that in the control group (P < 0.05). At 3, 7, and 14 days after transfection, the levels of collagen type I, alkaline phosphatase and osteocalcin mRNA in the co-transfection group were significantly higher than those in the control and Nell-1 groups, and these expression levels were also significantly higher in the Nell-group than in the control group (P < 0.05). At 14 days after transfection, there were more calcium nodules in the co-transfection group than in the Nell-1 group, while no calcium nodules were observed in the control group. The overall results indicate that Nell-1 can enhance the differentiation of ADSCs into osteoblasts, and interference with Noggin gene can upregulate the expression of Nell-1, thereby triggering a synergistic effect on osteogenic differentiation.

Key words: Nell-1, Noggin, co-transfection, adipose derived mesenchymal stem cells, osteogenic differentiation

中图分类号:

刘浩, 刘军, 芮永军, 汤峰林, 陆淼, 丁涛. 成骨诱导因子Nell-1及Noggin短发卡RNA联合转染脂肪间充质干细胞的促成骨分化能力[J]. 中国组织工程研究, 2020, 24(31): 4966-4970.

Liu Hao, Liu Jun, Rui Yongjun, Tang Fenglin, Lu Miao, Ding Tao. Co-transfection by Nell-1 and Noggin-shRNA promotes osteoblast differentiation of adipose derived mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2020, 24(31): 4966-4970.

图/表（结果） 3

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引言

临床上由于创伤、肿瘤或者骨感染导致的大段骨缺损越发常见，这也一直是困扰着骨科医师的难题之一^[1-3]。近年来，分子生物学及干细胞研究的不断深入，一系列成果有望从实验室走向临床，其中基因强化组织工程法修复大段骨缺损也是研究热点之一，成为目前实现临床转化很有前景的一种治疗手段^[4]。Nell-1(Nel-like molecule-1)是新近发现的一种具有促成骨作用的细胞因子^[5]，其在新生儿颅骨早闭中扮演了重要的角色^[6^-^7]。有相关实验报道，Nell-1可以促进骨折愈合、牵张成骨及脊柱融合。Nell-1相对于骨形态发生蛋白(bone morphogenetic proteins，BMPs)具有更强的促成骨分化能力，形成的新骨有更高的密度及强度。众所周知，在新骨形成过程中经典的骨形态发生蛋白信号通路发挥了巨大作用，而Noggin可以拮抗骨形态发生蛋白信号。Noggin在体内广泛表达，其起作用的方式主要是通过与骨形态发生蛋白特异性结合，从而导致骨形态发生蛋白和受体结合受阻，活性受到抑制，影响成骨^[8^-^9]。既往实验已经证明，Noggin可以抑制成骨细胞分化及其功能^[10-12]。作者猜想，既然Nell-1和骨形态发生蛋白一样，过表达能够促进骨再生，Wnt/β-catenin信号通路是否有可能也参与了这一过程？Noggin是否同样可以抑制Nell-1的活性？据此该实验试图双管齐下，上调Nell-1并抑制Noggin的表达，理论上可以最大程度地强化干细胞的成骨分化能力，并为以后探索新的细胞因子组合提供实验基础。如果实验结果理想，将来还可以制备一种具有转化价值的生物制品，为临床修复大段骨缺损带来新的希望。故作者从正反2个方面着手，分别构建可以在脂肪间充质干细胞(adipose derived stem cells，ADSCs)中高表达的Nell-1慢病毒载体及Noggin干扰载体，包装成重组慢病毒。将双基因重组慢病毒转染大鼠脂肪间充质干细胞，测定转染后成骨相关基因的表达，为基因强化组织工程法治疗大段骨缺损奠定实验基础。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

材料方法

1.1 设计细胞学体外观察对照研究。

1.2 时间及地点实验于2017年1月至2019年12月在南京医科大学附属无锡人民医院中心实验室完成。

1.3 材料

1.3.1 实验动物及细胞株健康成年雄性SD大鼠，体质量275 g左右，由南京医科大学实验动物中心提供。293FT细胞株由南京医科大学附属无锡人民医院中心实验室冻存。

1.3.2 实验试剂 Ⅰ型胶原酶、茜素红、抗坏血酸、胰岛素、地塞米松(美国Sigma公司)；慢病毒表达质粒(北京英茂盛业生物科技有限公司)；Nell-1单克隆抗体、Noggin多克隆抗体、β-actin(美国Santa Cruz公司)；SYBR Green PCR Master Mix(苏州木芮生物科技公司)。

1.4 实验方法

1.4.1 分离、培养、鉴定大鼠脂肪间充质干细胞将SD大鼠拉颈处死后，取腹股沟脂肪组织，用PBS清洗后去除小血管及浅筋膜等结缔组织。将组织剪碎，转移至5 mL离心管，加入5倍于组织体积的0.1%Ⅰ型胶原酶，放入37 ℃的恒温振荡箱中消化1.0-1.5 h，加入含体积分数为10%胎牛血清的DMEM等体积中和，终止消化；1 500 r/min离心10 min，弃上清，沉淀物用含体积分数为10%胎牛血清的DMEM重悬，细胞计数，按2×10⁵/瓶的密度种植于35 cm²培养瓶内，置于培养箱中培养。采用流式细胞技术分析原代细胞表面抗原CD34、CD44、CD29、CD45、CD90、CD105的表达。

1.4.2 构建Nell-1表达载体收集脂肪间充质干细胞，抽提RNA，进行体外反转录获得cDNA，结合引物进行PCR扩增，得到产物片段1 800 bp；用XbaⅠ/EcoRⅠ酶切，将Nell-1连接到质粒相应位点。酶切鉴定图谱见图1。

1.4.3 构建Noggin shRNA干扰载体根据目的基因Noggin的序列，合成1对shRNA寡核苷酸序列，经退火使其变为双链。AgeⅠ/EcoRⅠ双酶切后，将Noggin shRNA连接到质粒的AgeⅠ/EcoRⅠ位点，连接转化后测序鉴定。序列见图2。

1.4.4 病毒包装将慢病毒包装辅助质粒与构建的Nell-1、Noggin shRNA表达质粒共转染293FT细胞，转染后48 h及72 h收集培养上清进行浓缩，将上清离心后过滤，包装出携带Nell-1以及Noggin shRNA的重组慢病毒(Lv-Nell-1、Lv-Noggin shRNA)。

1.4.5 基因转染实验分为3组，分别为对照组(Lv-EGFP转染脂肪间充质干细胞)、Nell-1组(Lv-Nell-1转染脂肪间充质干细胞)和Nell-1+Noggin shRNA组(Lv-Nell-1+Lv- Noggin shRNA转染脂肪间充质干细胞)。取生长状态良好的第3代脂肪间充质干细胞，铺于6孔板上(3×10⁵/孔)，37 ℃培养过夜，吸去细胞原培养基，加入1/2体积的慢病毒液，8字混匀，4-6 h后更换新鲜培养液，两三天后观察荧光表达及细胞生长情况。转染前将病毒在冰上缓慢融化，用含体积分数为10%胎牛血清的DMEM培养液稀释。

1.5 主要观察指标

1.5.1 Nell-1及成骨相关基因表达转染后3，7，14 d收集各组细胞，抽提RNA，反转录为cDNA，设计各基因相应引物。以GAPDH为内参，采用2^-^ΔΔCt法计算Nell-1及成骨相关标志物Col-Ⅰ、ALP、OCN mRNA相对表达量，Nell-1的相对表达量间接反映了Noggin基因沉默的效率。

引物：GAPDH，上游5'-ACT TTG TCA AGC TCA TTT CC-3'，下游，5'-TGC AGC GAA CTT TAT TGA TG-3'；Nell-1，上游5'-CTG TGT GGC TCC TAA CAA GTG TG-3'，下游，5'-GGA TTC TGG CAA TCA CAA GCT GCT-3'；Col-Ⅰ，上游5'-TGA ACG TGG TGT ACA AGG TC-3'，下游5'-CCA TCT TTA CCA GGA GAA CCA T-3’；ALP，上游5'-CCC CAT GTG ATG GCG TAT-3'，下游5'-CGG TAG GGA GAG CAC AGC-3'；OCN，上游5'-CCA AGC AGG AGG GCA ATA-3'，下游5'-TCG TCA CAA GCA GGG TCA-3'。

1.5.2 钙结节观察转染后14 d，各组细胞经PBS清洗后，体积分数为95%乙醇固定，0.1%茜素红染液染色，再予以ddH₂O清洗，显微镜下观察钙结节发生情况。

1.6 统计学分析采用SPSS 19.0统计软件进行分析。所有实验数据以x(_)±s表示；组间比较采用单因素方差分析，两两比较采用q检验。P < 0.05为差异有显著性意义。

讨论

近年来，由于创伤、肿瘤甚至骨髓炎等原因引起的大段骨缺损在临床上越来越常见，大面积骨缺损的修复始终是创伤骨科医师面临的难题，也是患者致残的主要原因之一^[13^-^14]。治疗骨缺损最常用的方法是自体骨移植及同种异体骨移植，但往往因为供区骨量不足、机体排斥反应等并发症使得临床疗效不佳。目前，基因强化组织工程的发展日新月异，在解决大段骨缺损修复这方面具有巨大潜力^[15-18]。该实验选择的脂肪间充质干细胞是由Zuk等首次提出，具有取材方便、生长增殖能力强且具有多向分化潜能等优点^[19^-^20]。实验成功分离、培养了脂肪间充质干细胞进行细胞来源鉴定。骨诱导因子是指一种能够促进间充质干细胞生长、增殖、成骨分化和基质合成的一种蛋白质或多肽。Nell-1就是新近发现的一种骨诱导因子，引起了学者的广泛关注^[21-23]。动物实验已经证明Nell-1可以修复颅骨及颌骨缺损甚至促进脊椎椎间融合。实验结果显示，Nell-1组成骨相关标志物的表达量高于对照组，也初步验证了Nell-1能够促进大鼠脂肪间充质干细胞诱导成骨分化。Noggin是与骨形态发生蛋白最具亲和力的拮抗剂，通过阻挡骨形态发生蛋白与其受体结合，从而抑制骨形成的过程^[24-26]。作者猜想，Nell-1作为新发现的成骨诱导因子，是否与骨形态发生蛋白存在共同的信号通路？Noggin是否对其同样具有拮抗作用？该实验通过构建慢病毒干扰载体Lv-Noggin shRNA来下调Noggin基因的表达，并且联合Nell-1基因过表达的方式，结果发现Nell-1+Noggin shRNA组Nell-1 mRNA相对表达量明显上升。但该实验设计仍有不足之处：没有设立单纯下调Noggin的实验组，使得对照结果的说服力欠佳^[27^-^28]。接下来将进一步完善实验设计，并行Western blot检测Nell-1蛋白的表达及碱性磷酸酶组织化学染色^[29-31]，增加实验结论的可靠性。既往研究中大多数学者采用单纯转染1种骨诱导因子的方式，而该实验中联合转染2种基因，可以产生协同诱导成骨作用。另外没有选择2种正向基因简单叠加，而是从正、反2个方向同时干预，初步实验结果令人满意。作者认为：基因强化组织工程法治疗大段骨缺损具有走出实验室应用于临床的巨大潜力，值得进一步深入研究。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

成骨诱导因子Nell-1及Noggin短发卡RNA联合转染脂肪间充质干细胞的促成骨分化能力

Co-transfection by Nell-1 and Noggin-shRNA promotes osteoblast differentiation of adipose derived mesenchymal stem cells

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