BMP2重组慢病毒转染兔BMSCs后细胞矿化的实验研究

doi:10.11958/20221496

天津医药 ›› 2023, Vol. 51 ›› Issue (5): 454-459.doi: 10.11958/20221496

BMP2重组慢病毒转染兔BMSCs后细胞矿化的实验研究

宁寅宽(), 刘林志^△(), 陈贤平

邵阳市中心医院血管外科（邮编422000）

收稿日期:2022-09-16 修回日期:2022-11-18 出版日期:2023-05-15 发布日期:2023-05-05
通讯作者: △E-mail:liulinzhi1010@163.com
作者简介:宁寅宽（1986），男，主治医师，主要从事生物材料、血管与骨组织工程、生物矿化方面研究。E-mail：nyk2004@163.com
基金资助:
广西自然科学基金资助项目(2014GXNSFAA118263)

Experimental study on the mineralization of rabbit bone marrow mesenchymal stem cells after transfection with BMP2 recombinant lentivirus

NING Yinkuan(), LIU Linzhi^△(), CHEN Xianping

Department of Vascular Surgery, Shaoyang Central Hospital, Shaoyang 422000, China

Received:2022-09-16 Revised:2022-11-18 Published:2023-05-15 Online:2023-05-05
Contact: △E-mail:liulinzhi1010@163.com

宁寅宽, 刘林志, 陈贤平. BMP2重组慢病毒转染兔BMSCs后细胞矿化的实验研究[J]. 天津医药, 2023, 51(5): 454-459.

NING Yinkuan, LIU Linzhi, CHEN Xianping. Experimental study on the mineralization of rabbit bone marrow mesenchymal stem cells after transfection with BMP2 recombinant lentivirus[J]. Tianjin Medical Journal, 2023, 51(5): 454-459.

摘要/Abstract

摘要：

目的探讨骨形态发生蛋白2（BMP2）重组慢病毒转染兔骨髓间充质干细胞（BMSCs）成骨分化后细胞矿化的能力。方法密度梯度离心及贴壁培养法获取第5代兔BMSCs，流式细胞仪检测细胞表面标记。将BMSCs分为空白对照组（未转染）、Lv-EGFP组[转染仅携带增强绿色荧光蛋白（EGFP）基因的慢病毒]和Lv-BMP2/EGFP组（转染携带BMP2和EGFP基因的慢病毒）。免疫组织化学染色、RT-PCR、Western blot检测BMP2蛋白和基因表达情况；SP法检测Ⅰ型胶原蛋白表达；茜素红染色法检测矿化结节形成；于转染第7、14、21天检测细胞碱性磷酸酶（ALP）水平；通过扫描电镜和能谱分析进一步观测矿化结节表面微观形貌及其主要元素构成。结果流式细胞仪检测显示第5代BMSCs的细胞表面CD44、CD29表达呈阳性，CD45表达呈阴性；免疫组织化学染色、RT-PCR、Western blot显示Lv-BMP2/EGFP组较Lv-EGFP组及空白对照组能高效表达BMP2目的蛋白和基因，转染第7、14、21天ALP水平较其余2组升高，Ⅰ型胶原染色及茜素红染色均呈阳性，扫描电镜下见矿化结节散布于成骨方向分化的细胞中，细胞叠加生长，基质分泌旺盛，能谱分析显示其表面为钙、磷沉积物，其钙磷比值为1.52±0.13，发生了细胞矿化。结论 BMP2重组慢病毒转染兔BMSCs能成功诱导其向成骨方向分化并发生细胞矿化。

关键词: 骨形态发生蛋白质2, 慢病毒载体属, 转染, 间充质干细胞, 生物矿化, 基因治疗

Abstract:

Objective To investigate the cell mineralization ability of rabbit bone marrow mesenchymal stem cells (BMSCs) transfected with recombinant lentivirus BMP2 after osteogenic differentiation. Methods The 5th generation rabbit BMSCs were obtained by density gradient centrifugation and adherent culture, and cell surface markers were detected by flow cytometry. BMSCs were divided into the blank control group (untransfected), the Lv-EGFP group (transfected lentivirus without BMP2 gene) and the Lv-BMP2/EGFP group [transfected lentivirus carrying BMP2 and enhanced green fluorescent protein (EGFP) gene]. Immunohistochemical staining, RT-PCR and Western blot assay were used to detect the expression of BMP2 protein and gene. The expression of type Ⅰ collagen was detected by SP method. Alizarin red staining was used to detect the formation of mineralized nodules. Alkaline phosphatase (ALP) levels were detected at 7, 14 and 21 days after transfection. The surface morphology and main element composition of mineralized nodules were further observed by scanning electron microscopy and energy spectrum analysis. Results Flow cytometry showed that the expressions of CD44 and CD29 on the surface of BMSCs of the 5th generation were positive, while the expression of CD45 was negative. Immunohistochemical staining, RT-PCR and Western blot assay showed that the Lv-BMP2/EGFP group could efficiently express BMP2 target protein and gene compared with the Lv-EGFP group and the blank control group, and the level of ALP was higher in the Lv-BMP2/EGFP group than that of the other two groups on the 7th, 14th and 21st days after transfection, and type Ⅰ collagen staining and alizarin red staining were positive. Under scanning electron microscope, mineralized nodes were scattered in osteoblast differentiated cells, cells grew superposition, and the matrix secreted vigorously. Energy spectrum analysis showed that the surface of cells was calcium and phosphorus deposits, and the ratio of calcium to phosphorus was 1.52±0.13. The cell mineralization occurred. Conclusion After transfecting rabbit BMSCs with BMP2 recombinant lentivirus, rabbit BMSCs can be successfully induced to differentiate into osteogenic direction and mineralization in vitro.

Key words: bone morphogenetic protein 2, lentivirus, transfection, mesenchymal stem cells, biomineralization, genetic therapy

中图分类号:

R394.2

图/表 11

图1 BMSCs细胞表面标志物表达特征

Fig.1 The characteristics of cell surface markers in BMSCs

图2 BMSCs在倒置荧光显微镜下细胞形态（×100）

Fig.2 The cell morphology of BMSCs under fluorescence microscope (×100)

图3 免疫组织化学染色SP法检测BMP2表达（DAB染色，×200）

Fig.3 The SP immunochemical staining of BMP2 expression (DAB stanining, ×200)

图4 RT-PCR检测BMSCs中BMP2基因表达水平 M：Mark；A：空白对照组；B：Lv-EGFP组；C：Lv-BMP2/EGFP组。

Fig.4 The BMP2 gene expression in BMSCs measured by RT-PCR

图5 Western blot检测BMP2蛋白表达 A：空白对照组；B：Lv-EGFP组；C：Lv-BMP2/EGFP组。

Fig.5 The protein expression of BMP2 measured by Western blot assay

图6 BMSCsⅠ型胶原免疫组织化学染色（×100）

Fig.6 The I collagen immunohistochemical staining of BMSCs (×100)

图7 BMSCs茜素红染色（×50）

Fig.7 The alizarin red S staining of BMSCs (×50)

表1 各组不同时间点ALP水平比较（n=9,U/gprot,$\bar{x}\pm s$）

Tab.1 Comparison of ALP levels at different time points between the three groups

组别	第7天	第14天	第21天
空白对照组	2.88±0.57	4.99±0.47	5.76±0.50
Lv-EGFP组	2.76±0.50	4.76±0.50	5.71±0.44
Lv-BMP2/EGFP组	6.76±0.91^ab	16.42±2.22^ab	24.39±2.54^ab
F	98.659^＊＊	222.143^＊＊	452.715^＊＊

图8 扫描电镜观察矿化结节表面微观形貌

Fig.8 The microscopic morphology of the mineralized nodule surface observed by SEM

图9 矿化结节表面扫描区域能谱分析图

Fig.9 The energy spectrum analysis of the scanned area on the surface of the mineralized nodule

表2 矿化结节表面扫描区域元素分析

Tab.2 The elemental analysis of the scanned area on the surface of the mineralized nodule

元素	原子质量百分比（%）	原子个数百分比（%）
C K	0.41	13.08
N K	0.95	25.79
O K	2.19	51.93
P K	0.23	2.78
S K	0.18	2.16
Ca K	0.45	4.27
总量	4.42	-
Ca/P	1.52	-

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BMP2重组慢病毒转染兔BMSCs后细胞矿化的实验研究

Experimental study on the mineralization of rabbit bone marrow mesenchymal stem cells after transfection with BMP2 recombinant lentivirus

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