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

doi:10.11958/20221496

Abstract

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

CLC Number:

R394.2

Figures/Tables 11

Fig.1 The characteristics of cell surface markers in BMSCs

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

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

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

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

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

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

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^＊＊

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

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

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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