辛伐他汀联合岩藻多糖给药对大鼠BMSCs成骨分化的影响

摘要/Abstract

摘要：

目的探究在体外成骨诱导环境下，辛伐他汀（simvastatin， SIM）联合岩藻多糖（fucoidan， FD）对SD大鼠骨髓间充质干细胞（bone mesenchymal stem cells， BMSCs）向成骨细胞分化中期的影响。方法（1）从SD大鼠双侧股骨和胫骨的骨髓腔中提取原代细胞，体外培养至P3代进行细胞鉴定。鉴定方法包括细胞形态学观察、流式鉴定、成脂肪鉴定和成软骨鉴定。（2）取P3代BMSCs，分为空白组（DMEM完全培养基）和试验组（含有不同FD浓度的DMEM完全培养基）。①采用CCK-8法检测各组培养1、2、3天的细胞活性；②培养至第7天，提取细胞中的总RNA，采用qPCR法检测碱性磷酸酶（alkaline phosphatase， ALP）和骨形态发生蛋白-2（bone morphogenetic protein-2， BMP-2）mRNR的表达情况。③培养至第7天，进行ALP定性和定量分析。综合以上结果确定FD的给药浓度。（3）取P3代BMSCs，分为对照组（OM组）、SIM组、FD组和SIM + FD组。①采用CCK-8法检测各组培养1、2、3天的细胞活性；②培养至第7天，观察细胞形态和生长状况，并拍照记录；③培养至第7天，提取细胞中的总RNA，采用qPCR法检测BMP-2、Ⅰ型胶原（Type I collagen，COLⅠ）、RUNX2以及ALP mRNA的表达情况；④培养至第7天，进行ALP定性和定量分析。结果（1）细胞鉴定。①细胞形态观察：细胞在培养过程中慢慢延伸并舒展开。3代以后，细胞形态基本趋于一致，成纤维样贴壁生长。②流式鉴定：CD29、CD90和CD44呈阳性，表达率分别为99.1%、99.5%、99.4%，CD45呈阴性，表达率为0.4%。③成脂肪鉴定：细胞在诱导过程中逐渐变圆，胞内出现折光性高、大小不一的脂滴，经油红O染色呈红色。④成软骨鉴定：细胞逐渐由长梭形变为三角形或多边形，经阿尔新兰染色，软骨基质呈蓝色，细胞核呈红色。（2）FD的给药浓度。①细胞活性：BMSCs的活性随培养时间和FD的浓度增加而增加。与空白组比较，差异均具有统计学意义（P< 0.001）。②qPCR ：浓度为1 μg/mL的FD组BMP-2和ALP mRNA表达量均高于空白组和其他实验组，差异具有统计学意义（P< 0.05）。③ALP定性和定量：当FD浓度为1 μg/mL时，染色最深，ALP活性最高（P< 0.001），说明此浓度下的FD适宜BMSCs的生长和分化。（3）两种药物联合后的促成骨效果。①细胞活性：SIM + FD组的细胞活性明显高于OM组和其他实验组（P< 0.001），说明两种药物联合后对细胞增殖有促进作用；②细胞形态观察：SIM + FD组细胞生长较快，细胞数量明显多于OM组和其他实验组；③qPCR：SIM + FD组成骨相关基因mRNA的表达量均高于OM组、SIM组和FD组（P< 0.001）；④ALP定性和定量：定量结果表明，SIM + FD组ALP染色结果最深。定量数据同样显示联合组ALP表达量最高（P< 0.001）。结论辛伐他汀联合岩藻多糖给药可明显促进大鼠BMSCs向成骨细胞分化，使得细胞活性升高，分化中期的成骨相关因子的表达量增加。

关键词:辛伐他汀,岩藻多糖,骨髓间充质干细胞,成骨诱导

Abstract:

ObjectiveTo investigate the effect of simvastatin （SIM） combined with fucoidan（FD）administration on the middle-stage differentiation of BMSCs from SD rats to osteoblasts in the osteogenic induction environment in vitro.Methods（1） Primary cells were extracted from the bone marrow cavity of bilateral femurs and tibias of SD rats， and cultured in vitro to P3 passage for cell identification. The identification methods included cell morphology observation， flow identification， adipogenic identification and chondrogenesis identification. （2） P3 generation BMSCs were taken and divided into blank group （DMEM complete medium） and experimental group （DMEM complete culture containing different FD concentrations base）. ① CCK-8 method was used to detect the cell activity of each group on 1， 2， and 3 days of culture. ② To the 7th day of culture， the total RNA in the cells was extracted， and the qPCR method was used to detect the expressions of mRNR in Alkaline phosphatase （ALP） and bone morphogenetic protein-2 （BMP-2）.. ③ To the 7th day， the qualitative and quantitative analysis of ALP were carried out. Based on the above results， the administration concentration of FD was determined. （3） P3 generation BMSCs were taken and divided into control group （OM group）， SIM group， FD group and SIM+FD group. ① CCK-8 method was used to detect the cell viability of each group for 1， 2， and 3 days of culture. ② The cell morphology and growth status were observed on the 7th day of culture， and photographed and recorded. ③ To the 7th day of culture， the total cells in the cells were extracted. RNA， qPCR method was used to detect the expressions of BMP-2， Type I collagen （COLⅠ）， RUNX2 and ALP mRNA. ④ To the 7th day of culture， the qualitative and quantitative analyses of ALP wwere carried out.Results（1） ① Observation of cell morphology showed that cells slowly extended and stretched out during the culture process. After 3 generations， the shape of the cells was basically the same， and the fibroblast-like growth adhered to the wall. ② The detection of flow cytometry showed that CD29， CD90 and CD44 were positive， and the expression rates were 99.1%， 99.5%， and 99.4%， respectively. CD45 was negative， and the expression rate was 0.4%. ③ The identification of adipogenesis showed that during the induction process， the cells gradually became round， and lipid droplets with high refractive index and different sizes appeared in the cells， which turned red after staining with Oil Red O. ④ The identification of cartilage formation showed that cells gradually changed from long spindle to triangle or polygon. After Alcian blue staining， the cartilage matrix was blue， and the nucleus was red. （2） The administration concentrations of FD were as follows. ① In the aspect of cell viability， the viability of BMSCs increased with the culture time and the concentration of FD. Compared with the blank group， the experimental group had statistical significance （P< 0.001）. ② qPCR showed that the expressions of BMP-2 and ALP mRNA in the FD group at a concentration of 1 μg/ml were higher than those in the blank group and other experimental groups， and the difference was statistically significant （P< 0.05）. ③ ALP qualitative and quantitative analysis showed that when the concentration of FD was 1 μg/mL， the staining was the deepest， and the activity of ALP was the highest （P< 0.001）， indicating that the concentration of FD was suitable for the growth and differentiation of BMSCs. （3） The osteopromoting effect of the combination of the two drugs was as follows. ① In the aspect of cell activity， the cell activity in SIM+FD group was significantly higher than that in OM group and other experimental groups （P< 0.001）， indicating that the combination of the two drugs could promote cell proliferation； ② Cell morphology observation of cell growth showed that the number of cells was significantly higher in SIM+FD group than that in the OM group and other experimental groups； ③ qPCRshowed that the expression of bone-related gene mRNA in the SIM+FD group was higher than that in the OM group， SIM group and FD group （P< 0.001）； ④ In the aspect of ALP qualitative and quantitative analysis， the quantitative results showed that the ALP staining results were the deepest in the SIM+FD group. Quantitative data also showed that the combined group had the highest ALP expression （P< 0.001）.ConclusionSimvastatin combined with fucoidan administration can significantly promote the differentiation of BMSCs into osteoblasts， and increase cell activity and the expression of osteogenesis-related factors in the middle stage of differentiation.

Key words:simvastatin,fucoidan,bone marrow mesenchymal stem cells,osteogenic induction

王莹, 詹玉林. 辛伐他汀联合岩藻多糖给药对大鼠BMSCs成骨分化的影响[J]. betway必威登陆网址（betway.com ）学报, 2022, 43(12): 897-905.

Ying WANG, Yulin ZHAN. Effects of simvastatin combined with fucoidan administration on osteogenic differentiation of BMSCs[J]. Journal of Shandong First Medical Unversity & Shandong Academy of Medical Sciences, 2022, 43(12): 897-905.

图/表17

图1实验流程示意图

表1设计的引物序列

基因	序列（5΄-3΄）	长度（bp）
BMP-2	正向引物： CTGCGGTCTCCTAAAGGTCG	375
BMP-2	反向引物： CCTCGATGGCTTCTTCGTGA	375
COLⅠ	正向引物： TGGACCCCGAGGAAACAATG	492
COLⅠ	反向引物： TTCGATGACTGTCTTGCCCC	492
RUNX2	正向引物： TACTCTGCCGAGCTACGAAA	135
RUNX2	反向引物： GGGAGGATTTGTGAAGACCGT	135
ALP	正向引物： GCTTCAGTTCCCCCTCAGTC	456
ALP	反向引物： CGCATGCGTGAATGAGTGTT	456
GADPH	正向引物： AGGATTACGGCCCGGAGATA	632
GADPH	反向引物： GACCGGGCTAACGGTTGAT	632

A为培养3 d后；B为培养5 d后图2　P0代BMSCs细胞形态（× 50）

图3P3代BMSCs细胞形态（A：× 50；B：× 100）

图4P3代BMSCs表面抗原表达情况

图5P3代BMSCs油红O染色（× 40）

图6P3代BMSCs阿尔新蓝染色（× 40）

图7培养1、2、3 d时空白组和实验组的细胞活性（A：第1天；B：第2天；C：第3天）A：第1天；B：第2天；C：第3天。与空白组比较，*P < 0.001

图8空白组和实验组培养至第7天BMP-2、ALP mRNA的表达情况（A：BMP-2；B：ALP）A：BMP-2；B：ALP。与空白组和其他实验组比较，*P < 0.001

图9空白组和实验组培养至第7天的ALP染色结果（× 50）

图10空白组和实验组培养至第7天的ALP的定量结果与空白组和其他实验组比较，*P < 0.001

表2OM组、SIM组、FD组和SIM+FD组BMSCs细胞培养1、2、3天的细胞活性（xˉ ± s，n=3）

组别	培养时间
组别	1 d	2 d	3 d
OM	0.6397 ± 0.018	0.9497 ± 0.035	1.416 ± 0.054
SIM	0.722 ± 0.016^a	1.453 ± 0.042^a	1.745 ± 0.046^a
FD	0.74 ± 0.022^a	1.535 ± 0.040^a	1.932 ± 0.055^ab
SIM+FD	0.8207 ± 0.022^abc	1.649 ± 0.038^abc	2.072 ± 0.027^abc
F	44.010	189.100	110.200
P	< 0.001	< 0.001	< 0.001

组别	培养时间
OM	0.6397 ± 0.018	0.9497 ± 0.035	1.416 ± 0.054
SIM	0.722 ± 0.016^a	1.453 ± 0.042^a	1.745 ± 0.046^a
FD	0.74 ± 0.022^a	1.535 ± 0.040^a	1.932 ± 0.055^ab
SIM+FD	0.8207 ± 0.022^abc	1.649 ± 0.038^abc	2.072 ± 0.027^abc
F	44.010	189.100	110.200
P	< 0.001	< 0.001	< 0.001

图11OM组、SIM组、FD组和SIM + FD组培养至第7天BMSCs的形态（× 50）

表3OM组、SIM组、FD组和SIM + FD组BMSCs细胞培养至第7天的qPCR结果（用2-△△Ct表示， xˉ ± s，n = 3）

组别	BMP-2	COLⅠ	RUNX2	ALP
OM	1	1	1	1
SIM	1.353 ± 0.008^a	1.682 ± 0.014^a	1.829 ± 0.014^a	1.667 ± 0.031^a
FD	1.385 ± 0.012^ab	1.775 ± 0.011^ab	1.874 ± 0.012^ab	1.816 ± 0.020^ab
SIM+FD	1.463 ± 0.010^abc	2.051 ± 0.016^abc	2.135 ± 0.009^abc	1.977 ± 0.014^abc
F	1643.000	3946.000	6947.000	1407.000
P	< 0.001	< 0.001	< 0.001	< 0.001

图12OM组、SIM组、FD组和SIM+FD组培养至第7天的ALP染色结果（A： × 50；B × 100）

图13OM组、SIM组、FD组和SIM+FD组培养至第7 天的ALP染色结果

表4OM组、SIM组、FD组和SIM+FD组BMSCs细胞培养至第7天的ALP定量结果（n = 3）

组别	ALP相对表达量
OM	39 719 ± 9.603
SIM	51 083 ± 2.568^a
FD	69 115 ± 5.851^ab
SIM+FD	137 103 ± 5.508^ac
F	140 731 811.000
P	< 0.001

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