目的探讨全反式维A酸(AT R A)与骨形态发生蛋白(B M Ps)单独或联合应用对小鼠成骨前体细胞(MC3T3-E1)和大鼠骨髓间充质干细胞(BMSCs)的骨钙素(OCN)以及成骨相关基因ALP、OCN、Runx2表达的影响。方法传代培养MC3T3-E1细胞及BMSCs,分别经...目的探讨全反式维A酸(AT R A)与骨形态发生蛋白(B M Ps)单独或联合应用对小鼠成骨前体细胞(MC3T3-E1)和大鼠骨髓间充质干细胞(BMSCs)的骨钙素(OCN)以及成骨相关基因ALP、OCN、Runx2表达的影响。方法传代培养MC3T3-E1细胞及BMSCs,分别经不同浓度细胞因子刺激培养后分为10个组:空白对照组、ATRA组、5ng/ml BMP2/7组、50ng/ml BMP2/7组、5ng/ml BMP2组、50ng/ml BMP2组、ATRA+5ng/ml BMP2/7组、ATRA+50ng/ml BMP2/7组、ATRA+5ng/mlBMP2组、ATRA+50ng/mlBMP2组,采用免疫荧光染色观察细胞形态,免疫酶联吸附实验(ELISA)检测两种细胞OCN的表达,实时荧光定量聚合酶链式反应(RT-qPCR)检测ALP、OCN、Runx2基因的表达。结果免疫荧光染色观察结果显示,MC3T3-E1及BMSCs细胞生长状态良好,结构完整。单独应用ATRA可明显抑制两种细胞(MC3T3-E1及BMSCs)的OCN表达,第7天尤为明显:MC3T3-E1细胞ATRA单独组[(20.97±0.31)ng/ml]明显低于空白对照组[(33.45±0.55)ng/ml],BMSCs细胞ATRA单独组[(9.90±0.16)ng/ml]也明显低于空白对照组[(14.30±0.53)ng/ml],差异均有统计学意义(P<0.05)。单独应用BMPs可浓度依赖性地促进细胞OCN表达,且BMP2/7促进作用更强,50ng/ml BMP2/7作用后MC3T3-E1细胞的OCN浓度为(47.13±0.59)ng/ml,BMSCs的OCN浓度为(49.92±0.53)ng/ml,50ng/ml BMP2/7能够完全拮抗ATRA对细胞OCN表达的抑制作用(P<0.05)。RT-qPCR检测结果显示,单独应用ATRA可明显抑制MC3T3-E1细胞ALP、OCN基因的表达,但可促进Runx2基因的表达(P<0.05),单独应用BMPs呈浓度依赖性地促进MC3T3-E1细胞成骨相关基因的表达(P<0.05),ATRA可显著抑制BMPs对OCN基因表达的促进作用,但ATRA与50ng/ml BMP2/7可协同促进ALP基因的表达(P<0.05);单独应用ATRA第7天可显著促进BMSCsRunx2基因的表达,但可抑制ALP、OCN基因的表达(P<0.05),50ng/mlBMPs可显著促进BMSCsALP、Runx2基因的表达(P<0.05),50ng/mlBMP2/7可拮抗ATRA对ALP基因表达的抑制作用并促进其表达(P<0.05)。结论单独应用ATRA可抑制细胞OCN蛋白的表达,抑制细胞ALP、OCN基因的表达;BMP2/7可显著促进细胞OCN蛋白的表达,BMPs可促进成骨相关基因的表达;50ng/ml BMP2/7可完全拮抗ATRA对细胞的成骨抑制作用。展开更多
Hydroxyapatite ceramics (HA) has been proved to be excellent in biocompatibility and bioactivity. However, limited information is available concerning how HA ceramics affects the maturation of osteoblasts in molecular...Hydroxyapatite ceramics (HA) has been proved to be excellent in biocompatibility and bioactivity. However, limited information is available concerning how HA ceramics affects the maturation of osteoblasts in molecular biological level in vitro. This study examines the mRNA expression and protein production of bone-related genes in osteoblast-like cell line (Saos-2) cultured on HA disks. Saos-2 cells are seeded onto the substrates and cultured for 18 days. Harvested cells are tested for the cell growth rate, expression of mRNAs for osteocalcin and alkaline phosphatase, and protein production of bone sialoprotein and osteocalcin. MTS assay shows that cell proliferates well on HA ceramic substrate. After 9d, bone sialoprotein and osteocalcin protein production in SaPS-2 increases more on HA surfaces than on control material. As bone sialoprotein and osteocalcin are the genes to be highly expressed at the late stage of osteoblast differentiation, this study reveals that after long time culture in HA, HA can induce Saos-2 maturation. The behavior of Saos-2 on HA surfaces revealed in this study provides valuable information for the understanding of the biocompatibility and bioactivity of HA ceramics.展开更多
文摘Hydroxyapatite ceramics (HA) has been proved to be excellent in biocompatibility and bioactivity. However, limited information is available concerning how HA ceramics affects the maturation of osteoblasts in molecular biological level in vitro. This study examines the mRNA expression and protein production of bone-related genes in osteoblast-like cell line (Saos-2) cultured on HA disks. Saos-2 cells are seeded onto the substrates and cultured for 18 days. Harvested cells are tested for the cell growth rate, expression of mRNAs for osteocalcin and alkaline phosphatase, and protein production of bone sialoprotein and osteocalcin. MTS assay shows that cell proliferates well on HA ceramic substrate. After 9d, bone sialoprotein and osteocalcin protein production in SaPS-2 increases more on HA surfaces than on control material. As bone sialoprotein and osteocalcin are the genes to be highly expressed at the late stage of osteoblast differentiation, this study reveals that after long time culture in HA, HA can induce Saos-2 maturation. The behavior of Saos-2 on HA surfaces revealed in this study provides valuable information for the understanding of the biocompatibility and bioactivity of HA ceramics.
