Tetracycline hydrochloride induces the osteogenic differentiation of rat bone marrow mesenchymal stem cells

doi:10.3969/j.issn.2095-4344.2017.29.003

Abstract

Abstract:

BACKGROUND: Tetracycline hydrochloride not only promotes the proliferation of bone marrow mesenchymal stem cells (BMSCs), but also has a strong bone activity. Studies on the osteogenic differentiation of BMSCs induced by tetracycline hydrochloride will provide a new cell source for bone tissue engineering.
OBJECTIVE: To observe the induction and differentiation activity of BMSCs into osteoblasts cultured by tetracycline hydrochloride in vitro.
METHODS: BMSCs were separated and expanded by whole bone marrow adherent method and identified by cell morphology and BMSCs cell markers CD34, CD45, CD90, CD29. Passage 3 BMSCs were collected and cultured with tetracycline hydrochloride (experimental group) or cultured in L-DMEM containing 10% fetal bovine serum (control group). Osteogenic induction and differentiation activity of BMSCs cultured by tetracycline hydrochloride were detected by alkaline phosphatase activity detection, collagen I and osteocalcin immunohistochemistry staining, alizarin red staining of mineralized calcium nodules, and detection of collagen I and osteocalcin gene expression by RT-PCR.
RESULTS AND CONCLUSION: Tetracycline hydrochloride continuously promoted alkaline phosphatase activity in the BMSCs, significantly different from that in the control group. After 21 days of culture, positive expression of collagen I and osteocalcin proteins, red calcium nodules shown by alizarin red staining, and mRNA expression of collagen I and osteocalcin were observed in the experimental group, while the negative expression was found in the control group. In conclusion, tetracycline hydrochloride has a significant role to promote the osteogenic differentiation of BMSCs.

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

Key words: Stem Cells, Tetracycline Hydrochloride, Mesenchymal Stem Cells, Osteoblasts

CLC Number:

R394.2

Zhang Jue, Xue Si-liang, Luo Yuan, Zhi Wei. Tetracycline hydrochloride induces the osteogenic differentiation of rat bone marrow mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(29): 4605-4610.

Figures/Tables 1

2.1 骨髓间充质干细胞的分离培养及表面标记物检测结果骨髓间充质干细胞经体积分数10%FBS的L-DMEM完全培养基培养24 h后贴壁，呈棱形、圆形或多角形；6 d后细胞数目明显增多，以梭形细胞为主，涡旋状排列，八九天后细胞融合达90%，且间边界不清，呈纺锤形。流式细胞仪检测骨髓间充质干细胞表面标记物抗原CD34阴性表达率99.7%(图1A)，CD45阴性表达率99.4%(图1B)，CD90阳性表达率99.2%(图1C)，CD29阳性表达率99.8%(图1D)，符合SD大鼠骨髓间充质干细胞的特征。 2.2 碱性磷酸酶活性检测结果随着培养时间的延长，对照组和盐酸四环素诱导组细胞的碱性磷酸酶活性均持续增高。盐酸四环素诱导组细胞在各时间点碱性磷酸酶活性均高于对照组，差异有显著性意义(t 检验，P < 0.05，表1)。 2.3 胶原蛋白免疫组织化学染色结果盐酸四环素诱导组行Ⅰ型胶原蛋白免疫组织化学染色后，绝大多数细胞呈现阳性反应，即细胞胞浆内有较多棕黄色颗粒出现(图2A)，这说明细胞中有大量Ⅰ型胶原蛋白表达。细胞核苏木精染色呈蓝色；对照组胞核呈蓝色，胞浆不着色，表明Ⅰ型胶原蛋白几乎不表达，反应阴性(图2B)。 2.4 骨钙素免疫组织化学染色结果盐酸四环素诱导组行骨钙素免疫组织化学染色后，绝大多数细胞呈现阳性反应，即细胞胞浆内有较多棕黄色颗粒出现(图3A)，这说明细胞中有大量骨钙素表达。对照组细胞胞浆不着色，核呈蓝色(图3B)。 2.5 茜素红矿化钙结节组织化学染色结果盐酸四环素诱导组细胞培养第16 d可见细胞呈集落式生长，呈团状向四周放射，形成钙结节(图4A)，21 d经茜素红染色钙结节呈红色，结节边缘光滑，立体感强。茜素红对钙离子特征性红染，当骨向分化的细胞进入分化末期时，细胞停止增殖，大量分泌类骨质掩埋自己，并释放骨钙素促进钙磷沉积，最终成为骨细胞(图4B)；对照组细胞培养随时间延长聚集生长，21 d染色未见钙结节出现(图5A、图5B)。 2.6 Ⅰ型胶原蛋白和骨钙素的RT-PCR鉴定结果盐酸四环素诱导组可见大小为328 bp的Ⅰ型胶原蛋白条带和221 bp的骨钙素条带及692 bp的β-actin条带；对照组中未见Ⅰ型胶原蛋白条带和骨钙素条带(图6)。"

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