Teja Kavalipurapu Venkata, Ramesh Sindhu, Priya Vishnu
Department of Conservative Dentistry and Endodontics, Saveetha Dental College, Saveetha Institute of Medical and Technical Sciences, Saveetha University, Chennai, Tamil Nadu, India.
Department of Biochemistry, Saveetha Dental College, Saveetha Institute of Medical and Technical Sciences, Saveetha University, Chennai, Tamil Nadu, India.
J Conserv Dent. 2018 Nov-Dec;21(6):592-596. doi: 10.4103/JCD.JCD_154_18.
Matrix metalloproteinases (MMPs) play a significant role in the efficient tissue turnover and remodeling. This study focuses on the regulation of the MMPs by the protein kinases at the level of gene expression and their signaling pathways.
Lipopolysaccharide-induced murine macrophage-like RAW 264.7 cell lines were obtained and maintained in Dulbecco's modified Eagle's medium plus 10% fetal bovine serum under optimal temperatures. Primers used were MMP-3 forward primer, MMP-3 reverse primer, and glyceraldehyde-3-phosphate dehydrogenase forward primer and glyceraldehyde-3-phosphate reverse primer. Total RNA was isolated, the sample was prepared, and electrophoresis was performed. The first strand of cDNA was synthesized and amplification of specific isolated gene using polymerase chain reactor (PCR). The amplified products were then separated on a 1.0% agarose gel in 1XTBE at 75 V for 3 h. The gel was stained with ethidium bromide, and the amplified product was visualized and photographed on Gel Doc system.
Real-time PCR showed only bands at expected size of 595 bp for internal control amplification of glyceraldehyde-3-dehydrogenase gene. Analysis was done with densitometry, and these values are compared with the negative control. Results showed a statistically significant rise in the relative levels of MMP-3-mRNA when compared with negative control at 1, 2, and 3 h.
This study proved the significantly increased levels of MMP gene at different period, thereby it can be concluded that MMP-3 levels are higher in inflammatory conditions.
基质金属蛋白酶（MMPs）在有效的组织更新和重塑中发挥着重要作用。本研究聚焦于蛋白激酶在基因表达水平对MMPs的调控及其信号通路。
获取脂多糖诱导的小鼠巨噬细胞样RAW 264.7细胞系，并在最佳温度下于含10%胎牛血清的杜氏改良 Eagle 培养基中培养。所用引物为MMP-3正向引物、MMP-3反向引物、甘油醛-3-磷酸脱氢酶正向引物和甘油醛-3-磷酸反向引物。提取总RNA，制备样品并进行电泳。合成cDNA第一链，使用聚合酶链反应（PCR）扩增特定分离基因。扩增产物随后在1XTBE缓冲液中的1.0%琼脂糖凝胶上，于75 V电压下电泳3小时。凝胶用溴化乙锭染色，在凝胶成像系统上观察并拍摄扩增产物。
实时PCR显示，甘油醛-3-脱氢酶基因内参扩增仅出现预期大小595 bp的条带。采用密度测定法进行分析，并将这些值与阴性对照进行比较。结果显示，与阴性对照相比，在1、2和3小时时，MMP-3-mRNA的相对水平有统计学意义的升高。
本研究证明了不同时期MMP基因水平显著升高，从而可以得出结论，炎症状态下MMP-3水平更高。
