Nanoworld Institute, Inter-University Center for Research and Teaching activity on Organic and Biological Nanoscience and Nanotechnology, University of Genoa, Italy.
J Periodontol. 2011 Jul;82(7):1018-24. doi: 10.1902/jop.2010.100312. Epub 2010 Dec 28.
Periodontitis is a complex multifactorial disease and is typically polygenic in origin. Genes play a fundamental part in each biologic process forming complex networks of interactions. However, only some genes have a high number of interactions with other genes in the network and may, therefore, be considered to play an important role. In a preliminary bioinformatic analysis, five genes that showed a higher number of interactions were identified and termed leader genes. In the present study, we use real-time quantitative polymerase chain reaction (PCR) technology to evaluate the expression levels of leader genes in the leukocytes of 10 patients with refractory chronic periodontitis and compare the expression levels with those of the same genes in 24 healthy patients.
Blood was collected from 24 healthy human subjects and 10 patients with refractory chronic periodontitis and placed into heparinized blood collection tubes by personnel trained in phlebotomy using a sterile technique. Blood leukocyte cells were immediately lysed by using a kit for total RNA purification from human whole blood. Complementary DNA (cDNA) synthesis was obtained from total RNA and then real-time quantitative PCR was performed. PCR efficiencies were calculated with a relative standard curve derived from a five cDNA dilution series in triplicate that gave regression coefficients >0.98 and efficiencies >96%. The standard curves were obtained using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and growth factor receptor binding protein 2 (GRB2), casitas B-lineage lymphoma (CBL), nuclear factor-KB1 (NFKB1), and REL-A (gene for transcription factor p65) gene primers and amplified with 1.6, 8, 40, 200, and 1,000 ng/μL total cDNA. Curves obtained for each sample showed a linear relationship between RNA concentrations and the cycle threshold value of real-time quantitative PCR for all genes. Data were expressed as mean ± SE (SEM). The groups were compared to the analysis of variance. A probability value <0.01 was considered statistically significant.
The present study agrees with the preliminary bioinformatics analysis. In our experiments, the association of pathology with the genes was statistically significant for GRB2 and CBL (P <0.01), and it was not statistically significant for REL-A and NFKB1.
This article lends support to our preliminary hypothesis that assigned an important role in refractory aggressive periodontitis to leader genes.
牙周炎是一种复杂的多因素疾病，通常具有多基因遗传基础。基因在构成复杂相互作用网络的每个生物学过程中起着根本作用。然而，只有少数基因与网络中的其他基因有大量的相互作用，因此可能被认为起着重要作用。在初步的生物信息学分析中，鉴定出了五个具有较高相互作用数量的基因，并将其命名为“先导基因”。在本研究中，我们使用实时定量聚合酶链反应（PCR）技术评估了 10 例难治性慢性牙周炎患者白细胞中先导基因的表达水平，并将其与 24 例健康患者的相同基因的表达水平进行了比较。
采集 24 名健康受试者和 10 例难治性慢性牙周炎患者的血液，置于肝素化采血管中，由经过采血技术培训的人员采用无菌技术采集。立即使用从人全血中提取总 RNA 的试剂盒裂解血液白细胞细胞。从总 RNA 获得互补 DNA（cDNA）合成，然后进行实时定量 PCR。通过从 5 个 cDNA 稀释系列重复获得的相对标准曲线计算 PCR 效率，该曲线的回归系数> 0.98，效率> 96%。使用甘油醛-3-磷酸脱氢酶（GAPDH）和生长因子受体结合蛋白 2（GRB2）、 Casitas B 细胞淋巴瘤（CBL）、核因子-KB1（NFKB1）和 REL-A（转录因子 p65 的基因）基因引物获得标准曲线，并以 1.6、8、40、200 和 1000ng/μL 总 cDNA 扩增。对于所有基因，每个样本的曲线都显示了 RNA 浓度与实时定量 PCR 循环阈值之间的线性关系。数据表示为平均值± SE（SEM）。通过方差分析比较组间差异。概率值<0.01 被认为具有统计学意义。
本研究与初步的生物信息学分析一致。在我们的实验中，基因与病理学的关联对于 GRB2 和 CBL 具有统计学意义（P<0.01），而对于 REL-A 和 NFKB1 则无统计学意义。
本文支持了我们的初步假设，即先导基因在难治性侵袭性牙周炎中起着重要作用。
