Hawley Dillon, Aluri Hema, Armaos Helene, Kim Gina, Kublin Claire, Zoukhri Driss
Department of Comprehensive Care, Tufts University School of Dental Medicine, Boston, MA.
Department of Comprehensive Care, Tufts University School of Dental Medicine, Boston, MA; Department of Ophthalmology, Tufts University School of Medicine, Boston, MA.
Mol Vis. 2016 Oct 17;22:1221-1228. eCollection 2016.
Gene expression and protein analysis studies require high-quality human tissue which is a challenge and difficult to obtain through live human biopsies. Human postmortem lacrimal gland (LG) and submandibular gland (SMG) tissues have the potential to provide an invaluable source for studying the mechanisms involved in LG and SMG dysfunction. Therefore, we aimed to test the suitability of post-mortem LG and SMG for molecular, biochemical, and cell biological studies.
LG and SMG tissue from healthy donors was collected and immediately placed in RNA solution and then shipped overnight at 4 °C. After receipt, each gland was divided into three pieces for RNA, protein, and histological analysis, respectively. Total RNA isolated from each LG and SMG was analyzed for RNA integrity using an Agilent Bioanalyzer and reverse transcription-PCR (RT-PCR). For histology, tissues were embedded in paraffin and stained with hematoxylin and eosin. For protein analysis, lysates were prepared and processed for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting.
When the LG and SMG samples were preserved in RNA, the RNA integrity number (RIN) values from the LG and SMG were >7.0 from all three donors, while the RNAs from tissue not preserved in RNA were of poorer quality. The gene and/or protein expression of E-cadherin, aquaporin 5, alpha-smooth muscle actin (α-SMA), β-actin, and GAPDH was preserved in all samples. In addition, histological analyses showed normal tubuloacinar structures of all glands with serous and mucous producing acini within lobules interspersed with adipose fat.
In this study, we determined that RNA, protein, and histological sections obtained from postmortem human LG and SMG tissue preserved in RNA were of high quality. This would provide a viable source of human LG and SMG tissue suitable for studies of diseases that affect these glands, such as Sjögren's syndrome.
基因表达和蛋白质分析研究需要高质量的人体组织，而通过活体人类活检获取此类组织具有挑战性且难度较大。人类尸检泪腺（LG）和颌下腺（SMG）组织有可能为研究LG和SMG功能障碍所涉及的机制提供宝贵资源。因此，我们旨在测试尸检LG和SMG用于分子、生化和细胞生物学研究的适用性。
收集健康供体的LG和SMG组织，立即置于RNA溶液中，然后在4℃下 overnight 运输。收到后，将每个腺体分成三块，分别用于RNA、蛋白质和组织学分析。使用安捷伦生物分析仪和逆转录聚合酶链反应（RT-PCR）分析从每个LG和SMG分离的总RNA的RNA完整性。对于组织学，将组织包埋在石蜡中，并用苏木精和伊红染色。对于蛋白质分析，制备裂解物并进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和蛋白质印迹分析。
当LG和SMG样本保存在RNA中时，来自所有三位供体的LG和SMG的RNA完整性数（RIN）值均>7.0，而未保存在RNA中的组织的RNA质量较差。所有样本中E-钙黏蛋白、水通道蛋白5、α-平滑肌肌动蛋白（α-SMA）、β-肌动蛋白和甘油醛-3-磷酸脱氢酶（GAPDH）的基因和/或蛋白质表达均得以保留。此外，组织学分析显示所有腺体的小管泡状结构正常，小叶内有分泌浆液和黏液的腺泡，并散布着脂肪。
在本研究中，我们确定从保存在RNA中的尸检人类LG和SMG组织获得的RNA、蛋白质和组织学切片质量很高。这将为人类LG和SMG组织提供一个可行的来源，适用于研究影响这些腺体的疾病，如干燥综合征。
