In this study, six cases of LGBLEL and six cases of orbital CH, diagnosed by histopathological examination by two experienced pathologists in Beijing Tongren Hospital, Capital Medical University, between July 2010 and October 2013, were randomly selected for proteomic analysis. The LGBLEL group had a male-female ratio of 1:5 and ranged in age from 28 to 64 years, with a median age of 38.5 years. In the CH group, the male-to-female ratio was 1:2 and ranged in age from 31 to 55 years, with a median age of 51.5 years. For the verification experiment, four cases of LGBLEL diagnosed by pathologic examination by two experienced pathologists in Beijing Tongren Hospital, Capital Medical University, between October 2018 and August 2019, were randomly selected as the experimental group, and three cases of orbital CH were selected as the control group. In the experimental group, the male to female ratio was 1:3 and ranged in age from 45 to 50 years, with a median age of 46 years. In the control group, the male to female ratio was 2:1 and ranged in age from 45 to 53 years, with a median age of 48 years. All patients were fully aware of the purpose of this study, and informed consent was received. This study was approved by the Ethics Committee of Beijing Tongren Hospital, Capital Medical University.
Tissue and blood samples
Specimens of LGBLEL and orbital CH were collected intraoperatively by clinicians and quickly transferred to a standardized laboratory for processing and storage. Portions of the pathological tissue specimens were cryopreserved for later use, while the other parts were soaked in 10% formalin for paraffin embedding and sectioning.
Proteins were extracted from tissues and were identified and quantified by secondary mass spectrometry after quality inspection. Samples were processed and labeled according to the instructions in the Pierce TMT ® Mass Tagging and Reagents kit and the AB Sciex iTRAQ™ Reagents kit. Mixed labeled samples were used for C18 column grading. After the samples were fully dissolved, they were loaded into the liquid mass spectrometry system for secondary mass spectrometry sequencing, and the original files were generated for GO and KEGG analysis.
Reverse transcription polymerase chain reaction (RT-PCR)
The pathological tissues were cut, lysate was added and RNA was extracted. PCR primers were designed, and cDNA of total RNA was synthesized via reverse transcription, according to RT kit instructions, and the cDNA was used as a template for RT-PCR. The PCR reaction conditions were 95°C/3 min, 95°C/30 s, 55°C/20 s, 72°C/20 s, 40 cycles, with GAPDH used as the internal reference. Primer sequence information is shown in Table 1.
Diseased tissue was dewaxed, incubated at room temperature for 5–10 min, washed with distilled water and soaked in PBS for 5 min. Drops of primary antibody were added, and the tissue incubated overnight at 4°C. The tissue was washed three times with PBS, biotin-labeled secondary antibody was added after 5 min, and the tissue was incubated at 37°C for 30 min. The tissue was again washed three times with PBS, 5 min each time, DAB stained, rinsed with water, hematoxylin stained for mounting, imaged under a microscope (Olympus CX41, Japan). The primary antibodies used for protein immunohistochemistry related to the signaling pathway of the complement system were C3, C5, C9 and C1qA.
The diseased tissue was cut into pieces, and lysate was added. The tissue was then centrifuged, and the protein concentration was determined. An electrophoretic gel was prepared as follows: the protein samples were mixed with 5× loading buffer and treated at 100°C for 5 min. One-hundred micrograms of total cell protein was collected from each well, and electrophoresis was performed with a constant current power source of 15 mA per gel. The transfer tank was filled with electrotransfer solution to start the film transfer, and the ECL system was used for Western blotting.
Data processing and statistical analysis
Statistical analysis was performed using SPSS Version 18.0 software (SPSS Inc., Chicago, IL, USA) and GraphPad Prism 8 (Graph Pad SoftareInc., La Jolla, CA, USA). An unpaired t-test was used to analyze differences in RNA content and protein concentration between the LGBLEL group and control group. P<0.05 was considered statistically significant.