Vascular endothelial growth factor (vegf)

The deoxyribonuclease I (DNase I) were obtained from solarbio (Beijing, China, http://www.solarbio.com). The MB, CD63, BSA, EPCAM and VEGF were purchased from Cusabio Biotech Co. Ltd. (http://www.cusabio.cn/). The MB aptamer (5′-CCCTCCTTTCCTTCGACGTAGATCTGCTGCGTTGTTCCGA-3′-FAM) were synthesized by Sangon Biological Engineering Technology Co., Ltd. (Shanghai, China, http://www.sangon.com) and purified using high performance liquid chromatography. All reagents employed were of analytical grade and used without further purification. And were diluted to the required concentration with working buffer (10 mM Tris, 100 mM NaNO₃, pH 7.4) before usage. This study all experiments were performed in compliance with the relevant laws and institutional guidelines (National Health Commission of the people's Republic of china, ethical review of biomedical research involving human beings), healthy human serum, urine and saliva were provided by Affiliated Dongfeng Hospital, Hubei University of Medicine, and approved by Dongfeng Hospital's Ethics Committee, informed consent was obtained for any experimentation with human subjects. Ultrapure water obtained from a Millipore water purification system (18.2 MΩ cm resistivity, Milli-Q Direct 8) was used in all runs.

All herbs were provided by the First Affiliated Hospital of Jinan University (China). Blank matrix and RUc (1 g of high or low dose RUc respectively contained 4 g or 2 g herbal drugs) was prepared in the School of Traditional Chinese Medicine, Jinan University.^{14 (link)-16 (link, link)} The effective components and quality control of drugs have been analyzed by HPLC-MS in our previous studies.^{14 (link)}
7,12-dimethylbenz(a)anthracene (DMBA) was purchased from Tokyo Chemical Industry Co., Ltd (Tokyo, Japan). Tamoxifen was purchased from Chi-Fei Chemical Co., Ltd (Wuhan, China). The DMEM/F-12 medium, DMEM High Glucose medium, horse serum, fetal bovine serum (FBS) and rhEGF were purchased from Gibco BRL (Grand Island, NY, USA). Matrigel was purchased from BD Biosciences (San Jose, CA, USA). VEGF, MMP2, and MMP9 ELISA kits were purchased from Cusabio Biotech Co., Ltd (Wuhan, China). H-Ras, c-Raf, MEK, p-MEK, ERK, p-ERK, c-jun, and c-fos primary antibodies were purchased from Cell Signaling Techniques (Beverly, MA, USA). VEGF, MMP2, MMP9, CD34, and β-actin were purchased from Abcam (Cambridge, MA, USA).

IL-6, VEGF, VCAM-1 and ICAM-1 expression levels in each cultured OLP-MFs and NFs were evaluated using ELISAs. NFs and OLP-MFs cell lines were seeded into 6-well plates at a density of 4x10⁵ cells/ml. Following overnight incubation at 37˚C, cell lines were exposed to the conditions described in the following section and incubated with 10% FBS in DMEM. After 12, 24, 36 or 48 h, culture supernatants were collected, centrifuged at 1,000 x g at room temperature for 20 min to pellet any detached cells and measured using IL-6 ELISA Kit (cat. no. CSB-E04638h; Cusabio Technology LLC), VEGF (cat. no. DVE00; R&D Systems, Inc.), VCAM-1 (cat. no. DVC00; R&D Systems, Inc.) and ICAM-1 (cat. no. DCIM00; R&D Systems, Inc.) ELISA kits, according to the manufacturer's protocols.