Effects of recombinant human granulocyte colony-stimulating factor on central and peripheral T lymphocyte reconstitution after sublethal irradiation in mice

摘要

Female BALB/c (H-2d/d) mice between 8 to 12 weeks of age were purchased from the Animal Center of the Chinese Academy of Military Medical Sciences (Beijing, China). Mice were housed in sterilized cages and were maintained in a temperature-controlled (21–23°C), specific pathogen-free environment with a relative humidity between 50% and 60%. Mice received sterilized, commercial rodent chow and filtered water ad libitum. All experiments were approved by the local review board of the Chinese Academy of Military Medical Sciences in Beijing, China and were performed in accordance with national and international guidelines for laboratory animal care. Recombinant human G-CSF (Sihuan Inc., Beijing, China) was diluted in phosphate-buffered saline (PBS) at a concentration of 10 μg/ml immediately before injection. BALB/c mice were sublethally γ-irradiated with a single dose of 600 cGy administered at a rate of 187 cGy/min using a cobalt source (GWXJ80 Theratron; Nuclear Power Institute of China, Sichuan, China). Immediately after irradiation, mice were injected subcutaneously with G-CSF (100 μg/kg) or with a similar volume of PBS once daily for 14 days. For all data, Day 0 refers to the day on which the mice were irradiated and received their first treatment. This therapeutic regimen for G-CSF was determined based on previous human clinical trials and scaled accordingly [13]. Approximately 30 μl of peripheral blood was collected from the lateral tail vein and was mixed with the anticoagulant Na₂-EDTA (Beijing Chemical Agent Co., Ltd, Beijing, China). Automated hematological analysis was performed on the blood samples using a Sysmex MEK-7222K automated hematology analyzer (Nihon Kohden, Japan) with settings appropriate for the analysis of mouse samples. The following blood components were determined: white blood cell (WBC) count, platelet count, hemoglobin (HGB) level and absolute number of neutrophils and lymphocytes. Thymi were harvested from BALB/c mice at Days 7, 14, 21, 28 and 60 after irradiation. Thymocytes were gently passed through sterile stainless steel mesh strainers into cold RPMI 1640 medium (GIBCO, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FCS). Thymocytes in suspension were centrifuged at 200 × g for 10 min and then were resuspended in cold PBS at a concentration of 5 × 106 cells/ml. The cells were stored at 4°C until further use. Rat anti-mouse CD4-FITC (GK1.5), CD8-PE (53-6.7) and CD3-PerCP (145-2c11) antibody conjugates were used for peripheral T cell subset analysis by flow cytometry. Rat anti-mouse CD4-FITC (GK1.5) and CD8-PE (53-6.7) were used for thymocyte subset analysis. All fluorochrome-conjugated monoclonal antibodies (mAbs) and their appropriate isotype controls were purchased from eBioscience (San Diego, CA, USA). Fifty microliters of whole blood or 1 × 106 thymocytes were incubated with anti-mouse CD16/CD32 FcR (2.4G2) (eBioscience Inc.) for 20 min at 4°C to prevent nonspecific binding. The samples were treated with FACS lysis solution (Becton Dickinson, San Jose, CA, USA) to remove red blood cells and then were washed twice with PBS. Cells were stained as previously described [14] and then analyzed using a Coulter EPICS XL-MCL flow cytometer operated with System II software (Coulter). For each sample, a lymphocyte gate was established based on the linear forward scatter and side scatter. Thymocytes were precisely counted and centrifuged for 2 min at 12000 rpm. Cell pellets were immediately resuspended in a 0.8 mg/ml solution of proteinase K (Boehringer Mannheim, Germany) digestion buffer (1 × 105 cells/10 μl of buffer). Samples were incubated for 1 h at 56°C followed by 10 min at 95°C to inactivate the proteinase K. Real-time quantitative PCR was performed on 5 μl of cell lysate (equivalent to 50 000 cells) using the mδRec primer 5′-CAAAAGAGGAAAGGAAGGCAGTC, the ψJα primer 5′-AAGGCATAAAGCGACACGAAGA and the mδRec-ψJα probe 5′-FAM-CTGCTGTGTGCCCTACCCTGCCC-TAMRA-3′. Primers and fluorescent probes were synthesized by Invitrogen (Carlsbad, CA, USA). PCR reactions contained 0.5 μM each of primer, 0.3 μM fluorescent probe, 1x Platinum Quantitative PCR Supermix-UDG (Invitrogen), and 1x Blue-636 reference dye (MegaBases, Evanston, IL, USA). Amplifications were performed in duplicate on a STRATAGENE MX3500P sequence Detection System (Agilent Technologies, Inc. Santa Clara, CA, USA) and were analyzed using the associated software as previously described [15]. PCR conditions were as follows: 95°C for 5 min, then 40 cycles of 95°C for 30 s, 59°C for 30 s and 72°C for 30 s. The number of molecules of murine TRECs was doubled to normalize the values per 100 000 cells. T cell proliferation was assessed by using the Cell Counting Kit-8 (Dojindo, Kumomoto, Japan) according to the manufacturer's instructions. Briefly, the splenic mononuclear cells (MNCs) were collected and were suspended in RPMI 1640 medium supplemented with 10% FCS to a final