Study On Apoptotic Peripheral Blood CD4～+T Lymphocytes In Chinese HIV/AIDS Patients Correlated With Disease Progression

ObjectiveIn HIV infection, the gradual decline of CD4+ T lymphocytes is associated with host immune dysfunction. The deficiency of CD4+ T lymphocytes is the key of AIDS pathogenesis. However it isnt still distinct what the definite mechanism of the deficiency of CD4+ T lymphocytes is in HIV infection. There were a large quantity of apoptotic cells in HIV infectors and T cell lines infected HIV ex vivo in many researches. Therefore we infer it is possible that apoptosis cause the death of T lymphocytes. Apoptosis is one of essential physiological mechanisms, by which the organisms replace normal cells and eliminate abnormal cells, and maintains normal physiological progress and functional expression. But in some pathological conditions, the balance between production and death is destroyed. Apoptosis regulated abnormally causes abnormal cells survive and normal cells excessively die, which leads to multi - systemic diseases. At present there are different researches in HIV infection. Some researches show apoptosis caused by HIV infection increases with disease progression. Contrarily another researches show nothing. Furthermore there are no related documents in China. To determine the apoptotic function in HIV infection, we study apoptotic peripheral blood CD4+ T lymphocytes in Chinese HIV/AIDS patients to explore the relationship between apoptosis and disease progression.Material and MethodsStudy population32 HIV/AIDS patients are diagnosed by AIDS research institution of China Medical University, the Center for disease prevention and control in Iiaoning and Jilin province. Diagnosis of HIV - 1 infection was made on the basis of a positive anti-HIV ELISA ( Vironostika, Organon Tednika, The Netherlands) and confirmed by a positive Western immunoblot ( Genelab Diagnostics, Singapore ). There are 19 men and 13 women. The average age is 41 (18 - 64). All samples are collected before antiviral treatment. HIV - seronegative controls were randomly selected from healthy population with normal blood routine test, liver and renal function. There are 7 men and 6 women. The average age is 38 (20-58).2. Determination of lymphocyte countsPipette 20μl of TriTEST CD4FITC/CD8PE/CD3PerCP reagent and add 50μl anticoagulated whole blood into the bottom of the TruCOUNT Tube using reverse pipetting and incubate for 15 minutes in dark at room temperature (20 -25℃). Add 450μl 1X FACS Lysing Solution to the tube, incubate for 15 minutes in the dark at room temperature and analyze the samples on the flow cytometer using FACS MULTISET software to acquire T lymphocyte counts.3. The rate of apoptotic peripheral blood CD4+ T lymphocytesCollect 2 ml heparin anticoagulated blood and isolate peripheral blood lym-phocytes by density gradient centrifugation. Wash lymphocytes twice by PBS at 4℃. Regulate suspended liquid to 1 × 10~6/ mL by 1 × Binding Buffer. Add 100μL to the tube. Add 5μL FITC - Annexin V,5μL PI and 6μL PerCP - CD4 into the tube. Mix them and culture for 15 min in dark at 4℃.. Add 400μL 1 × Binding Buffer and analyze using CELLQUEST software by FACSCalibur flow cytometer. Control tubes are nude cells, cells only stained FITC - Annexin V and only stained PI.4. Isolation of nucleic acidViral RNA or human leucocyte DNA were isolated using automatic nucleic acid extraction system ( MagNApure LC) and nucleic acid extraction kits (ROC).5. Determination of viral load ( VL)Viral load was performed by Roch viral load kits and preformed accordingto the kit instruction.Results1. The rate of apoptotic peripheral blood CD4+ T lymphocytes in Chinese HIV/AIDS patientsThe rate of apoptotic peripheral blood CD4+ T lymphocytes in 32 HIV/ AIDS patients is 6.75 ±2. 87 (1.12-11. 86) and is significantly higher than the normal control 2.64 ± 1.01 (1.15 ~4.33) (P <0.01).2. The comparison of apoptotic peripheral blood CD4+T lymphocytes in long - term nonprogressive patients, asymptomatic HIV infectors, AIDS patients andnormal controls2. 1 The rate of apoptotic peripheral blood CD4 + T lymphocytes in AIDS patients is significantly higher than it in normal controlsThe rate of apoptotic peripheral blood CD4 + T lymphocytes in 7 AIDS patients is 11.08 ± 1.02(9. 57 ~ 11. 86) and is significantly higher than it in normal controls, long - term nonprogressive patients and chronic HIV infectors( P < 0.01).2.2 The rate of apoptotic peripheral blood CD4 + T lymphocytes in asymptomatic HIV infectors is significantly higher than it in normal controlsThe rate of apoptotic peripheral blood CD4 + T lymphocytes in 18 asymptomatic HIV infectors is 6. 49 ± 2. 09 (2. 63 ~ 9. 72) and is significantly higher than it in normal controls ( P <0. 01) ; it is higher than it in long - term nonprogressive patients, but there are no statistic difference between them(P =0.02 > 0.01).2. 3 There are no significantly difference in the rate of apoptotic peripheral blood CD4 + T lymphocytes between long - term nonprogressive patients and normal controlsThe rate of apoptotic peripheral blood CD4 + T lymphocytes in 7 long - term nonprogressive patients is 4.97 ±3.05( 1.12 ~9.95) . And there are no significantly difference between it in long - term nonprogressive patients and in normal controls( P = 0.02 >0.01).