Apoptosis And Expression Of Caspase-3 Of Peripheral Blood Mononuclear Cells In Patients Infected With HBV

ABMCircumstantial evidence indicates that hepatitis B virus ( HBV) is not directly cytopathic for the infected hepatocytes. Activation-induced cell death ( AICD ) of mature T cells caused by HBV may lead to cellular immune response decreased, which may be an important factor for chronic infection of HBV persistent. Programmed cell death, or Apoptosis, is important in homeostasis in vivo. A pivotal biochemical event of apoptosis is the activation of cysteinyl aspar-tate-specific proteases or caspases. Caspases recognize a four-amino acid sequence on their substrate proteins; the carboxyl end of aspartic acid within this sequence is the target for cleavage. Caspase-3 is a member of the caspase-family and acts as the effecter or executioner of apoptosis.Peripheral blood mononuclear cells ( PBMCs) including T lymphocytes, B lymphocytes, monocytes, and NK cells, which are the main immune cells of the body and the main location of HBV infection in extrahepatocyte. To investigate the cell immune of patients infected with HBV, this article studies the apoptosis of PBMCs and the role of caspase-3 in HBV infection.MATERIAS AND METHODSThirty-five patients with HBV carrier and chronic hepatitis B patients (22 men and 13 women; age from 18 years to 64 years; Mean age 38 years) were studied. All patients had been grouped. ASC (The chronic asymptomatic HB-sAg carrier, ASC) 10, mild type of chronic hepatitis B 10, moderate type of chronic hepatitis B 8, severe type of chronic hepatitis B 7. All subjects were diagnosed according to the criterion of Xi'an 2000's conference. All subjectsshowed no evidence of alcoholic, autoimmune or drug-induced liver disease. None had undergone antiviral or immunomodulatory therapy. Health donors 10 (5 men and 5 women; age from 18 years to 32 years; mean age 26 years) were as control group. PBMCs collection and cells culture:PBMCs were prepared from fresh, heparinezed blood by centrifugation through a Ficoll-Hypaque density gradient. PBMCs were cultured in 24-well culture plates using 4 x 10 cells/ml in complete medium with fetal calf serum for 72h with PHA. After culture, cells were collected and froze by -80TI. Detection of apoptosis:1. TUNEL (terminal deoxyribonucleotidyl transferase-mediated biotinylated dUTP nick-end labeling) :Cells were smeared on a glass slide, and then fixed by 4% paraformalde-hyde. Detection was performed as the illumination of the kit. Cells, which nuclear stained in yellow brown, were the positive pattern.2. FCM (flow cytometry) :PBMCs were fixed by 10% ethanol at -30T! overnight. Washed twice with PBS (phosphate-buffered saline, PBS) . Cells were incubated with about 500jxl of the solution (included Rnase A, PI). Incubated in room temperature about 30 min. Then analyzed using Lysis II software in a FACScan flow cytometer. Detection of caspase-3:RT-PCR; Total RNA was extracted from cells. Reverse-transcribed DNA was amplified in AmpliTaq DNA polymerase, paired primers, and four deoxybi-bonucleoside triphosphates. We performed 35 amplification cycles using a step-wise program.The primers: caspase-3-F: 5'-AGG ACT CAA ATT CTG TTG CCA CC-3' (sense); caspase-3-R: 5'-TGG AAC AAA TGG ACC TGT TGA CC-3'(anti-sense) . The size of product ;365bp. The primers were checked with the Gene bank AF052647.-actin-F:5'-GTG GGG CGC CCC AGG CAC CA-3';3-actin-R: 5'- CTT CCT TAA TGT CAC GCA CGA TTT C-3'. The size of product :540bp.The amplified products were then electrophoresed through 2% agrose gels.We use tannon analysis software to analyze the caspase-3 DNA. Caspase-3 mR-NA = caspase-3 density/p-actin density x 100%.RESULTSThere's apoptosis in PBMCs of HBV infections. The apoptosis level is the highest in the patients of mild type of chronic HBV infected. TUNEL and FCM had the correlation on detecting the apoptosis in PBMCs.Caspae-3 mRNA was expressed in PBMCs. We detected caspase-3 mRNA using RT-PCR showed that caspase-3 expressed the highest in mild type of chronic hepatitis B. It has correlation with the apoptos...