The Study On The Expression Of Bcl-2, Bax, Caspase-1, Caspase-3, NF-κB And Cell Apoptosis After Experimental Brain Contusion Of Different Severity In Rats

IntroductionA series of pathological, physiological and biochemical changes occur after brain injury. All these changes are called secondary changes and can do secondary injury to the brain. Several genes and cytokines take part in the accommodation of these changes and influence the severity of brain injury, Such as tumor necrosis factor a, Bel - 2 gene family and ced gene family ( cystein - dependent aspartate - specific protease, caspase). They are all has relationship with cell death after TBI and control the different cascade of cell death.There are two kinds of cell death after traumatic brain injury (TBI) that is necrosis and apoptosis. Many experiments certificate that markedly lesion of the brain after TBI are mainly caused by secondary injury, that is to say secondary injury is very important to the severity of brain injury. Studies suggest that programmed cell death (PCD; Cells that occur PCD have a strict morphological change called apoptosis) occurs after TBI. Genes that control apoptosis are included Bel - 2 gene family and caspase family. Cytokines in Bel - 2 family may both promote ( such as Bax) and prevent ( such as Bel -2 and bcl - d) neurons occur apoptosis, and caspase family promote cells occur apoptosis. Nuclear factor - KB ( NF - KB) is one kind of cell transcription factor that exist in many kinds of cells in nervous system. It is also has great association with the process of neuronal degeneration and apoptosis.From the above we can see that TBI, secondary brain injury, apoptosis and cytokines associated tightly. But the relationship among the severity of injury and changes of cytokines are not clear.The mechanisms, post - intervals, severity and prognosis of brain injuryare very important in forensic and clinical practice. In this study, brain contusion model of different severity and post - traumatic intervals were made. Pathological , biochemical and molecular biological technique were used to study the changes of Bcl - 2, Bax, caspase - 1, caspase - 3 and NF - KB after brain contusion aim to investigate the relationship among them and apoptosis and try to provide some objective marker and theory basis to the judgment on post - traumatic intervals and severity of brain injury.Materials and Methodes1. Animals: Adult healthy Wistar rats of either sex, weighing 180g ~280g were provided by the Animal Breeding Laboratory of China Medical University).2. Reagents: Mouse anti -human Bel -2(sc -7382) , Bax (sc -7480) and NF ?KB p65 ( sc ?8008 ) mono ?clone antibody, rabbit anti - human caspase -l(sc-514) poly - clone antibody, horseradish peroxidase ( HRP) marked or alkaline phosphatase marked goat anti - mouse or goat anti - rabbit IgG, immunohistochemical staining kit and DAB were purchased from Santa Cruz Biotechnology U. S. A. ; rabbit anti - human poly - clone antibody caspase -3 (BA0588) was purchased from Boster Biotechnology co. , LTD; Bel -2, Bax, caspase - 1, caspase - 3 and NF - KB mRNA primer were synthesized by Beijing Auke Biotechnology co. , LTD. ; RNA splitting liquor was purchased from Huamei biotechnology co. , LTD. ; PCR kit from Takara; Terminal deixyl-nucleotidyl transferase (TdT) was from Promega co. , LTD. ; DIG -dUTP was from B. M co. , LTD. ; Anti - DIG - Biotin and Western ?blotting developing a-gent were from Sigma co. , LTD. ; SABC and TUNEL kit were from Boster co. , LTD.3. EquipmentOlympus microscope (BX51, Japan)Image analyzer( Metamorph/DP10/BX51, U. S. A/Japan)Even oar machine (Heidolph DIAX900,Germany)Hypothermia high speed centrifugal apparatus (Sigma2K15C,Germany)LKB electrophresis apparatus(Beomma2301,Sweden)BIO - RAD electrophoresis chamber (U. S. A)Inversio -transcription PCR apparatus (PTC100,U. S. A)Gradient PCR apparatus ( Biometra, Germany )Electrophresis apparatus( Bio - RAD,PAC3000,U. S. A)Spectrophotomete -722 (China)ID Kodak image analysis system (U. S. A)Electrophoresis gel image analysis workstation ( T - software, UICMetamorts USA)GDS8000 gel auto - imaging appara...