OBJECTIVETo observe the cell apoptosis after tractive spinal cord injury in rats, determine expression of apoptosis correlative genes, and study the molecular mechanism of cell apoptosis.

METHODSThe T(13)-L(2) spinal cord of rats was injured by traction after the amplitude of P1-N1 wave decreased to 70% in postoperation than in preoperation through cortical somatosensory evoked potential (CSEP) monitor. Then rats were killed in 30 min, 6 h, 1, 4, 7, 14 and 21 d respectively after operation (n = 4). Cell apoptosis was examined by the flow cytometer and terminal deoxynucleotidyl transferase-mediated DUTP-biotin nick end labeling (TUNEL) reaction, the expression of p53, bax and bc1-2 genes was tested with immunohistochemistry.

RESULTSThe flow cytometer test and TUNEL method showed that the apoptosis cell ratio raised in 6 h and reached at peak in 7 d after injury, and then declined till 21 d, they showed significant difference (P < 0.05, 0.01). TUNEL method showed that injured group had a large number of apoptosis glial cells in white matter. Immunohistochemical staining showed that the positive expression of p53, bax and bc1-2 protein raised at 6 h, expression of p53 protein reached at peak in 4 d, bax and bc1-2 protein reached at peak in 7 d after injury. Compared with control group and laminectomy group, the injured group showed significant difference (P < 0.05, 0.01).

CONCLUSIONThere is cell apoptosis phenomenon after tractive spinal cord injury in rats. Morphology indicates that apoptosis includes neurons and glialcytes, which is an important form of cell death and pathological changes in secondary lesion period after tractive spinal cord. There exist high expression of apoptosis correlative gene p53 and bax after spinal cord injury, they may play an important role in reduction of cells to apoptosis.

Animals ; Apoptosis ; genetics ; Disease Models, Animal ; Female ; Gene Expression ; Genes, bcl-2 ; genetics ; Genes, p53 ; genetics ; Male ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; Rats ; Rats, Sprague-Dawley ; Spinal Cord Injuries ; genetics ; pathology ; bcl-2-Associated X Protein

Genes, Reporter ; Genetic Vectors ; Luciferases ; genetics ; Transcription, Genetic ; bcl-2-Associated X Protein ; genetics

OBJECTIVETo assess the protection against cisplatin-induced ototoxicity by adenovirus-mediated overexpression of the bcl-2 gene in cultured spiral ganglion cells (SGC).

METHODSSGC from P3 rats were cultured in vitro and exposed to adenovirus vector carrying green fluorescent protein gene (Ad-GFP), followed by immunocytochemical analysis for expression of the neuron-specific marker Neurofilament 200 (NF200) and detection under laser scanning confocal fluorescence microscope. Then, SGC were transduced by Ad-bcl-2 and the expression of human bcl-2 protein was evaluated by Western Blot. Finally, the cultures of SGC were divided into 4 groups: the group of Ad-bcl-2 transfection followed by cisplatin treatment, the group of Ad-GFP transfection followed by cisplatin treatment, the group of cisplatin treatment only and the untreated group. Cisplatin worked for 48 hours at a concentration of 2 microg/ml. Outcome measures included survival number of SGC and longest neurite length by using ImageJ software.

RESULTSSGC were cultured successfully in vitro and transfected by adenovirus vector safely and efficiently. By Western Blot, human bcl-2 protein was expressed in the group after exposure to Ad-bcl-2, but not in the Ad-GFP transfected SGC. Cisplatin exposure resulted in shrinking of neuritis and pyknosis of cell body, even cell death. Expression of bcl-2 in the SGC provided a significant level of protection against cisplatin-induced SGC degeneration.

CONCLUSIONSOur results suggest that SGC can be transduced by adenovirus vector safely and efficiently in vitro. Adenovirus-mediated delivery of the bcl-2 gene attenuates cisplatin-induced SGC degeneration.

Adenoviridae ; genetics ; Animals ; Apoptosis ; drug effects ; Cisplatin ; pharmacology ; Genes, bcl-2 ; Humans ; Spiral Ganglion ; cytology

The study was aimed to explore the apoptosis effect of ouabain on Jurkat cells and its mechanism. MTT method was used to observe the inhibitory effect of ouabain on Jurkat cell proliferation. Apoptosis was detected by using flow cytometry (FCM) and the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling reaction method (TUNEL). The protein expressions of Bax, Bcl-2 and active subunits of caspase-3 were measured by Western blot. Activities of caspase-3 were determined by colorimetry method. The results showed that ouabain could induce apoptosis of Jurkat cells, the expression of Bax protein in process of cell apoptosis, caspase-3 activity of Jurkat cells were remarkably enhanced after ouabain treatment. It is concluded that ouabain may induce apoptosis of Jurkat cells due to the activation of caspase-3 resulting from regulation of Bax protein and Bcl-2 gene expressions.
Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Cell Proliferation ; drug effects ; Genes, bcl-2 ; genetics ; Humans ; Jurkat Cells ; Ouabain ; pharmacology ; bcl-2-Associated X Protein ; biosynthesis ; genetics

OBJECTIVETo investigate the therapeutic effect of Bcl-2 fusion protein on apoptosis in brain following traumatic brain injury.

METHODSBcl-2 gene was cloned by RT-PCR. Bcl-2 and EGFP genes were linked together and inserted into pAdeno-X vector. This recombinant vector was packaged into infectious adenovirus in HEK293 cells. Ninety Wistar rats were assigned randomly into experimental group (n=45) and control group (n=45). All rats were subjected to traumatic brain injury. Then recombinant adenovirus (for experimental group) or saline (for control group) was injected into the traumatic brain. The expression of Bcl-2 fusion protein was investigated by Western blotting, immunohistochemistry and fluorescence microscopy. Apoptosis in the injured brain was studied by TUNEL. Animals' behavior capacity was evaluated by tiltboard test.

RESULTSIn the experimental group, many fluorescent cells were found around the traumatic locus, which were also proven to be Bcl-2 positive by immunohistochemistry. On the contrary, few Bcl-2 positive cells and no fluorescent cell were detected in the control group. Bcl-2 expression of experimental group was much higher than that of control group, which was illustrated by Western blotting. The apoptosis index of experimental group was 0.027+/-0.005, and that of control group was 0.141+/-0.025 (P < 0.01). Two weeks after injury, animals of the experimental group behaved better than those of the control group.

CONCLUSIONSA recombinant adenovirus vector expressing Bcl-2 fusion protein has been constructed. Bcl-2 fusion protein can suppress apoptosis and promote cell survival. Moreover, the behavior recovery of the injured animal is promoted. Bcl-2 fusion protein provides a way to track the target cells in vivo.

Adenoviridae ; genetics ; Animals ; Apoptosis ; Base Sequence ; Brain Injuries ; therapy ; Cloning, Molecular ; Genes, bcl-2 ; Genetic Therapy ; Proto-Oncogene Proteins c-bcl-2 ; analysis ; Rats ; Rats, Wistar

Alveolar Epithelial Cells ; metabolism ; pathology ; Animals ; Apoptosis ; physiology ; Cell Survival ; physiology ; Cells, Cultured ; Epithelial Cells ; cytology ; physiology ; Genes, bcl-2 ; genetics ; Genes, p53 ; genetics ; Oxidative Stress ; genetics ; Rats

OBJECTIVE: To investigate the effects of long-term estrogen replacement treatment (ERT) on the expression of bcl-2 and H-ras in rat endometrium. METHODS: Thirty 5-month-old SD female rats were randomly divided into 3 groups: Control group ( sham operated and vehicle injected, n 10) , OVX group (OVX operated and vehicle injected, n = 10) , and ERT group (OVX operated and 17 beta-estradiol injected, n = 10). The rats were killed in the 13th week and the uteri were isolated and weighed, pathologically analyzed, and we measured the thickness of the endometrium. Immunochemistry and in situ hybridization analysis were used to examine the changes of bcl-2 and H-ras mRNA and Bcl and H-ras protein expression in the endometrium of the rats. RESULTS: Uterine weight and endometrial thickness of OVX decreased much more than those of the control (P <0.01 ) and ERT rats (P < 0.01). One simple hyperplasia and one squamous metaplasia of endometrium were found in ERT rats. Quantitatively, bcl-2 and H-ras mRNA and Bcl and H-ras protein level of ERT were higher than those of OVX rats (P < 0.01 ), and there were no statistical significances between the ERT group and the control rats. CONCLUSION Long-term estrogen replacement can keep the endometrium from atrophy, and lead to the genesis of simple hyperplasia and squamous metaplasia of the endometriun, which can increase the risk of endometrial carcinomas. Estrogen may inhibit apoptosis and promote the proliferation of endometrial cells through increasing the expression of bcl-2 and H-ras mRNA and Bcl-H-ras proteins.
Animals ; Endometrium ; metabolism ; Estradiol ; pharmacology ; Estrogen Replacement Therapy ; Female ; Genes, bcl-2 ; Genes, ras ; Ovariectomy ; Proto-Oncogene Proteins c-bcl-2 ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Time Factors ; ras Proteins ; biosynthesis ; genetics

The aim of this study was to investigate the bcl-2/IgH expression levels in patients with follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL) and its clinical significance. The bcl-2/IgH expression levels in bone marrow (BM) and/or peripheral blood (PB) of 20 patients were detected by using SYBR Green I real-time polymerase chain reaction, and the dynamic monitoring for bcl-2/IgH expression level in 4 of these patients was performed. The results showed that in patients with bcl-2/IgH-positive there was no statistically significant difference in the relative copy-numbers of bcl-2/IgH fusion gene in BM and PB (4.23 and 2.73 respectively, p = 0.107), but the difference was significant before and after treatment (3.61 and 2.69 respectively, p = 0.000), the expression level of bcl-2/IgH fusion gene in newly diagnosed and relapsed group was remarkably higher than that in remission group (p = 0.008). The bcl-2/IgH expression level in PB increased evidently 3 months prior to the clinical relapse in one case out of dynamically monitored 4 cases. It is concluded that the bcl-2/IgH expression level is associated with the disease status, the expression level is high in newly diagnosed and relapsed patients and low in those who achieved remission, the bcl-2/IgH fusion gene expression level decreased evidently after therapy, this change may be related to the clinical disease progression, the results suggest that peripheral blood can be regarded as the resource for detection of bcl-2/IgH fusion gene.
Adult ; Aged ; Female ; Gene Expression ; Genes, Immunoglobulin Heavy Chain ; Genes, bcl-2 ; Humans ; Lymphoma ; genetics ; Male ; Middle Aged ; Oncogene Proteins, Fusion ; genetics ; Polymerase Chain Reaction ; methods ; Translocation, Genetic

OBJECTIVETo investigate the effect of lentivirus-mediated Bcl-2 gene transfection in protecting human primary ovarian granulosa cells against phosphoramide mustard (PM)-induced apoptosis.

METHODSGranulosa cells were isolated from the follicle fluid of women undergoing in vitro fertilization and embryo transfer. The lentiviral vectors carrying Bcl-2 gene (pGC-FU-Bcl-2) and enhanced green fluorescence protein (pGC-FU-EGFP) were constructed and packaged into high-titer lentiviruses. The resulting recombinant lentivirus carrying Bcl-2 and EGFP genes or the empty vector were used to infect the primary human ovarian granulosa cells, followed by addition of PM in the cell culture, with untreated granulosa cells as the control. The cell apoptosis was detected by Annexin V and Hochst 33258 staining, and the expression of Bcl-2 protein was assessed using Western blotting.

RESULTSThe control granulosa cells showed an apoptotic rate of (1.93±0.28)%. The cells infected with pGC-FU-Bcl-2 prior to PM exposure had a apoptotic rate of (6.99±10.55)%, significantly higher than that of the control cells, but significantly lower than that of the cells with PM exposure only and those infected with the empty vector before PM exposure (P<0.05). The expression of Bcl-2 was the highest in the cells infected with pGC-FU-Bcl-2 prior to PM exposure (P<0.05).

CONCLUSIONLentivirus-mediated Bcl-2 gene transfection can protect human ovarian granulosa cells against PM-induced apoptosis by upregulating Bcl-2 protein expression.

Adult ; Apoptosis ; drug effects ; Female ; Genes, bcl-2 ; Genetic Vectors ; Granulosa Cells ; drug effects ; Humans ; Lentivirus ; genetics ; Phosphoramide Mustards ; Transfection

OBJECTIVETo construct a lentiviral vector carrying human bcl-2 gene and investigate its expression in human ovarian granulosa cells (GCs).

METHODSHuman bcl-2 gene was amplified from the plasmid pCMV-SPORT6 using PCR and subcloned into the lentiviral vector pGC-FU to construct the lentiviral expression vector pGC-FU- bcl-2. The bcl-2 gene insert was confirmed by restriction enzyme digestion and sequencing. The recombinant lentiviruses generated by 293T cells co-transfected with pGC-FU-bcl-2 and the packaging plasmids pHelper1.0 and pHelper2.0. The resulting recombinant lentivirus GC-FU-bcl-2 carrying bcl-2 and EGFP genes were then used to infect human ovarian granulosa cells. EGFP and bcl-2 protein expressions in 293T and human ovarian GCs were detected by fluorescent microscope and Western blotting.

RESULTSThe plasmid pGC-FU-bcl-2 carrying the correct bcl-2 gene sequence could be expressed in human ovarian GC cells, and the recombinant lentivirus GC-FU-bcl-2 was generated by the packaging 293T cells. Stable expression of EGFP and bcl-2 proteins were detected by fluorescent microscope and Western blotting in 293T and human ovarian GCs after the infection. The recombinant lentivirus efficiently delivered bcl-2 gene into human ovarian GCs, in which bcl-2 expression was expressed efficiently and stably.

CONCLUSIONThe recombinant lentivirus GC-FU-bcl-2 has been successfully constructed, which is capable of delivering the target gene bcl-2 into human ovarian GCs for its stable expression.

Cells, Cultured ; Female ; Genes, bcl-2 ; genetics ; Genetic Vectors ; Granulosa Cells ; metabolism ; Humans ; Lentivirus ; genetics ; metabolism ; Ovary ; cytology ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Transfection