Objective To explore the effect of artemisinin on the apoptosis of pancreas acinar cells in acute pancreatitis (AP), and to study whether artemisinin can relieve the severity of AP. Methods ① In vivo experiment: twenty one Wistar rats were divided into the following 3 groups randomly: the normal control group, the AP group and the artemisinin group. The model of AP was established by injecting cerulein into the peritoneal cavity of rat. After establishment of AP in the artemisinin group, artemisinin was injected into the peritoneal cavity. Meanwhile normal saline was injected into the peritoneal cavity of rats of the normal control group and the AP group. The apoptosis of pancreas acinar cell was detected by terminal deoxynucleotidyl transferase mediated nick end labeling (TUNEL). The activity of myeloperoxidase was detected by absorption spectrometry. ② In vitro experiment: the pancreas acinar cells of normal rats were isolated through twostep enzyme digestion, and cultured. These acinar cells were divided into 3 groups: the normal control group, the AP group and the artemisinin group. Then, the cells of AP group were cocultured with cerulein, and those of the artemisinin group were cocultured with cerulein and artemisinin. The apoptosis of pancreas acinar cells were detected by AO dyeing and the measurement of the activity of caspase3. And the activity of LDH and AMS in the culture medium of each group were measured. Results ① In vivo: the apoptosis index of the artemisinin group was sigificantly increased and the activity of myeloperoxidase was obviously decreased compared with the AP group (P＜0.05). ② In vitro: the apoptosis index and the activity of caspase3 of the artemisinin group were significantly increased compared with the AP group (P＜0.05); the activities of LDH and AMS of the artemisinin group were more decreased than those of the AP group (P＜0.05). Conclusion Artemisinin could contribute to the apoptosis of rat pancreas acinar cells, decrease the releasing of trypsogen, alleviate the activation of neutrophil and relieve the severity of AP.
【Abstract】 Objective To investigate the effect of retinoic acid (RA) on cell apoptosis and gene regulation of IGF-2in chondrocyte. Methods One 1-month-old Chinese rabbit weighted 500 g was used in this experiment. The chondrocyte from rabbit knee were cultured by enzyme digestion. Twenty-five μL all-trans-retinoic acid (ATRA) (1×10-6 mol/L) were added in the media of cultured chondrocyte for 24 hours as experimental group, while 25 μL DMEM were added as control group. The secretion of collagen Ⅱ was observed by immunohistochemistry method, cell apoptosis was detected by flow cytometry, IGF-2 mRNA and protein expression in chondrocyte were detected by RT-PCR and Western blot analysis. Results The expression of collagen Ⅱ was down-regulated by ATRA in the experimental group. The cell apoptosis in chondrocyte exposed to ATRA at 1 ×10-6 mol/L was 21% ± 2%, which increased 5 times compared with the control group（5% ± 1%）. The IGF-2 mRNA and protein level in the experimental group were decreased 75% and 57%, respectively, compared to the control group. There weresignificant difference between the experimental group and control group in each index (P lt; 0.05). Conclusion RA may down-regulate the secretion and cell prol iferation, but up-regulate the cell apoptosis in chondrocyte. The apoptotic effect may carry out through inhibiting the IGF-2 expression of chondrocyte.
ObjectiveTo explore the new gene therapy method for tumor, the recombinant Caspase3 gene (rcaspase3) eukaryotic expression plasmid was constructed by molecular biologic method. MethodsThe eukaryotic expression plasmid pcDNA3.1(+)/rCaspase3 was constructed by rearrangement of the large subunit and small subunit of Caspase3 and it was transfected into pancreatic carcinoma cells(PCⅡ). After being transfected, the expression of rCaspase3 mRNA in pancreatic carcinoma cells was detected by RTPCR and it’s apoptotic activity was detected by FCM. ResultsThe sequence of rCaspase3 showed that the recombinant molecules (rCaspase3) now had its’ small subunit preceding its’ large subunit. After pancreatic carcinoma cells being transfected with the pcDNA3.1(+)/rCaspase3 by liposomes, a 894 bp strap was observed by RTPCR. No strap was found in control groups. A transparent hypodiploid karyotype peak was found by FCM.ConclusionThe plasmid of pcDNA3.1(+)/rCaspase3 has been constructed successfully. rCaspase3 has apoptotic activity and can be used as target gene in gene therapy for pancreatic carcinoma.
Objective To observe the effect of a wide range of concentration of arsenic trioxide on hepatoma cell line BEL-7402 with variable duration. Methods The cell activity and morphologic changes were studied after treated with different concentration. The apoptosis were detected by flow cytometry assay and DNA Gel electrophoresis. Results The effect of arsenic trioxide on hepatoma cell lines were dependent on the time and concentration obviously. Hepatoma cells cultured with different concentration presented apoptosis features: i.e. intact cell membrane, chromatin condensation, nucleic fragmentation and apoptotic body formation; flow cytometry analysis showed an arrestment at G2/M phase and a subG1 cell peak, DNA gel electrophoresis showed a marked DNA ladder. Conclusion Arsenic trioxide can obviously inhibit the growth of hepatoma cell lines through inducing hepatoma cell apoptosis.
OBJECTIVE: To explore the effect of Fas/Apo-1 and Bcl-2 gene expression on mechanism of scar formation. METHODS: Immunohistochemical method was applied to defect the expression of Fas and Bcl-2 protein in fibroblasts from 10 cases with normal skin, 10 cases with hypertrophic scar and 10 cases with keloid. RESULTS: The positive expression rate of Bcl-2 protein in keloid was 83.2%, significantly higher than that in hypertrophic scar (38.6%), (P lt; 0.01), and the positive expression rate in hypertrophic scar and keloid was higher than that in normal skin (6.78%), (P lt; 0.01). But the positive expression rate of Fas/Apo-1 protein was 78.4% in normal skin 80.4% in hypertrophic scar, 84.4% in keloid respectively, which showed no significant difference among them (P gt; 0.05). CONCLUSION: Bcl-2 gene but Fas gene may take part in the formation of pathologic scar.
Objective To observe the expression of cyclin dependent kinase 5 (Cdk5) and p25 in the pathogenesis of retinitis pigmentosa (RP) in Royal College of Surgeon (RCS) rats and its relationships with apoptosis. To explore the mechanism of Cdk5 and p25 induced photoreceptor apoptosis in the pathogenesis of RP. Methods Retinas of RCS and RCS-rdy+ rats were obtained at the ages of postnatal day 17, 25, 35, 60. The retinal structure and thickness of outer nuclear layer were measured by optical microscopy. The expression of Cdk5, p25, cleave-caspase 3 in the retina was evaluated by immunohistochemistry. The protein expression of cleave-caspase 3 in the retina was determined by Western blot. The apoptosis of retinal cells was examined by terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL). The mean absorbance value of apoptotic cells was analyzed by SPSS 17.0 software. Results The retinal thickness of the RCS rats was significantly reduced in comparison to the RCS-rdy+ rats as the postnatal days progressed, particularly in the layer of rods and cones and the outer nuclear layer. The expression level of Cdk5, p25, cleave-caspase 3 of RCS rats increased from postnatal 17 days to postnatal 35 days, while decreased on postnatal 60 days; but there was no obvious change of above indexes in RCS-rdy+ rats. The protein expression of cleave-caspase 3 in the RCS rats was significantly increased with progression of postnatal days to postnatal 35; but there was no obvious similar change in RCS-rdy+ rats. The results of TUNEL showed that the apoptotic cells significantly increased in the outer nuclear layer of RCS rats from postnatal 17 days to postnatal 35 days, while decreased on postnatal 60 days; but there was no obvious change of above index in RCS-rdy+ rats. This study showed that there were significant correlations between the following variables: Cdk5 expression and p25 expression, Cdk5 expression and cleave-caspase 3 expression, Cdk5 expression and apoptotic cells, p25 expression and cleave-caspase 3 expression, p25 expression and apoptotic cells, cleave-caspase 3 expression and apoptotic cells. The partial correlation coefficients were 0.949, 0.808, 0.959, 0.887, 0.979, 0.852, respectively and the P value was 0.000. Conclusions The apoptotic cells significantly increases and the expression level of Cdk5, p25, cleave-caspase 3 of RCS rats increases from postnatal 17 days to postnatal 35 days. The tendency of apoptotic cells to increase is consistent with the change of Cdk5, p25, cleave-caspase 3 expression. The apoptosis of photoreceptor cells is related to increasing expression of Cdk5 and p25 in RCS rats. Cdk5 may be involved in the development of RP in RCS rats.
To evaluate the significance of bcl-2 protein, estrogen receptor (ER), progesterone receptor (PR) and p53 oncogene in invasive breast cancer of ductal and lobular type. Tumor tissues were examined immunohistochemically in paraffin embedded tissues from 125 patients. Results: The invasive ductal breast carcinomas expressed bcl-2 protein significantly less frequently than the lobular type (Ｐ＜0.001). And the expression of bcl-2 protein was significantly correlated with ER, PR (Ｐ＜0.001) and p53 (Ｐ＜0.001), also correlated with primary tumor size and grade. No statistical evidence was found to indicate the relationship between bcl-2 protein expression and anxillary lymph node metastasis. Conclusions: The expression of bcl-2 protein may be regarded as a biological index and may play important role in evaluating the biological characteristics of breast cancer.
Objective:To observe the inhibited effect and its mechani sm of estro gen on lightinduced apoptosis of retinal photoreceptor cells. Methods:Twenty female Sprague-Dawley rats were randomly divided into two groups: ovariectomized (OV) group and OV and estrogen (E2) replacement (OV+E2) group, with 10 rats in each group. All of the rats were exposed to the cyclic illumination under 12 hou r light and 12 hour dark condition with the light intensity of (600plusmn;35.4) lx ( a total of 14 times). All of the right eyes were extracted and the thickness of r etinal outer nuclear layer (ONL) was measured. Terminal deoxynucleotidyl Transfe rasemediated dUTP nick end labeling (TUNEL) method was used to evaluate positi v e apoptosis cells in ONL. The expression of nitric oxide synthase (NOS) in retin al cells was detected by immunohistochemistry with image analysis method. Results:The thickness of ONL in OV group was obviously thinner than that in the OV+ E2 group. The number of positive apoptosis of the cells was (0.0275plusmn;0.0069) c el ls/mu;m2 in OV group and (0.0162plusmn;0.0054) cells/mu;m2 in OV+E2group; the di fferen ce between the two groups was significant (t=4.1370，P=0.0012). The values o f in tegral optical density of NOS positively stained cells in retinal inner nuclear layer was (0.3675plusmn;0.0662) in the OV group and (0.2941plusmn;0.0350) in OV+E2 group ; the difference between the two groups was significant (t=3.4885, P=0.0031). Conclusion:Estrogen can protect retina from light injuries by regu lating NOS synthesis and inhibiting apoptosis of photoreceptor cells.
Objective To further investigate pathologic mechanism of retinal phototrauma. Methods Twenty Wistar rats were divided into control and experimental groups.Their eyes were extracted in 12，24 and 36 hours after light exposure.HE stained retina samples were examined and TDT-mediated dUTP nick end labelling(TUNEL)method was employed to distinguish apoptotic cells. Results After 12-hour light exposure,slight vesiculation was observed in the rod outer segment of the retinas.After 24-hour light exposure,the outer nuclear layer showed predominant fractured and condensed nuclei and fragmented DNA.After 36-hour light exposure,the rod outer and inner segments were lysed and most of the nuclei in the outer nuclear layer were disappeared. Conclusions Apoptosis of photoreceptor cell is one of the important mechanisms which cause experimental retinal photoinjury of rats. (Chin J Ocul Fundus Dis, 1999, 15: 167-169)
Objective To observe the proapoptotic effect ofthe homogenate of different parts of pig’s full thickness dermal wounds on cultured fibroblasts. Methods The tissues were dissected from the wound center and subneoepithelium separately 15 days after homogenization and sterilization, the specimens stored at -70℃. The forth passage of the fibroblasts were cultured for 16 hours in different culture solutions and were grouped into 7 groups: DMEM containing 5% fetal bovine serum as Group Ⅰ, DMEM containing 5% homogenate of tissue from wound center as GroupⅡ, DMEM containing 5% homogenate of tissue from subneoepithelium as Group Ⅲ, the culture solution of Group Ⅱmixed with 10 μg/ml GM6001 in Group Ⅳ, with the culturing medium of Group Ⅲplus 10 μg/ml GM6001 as Group Ⅴ, the culture solution of Group Ⅱ mixed with 10 ng/ml aFGF as Group Ⅵ, and the culture solution of Group Ⅲ mixed with 10 ng/ml aFGF as Group Ⅶ. In all groups except Group Ⅰ, the fibroblasts of the 6 pigs were treated with the homogenate derived from the same animal respectively. After being incubated in Annexin Ⅴ-FITC and PI, cells were analyzed by Flow Cytometry and the rate of apoptotic cells was acquired. The data were analyzed by SPSS 11.0 using Leastsignificant Difference test(LSD). Results The apoptotic rate of the 7 groups were as follows:4.39%±0.41% in Group Ⅰ，10.98%±1.42% in Group Ⅱ，13.47%±1.44% in Group Ⅲ，7.2%±0.46% in Group Ⅳ，12.1%±0.85% in Group Ⅴ，3.9%±0.63% in Group Ⅵ，9.8%±0.50% in Group Ⅶ； there were significant differences between every two groups except Group Ⅰand Group Ⅵ. Conclusion Homogenate of the tissue derived from the subneoepithelium has greater proapoptotic effect than that from the wound center; the proapoptotic effect of homogenate of the tissue both under neoepithelium and in wound center can be significantly alleviated by acid fibroblast growth factor, partly because of MMPs.