Objective To construct human secreted apoptosis-related protein 1 (SARP1) gene yeast two-hybrid bait vector so as to study the biological functions of the SARP1 gene in the scar tissue. Methods The target gene from SARP1 gene full-length DNA segment was amplified by PCR, the upstream and downstream primers of the SARP1 gene with restriction enzymes Nde I and Sal I were designed. pGBKT7-SARP1 recombination plasmid was constructed by ligating the vector and the PCR production and identified by PCR and sequencing. Further more, pGBKT7-SARP1 was transformed into competent AH109 which contained kanamycin for selecting positive clones and screened the positive clony on the plate of SD/-Trp. The toxicity and transcriptional activation were tested by the phenotype assay. Results SARP1 was amplified and cloned into pGBKT7 successfully, SARP1 gene sequence in recombinant plasmid pGBKT7-SARP1 was verified by gel electrophoresis and DNA sequencing analysis. The sequence of inserted SARP1 gene was the same as the corresponding sequence found in GenBank database. The recombinant pGBKT7-SARP1 plasmids and empty pGBKT7 vector could form white colonies on SD/-Trp plates and none could survive on SD/-Leu plates. Conclusion The recombinant pGBKT7-SARP1 gene yeast two-hybrid bait vector is successfully constructed.
ObjectiveTo introduce the relationship between the apoptosis hepatocyte and its genic mediation and the ischemia of portal vein. MethodsThe combination of related literatures and our research findings were made.ResultsPortal vein ischemia may induced hepatocyte apoptosis, p53 and bcl2 gene alternatively adjust hepatocyte apoptosis. Expression of p53 gene is enhanced in hepatic tissue when hepatocyte apoptosis is not obvious, but after 24-72 h of portal vein ischemia, when hepatocyte apoptosis is obvious, enhanced expression of p53 gene or reduced expression of bcl2 gene occur. There exists close relationship between portal vein ischemia and hepatocyte apoptosis. Conclusion Apoptosis hepatocyte is involved in organic atrophy after ischemia of portal vein, and p53 and bcl2 gene alternatively adjust hepatocyte apoptosis. At present, the mechanism of apoptosis of hepatocyte induced by ischemia of portal vein is not clear, which needs further study.
ObjectiveTo establish the co-culture model of mice's early embryo and tumor cells in Vitro to observe the embryonic development and biologic behavior of tumor cells in the same microenvironment and discuss their interaction. MethodsWe acquired 2-cell embryos from mice, and then co-cultured them with tumor cell lines of mice in Vitro. We observed the development of embryos in Vitro and the rates of 4-cell embryos, morula and blastocyst formation. The transwell chamber was used for culture. Methylthiazolyldiphenyl-tetrazolium method was used to test the proliferative activity of tumor cells, while the flow cytometry was used to test its apoptosis. The interaction of co-cultured embryos and tumor cells was analyzed by propidium iodide staining and immunohistochemical technique. ResultsThe co-cultured 2-cell embryos could continue surviving and developing. The rates of 4-cell embryos, morula and blastocyst formation increased significantly in the co-cultured group (P<0.05). There was no significant difference in the proliferative activity and apoptosis of tumor cells between the co-cultured group and the control group (P>0.05). Tumor-free ring formed between the trophoblast and tumor cells. We could observe tumor cells stacked around the tumor-free ring. However, no difference in expression of proliferating cell nuclear antigen and B-cell lymphoma leukemia-2 was observed in tumor cells stacking around the tumor-free ring compared with those elsewhere. ConclusionThe development of 2-cell embryos is enhanced in the co-culture model. The proliferative activity and apoptosis of tumor cells are not affected in this model. A tumor-free ring can form between trophoblast and tumor cells. However, the proliferative activity of tumor cells is not affected by this ring.
ObjectiveTo study the expression of apoptosissuppressing gene (bcl2) and apoptosispromoting gene (bax) in colorectal cancerous tissue and transitional mucosas. MethodsColorectal cancerous tissue, transitional mucosas (3 cm from the cancerous tissue) and normal tissue were taken respectively in thirtyone cases. Immunohistochemical technique SP method was used to detect the expression in those tissues. ResultsThe positive expression rate of bcl2 protein in cancerous tissue and transitional mucosa were 64.5%and 60.0% respectively and significantly higher than that in normal tissue (P＜0.05). The positive expression rate of bcl2 protein in normal tissue was 35.0%. The positive expression rate of bax protein in cancerous tissue and transitional mucosa were 45.2%and 45.0% respectively and significantly lower than that in normal mucosa (P＜0.05). The positive expression rate of bax protein in normal tissue was 60.0%. There was no obvious difference in the positive rate of bax and bcl2 protein between cancerous tissue and transitional mucosa (Pgt;0.05). The expression rate of bax and bcl2 protein in colorectal cancer was irrelative to clinical pathological gradation and clinical stage (Pgt;0.05). ConclusionThere is over expression of bcl2 protein and low expression of bax protein in colorectal cancer and transitional mucosa. bcl2 protein and bax protein can affect the generation of colorectal cancer by participating in the regulation of apoptosis. But it is irrelative to clinical pathological gradation and clinical stage. Transitional mucosas should be viewed as precancerous lesion and resected during operation.
【Abstract】 Objective To investigate the expression and significance of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors in pancreatic cancer. Methods Thirty-two samples of pancreatic cancer tissue were collected from year 2002 to 2004. All of them were verified by histopathology and there were 9 cases of well-differentiated, 12 of moderately differentiated, and 11 of poorly differentiated, in which 12 cases were in the stage of Ⅰor Ⅱand 20 in the stage of Ⅲ or Ⅳ according to the TNM staging method. Eighteen normal pancreatic tissues were used as control group. The expressions of TRAIL receptors (death receptor 4, death receptor 5, decoy receptor 4 and decoy receptor 5) mRNA were assayed by semi-quantitive reverse transcription polymerase chain reaction (RT-PCR) in the pancreatic cancer tissues and the normal pancreatic tissues. Results The expressions of death receptor 4 (DR4) and death receptor 5 (DR5) were detected in all the pancreatic cancer tissues and the normal pancreatic tissues and the levels of DR4 and DR5 were significantly higher than those of the normal pancreatic tissues (P＜0.01). Decoy receptor 1 (DcR1) and decoy receptor 2 (DcR2) were also expressed in normal pancreatic tissues, whereas DcR1 and DcR2 were only expressed in 18 and 20 pancreatic cancer tissues, respectively. However, there were no significant difference of the expression of DcR1 and DcR2 between the pancreatic cancer tissues and the normal pancreatic tissues （Pgt;0.05）. The expression level of DR5 in pancreatic cancer tissue was correlated with tumor differentiation and clinical stage, and the levels in stage Ⅲ and stage Ⅳwere significantly lower than those of stageⅠand stageⅡ(P＜0.05). The expressions of DR4, DcR1 and DcR2 were not correlated with tumor differentiation and clinical stage (Pgt;0.05). Conclusion ①The expression of TRAIL receptors in pancreatic cancer tissues is prevalent, but the types of receptors expressed in different tissues were also different. High expression of death receptors may play an important role in TRAIL recptors regulated pancreatic cancer apoptosis. ②The expression of DR5 is correlated with the differentiation degree of pancreatic cancer cell and clinical stage of tumor. The expressions of DR4, DcR1 and DcR2 should not be considered as related indexes of differentiation degree or clinical stage of pancreatic cancer.
Objective To observe the outcomes of using different concentrations of arsenic trioxide at varying phases on the breast cancer cell line MCF-7 and to study the mechanism of this effect. Methods The effect of arsenic trioxide on the growth of breast cancer cell line MCF-7 was observed after applying arsenic trioxide of different concentrations （0.5-16 μmol/Ｌ）. The inhibitory effect of arsenic trioxide on the cell proliferation was investigated with 3-(4，5-dimethyl-thizazol-2-yl)-2， 5-diphenyl tetrazolium bromide (MTT) and the induction of arsenic trioxide on cell apoptosis was detected by DNA ladder and terminal deoxynucleotidyl transferase mediated nick end labeling (TUNEL). Results The effect of arsenic trioxide on breast cancer cell line MCF-7 depended on the phase and the dose. The number of cell decreased significantly and there were conspicuously typical morphological changes of apoptosis after the use of arsenic trioxide， including membrane blebbing， chromatin pyknosis, nuclear fragmentation and the formation of apoptotic body. The typical DNA ladders were observed in the MCF-7 cells after 48 h administration of arsenic trioxide at concentrations 1-8 μmol/Ｌ. Significant elevations of apoptosis index at 24 h, 48 h and 72 h were all detected by TUNEL after incubating with 4 μmol/L arsenic trioxide. Conclusion Arsenic trioxide may inhibit the growth of breast cancer cell line MCF-7 significantly by inducing the apoptosis of breast cancer cell.
Objective To investigate the expressions of tumor necrosis factor related apoptosis inducing ligand （TRAIL） and its receptors (DR4, DcR1) in human rectal cancer tissues and normal rectal tissues. MethodsThe expressions of TRAIL and its receptors (DR4, DcR1) in 31 cases of human rectal cancer tissues and 20 cases of normal rectal tissues were detected by immunohistochemical staining. ResultsThe positive expression rates of TRAIL, DR4 and DcR1 （32.26%， 29.03%， 0） were lower than those of normal rectal tissues （55.00%， 70.00%， 65.00%）, the difference was statistically significant(P=0.015， P=0.000， P=0.000). There were no relation between the expressions of TRAIL, DR4 and DcR1 and clinicopathologic characteristics (Pgt;0.05). ConclusionThe expressions of TRAIL and its receptors (DR4, DcR1) in human rectal cancer tissues were lower than those of normal rectal tissues, which may suggest that the apoptotic effect induced by the interaction between TRAIL and its receptors has attenuated in human rectal cancer.
ObjectiveTo explore the influence of different radiation doses of 125Ⅰseed on human poorly differentiated gastric cancer cells (BGC823). MethodsSixty four male nude mice of BLAB nu/nu inoculated with human poorly differentiated gastric cancer cells (BGC823) were took as animal models. When tumors of nude mice grew to 0.7-1.2 cm, the nude mice were randomly divided into blank control group, low dose group, middle dose group, and high dose group (n=16). The tumors of nude mice in blank control group were implanted with blank seed, but tumors of nude mice in low dose group, middle dose group, and high dose group were implanted with 125Ⅰseed (the radiation doses of 3 groups were 1.48×10-7 Bq, 2.22×10-7 Bq, and 2.96×10-7 Bq respectively). Four nude mice of 4 groups were randomly collected to measure the tumor volume and weight before implantation, and on 7, 14, 21, and 28 days after implantation, but apoptosis rates and expression levels of cyclinE mRNA were measured on 14 days and 28 days after implantation, by using flow cytometer and semi quantitative RT-PCR method respectively. Results①Except for the blank control group, the tumor volume and weight decreased over time in low dose group, middle dose group, and high dose group. The tumor volume and weight in blank control group were higher than those of other 3 groups on 7, 14, 21, and 28 days after implantation (P < 0.05);in addition, on 28 days after implantation, tumor volume and weight in low dose group were lower than those of middle dose group and high dose group (P < 0.05).②On 14 days and 28 days after implantation, the apoptosis rates of blank control group were lower than those of other 3 groups (P < 0.05), but expression levels of cyclinE mRNA were all higher than those of other 3 groups (P < 0.05). In addition, on 28 days after implantation, apoptosis rate of low dose group was higher than both of middle dose group and high dose group (P < 0.05), but expression level of cyclinE mRNA was lower (P < 0.05). Compared with 14 days in the same group, except for the blank control group (P > 0.05), the apoptosis rates in other 3 groups on 28 days were higher (P < 0.05), with the lower expression levels of cyclinE mRNA (P < 0.05). Conclusions125Ⅰseed in organizational implantation can effectively inhibit the expression of cyclinE mRNA and the growth of human poorly differentiated gastric cancer tissue. Compared with doses of 2.22×10-7 Bq and 2.96×10-7 Bq of 125Ⅰ, low dose (1.48×10-7 Bq) contributes to the apoptosis of human poorly differentiated gastric cancer cells (BGC823).
Objective To investigate the effect of mitochondrial fission mediated by mitochondrial dynamics related protein 1 (DRP1) on glucose metabolism reprogramming in lung cancer cells, and the regulatory mechanism on phosphatidylinositol-3-kinases (PI3K)/protein kinase B (Akt) signaling pathway. Methods Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to detect the mRNA expression of DRP1 in lung cancer tissue and lung cancer cells. Mitochondrial fission inhibitor (Mdivi-1) and Mdivi-1+PI3K/Akt signaling pathway activator 740Y-P were used to treat H1299 cells. Mitochondrial fission agonist (WY14643) + signal inhibitor LY294002 were used to intervene PC14 cells. The reagent kit was used to detect the glucose consumption, lactate release, and ATP production of each group of cells. 5-ethynyl-2-deoxyuridine (EdU) labeling experiment was used to detect the proliferation of cells in each group, and acridine orange/ethidium bromide (AO/EB) staining was used to detect the apoptosis of cells in each group. MitoTracker Red CMXRos was used to detect the mitochondrial morphology of each group of cells. Tetramethylrhodamine ethyl ester (TMRE) staining was used to detect the mitochondrial membrane potential of cells. Dihydroethidium (DHE) staining was used to detect the level of reactive oxygen species (ROS) in cells. Western blot was used to detect was used to detect the expression of pyruvate kinase M2 (PKM2), hexokinase 2 (HK2), phosphofructokinase-1 (PFK1), DRP1, phosphorylated DRP1 (p-DRP1), PI3K, Akt, phosphorylated PI3K (p-PI3K), and phosphorylated Ak t(p-Akt) in each group of cells. Results The mRNA expression of DRP1 was significantly increased in lung cancer tissue and lung cancer cells. Mdivi-1 promoted the development of lung cancer and exerts anticancer effects, while activating PI3K/Akt signaling could partially reverse the anticancer effects of Mdivi-1. WY14643 exerted a pro-cancer effect, and inhibiting PI3K/Akt signaling could partially reverse the pro-cancer effect of WY14643, and the differences were statistically significant (all P<0.05). Conclusions In lung cancer, the expression of DRP1 is significantly increased, and DRP1 affects the glycolysis process and proliferation performance of lung cancer cells by regulating the activation of PI3K/Akt signaling.
摘要:目的:探讨卡配因抑制剂3（MDL28170）对新生大鼠缺氧缺血性脑损伤(HIBD)神经细胞凋亡的影响。方法:建立新生SD大鼠HIBD模型,治疗组于缺养缺血后即刻、2 h、4 h腹腔内注射MDL28170,对照组及手术组同时予生理盐水。缺氧缺血后24 h用免疫组化方法观察大脑皮质及海马CA1区Caspase3 蛋白表达、TUNEL法检测细胞凋亡,观察组织病理改变并计算海马神经元死亡数,透射电镜观察细胞超微结构。结果:缺氧缺血后24 h缺血侧大脑皮质及海马CA1区Caspase3和TUNEL阳性细胞数较对照组明显增加,透射电镜证实有凋亡细胞;MDL28170可减少阳性细胞数量,抑制神经元死亡,差异有显著性(Plt;0.05)。结论:MDL28170可通过抑制神经凋亡而对新生大鼠HIBD具有一定保护作用。Abstract: Objective: To investigate the effect of (Calpain inhibitor3) MDL28170 on neural apoptosis in a neonatal model of hypoxicischemic brain damage (HIBD). Methods: A neonatal model of HIBD was established, 7dayold SD rats were divided into three groups. The treatment group received MDL28170(ip) at 0 h,2 h,4 h after HI, whereas the other two groups were administered normal saline simultaneously. The expression of caspase3 (by immunohistochemistry), neural apoptosis (by TUNEL) in cortex and hippocampus ipsilateral to the insult were observed 24 h after HI; hippocampal CA1 neural loss and electromicroscopic changes were assessed at the same time. Results: Apoptotic body was observed by electromicroscopy. Caspase3 positive cells and apoptotic cells increased significantly in the ipsilateral cortex and hippocampal CA1 region compared to the control, and MDL28170 reduced the number of positive cells, attenuated CA1 neural loss with significance (Plt;0.05). Conclusion: It is suggested that MDL28170 may protect the brain of neonatal rats after HIBD by suppressing neural apoptosis.