BACKGROUND ＆ AIM: To study the effects of deltamethrin(DM) on mitochondrial membrane potential and membrane fluidity in rat brain tissue. MATERIAL AND METHODS: Male adult Wistar rats were treated intraperitonealy with 12.5 mg/kg DM. At 5, 24, 48 and 72 h after injection, we extracted the mitochondria of rat brain tissue to measure the membrane potential, membrane fluidity, activities of Na＋-K＋-ATPase、Ca2＋-Mg2＋-ATPase and succinic dehydrogenase.At the same time the control group were merely injected intraperitonealy with 0.5 mg/100 g salad oil and then executed after 5 h. RESULTS: After treatment with deltamethrin, the mitochondrial membrane potential decreased, membrane fluidity was reduced, the activities of Na＋-K＋-ATPase,Ca2＋-Mg2＋-ATPase and succinic dehydrogenase were inhibited (P<0.01). Moreover, there were correlations between those indexes and time after treatment. CONCLUSION: DM had obvious effects on inhibiting the mitochondrial function of rat brain tissue, then caused the impairment of oxidative phosphorylation in the mitochondria.

BACKGROUND ＆ AIM: To observe the effects of deltamethrin(DM) on the neuroimmunological functions of rats by the changes of IL-1β in the cortex, hippocampus and hypothalamus. MATERIAL AND METHODS: 65 male adult SD rats were separated into 3 groups，one group included 15 rats for RT-PCR, the second group included 25 rats for immunohistochemistry, and the third included 25 rats for detecting the activities of ATPase. Each group was separated randomly into 5 sub-groups including 1 control and 4 DM treated. The DM treated rats received intraperitonea injection of 12.5 mg/kg DM. At 1, 6, 24 and 48 h after injection,we used RT-PCR and immunohistochemistry to characterize the changes of IL-1β mRNA expression and the protein content in different encephalic regions. Then we detected the activities of Na＋－K＋－ATPase as biomarker to explore the relationship between IL-1β and neural toxicity of DM. RESULTS: From 1 h to 6 h after DM administration, the IL-1β protein content in cortex increased while the gene expression of IL-1β rose after 24 h to 48 h, without neural toxicity in cortex. In the hippocampus, after IL-1β protein increased at 1 h with transient increase of IL-1β mRNA. The activity of Na＋－K＋－ATPase decreased at 6 h, and recovered at 24 h, but decreased again at 48 h. The IL-1β mRNA of hypothalamus increased rapidly in 1 h, reaching maximum at 6 h, whilst the IL-1β protein remained higher than that of the control group until 24 h. The activity of Na＋－K＋－ATPase only showed an apparent reduction at 1 h after injection. CONCLUSION: IL-1β may play different roles in above-mentioned cerebral regions in mediating the neural toxicity of DM in rats.

BACKGROUND ＆ AIM: To investigate the antioxidative activity of high doses vitamin E(VE) and C(VC) and its effects on DNA oxidative and alkyl damages in rats. MATERIAL AND METHODS: 72 Wistar rats were randomly divided into six groups including control,VC,VE1,VE2,VE1＋VC and VE2＋VC groups. The trial lasted 8 weeks,and urine and blood samples of each rat were collected at the end of the trial. The activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), level of malondialdehyde (MDA) and DNA damage were assessed. RESULTS: Plasma SOD and GSH-Px activities in VE1 group were 425.21 NU/ml and 440.08 U/ml,respectively which were significantly higher than those of the control group while MDA in this group was significantly lower than that of the control. The same antioxidative enzyme activities in VE1＋VC group were significantly lower than that of the VE1 group. Lymphocyte DNA oxidative damage induced by 10 μmol/L H2O2 in VE1 group was significantly lower than those in other groups and O6-methyl guanine (O6-meG) level in this group was 0.89 mg/g creatinine which was significantly lowered by 50.28％,50.00％ compared with that of control group and that of the VE2 group,respectively. CONCLUSION: Relatively high dose VE could effectively increase antioxidative activity and decrease DNA oxidative and alkyl damages. Using the same dose of VE and VC supplements in combination was found to confer no such advantageous effect. Too high dose VE and VC could decrease the genetic stability.

北豆根提取成分PE2诱导胃癌细胞凋亡的实验研究 单保恩1/梁文杰1/刘东青2/任风芝3 (1.河北医科大学第四医院科研中心，河北　石家庄 050011；2.邯郸市第一医院；3.华北制药集团新药研究中心) () 　　【摘要】 　　【关键词】 北豆根PE2成分； 肿瘤细胞； 细胞凋亡 　　中图分类号： R730.32　　　　文献标识码： A　　　　文章编号： 1004－616X(2006)04－0269－04 　　【ABSTRACT】 BACKGROUND ＆ AIM: To study the effects of tumor cell apoptosis induced by Rhizoma Menipermi Extracts-PE2 and their mechanisms. MATERIAL AND METHODS: Rhizoma Menipermi extract with ethanol was preliminary purified using three steps method. PE2 was isolated and purified further by Pole Chromatogram. The apoptosis of tumor cells BGC823 induced by PE2 was studied by fluorescent staining, flow-cytometry (FCM), electron microscopy and DNA agarose gel electrophoresis technique. RESULTS: PE2 could induce apoptosis of BGC823 cells in a dose-and time-dependent relationship. After treatment with PE2, the BGC823 cells received some typical morphologic features and super-microstructural changes of apoptosis, including cell shrinkage chromatin concentrating on nuclear membrane or forming crescent, nuclear condensation, nuclear fragmentation and formation of apoptotic bodies. Some typical subdiploid peaks before G0/G1 phase were observed. DNA agarose gel electrophoresis showed characteristic "DNA ladder" pattern. After treatment with 5 μg/ml PE2, the cells in G0/G1 phase were blocked and increased markedly. But the cells in S phase were blocked after treatment with 50 μg/ml PE2. And the expression of bax was up-regulated. CONCLUSION: Rhizoma Menispermi Extract-PE2 induced tumor cell apoptosis, which was an important mechanism of its anti-tumor effect. Futher purification and study of their mechanisms can produce the scientific evidence for developing and manufacturing new antitumor drugs. 【KEY WORDS】 收稿日期： ； 修订日期： 基金项目： 国家自然基金，No.3037153；河北省自然基金，No.C2004000610 作者简介： 单保恩(1962－　)，男，河北省广平县人，教授，博士，博士生导师，研究方向：肿瘤免疫学。电话：0311(86033941－283)；传真：0311－86992004；Email:

BACKGROUND ＆ AIM: To study the effect of sulindac on growth inhibition of human gastric cancer cells BGC-823 in vitro and its antineoplastic mechanisms. MATERIAL AND METHODS: Human gastric cancer cells BGC-823 were incubated with various concentrations of sulindac for different periods. Antiproliferation effects were measured by MTT colorimetric assay, flow cytometry was used for cell cycle distribution, transmission electron microscopy was used to examine cell apoptosis morphology, immunohistochemical staining was used to measure the expressions of ki-67, bcl-2 and COX-2 in cells. RESULTS: Sulindac could inhibit the growth of BGC-823 cells, increased the proportion of cells in the G0/G1 phase, and decreased the proportion of cells in the S phase. Some morphologic features of apoptosis and the apoptosis bodies were examined by transmission electron microscopy, showing a decreased expressions of ki-67, bcl-2 and COX-2 in cells. All the effects demonstrated a time-and dose-dependent relationship. CONCLUSION: Sulindac could significantly inhibit the growth of human gastric cancer cells BGC-823 in vitro and the antitumor mechanism may be related to affecting cell cycle distribution, inducing cell apoptosis and inhibiting the expression of COX-2, bcl-2, and ki-67.

BACKGROUND ＆ AIM: To investigate the efficacy of transfection of spermatogonial stem cells with pDsRed2-N1 reporter by liposomes. MATERIAL AND METHODS: Red-fluorescent protein (DsRed2) genes were mediated by DOSPER liposomes to spermatogonial stem cells of donor mice in vitro and in vivo. The DsRed2 gene-transfected cells, after isolation and purification, were transplanted into the seminiferous tubules of anesthetized recipient mice depleted of germ cells by busulfan treatment. RESULTS： The expression rate of DsRed2 gene was 20％ of in vitro transfected cells and higher than 8％ of in vivo transfected cells (P<0.01). Spermatogonial stem cells with DsRed2 expression were detected on frozen section of testis from the recipient mouse 6 wafter transplantation. CONCLUSION: This may provide a simple and effective approach to study spermatogenesis mechanism and to produce transgenic animals using combination of DOSPER-mediated gene transfer in vitro with spermatogonial cell transplantation.

BACKGROUND ＆ AIM: To study protein kinase C(PKC) expression in breast cancer cells at differentiation stage and their sensitivity to TNFα. MATERIALS AND METHODS: Protein immunoblot and MTT analysis were used to determine the expression profile of PKC isoforms in 12 breast cancer cell lines in various differentiation stages. RESULTS: The results suggested that expression of PKCε increased in stagesⅢ ＆ Ⅳ cell lines differentiations, compared to those in stages I and II. There was no association between differentiation stage of breast cancer cells and expression of PKCα、η、δ and μ. IC50 of breast cancer cell lines to TNFα at stage IV was higher than cells at other stages. CONCLUSION: PKCε might be useful markers to evaluate the differentiation in human breast carcinoma.

BACKGROUND ＆ AIM: To investigate the inactivation of Fhit gene in primary laryngocarcinoma, to discover the association of Fhit gene inactivation and the biological behavior of laryngocarcinoma, and the influence of environmental factors on Fhit gene. MATERIAL AND METHODS: RT_PCR was used to determine Fhit gene expression in the laryngocarnioma tissue and peripheral cancer tissue of 29 patients. If abnormal expression of Fhit gene was found, further sequencing was performed. We prepared the different profiles of Fhit gene expression in cancer tissue and non_cancer tissue, and in various clinical stages of laryngocarcinoma. Furthermore, we studied the effects of environmental factors (e.g. smoking and alcohol intake) on Fhit gene expression. RESULTS: Abnormal expression of Fhit gene was found in 22/29 cases. Among these, 4 did not express Fhit gene, and 18 had abnormal transcription of Fhit gene. There was a significant difference of abnormal Fhit expression between laryngocarnoma tissue and non_laryngocarnoma tissue (P<0.01). However, the abnormal expression of Fhit gene did not show significant difference during the different clinical stages of laryngocarcinoma (P>0.05). Between the smoking and non_smoking patiens, abnormal expression of Fhit showed significant difference (P<0.05). CONCLUSION： Abnormal expression of Fhit gene was a common event in laryngocarcinoma. Abnormal Fhit gene expression might be associated with oncogenesis of laryngocarcinoma, but did not worsen further with disease progression. Excessive smoking might result in structural change of the Fhit gene, and induce the occurrence of tumor.

BACKGROUND ＆ AIM: To investigate the effects of grape procyanidins on the proliferative activity and the concentration of intracellular calcium of hepatoma cells. MATERIAL AND METHODS: Human hepatoma cells were cultured with differrent concentrations of GPC. The proliferative activity and concentration of intracellular calcium of the hepatoma cells were measured by methyl thiazolyl tetrazolium(MTT) assay and microfluorimetric techniques based on calcium indicator Fura-2/AM. RESULTS: The proliferation level and concentration of intracellular calcium of the control group were (0.045±0.024) nmol/L and (130.53±17.48) nmol/L, respectively. The cell proliferation activitives of 3 GPC groups were lower, but the concentration of intracellular calcium was higher with increasing amounts of GPC(t>2.78, P<0.05). The concentration of intracellular calcium of both 50 mg/L and 100 mg/L GPC group were overloaded. CONCLUSION:GPC could inhibit the proliferative activity and abnormally elevate the concentration of intracellular calcium of hepatoma cells .

BACKGROUND ＆ AIM: The study investigated the effect of tributyltin (TBT) on the activities of acetylcholinesterase(AChE)in brain of rats. The possible relationship between AChE and the toxicity of TBT was discussed. MATERIAL AND METHODS:48 SD rats were divided randomly into 4 groups,12 rats in each group.Experimental groups received 0.1,1 or 10 mg/kg TBT by oral gavage and same volume olive oil was given to control group(0 mg/kg TBT).The brain tissues were obtained from 4 rats in each group at the 4th,8th and 12th day and the AChE activities were measured. RESULTS: TBT exposure could lead to AChE activity elevation in rat brains, and in a dose-related and time-depending manner. CONCLUSION: The increase of AChE activity could possibly be related to the neurotoxicity of TBT.

BACKGROUND ＆ AIM: To study the function and role of two subclasses in MPs family, MMPs and ADAMs, for processing of precursor TNF-α, and explore the anti-inflammatory mechanisms of Chinese herbal preparation Reduqing (RDQ). MATERIAL AND METHODS: The in vitro study was carried out on HL-60、U937 cells and macrophages of murine abdominal cavity treated by LPS and RDQ. Using MTT colorimetry,in situ hybridization and Gelatin-PAGE to detect transcriptional levels of MMPs,ADAM17, TNF-α mRNA and the changes of enzymatic activity. RESULTS: ①After stimulation by LPS, the expression level of MMPs and electrophoresis zymogram on gelatin-PAGE decreased with increased stimulation time; ②ADAM17 (TACE) played a more important role in precursor processing of proTNF-α of HL-60 cells compared with MMPs; ③RDQ had obvious inhibitory effects on enhancing secretion of TNF-α induced by LPS stimulation (P<0.01) at the transcriptional level of ADAM17 mRNA in HL-60 cells. CONCLUSION: ①ADAM17 of MPs family had a main role in TNF-α secretion induced by LPS. It is a key step to choose and design the correct probe or primers aiming specifically at the disintegrin domain for ADAM17, in order to eliminate the interference of MMPs and reflect the true expression level of ADAM17 gene; ②The mechanism of RDQ against inflammation may be its inhibitory effect on ADAM17 mRNA expression activated by LPS. ADAM17 is able to represent the novel target for studing the mechanism and the therapeutic pathway for inflammation.

BACKGROUND ＆ AIM： To study the effect of (Haishen polysaccharide peptide,HPP) on the apoptosis and apoptosis gene of mice hepatic cells induced by 60Co-γ irradiation . MATERIAL AND METHODS: Mice were fed 150 mg/kg and 100 mg/kg HPP for four weeks. Mice were then irradiated with 60Co-γ to induce hepatic cells apoptosis once a day five days a week. Four weeks later, in situ cell death detection kit and in situ hybridization were applied to measure qualitative changes at expression level of POD and Caspase-3 positive cells rate to analyze the apoptosis of mice hepatic cells. RESULTS: The rates of POD and Caspase-3 expression in the radiation-injury group were(40.0±3.5)％ and(26.0±1.2)％, respectively,and the rates of the high dose group were(23.9±3.8)％ and (6.0±1.5)％, respectively，the difference between the radiation-injury group and high dose group showed statistical significance (P<0.01).CONCLUSION: HPP had a significant inhibitory effect on the abnormal hepatic expression of POD and Caspase-3 induced by radioactive damage.

BACKGROUNG ＆ AIM: Previous studies showed a gene fragment was identified as being over-expressed in 16HBE-C cells when compared to 16HBE cells, which is named brg (anti-BPDE related gene). The aim of this study was to obtain the full-length cDNA of brg based on its EST fragment and to provide basic data for the functional research of the novel gene. MATERIAL AND METHODS: Based on the rapid amplification of cDNA ends (RACE) method, gradient and nested PCR protocols were used to obtain full-length cDNA in order to get a discrete and bright band on an agarose gel electrophoresis. The nested PCR products were then sequenced and the bioinformatic analysis was carried out. RESULTS: Full-length cDNA of the gene was successfully cloned by both 3＇-end RACE and 5＇-end RACE. The cDNA had a total length of 1 214 bp. Sequence analysis of the full-length cDNA of the gene revealed that the cDNA encoded an open reading frame of 1 032 bp; with the deduced protein containing 344 amino acids. CONCLUSION: The general and simple protocol was useful for cloning of full-length gene based on EST fragment. The results suggest that brg gene may be involved in carcinogenesis during anti-BPDE related tumor development.

BACKGROUND ＆ AIM：The fluorescent differential display technique was used to clone differentially- expressed genes in gastric cancer, its premalignant lesions and normal gastric tissue. This might be helpful to understand the molecular mechanisms of tumor formation, early diagnosis and treatment of gastric cancer. MATERIAL AND METHODS: The differentially-expressed cDNA bands were isolated and identified by fluorescent differential display in 3 gastric cancers,3 matched normal gastric mucosa, 3 premalignant lesions These were then recovered, amplified, cloned and sequenced. The ribosomal protein S24 (RPS24) gene was one of the up-regulated genes and was confirmed by Northern hybridization. RPS24 gene expression in different tissue was analyzed in serial analysis of gene expression(SAGE)and UniGene databases. RESULTS: A differentially-expressed cDNA fragment was expressed highly in gastric cancers, This cDNA fragment was homologous to RPS24 gene and Northern hybridization confirmed the differential expression. RPS24 was highly expressed and extensively distributed in various tumor tissues. CONCLUSION: RPS24 gene expression was significantly higher in gastric cancer, and could be correlated with gastric cancer development.

BACKGROUND＆AIM： This study was to screen an esophageal cancer cell line (SHEEC) cDNA library for the cDNA that expressed a protein that can specifically bind the NF-kappa B element, for further understanding the expression and regulation mechanism of the NGAL(neutrophil gelatinase-associated lipocalin) gene, and the course for the over-expression of the NGAL gene in the esophageal epithelial squamous carcinoma. MATERAL AND METHODS：A tandem three oligo-nucleotides of NF-kappa B was employed as a bait in a yeast one hybrid system to screen the cDNA library of the human esophageal carcinoma cell line (SHEEC). The cDNA clones obtained were sequenced and analyzed with bioinformatics tools. RESULTS： Among the thirty clones obtained, four types of clones could encode proteins, two clones were hypothetical proteins, two types of clones were mitochondrial genes, and three types of clones were non-encoding sequences. CONCLUSION： Using the yeast one hybrid system to screen the cDNA library for the NF-kappa B core element is a preliminary work that needs confirmation by further study.

BACKGROUND＆AIM: To study the expression of tankyrase in human bronchial epithelial malignantly transformed cells induced by crystalline nickel sulfide. MATERIAL AND METHODS: The method of reverse transcription-polymerase chain reaction(RT-PCR)was used to measure the expression of tankyrase mRNA in 16HBE and malignantly transformed cells. RESULTS: The expression of tankyrase in malignantly transformed cells was higher than that in normal cells. CONCLUSION: The overexpression of tankyrase in cell transformation might be related to the carcinogenesis of crystalline nickel sulfide.

BACKGROUND ＆ AIM: To elucidate the effects of ultrashort waves on the in vitro fertilization and embryo transfer(IVF-ET)cycles through the measurement of cytokines level in follicular fluid and peritoneal fluid of the patients. MATERIAL AND METHODS: Two hundreds and seven infertility patients of simple pelvic adhesion, pelvic endometriosis and tuberculosis were divided into two groups, one with physiotherapy of ultrashort waves,and the other as control. The concentrations of tumour necrosis factor α and interleukin-1β in peritoneal fluid and follicular fluid in both groups were detemined; gnadal hormone E2 in blood serum and the thickness of the endometrium were measured as well. RESUITS: ① There were no significant differences of the number of follicle recruitment,the dosage of GnRH_a and Gn between two groups. In physical therapy group, E2 on the day of hCG administration was higher than that of the control group (P<0.05). There was significant diffirence in the thickness of the endometrium on ET day. ② TNFα in follicular fluid had negative correlation with E2(r=－0.27,P=0.041); IL-1β had no correlation with E2. ③ In physical therapy group, TNFα and IL-1β levels in peritoneal fluid were lower than those of the control group (P<0.05). ④ In physical therapy group, TNFα and IL-1β levels of follicular fluid were significantly higher than those of peritoneal fluid(P<0.05). CONCLUSION: Through regulating expression of cytokines, treatment with physiotherapy of ultrashort waves can promote the level of E2 in blood serum and improve plant environment of endometrium, which can lead to a good result.

BACKGROUND ＆ AIM: To investigate the expression of early growth response gene-1 (Egr-1 gene) and PTEN in esophageal epitheliosis and carcinogenesis, and the relation with the development of esophageal carcinoma. MATERIAL AND METHODS: Egr-1 and PTEN protein expression in 86 fresh tissue specimens, including esophageal mucosa above the upper surgical margin, carcinoma in situ and mucosa adjacent to tumor, were studied using immunohistochemical EnVision two step method. RESULTS: Egr-1 and PTEN protein were expressed in normal, proliferating and transformed malignant cells of esophageal epithelium. The expression of PTEN protein showed a different distribution pattern to that of Egr-1.The Egr-1 protein expression rose gradually and PTEN protein expression decreased gradually. CONCLUSION: Egr-1 and PTEN protein expression changes were closely related to the malignant transformation of esophageal epithelium. Egr-1 expression up-regulation and loss of PTEN expressions may be favorable markers and an early diagnostic indicator in patients with esophageal carcinoma.

BACKGROUND ＆ AIM: To investigate a possible mechanism of arsenic trioxide-induced apoptosis in K562 cells. MATERIAL AND METHODS: The K562 cells were treated with As2O3 at 1, 2, 4, 8 μmol/L. Light and transmission electron microscope were used to examine the morphologic changes of K562 cells every 24 hours. Methyl thiazolyl tetrozolium(MTT) was used to establish the dose-effect curves of cell proliferation and apoptotic rates were measured by flow cytometry(FCM),RT-PCR was used to detect the expression of Evi-1 mRNA and cellular JNK was detected by ELISA. RESULTS: MTT test showed K562 cell proliferation was significantly inhibited by As2O3 in time and dose-dependent fashion. Marked apoptosis was detected by FCM in K562 cells after treatment by As2O3 at 1,2,4, 8 μmol/L and increased in a time-dependent manner within 48 hours and the optimal concentration was 4 μmol/L. As2O3 could down-regulate significantly the expression of Evi-1 mRNA and up-regulate JNK protein in K562 cells in time- and dose-dependent manner. CONCLUSION: As2O3 could obviously inhibit the expression of Evi-1 mRNA and activate JNK signal transduction pathway, which may be a possible mechanism of As2O3 inducing apoptosis in K562 cells.

BACKGROUND ＆ AIM: To study the inhibition of lycopene on the genetic damage induced by mutagens MATERIAL AND METHODS： In accordance with the “Procedures and Methods for Toxicological Assessment on Food Safety”, and the modified micronucleus test and Ames test were used. The numbers of micronuclei and bacteria colonies were scored, then the micronucleus frequencies in polychromatic erythrocytes(fMPCE), relative bacteria colonies and the rates of inhibition were calculated. RESULTS: To compare with the positive control group treated with CP(Cyclophosphamide), the fMPCE in test groups pretreated with lycopene was significantly decreased(P<0.01). The rate of inhibition of lycopene on fMPCE induced by CP was over 59％. Compared with the positive group, the relative bacteria colonies of TA98、TA100 were reduced in Ames test in which the mutagens were pretreated by lycopene, and there was adose-related were responses. But only the relative bacteria colonies of TA98 showed significant rise at 140 μg/dish in Ames test in where the mutagens and lycopene were added at the same time. CONCLUSION: Lycopene could effectively inhibit the genetic damage induced by mutagens, and to some degree it could play an anti-mutation role.