Establishment of a mdr1 Multidrug Resistant Model of Orthotopic Transplantation of Liver Carcinoma on Nude Mice

To develop a new method of inducing mdrl multidrug resistance by establishinga nude mice model of orthotopic transplantation of liver carcinoma by sporadic abdominalchemotherapy at intervals. Methods: Hepatocellular carcinoma HepG2 cell was cultured and injectedsubcutaneously to form the tumor-supplying mice. The tumor bits from the tumor-supplying mice wereimplanted under the envelope of the mice liver and induced by abdominal chemotherapy withPharmorubicin. Physical examination, ultrasonography, spiral CT and operative inspection were usedto examine tumor progression. RT-PCR and immunohistochemistry were adopted to detect the expressionof mdr1-mRNA and its encoded protein P-gp protein (P-gp). Results: There was no operative dead, therate of implanting tumor successfully was 88% (22/25), the rate of implanting secondly successfullywas 100% (3/3), and the rate of inducing successfully was 80% (16/20). The expression of mdrl-mRNAand the P-gp in the inducing group was 23 folds and 13 folds in the control group respectively.Conclusion: We have established an in vivo model of mdr using nude mice transplanted with orthotopicliver neoplasm coupled to chemotherapy.

Construction and Application of Plasmid pUC19-CM-D

[Objective] The aims were to construct a new suicide plasmid of Lactobacillus and gene deletion engineering bacteria of Lactobacillus with pUC19 vector. [Methods] pUC19-CM was constructed by inserting a chloramphenicol resistant gene into the multi-cloning site of pUC19,and then two homologous fragments were cloned into each side of the pUC19-CM to construct suicide plasmid pUC19-CM-D. [Results] A replacement mutant strain,whose target gene was replaced by resistant gene,could be obtained by transforming the suicide plasmid pUC19-CM-D into Lactobacillus for resistance screening. [Conclusion] The construction and application of pUC19-CM-D provided a fast and efficient means of construction of gene deletion engineering bacteria of Lactobacillus,and laid a foundation for study of gene function of Lactobacillus.

Eradication of Helicobacter pylori significantly reduced gastric damage in nonsteroidal anti-inflammatory drug-treated Mongolian gerbils

AIM: To examine the effect of eradication of Hellcobacter pylori prior to usage of NSAIDs, by investigating gastric inflammatory activity, myeloperoxidase (MPO) activity, prostaglandin (PG) E_2 synthesis in H Pylori-infected, and H pylori-eradicated gerbils followed by administration of indomethacin and rofecoxib. METHODS: Six-week-old male gerbils were orally inoculated with H pylori. Seven weeks later, anti-H pylori triple therapy and vehicle were given to gerbils respectively and followed by oral indomethacin (2 mg/kg·d) or rofecoxib (10 mg/kg·d) for 2 wk. We examined the area of lesions, gastric inflammatory activity, PGE_2 synthesis and MPO activity in the stomach. RESULTS: In indomethacin and rofecoxib-treated gerbils, the following results were obtained in H pylori-infected group vs H pylori-eradicated group respectively: hyperplasia area of the stomach (mm^2): 82.4±9.2 vs 13.9±3.5 (P＜0.05), 30.5±5.1 vs 1.3±0.6 (P＜0.05); erosion and ulcer area (mm^2): 14.4±4.9 vs 0.86±0.5 (P＜0.05), 1.3±0.6 vs0.4±0.3 (P＜0.05); score of gastritis: 7.0±0.0 vs3.6±0.5 (P＜0.05), 7.0±0.0 vs 2.7±0.5 (P＜0.05); MPO activity (μmol H_2O_2/min/g tissue): 104.7±9.2 vs9.0±2.3(P＜0.05), 133.5±15.0 vs2.9±0.7 (P＜0.05); PGE_2 synthesis (pg/mg wet weight/min): 299.2±81.5 vs102.8±26.2 (P＜0.05), 321.4±30.3 vs 11.9±4.8(P＜0.05). CONCLUSION: Eradication of H pylori reduced gastric damage of NSAID-treated Mongolian gerbils. Rofecoxib caused less severe gastric damage than indomethacin in H pylori-eradicated gerbils.

宁夏沙鼠鼠疫40年流行动态分析

目的探讨宁夏沙鼠动物间鼠疫发生的时空分布,为鼠疫的预测及预警提供指导。方法收集整理40多年来宁夏沙鼠鼠疫的相关资料,采用描述流行病学方法进行分析。结果 1969─2013年宁夏沙鼠鼠疫疫源地共检出鼠疫野毒菌341株,其中动物检菌301株,媒介昆虫检菌40株;发生动物间鼠疫流行16年次,活鼠阳性检菌率1.54%,自毙鼠阳性检菌率高达60.32%;蚤的集组培养阳性检菌率为1.37%。结论春季为沙鼠动物间鼠疫的好发季节,应强化自毙鼠预警机制,建立以抗性鼠血清学为主的监测模式。

Apoptosis of erythrocytic stage parasites of Plasmodium berghei chloro-quine-resistant strain

Objective: To explore the characteristics of crisis state at erythrocytic stage of Plasmodium berghei chloroquine-resistant (RC) strain. Methods: Agarose electrophoresis, optical and transmission electron microscopes were used. Patterns of genomic DNA structures and ultra-structures of the erythrocytic parasites were observed in ICA mice (infected with the RC strain) during rising and declining of parasitemia. Results: During the declining parasitemia, the erythrocytic stage parasites of the RC strain showed round or oval appearance with intact plasma membrane and shrank nuclei with no metabolic window, mitochondria or other membranaceous structures. Their DNA electrophoretogram revealed a ladder pattern which evidently differed from the parasites of the RC strain in the rising parasitemia and the chloroquine-sensitive (N) strain.Conclusion: The crisis state of the erythrocytic stage parasites of the P. berghei chloroquine-resistant (RC)strain is characterized by apoptosis.

Screening Helicobacter pylori genes induced during infection of mouse stomachs

AIM:To investigate the effect of in vivo environment on gene expression in Helicobacter pylori(H.pylori) as it relates to its survival in the host.METHODS:In vivo expression technology(IVET) systems are used to identify microbial virulence genes.We modified the IVET-transcriptional fusion vector,pIVET8,which uses antibiotic resistance as the basis for selection of candidate genes in host tissues to develop two unique IVET-promoter-screening vectors,pIVET11 and pIVET12.Our novel IVET systems were developed by the fusion of random Sau3A DNA fragments of H.pylori and a tandem-reporter system of chloramphenicol acetyltransferase and beta-galactosidase.Additionally,each vector contains a kanamycin resistance gene.We used a mouse macrophage cell line,RAW 264.7 and mice,as selective media to identify specific genes that H.pylori expresses in vivo.Gene expression studies were conducted by infecting RAW 264.7 cells with H.pylori.This was followed by real time polymerase chain reaction(PCR) analysis to determine the relative expression levels of in vivo induced genes.RESULTS:In this study,we have identified 31 in vivo induced(ivi) genes in the initial screens.These 31 genes belong to several functional gene families,including several well-known virulence factors that are expressed by the bacterium in infected mouse stomachs.Virulence factors,vacA and cagA,were found in this screen and are known to play important roles in H.pylori infection,colonization and pathogenesis.Their detection validates the efficacy of these screening systems.Some of the identified ivi genes have already been implicated to play an important role in the pathogenesis of H.pylori and other bacterial pathogens such as Escherichia coli and Vibrio cholerae.Transcription profiles of allivi genes were confirmed by real time PCR analysis of H.pylori RNA isolated from H.pylori infected RAW 264.7 macrophages.We compared the expression profile of H.pylori and RAW 264.7 coculture with that of H.pylori only.Some genes such as cag A,vac A,lpx C,mur I,tlp C,trx B,sod B,tnp B,pgi,rbf A and inf B showed a 2-20 fold upregulation.Statistically significant upregulation was obtained for all the above mentioned genes(P < 0.05).tlp C,cag A,vac A,sod B,rbf A,inf B,tnp B,lpx C and mur I were also significantly upregulated(P < 0.01).These data suggest a strong correlation between results obtained in vitro in the macrophage cell line and in the intact animal.CONCLUSION:The positive identification of these genes demonstrates that our IVET systems are powerful tools for studying H.pylori gene expression in the host environment.

Antioxidant activity of chito-oligosaccharides on pancreatic islet cells in streptozotocin-induced diabetes in rats

AIM:To investigate the antioxidant activity of chitooligosaccharides(COSs)on pancreatic islet cells in diabetic rats induced by streptozotocin. METHODS:The antioxidant effect of COSs on pancreatic islet cells was detected under optical microscopy and with colorimetric assay and gel electrophoresis.The activities of glutathione peroxidase and superoxide dismutase,total antioxidant capacity,and content of malondialdehyde in serum and tissue slices of pancreas were examined after 60 d to determine the effect of COSs in streptozotocin-induced diabetes in rats. RESULTS:COSs can prohibit the apoptosis of pancreatic islet cells.All concentrations of COSs can improve the capability of total antioxidant capacity and activity of superoxide dismutase and decrease the content of malondialdehyde drastically.Morphological investigation in the pancreas showed that COSs have resulted in the reduction of islets,loss of pancreatic cells,and nuclear pyknosis of pancreatic cells. CONCLUSION:COSs possess various biological activities and can be used in the treatment of diabetes mellitus.

Effect of Therapeutic Ginger on Genotoxic of Taxol Drug （Anti-Cancer） in Bone Marrow Cell of Male Mice

Medicinal use of spices/herbs has been gradually increased in the developed countries, Zingiber officinale （Ginger） is known to possess potent antioxidant and anti inflammatory properties. Therefore, the aim of this study is to determine the possible anti-mutagenic effect of ginger against the genotoxic effect of anti-cancer drug Taxol 0.6 mg/kg. This study is conducted by using two types of cytogenetic studies in bone marrow cell of mal albino mice Mus musculus （average weight 25-30 g）. The animals were randomly distributed into six groups, each of 14 mi[ce, （GI） was given the solvent, （G2） treatment of the medical dose of Taxol drug, （G3） treatment of ginger, （G4） a pre-treatment of ginger prior to treatment of drug, （G5） a simultaneous treatment of ginger and treatment of drug, （G6） a post-treatment of ginger after treatment drug. The study results show that significant increase in total chromosomal aberrations and significant increase in the number of micronuclei were observed after treatment drug. The significant structural aberrations were in the form of end-to-end associations. The numerical chromosomal aberrations were endomitosis and polyploid. The results showed that the frequencies of chromosomal aberrations and micronuclei in ginger treated group were not significantly different from control. Simultaneous treatment of ginger was found to be effective in reducing the genotoxic effects induced by drug Taxol especially in the total number of the chromosomal aberrations and the number of micronuclei.

Antioxidation of quercetin against spinal cord injury in rats

Objective ： To observe the effect of quercetin on experimental spinal cord injury （SCI） in rats. Methods. Sixty Sprague-Dawley rats were randomly divided into four groups： Group A only for iaminectomy, Group B for iaminectomy with SCI, Group C for SCI and intraperitoneai injection with a bolus of 200 mg/kg quercetin and Group D for SCI and intraperitoneai injection of saline. SCI model was made by using modified Ailen＇s method on T12. Six rats of each group were killed at 4 h after injury and the levels of free iron and maiondiaidehyde （MDA） of the involved spinal cord segments were measured by bleomycin and thiobarbituric acid （TBA） assays separately. The recovery of hind limb function was assessed by Modified Tariov ＂s scale andinclined plane method at 7 d,14 d and 21 d after SCL The histological changes of the damaged spinal cord were also examined at 7 d after SCL Results ： After SCI, the levels of free iron and MDA were significantly increased in Groups B and D, while not in Group C. The Modified Tariov＇s score and the inclined plane angles were significantly decreased in Groups B, C and D. The histological findings were not improved. Conclusions： After SCI, quercetin can reduce the level of lipid peroxidation, but not improve recovery of function.

Effects of renal denervation on blood-pressure response to hemorrhagic shock in spontaneously hypertensive rats

Purpose： Renal denervation （RD） has been demonstrated to be an effective approach to reduce blood pressure for those with resistant hypertension. Yet, we aimed to explore the effect and possible mech- anism of RD on blood-pressure response to hemorrhagic shock in spontaneously hypertensive rats. Methods： A total of 48 male spontaneously hypertensive rats were randomized to three groups： study group, sham-operation group and control group. RD was achieved by cutting off renal nerves and swabbing phenol on it. Ten weeks after RD, 8 rats in each group were sacrificed to collect the kidney and heart tissues. The remaining rats were subjected to an operation to induce hemorrhagic shock which would lead to 40% loss of total blood volume, and observed for 120 min. The serum concentration of norepinephrine was measured before and three weeks after RD. Results： The blood-pressure and norepinephrine levels were reduced significantly after RD （p 〈 0.05）, Systolic blood pressure and diastolic blood pressure of the surgerygroup were higher than those in the sham and control groups at 15, 30 and 45 min after hemorrhagic shock （p 〈 0.05）, while no significant difference was observed at 60, 90 and 120 min （p 〉 0.05）. Additionally, the beta-1 adrenergic receptor （β1 -AR） in the study group was significantly higher than those in the other two groups （p 〈 0.05） after hemorrhagic shock. Conclusion： This study demonstrated that RD could to some extent improve blood-pressure response to hemorrhagic shock in an established model of severe hemorrhagic shock in spontaneously hypertensive rats. The mechanism might be associated with uo-regulation of β1-AR.

Effect of Wuziyanzong pill on levels of sex hormones, and expressions of nuclear- associated antigen Ki-67 and androgen receptor in testes of young rats

OBJECTIVE: To evaluate the effects of Wuziyanzong pill on the levels of serum follicle stimulating hormone(FSH), luteinizing hormone(LH), testosterone(T) and the expressions of nuclear-associated antigen Ki67(Ki67), androgen receptor(AR) in testes of young rats.METHODS: Sixteen 20-day-old Wistar male rats were randomly divided into control and treatment group(n = 8). Rats in treatment group were administered Wuziyanzong pill by gavage; rats in control group administered the same volume of saline. After 10 days of treatment, the rats were killed, and then serum and testes were taken. The levels of FSH, LH and T were measured by radioimmunoassay(RIA). The histology of seminiferous tubule was observed by hematoxylin-eosin(HE) staining. The expression of Ki67 was detected by immunohistochemical assay(IHC). The m RNA level of Ki67, ARand CK-18 was detected with quantitative real-time polymerase chain reaction(Q-RT-PCR), the protein level of AR and CK-18 were tested by Western blot.RESULTS: Compared with control group, T level in treatment group increased significantly(P < 0.05).HE staining showed that both leydig cells and germ cells increased in treatment group. Expressions of Ki67 and AR became higher after treatment. There were no changes in CK-18 expression.CONCLUSION: Wuziyanzong pill can up-regulate AR level to promote germ cell proliferation and differentiation in young male rats.