To silence the expression of K-RASAsn12 in human pancreatic cancer cell line by vector-based RNAi(RNA interference) technique,two single-strand DNA sequences encoding mutant-specific shRNA (short haipin RNA) for K-RASAsn12 were synthesized and then inserted into pSilenCircle. The recombinant plasmid was called pSC-K-RASAsn12. According to the same method, pSC-GFP encoding shRNA for GFP was gained. Both recombinant plasmids were transfected into human pacreatic cancer cell line AsPC-1 and BxPC-3. The expression level of K-RASAsn12 was detected by semi-quantitative RT-PCR and Western blot. The result indicated that the recombinant plasmid edcoding mutant-specific shRNA for K-RASAsn12 can inhibit significantly the expression of K-RASAsn12 without affection of wild-type K-RAS(K-RASWT)in Human Pancreatic Cancer Cell Line.

Abdominal Neoplasms ; complications ; metabolism ; pathology ; surgery ; Adult ; Dendritic Cell Sarcoma, Follicular ; complications ; metabolism ; pathology ; surgery ; Diagnosis, Differential ; Female ; Humans ; Ki-1 Antigen ; metabolism ; Leukocytosis ; complications ; metabolism ; pathology ; surgery ; Receptors, Complement 3b ; metabolism ; Receptors, Complement 3d ; metabolism ; Young Adult

OBJECTIVE: To investigate the diagnostic significance of basophil activation test (BAT) in anaphylaxis to non-ionic contrast media through testing the content of CD63, mast cell-carboxypeptidase A3 (MC-CPA3), and terminal complement complex SC5b-9 of the individuals by testing their levels in the normal immune group and the anaphylaxis groups to β-lactam drugs and non -ionic contrast media. METHODS: The CD63 expression of basophilic granulocyte in blood was detected by flow cytometry. The levels of MC-CPA3 in blood serum and SC5b-9 in blood plasma were detected by ELISA. RESULTS: The CD63 expression of basophilic granulocyte in blood, the levels of MC-CPA3 and SC5b-9 of anaphylaxis to non-ionic contrast media and β-lactam drugs were significantly higher than that in normal immune group (P < 0.05). CONCLUSION There is activation of basophilic granulocytes, mast cells and complement system in anaphylaxis to non-ionic contrast media. BAT can be used to diagnose the anaphylaxis to non-ionic contrast media.
Anaphylaxis/diagnosis* ; Basophils/cytology* ; Carboxypeptidases A/metabolism* ; Complement Membrane Attack Complex/metabolism* ; Contrast Media ; Flow Cytometry ; Granulocytes/cytology* ; Humans ; Mast Cells/cytology* ; Tetraspanin 30/metabolism*

Case-Control Studies ; Creatine ; urine ; Female ; Humans ; Hypoxia-Ischemia, Brain ; diagnosis ; urine ; Infant, Newborn ; Lactic Acid ; urine ; Male ; Predictive Value of Tests

AIM To establish an HPLC method for the content determination of six constituents in Shiyifang Vinum (Notoginseng Radix et Rhizoma,Dipsaci Radix,Carthami Flos,etc.).METHODS The content determination of notoginsenoside R1,ginsenoside Rg1,ginsenoside Rb1 and asperosaponin Ⅵ was performed on a 30℃ thermostatic Inertsil(R) ODS-3 C18column (4.6 mm ×250 mm,5 μm),with the mobile phase comprising of acetonitrile-0.1％ phosphoric acid flowing at 1.0 mL/min in a gradient manner,and the detection wavelength was set at 203 nm.The content determination of brucine and strychnine was conducted on a 30 ℃ thermostatic Geminni(R) C18 110(A) column (4.6 mm × 250 mm,5 μm),with the mobile phase comprising of acetonitrile-mixed solution of 0.01 mol/L sodium heptanesulfonate and 0.02 mol/L potassium dihydrogen phosphate flowing at 1.0 mL/min in an isocratic elution manner,and the detection wavelength was set at 260 nm.RESULTS Six constituents showed good linear relationships within their own ranges (r ＞ 0.999 0),whose average recoveries were 98.52％-99.96％ with the RSDs of 2.0％-2.3％.CONCLUSION This simple,accurate and reproducible method can be used for the quality control of Shiyifang Vinum.

AIM:To investigate the expression and localization of autophagy related protein microtublule associated protein 1 light chain 3 (LC3) at various stages of follicular development and atresia in the mice.METHODS:On 0,1,2,3,4 and 5 day after intraperitoneal injection of pregnant mare serum gonadotropin (PMSG),expression and positioning situation of autophagy related protein LC3 and apoptosis related protein cleaved caspase-3 were examined by the method of immunohistochemical staining.The protein levels of cleaved caspase-3 and LC3 were determined by Western blot in cultured mouse granulosa cells after incubation under serum-free conditions in the absence or presence of FSH.LC3 subcellular localization in granulosa cells were studied by the method of immunofluorescence.RESULTS:The LC3 protein expressed in granulosa cells during all developmental stages mainly.Granulosa cells of atretic follicles that showed intense staining of cleaved caspase-3 and LC3.The protein levels of cleaved caspase-3 and LC3-Ⅱ in the granulosa cells significantly decreased at 1 d and 2 d after intraperitoneal injection of PMSG (P ＜ 0.05).The protein levels of cleaved caspase3 and LC3-Ⅱ in the granulosa cells increased in turn on 3,4 and 5 day after intraperitoneal injection of PMSG.The positive correlation between LC3-Ⅱ and cleaved caspase-3 protein levels was observed (r2 =0.8299,P ＜ 0.05).The LC3-Ⅱ protein expressed with punctuate structures in granulosa cell cytoplasm cultured under serum-free conditions in the presence of FSH.CONCLUSION:LC3 is expressed in the follicular granulosa cells with cell specificity and regional specificity.Autophagy is induced mainly in granulosa cells during folliculogenesis and shows positive correlation with apoptosis.Ovarian granulosa cell autophagy and apoptosis are gonadotropic hormone dependent.

OBJECTIVETo determine the optimal cytokine combinations with hepatic growth factor (HGF) that results in the most significant simultaneous in vitro expansion of cc-kit(+)Lin(-) cells derived from the bone marrow.

METHODSC-kit(+)Lin(-) cells were isolated from mouse bone marrow using a high-gradient magnetic cell sorting system (MACS) and expanded in the presence of stem cell factor (SCF), FLt-3 ligand (FL), leukemia inhibitor factor (LIF) thrombopoietin (TPO) and different concentrations of HGF for 7days in a liquid culture system. The total cell number and Annexin-V-positive cell number were counted, and the antigen expressions were studied with fluorescence-activated cell sorting (FACS).

RESULTSIn each group, c-kit(+)Lin(-) cells were expanded effectively and rapidly by 2 to 8 folds. Addition of 10 ng/ml HGF into SCF+FL+LIF+TPO resulted in the most significant expansion of c-kit(+)Lin(-) and total cells by 8.00 and 45.43 folds, respectively, with cell apoptosis rate of 17.42 %. But as the concentration of HGF increased, the c-kit(+)Lin(-) cells and the apoptosis rate decreased.

CONCLUSIONHGF at10 ng/ml shows optimal synergistic effect with SCF, FL, LIF and TPO in expansion of c-kit(+)Lin(-) cells, and excessive HGF may induce cell differentiation.

Animals ; Bone Marrow Cells ; cytology ; drug effects ; metabolism ; Cell Count ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Dose-Response Relationship, Drug ; Flow Cytometry ; Hepatocyte Growth Factor ; pharmacology ; Mice ; Mice, Inbred BALB C ; Proto-Oncogene Proteins c-kit ; metabolism

Objective:Effect of gut microbiota on human health has become a hotspot in recent years with the emergence of new microbial technology.B.bifidum is a traditional probiotics and has been widely used in food and medicine.But the metabolism and function of B.bifidum ATCC 29521 has not been well documented.To investigate colonization and growth of B.bifidum ATCC 29521 in mice intestine,fecal B.bifidum concentrations were analyzed by real-time fluorescent quantitative PCR.Methods:Female C57BL/6 mice were orally gavaged with eigher a single dose of 1 × 109 CFU B.bifidum ATCC 29521 or continuous doses of 1 × 109/day for 3 weeks.Feces were collected at 0,2,4,6,8,10,12,16,20,24 h and at 10,7,14,21,24,28 d,respectively.Total DNA was isolated from the feces using the Qiagen DNA stool extraction kit according to the manufacturer's instructions.The linear standard curve was established by each dilution degrees of B.bifidum ATCC 29521 and their corresponding CT values.The curve equation was y =0.1835x + 9.0628 and R2 was 0.9994.The concentration of B.bifidum ATCC 29521 in mice feces were calculated by substituted CT values obtained by RT-PCR into the curve equation.Results:The curves of B.bifidum ATCC 29521 concentration rose at first and then reduced gradually.In single dose group,the concentration of B.bifidum ATCC 29521 began to increase at 4h after oral gavage,reaching its peak 6.0 × 107 CFU/g at 10h and then decreased gradually.The biggest drop occurred at the period between 12 h and 16 h after B.bifidum treatment.In successive administration group,the concentration rose at the highest rate in the first week when it achieved 2.0 × 107CFU/g and kept on inceasing to 1.0 × 108CFU/g in the next week.However,the concentration did not rose up significantly in the third week.It means that the concentration of B.bifidum ATCC 29521 in mice intestine reached platform in the second week after oral gavage.The concentration of B.bifidum ATCC 29521 significantly decreased at 24h and one week after B.bifidum treatment course in two group repectively and was still higher than baseline before oral gavage.Conclusion:Once the B.bifidum ATCC 29521 supplement was discontinued,the concentration fell down in short time.B.bifidum ATCC 29521 could not sustain colonization and growth in mice intestine without external supplement.It is necessary to provide daily supplemention for at least two weeks and to keep on in order to maintain sufficient concentration.

Objective To determine the epidemiological features of hand-foot-and-mouth disease (HFMD) outbreaks and the genetic characteristics of enterovirus type 71 (EV71) isolates from patients in Lianyungang, Jiangsu province in May, 2008. Methods Epidemiological, microbiological, cellular and molecular methods were performed to investigate pathogens and to describe the homogeneity of isolated strains. Results 21 cases were reported in this HFMD outbreak with the attack rate as 20.0%. 3 EV71 virus strains were isolated from 10 stool samples. The nucleotide and amino acid homogeneity of these 3 Jiangsu strain with Anhui Fuyang strains were 97.9%-100.0% and 99.7%-100.0%, respectively. These 4 Jiangsu strains were within genotype C sub-geno group C4 in phylogenetic tree. Data from the follow-up study showed that shedding of EV71 and Coxsackie A 16 virus (CA 16) in the latent period appeared in the outbreak of HFMD. Human beings could be infected by both EV71 virus and CA16 at the same time and could also carry the two viruses. We also discovered that EV71 virus could be expelled out of the human body through stool in the fast week and last for 10 weeks. Conclusion The recently identified EV71 isolates from this HFMD outbreak belonged to sub-geno group CA. Facts as: the release of viruses in the latent period, co-infection or coexisting of two viruses at the same time and super long period of expulsion of toxin exist in EV71 and CA16 did exist.

OBJECTIVETo compare the expression of pinopodes, the marker of endometrial receptivity, during the implantation window in Kunming mice stimulated with two different doses of raloxifene (RAL).

METHODSForty-eight 8-week-old female Kunming mice were randomly divided into 4 groups (n=12), namely saline group, clomiphene citrate (CC, 18 mg/kg) group, RAL (33 mg/kg) group and RAL (44 mg/kg group). In each group, the mice received intragastric administration of 1 mL of normal saline containing CC or RAL at the specified doses or saline only as indicated for ovulation induction, once daily for 2 days. The mice received then injection with 5 IU human chorionic gonadotropin (HCG) and mated and on day 4.5 of gestation, the pregnant mice were sacrificed for examination of the uterus with scanning electron microscopy.

RESULTSAbundant and well developed pinopodes were observed in the endometrium of the mice in the 2 RAL groups and in the saline control group. The mice in CC group showed obviously reduced endometrial pinopodes with poor development.

CONCLUSIONSRAL at two different doses does not obviously affect the expression of pinopodes in the uterine epithelium of mice, suggesting the safety of RAL at these two doses for ovulation induction without causing adverse effects on endometrial receptivity.