With the method of specific binding of DAPI and DNA, the mycoplasma contamination cell cultures are sensitively detected. In this way, the monolayer cells can be determinated if a certain cell culture has been contaminated by some mycoplasma. The process is simple and can be done within thirty minutes. This method can be widely applied as a routine determination for detecting mycoplasma contamination in laboratory cell cultures.

Objective To study the effects of tri-ortho-cresyl phosphate(TOCP) on mitochondrial membrane permeability in hen's nerve tissues and investigate the mechanism of the organophosphorus ester-induced delayed neurotoxicity(OPIDN).Methods Adult Roman hens were randomly divided into four groups,including three treated groups and one control group(24 in each group).The hens in the experimental groups were treated with TOCP by gavage at the single dosages of 185,375 and 750 mg/kg respectively.TOCP was dissolved in corn oil and administered at 0.65 ml/kg.The control hens received an equivalent volume of corn oil by gavage.The hens were sacrificed on the 1st,5th,15th and 21st day after treatment and the cerebrum,cerebellum,spinal cord were dissected and homogenized in ice bath.The mitochondria in these nerve tissues were extracted to determine the changes of the membrane permeability and membrane potential.Results Compared with the control group,no significant increase of the mitochondrial membrane permeability in the cerebrum was observed in treated groups.In the cerebellum,the membrane permeability in the 185 mg/kg group had no significant changes,while in the 375 and 750 mg/kg groups it increased significantly on the 1st and 5th day after TOCP treatment(P

Objective:To investigate relationships between the single nucleotide polymorphisms(SNPs) of interleukin-1B(IL-1B) and variable number of tandem repeat (VNTR)of interleukin-1RN(IL-1RN) promoter genes and susceptibility of the patients with gastric adenocarcinoma or the patients with gastric adenocarcinoma to helicobacter pylori (Hp) infection.Methods:The SNPs of the IL-1B(-31C/T and -511C/T) were determined by gene chip and The VNTR of IL-1RN were determined by agrose gel electrophoresis with a total of 130 cases of gastric adenocarcinoma and 142 healthy controls.The sera concentrations of IgG,IgM and IgA of Hp antibodies were measured by ELISA in all cases and controls.Results:H.pylori infection was detected in 69.2% of 130 patients and 46.5% of 142 controls (P=0.007,odds ratio [OR]=2.53).Frequencies of IL-1B-31TT genotype in patients with gastric adenocarcinoma or in patients with pooly-differentiated gastric adenocarcinoma were significantly higher than those in healthy controls or in well-differentiated groups,(P

To explore indications regularity of analogous decoctions of Erchen Decoction.323 analogous decoctions of Erchen Decoction were found in Concise Thesaurus of TCM Formula.Diseases and symptoms of TCM were analyzed.22 diseases and 118 symptoms of TCM were found.The main indication of analogous decoctions of Erchen Decoction included spleen-stomach and lung system,other symptoms were broadly involved.Erchen Decoction was the most appropriate for phlegm.

Aim To isolate cancer stem cells from human pan-creatic cancer cell line L3. 6pl and to identify their biological characteristics. Methods L3. 6pl cells were cultivated in com-mercial low adhesion plate with serum-free stem cell culture me-dium ( MEM/F12 1:1 ) supplemented with B27. The cancer stem cells reformed into floating spheres were isolated. The method of tumor sphere formation was used to isolate/enrich and characterize the cancer stem cells in pancreatic carcinoma cell line L3. 6pl. Cancer stem cell spheres were collected and sorted using magnetic cell sorting ( magnetic activated cell sorting, MACS) technology, with the cell surface markers of CD24 +CD44 + ESA+. Self-renewal and EMT-related oncogene expres-sion were measured with Western blot. Cancer stem cells differ-entiation potential and the expression of cancer stem cell related signs were checked with Immunofluorescence assay. To deter-mine tumorigenesis in vivo, Xenograft assay in NOD-SCID mice were performed respectively, then immunohistochemistry proto-oncogene c-Met and RON expression were also checked. West-ern blot was used to detect the changes of stemness relative tran-scriptional factors and epithelial markers expressed in spheres before and after differentiation. Drug resistance of pancreatic cancer stem cells to gemcitabine or paclitaxel was measured with MTT assay. Results CD24 + CD44 + ESA+ cells were signifi-cantly tumorigenic, and cultured in serum-free conditions to form spheroids, which had the characteristics of stem cells with self-renewal, EMT and drug-resistant capabilities, and had a posi-tive correlation with the c-Met, RON protein expression. Con-clusion Human pancreatic stem cells are successfully isolated, which provides a useful model for individualized therapy and e-valuation of the therapeutic efficacy for pancreatic cancer pa-tients.

Objective To investigate the effect of aurora kinase inhibitor VX-680 on cell apoptosis and Bcl-2 expressions in human bladder cancer T24 cells. Methods T24 cells were cultured and treated with VX-680 at various concentrations and time points in vitro. VX-680-induced apoptosis of T24 cells was calculated by flow cytometry. The morphological change of treated cells was observed by microscopy;Bcl-2 mRNA and protein expression in T24 cells was detected by RT-PCR and Western blot assay , respectively. Results The apoptosis rate of VX-680-induced T24 cells increased in a dose-and time-dependent manner. The increase of apoptosis rate and decrease of Bcl-2 mRNA and protein expression in VX-680-induced T24 cells were in a dose-dependent manner. Conclusion VX-680 can significantly induce the apoptosis of T24 cells by down-regulating Bcl-2expression in a dose-dependent manner.

BACKGROUND: Severe acute respiratory syndrome (SARS) is caused by a genetically novel coronavirus that is caused by acute infectious disease. It is not yet clear for the immunology function of SARS patients in their convalescent stage.OBJECTIVE: To study the effects on T lymphocyte, and the titer profiling of 24 T cell receptor (TCR) V β subfamilies expressions in SARS convalescent patients.DESIGN: A self-control observation.SETTING: Central Laboratory, Guangdong Provincial Hospital of Traditional Chinese Medicine.PARTICIPANTS: Seventy-six cured SARS patients who received treatment in the Second Hospital Affiliated to Guangzhou University of Traditional Chinese Medicine between January and April 2003. All the patients corresponded to "clinical diagnostic criteria of atypical pneumonia", " diagnostic criteria of severe atypical pneumonia and discharge criteria" and "clinical diagnostic criteria and discharge criteria of severe acute respiratory syndrome". The involved patients, 30 male and 46 female, averaged (32±11 (years old. Another 10 subjects who simultaneously received health examination in the same hospital, 5 male and 5 female, aged (32±7(years, were involved in the study. Informed consents of detected items were obtained from all the subjects.METHODS：①Detecting the expression of 24 T cell receptor(TCR)V β subfamilies in SARS convalescent patients：Peripheral blood(2 mL) was collected from the healthy convalescent subjects,and EDTA-K2 was used as anticoagulant．In the flow cytometry delection tubes．10 μL various fluorescein-labeled mAb，such as anti-CD3,anti-CD4，anti-CD8,anti-CD25，anti-CD28，anti-HLA-DR，anti-CD3mAb conjugated with PC5，TCR Vβ1(PE+FITC)．Vβ2(PE+FITC)。Vβ3 (FITC)，Vβ4(PE+FITC)，Vβ5．1(PE+FITC),Vβ5．2(PE),Vβ5．3(PE)，Vβ7．1(PE+FITC)，Vβ7．2(FITC),Vβ8(FITC),Vβ9 (PE)，Vβ11(PE)，Vβ12(FITC)，Vβ13．1(PE),Vβ13．2(PE),Vβ13．6(PE+FITC)，Vβ14(FITC),Vβ16(FITC),Vβ17 (PE+FITC)，Vβ18(PE)，Vβ20(FITC)，Vβ21．3(FITC),Vβ22(PE+FITC)and Vβ23(PE)，was added in special flow tubes,and then 50 μL whole blood was added．The mixed solution was incubated away from light for 15 minutes．After erythrocytolysin being added，mixed solution was washed．Finally．cell deposit was dissolved in 300 μl phosphate buffer solution (PBS)．Coulter ESP flow cytometer was used for detection．For the analysis of TCR expression,an electronic gate was set on these cells and at least 5000 events per sample were collected．Three-color cytofluodmetric analysis was performed using a Coulter ESP flow cytometer．②Detecting the T cell subset,activated T and B cells,and the percentage of Ts and Tc cells：5000 cells were collected and used to calculate the expression of T cells (CD3,CD4 and CD8),the activated T and B cells(CD3+／CD25+,CD3+/HLA-DR+ and CD3-／HLA-DR+),as well as the percentage of Ts and Tc cells by Coulter ESP flow cytometer and its software．MAIN OUTCOME MEASURES：①The change of T cell subset(CD3，CD4，and CD8)from SARS convalescent patients．②The change of activated T and B cells(CD3+／CD25+，CD3+／HLA-DR+ and CD3-／HLA-DR+)．③The percentage of Ts and Tc cells(CD8+／CD28+，CD8+/CD28-)in convalescent patients．④Analysis of the 24 TCR V β subfamilies from SARS patients in convalescence．RESULTS：All data were explored to analyze the expression profiling of 24 TCR Vβ subfamilies,the data from 74 SARSpatients and 10 healthy controls were explored to other result analysis．①The detecting results of T celI subset：The percentage of CD4+T cell mean value was lower than the reference value[(33．33±6．64)％ vs．(43±9)％，P＜0．01]．The percentage of CD8+T cell mean value was higher than the refefence value[(34．07±6．40)％ vs．(30±9)％,P＜0．01]．② The expression of activated T and B cells：Percentage of HLA-DR+ T and B cell was Increased while the percentage of CD25+ T-cell was decreased compared with reference values．In 53 out of 74 patients,the percentage of CD25+ T cells was lower than the reference value,and 64 patients had a lower percentage in CD3+/CD25+ T cells．The percentages of CD3+/HLA-DR+ and CD3-／HLA-DR+ cells were higher than the normal reference value．T cells expressing higher CD3+／HLA-DR+ were found in 36 patients，and T cells expressing higher CD3-/HLA-DR+ were found in 30 patients．③The ratios of Ts and Tc cells：The percentage of Ts cells which expressed CD8+／CD28- was increased compared with reference value [(28．75±7．31)％ vs．(15．99±5．1)％，P＜0．01],while the percentage of Tc cells which expressed CD8+／CD28+ was decreased [(5．99±3．60)％ vs．(13．2±4．1)％，P＜0．01]．Thirty-nine patients were found to possess the lower Tc cells and forty-eight patients were found to possess the higher Ts cells．The ratios of both CD4+ and CD8+ T cells were in the normal reference value．④24 TCR Vβ subfamilies expressions in T cells：It was noteworthy that Vβ14 had a highest percentage in all 24 Subfamilies,and followed by Vβ 5．3,and Vβ 23 in the convalescent patients．The percentage of Vβ 14 was the highest in the normal controls,which was consistent with the results of SARS patients．But the other subfamilies expression patterns were different．There were significant differences between Vβ1,Vβ5．2,Vβ5．3,Vβ7．2，Vβ9，Vβ11,Vβ13．1,Vv13．2，Vβ17,Vβ18，Vβ22 and Vβ23．In the convalescent period，each TCR Vβ expression of SARS patients was higher than that of controls(P＜0．05-0．01)．CONCLUSION:In SARS convalescent patients,the increased CD8+CD28- T cell may elevate CD8+ T cell number;Meanwhile.the reduced CD3+ and CD4+ T cell number may be corresponding to the increased Ts cell number．For some inhibiting factor secreted by Ts cell was also increased．The usage pattern of 24 TCR Vβ subfamilies in SARS patients is different from that of control group．The increase of percentage of CD3+／HLA-DR+ and CD3-／HLA-DR+ T cell may be related to the late response of activated T and B cells.

Objective To explore the effect of lysophosphatidic acid(LPA)on blood-brain barrier(BBB) permeability and its possible mechanism.Methods LPA or LPA+suramin(L+S)were stereotaxically injected into the right eaudate nucleus in SD rats in vivo.Evans blue(EB)was used to quantitatively measure the permeability of BBB at different time points.The expression of matrix metalloproteinase-9 was detected by immunohistochemistry technique.The pathological ultrastruetural changes of BBB were assessed by transmission electron microscopy.Results The BBB permeability began to increase after LPA administered into ipsilateral eaudate nucleus,and reached the peak at 24h.Then the permeability of BBB gradually lowered after 48h.In comparison with the same time points of control group,there were quite significant differences(P＜0.01).After L+S was injected,the change of BBB permeability had differences in comparison with those of LPA group in the same time points,(P＜0.05).MMP-9 positive cells were mainly vascular endothelial cells.The numbers of MMP-9 positive blood vessels grew at 6h in LPA group,and the expression of it reached maximum at 24h,then the number of it decreased at 48h,showing significant statistical differences in comparison with the L+S group(P＜0.01),It was observed microscopically that ultrastrueture of BBB of the LPA group was changed sharply,such as basement membrane roughed and fragmented,astroeyte end-feet swolled markedly and perivaseular space enlarged obviously.But there were no remarkable changes in BBB in L+S group.Conclusion LPA can induce increase of BBB permeability and its possible mechanism is the strong expression of MMP-9 protein produeted by endothelial cells through the mediation of LPA receptor,leading to degradation of basement membrane.

Heparin and low molecular weight heparin have been widely used in clinical therapy as anticoagulants in cardiovascular disease and in hemodialysis. Crude heparin is usually prepared from porcine intestinal mucosa. Purified heparin is a mixture of polysaccharides consisting mainly of repeating GlcNS(6S)-IdoA2S disaccharides and other disaccharides with different GlcNAc/GlcNS±3S±6S-GlcA/IdoA±2S residues. Heparin injections are drugs prepared from heparin active pharmaceutical ingredient ( API ) that is prepared from crude heparin. Low molecular weight heparins are dominant heparin-based drugs used clinically, which are prepared by degrading heparin into smaller sizes. As a result, low molecular weight heparins are sharing the same major disaccharides but have different reducing and non-reducing ends. In current study, we focused on the disaccharide compositional analysis of clinically used heparin and heparin-based drugs. HeparinaseⅠ,II, and Ⅲ were used to degrade all heparin and heparin-based drugs including heparin sodium injection, Enoxaparin sodium injection, Nadroparin calcium injection, Dalteparin sodium injection, Fondaparinux sodium into disaccharides. All the degraded products were analyzed by strong anion high perforance liquid chromatography ( SAX-HPLC) coupled with an UV-detector. Commercially available unsaturated disaccharide standards were then used for structral identification. Furthermore, unusual disaccharides present in Nadroparin, Dalteparin, and Fondaparinux were confirmed by reversed-phase ion pair HPLC coupled with mass spectrometry. The developed method produced detailed structural information, which should be useful for quality control of heparin and heparin-based drugs.

Objective:To study the positive expression rate of M2 subtype of macrophage cell surface molecules and the inflammatory factors of PPS in IL-4-induced M2 macrophage.Methods:The experiment was divided into 5 groups:blank control group, Model group,PPS groups(50 μg/ml,100 μg/ml and 200 μg/ml).The expression of CD206 and CD23 was used as bio-maker to confirm IL-4 induced macrophages by treating RAW264.7 with 20ng/ml of IL-4.IL-4 induced RAW264.7 cells were treated with PPS of 50μg/ml,100μg/ml and 200μg/ml for 24 h.Then the expression of CD206,CD16/32 and CD40 were analyzed by flow cytometry, and the mRNA expression of IL-1β,TNF-α,IL-10 and iNOS were detect by qRT-PCR.Results: After treated with IL-4,the positive rate of CD206 of RAW264.7 were high.After treated with PPS ,the rate of CD16/32 and CD40 in IL-4 induced RAW264.7 cells were high ,the expression of CD206 decreased,and the mRNA level of IL-1βand TNF-αincreased.Conclusion:RAW264.7 cells can be polarlized to M2 subtype macrophage by using 20 ng/ml IL-4.PPS enhances the mRNA of IL-1β,TNF-αand the expression of CD40, CD16/32 in IL-4-induced RAW264.7 cells .These results indicate that PPS can induce the M2 subtype to become M1 macrophages, can improve immune function of macrophages.