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【Objective】 To explore the effects of Ligusticum Chuanxiong extract on MPP+-induced SH-SY5Y cell damage and Parkinson’s syndrome. 【Methods】 1-methyl-4phenylpyridine ion (MPP+) interfered with SH-SY5Y to establish a cell model of elderly Parkinson’s syndrome (SH-SY5Y-MPP+). After intervention with Ligusticum Chuanxiong extract, cell proliferation and apoptosis as well as miR-23a-3p and SNCA expressions were detected. In addition, the changes of SH-SY5Y-MPP+ after regulating the expression of miR-23a-3p and SNCA were observed, and the relationship between miR-23a-3p and SNCA was verified by dual luciferase reporter. 【Results】 The cell proliferation capacity of SH-SY5y-MPP+ was significantly lower than that of SH-SY5Y, while the apoptosis rate was higher than that of SH-SY5Y (P<0.05). Under the intervention of Ligusticum Chuanxiong extract, the proliferation ability of SH-SY5Y-MPP+, and Bcl-2 and SNCA protein increased, the apoptosis rate, miR-23a-3p, and Bax proteins decreased (P<0.05). Both silencing miR-23a-3p and increasing SNCA could promote the proliferation of SH-SY5Y-MPP+ and inhibit apoptosis, while increasing miR-23a-3p and silencing SNCA were the opposite (P<0.05). The online target gene prediction website found that miR-23a-3p and SNCA had complementary sites that could bind, and the dual luciferase reporter enzyme showed that the firefly activity of SNCA-wt was significantly inhibited after the miR-23a-3p mimic sequence was transfected (P<0.05). After increasing miR-23a-3p, the expression of SNCA protein in SH-SY5Y-MPP+ decreased, while silencing miR-23a-3p was the opposite (P<0.05). Rescue experiments showed that the intervention effect of Ligusticum Chuanxiong extract on SH-SY5Y-MPP+ was completely reversed by increasing miR-23a-3p or silent SNCA (P>0.05); the effect of increasing miR-23a-3p on SH-SY5Y-MPP+ increased SNCA reversion (P>0.05). 【Conclusion】 Ligusticum Chuanxiong extract can affect the biological behavior changes of SH-SY5Y induced by MPP+ by regulating the miR-23a-3p/SNCA axis, which may be a new direction for the treatment of elderly Parkinson’s syndrome in the future.
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@#Objective To investigate the current status of occupational hazards and prevention and control of occupational ， - Methods ， diseases in micro small and medium sized manufacturing enterprises in Jiangxi Province. A total of 1 034 micro - small and medium sized manufacturing enterprises in Jiangxi Province were selected as the research subjects using a stratified cluster sampling method. The exposure of occupational hazards and the implementation of occupational hazards prevention and Results ， control measures were investigated. Among the 1 034 enterprises the small and micro enterprises accounted for ， 91.5%. The enterprises with serious occupational hazards were mainly concentrated in metallurgy building materials industry ， and machinery equipment and electrical appliance manufacturing industry. The exposure rate of occupational hazard factors in ， ， （ vs the categories of occupational hazard factors from high to low was physical factors dust and chemical factors 78.9% 52.8% vs ，P ） （ ）， ， 25.0% <0.01 . The exposure rate from high to low was metallurgy and building materials industry 60.8% machinery （ ）， ， equipment and electrical equipment manufacturing industry 42.9% light industry textile and tobacco processing industry （ ）， ， （ ）（P ） 32.0% chemical petrochemical and pharmaceutical industry 21.0% <0.01 . Noise exposure accounted for 98.3% in the workers exposed to physical factors. The implementation rate of prevention and control measures for occupational hazards ， in enterprises from high to low was the staffing of occupational health management personnel the establishment of ， ， occupational health management institutions the establishment of occupational health management systems the detection ， ， of occupational hazards the evaluation of the control effect of occupational hazards of construction projects and the - （ vs vs vs vs vs ，P ） pre evaluation of occupational hazards of construction projects 32.5% 25.7% 23.7% 16.2% 6.9% 4.2% <0.01 . Conclusion The focus of prevention and control of occupational hazards in manufacturing industry in Jiangxi Province is noise ， - and dust in small and micro metallurgy and building materials industry. Most of the micro small and medium sized manufacturing enterprises have not carried out the detection of occupational hazards and evaluation of occupational hazards in accordance with the law. The situation of occupational disease prevention and control is still challenging.
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@#Objective To investigate the current status of occupational hazards and prevention and control of occupational ， - Methods ， diseases in micro small and medium sized manufacturing enterprises in Jiangxi Province. A total of 1 034 micro - small and medium sized manufacturing enterprises in Jiangxi Province were selected as the research subjects using a stratified cluster sampling method. The exposure of occupational hazards and the implementation of occupational hazards prevention and Results ， control measures were investigated. Among the 1 034 enterprises the small and micro enterprises accounted for ， 91.5%. The enterprises with serious occupational hazards were mainly concentrated in metallurgy building materials industry ， and machinery equipment and electrical appliance manufacturing industry. The exposure rate of occupational hazard factors in ， ， （ vs the categories of occupational hazard factors from high to low was physical factors dust and chemical factors 78.9% 52.8% vs ，P ） （ ）， ， 25.0% <0.01 . The exposure rate from high to low was metallurgy and building materials industry 60.8% machinery （ ）， ， equipment and electrical equipment manufacturing industry 42.9% light industry textile and tobacco processing industry （ ）， ， （ ）（P ） 32.0% chemical petrochemical and pharmaceutical industry 21.0% <0.01 . Noise exposure accounted for 98.3% in the workers exposed to physical factors. The implementation rate of prevention and control measures for occupational hazards ， in enterprises from high to low was the staffing of occupational health management personnel the establishment of ， ， occupational health management institutions the establishment of occupational health management systems the detection ， ， of occupational hazards the evaluation of the control effect of occupational hazards of construction projects and the - （ vs vs vs vs vs ，P ） pre evaluation of occupational hazards of construction projects 32.5% 25.7% 23.7% 16.2% 6.9% 4.2% <0.01 . Conclusion The focus of prevention and control of occupational hazards in manufacturing industry in Jiangxi Province is noise ， - and dust in small and micro metallurgy and building materials industry. Most of the micro small and medium sized manufacturing enterprises have not carried out the detection of occupational hazards and evaluation of occupational hazards in accordance with the law. The situation of occupational disease prevention and control is still challenging.
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Objective:To establish a hydride generation atomic fluorescence method using ammonium persulfate as the digestion reagent for determination of arsenic in urine (hereinafter referred to as this method).Methods:The collected urine samples with ammonium persulfate were heated and digested on the tubular electric heating automatic control constant temperature digester (60 holes), with 5% hydrochloric acid solution as reaction medium and current carrier and 1.5% potassium borohydride solution as reducing agent. Arsenic content was determined with a four-channel atomic fluorescence spectrometer. The arsenic standard solution of 0 - 10 μg/L was prepared to determine the standard curve of this method, and the method was evaluated from the detection limit, linear range, correlation coefficient, precision, standard addition recovery experiment, and urine arsenic quality control sample detection. The standard method "Determination of Arsenic in Urine by Hydride Generation Atomic Fluorescence Spectrometry" (WS/T 474-2015, referred to as the standard method) was used for comparison experiments.Results:When the sampling volume was 1 ml, the detection limit of this method (digest with 1 ml 1.5 mol/L ammonium persulfate) was 0.03 μg/L. In the range of arsenic content from 0 - 10 μg/L, the linear relationship between arsenic content and fluorescence intensity was good, and the correlation coefficients ( r) were all 0.999 9. The relative standard deviations( RSD) of the three replicates of urine samples with different concentrations were 1.00%, 0.89% and 0.49%, respectively. Urine arsenic quality control samples were tested, and the test results were all within the range of public values; the overall average recovery was 102.29%, and the recovery range was 92.10% - 108.15%. Compared with the standard method in the determination results of 20 urine samples, the difference was not statistically significant ( t = - 0.40, P > 0.05). Conclusions:The hydride generation atomic fluorescence spectrometry using ammonium persulfate as digestion reagent for the determination of arsenic in urine has the advantages of low detection limit, good precision, high accuracy, small amount of sampling and digestion reagent, simple operation, and less harmful gas generation in sample pretreatment. It is suitable for rapid determination of arsenic in urine in large quantities.
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Glycosylation is one of the common post-translational modifications of proteins to regulate the ability of tumor invasion, metastasis and tumor heterogeneity by interacting with glycan-binding proteins such as lectins and antibodies. Glycan microarray can be constructed by chemical synthesis, chemical-enzyme synthesis or natural glycan releasing. Glycan microarray is an essential analytical tool to discover the interaction between glycan and its binding proteins. Here we summarize the standard techniques to construct glycan microarray for the application in cancer vaccine, monoclonal antibody and diagnostic markers.
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Antibodies, Monoclonal , Glycosylation , Lectins/metabolism , Microarray Analysis , Neoplasms , PolysaccharidesABSTRACT
Objective To understand the current status and influencing factors of public cognition, attitude, and behavior (KAP) for COVID-19, and to help the development of strategies for COVID-19 prevention and treatment. Methods Snowballing-based online questionnaire was used to conduct an anonymous survey. Results A total of 1 576 questionnaires were received, and 1 553 were effective (recovery rate 98.5%).The awareness rate for epidemiological knowledge was 87.3%, 93.1% for etiology knowledge and 85.9% for prevention and treatment knowledge.The average score for attitude towards COVID-19 fear was 15.47±3.15, agreement with relevant government regulations and policies was 11.28±1.58, and for preventive behavior was 24.47±2.61.Men′s knowledge scores in epidemiology and etiology were higher than women′s (P < 0.05).Women′s attitude towards epidemic fear and government identification were scored higher than men′s (P < 0.05).The public health prevention knowledge score was higher in subjects with urban household registration than in those with rural household registration (P < 0.05).Regression analysis showed that gender, age, occupation, level of attention on pandemic, epidemiological knowledge, and etiology knowledge were the influencing factors for the fear attitude to the epidemic (P < 0.05);the attention level and prevention knowledge were the influencing factors for prevention behavior (P < 0.05). Conclusion The public awareness rate of COVID-19 and attitude towards government identification are relatively high.The degree of pandemic fear and preventive behavior are above average.More targeted public education on COVID-19 is highly recommended.
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Previously, we reported that Y, a new epigallocatechin gallate derivative, is efficacious in reversing doxorubicin (DOX)--mediated resistance in hepatocellular carcinoma BEL-7404/DOX cells. In this study, we evaluated the efficacy of Y in reversing drug resistance both and by determining its effect on the adenosine triphosphate-binding cassette protein B1 transporter (ABCB1 or P-glycoprotein, P-gp). Our results showed that Y significantly sensitized cells overexpressing the ABCB1 transporter to anticancer drugs that are ABCB1 substrates. Y significantly stimulated the adenosine triphosphatase activity of ABCB1. Furthermore, Y exhibited a higher docking score as compared with epigallocatechin gallate inside the transmembrane domain of ABCB1. In addition, in the nude mouse tumor xenograft model, Y (110 mg/kg, intragastric administration), in combination with doxorubicin (2 mg/kg, intraperitoneal injection), significantly inhibited the growth of BEL-7404/DOX cell xenograft tumors, compared to equivalent epigallocatechin gallate. In conclusion, Y significantly reversed ABCB1-mediated multidrug resistance and its mechanisms of action may result from its competitive inhibition of the ABCB1 drug efflux function.
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Objective To evaluate the efficacy and adverse reactions of platinum-based combined chemotherapeutical regimens in treating relapsed or refractory non-Hodgkin lymphoma(NHL).Methods The clinical data of 68 patients with relapsed or refractory NHL treated with platinum-based combined chemotherapeutical regimens in the Affiliated Tumor Hospital of Guangxi Medical University from January 2008 to December 2014 were retrospectively analyzed.The curative effect of related regimens,adverse reactions and related influence factors were analyzed.Results Sixty-eight cases received 283 cycles of chemotherapy.In all cases,11 cases(16.18 ％) achieved the complete response(CR),31 cases(45.59 ％) achieved the partial response(PR),the overall response rate(ORR) was 61.76％;the median progression-free survival(PFS) was 6.51 months(95％CI:4.97-8.04 months).ORR and PFS in the cases of stage Ⅱ-Ⅲ,IPI score 0-2 and receiving only one chemotherapeutical regimen were superior to those in the cases of corresponding subgroup(P＜0.05);ORR and PFS had no statistical difference between the B cells lymphoma and Tcells lymphoma(P＞0.05).The medion PFS in the combined R group was 11.16 months,which was longer than 5.84 months in the non-combined R group(P =0.004).The major adverse events (stage Ⅱ-Ⅲ) included leukopenia (41.18 ％),thrombocytopenia (27.94％),hemoglobin decrease(11.76％),vomiting(8.82％) and diarrhea(1.47％).Conclusion The platinum-based combined chemotherapeutical regimens are effective with good safety in the treatment of relapsed or refractory NHL.
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Objective To establish disease-associated or cell type relevant neuron model generated from patients with Critical Illness Polyneuropathy (CIP) by making CIP patient-derived induced pluripotent stem (iPS) cell lines and neurons to provide a cell-based disease model of CIP.Methods Skin tissue of CIP patient was obtained clinically,and specific skin fibroblasts were isolated and cultured.The iPS cells were derived from CIP patient by introducing 4 transcription factors,namely Oct4,Klf4,Sox2,c-Myc,into patient-specific fibroblast cells by Millipore's Human STEMCCA Constitutive Polycistronic (OKSM) Lentivirus Kit.Colony morphology,alkaline phosphatase (AP) activity,immunofluorescence staining,quantitative reverse transcription polymerase chain reaction (RT-PCR),and differentiation ability were used to identify the pluripotency of these iPS cell lines.In addition,neurons were derived from these iPS cells by inhibiting SMAD pathway.Results The CIP-iPS cells presenting morphological and growth characteristics of human embryonic stem cell (hES) showed the presence of alkaline phosphatase detected by histochemical staining,and the expression of ESC-marker genes.The relative expressions of endogenous pluripotency genes,namely Sox2,REX1,NANOG and OCT4,in iPS cell lines were significantly increased compared with their primary fibroblasts (t values were-9.020,-10.753,-13.295,-12.677,P＜0.01).Subcutaneous injection of iPS cells into NOD-SCID mice resulted in teratomas containing tissues from all the 3 germ layers.Furthermore,cholinergic neurons were successfully induced from CIP-iPS cells.Conclusion The CIP patient-specific iPS cell line and cholinergic neurons were successfully established.Furthermore,the CIP-iPS cell line can be used as models for further elucidating the cellular pathology and developing therapeutic strategies for Critical Illness Polyneuropathy.
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AIM To investigate the toxicity of Buxue Shengru Granules (BSG,Astragali Radix,Angelicae sinensis Radix,Paeoniae Radix alba,etc.) to perinatal SD rats and their offspring to determine its safety.METHODS The respective BSG administration of 0,1,3,10 g/kg to the four groups rats,each with twenty-four female rats,started from the day 15 of gestation (GD15) through weaning when they were procured for the identification of the general condition,body weight and food ingestion.The offspring had their postnatal appearance observed the physiological development,reflexes development,behavior and fertility detected as well.RESULTS BSG gave neither significant influence on the rats' general condition,time of birth,body weight,and histological changes of organs during perinatal period;nor remarkable impact on the offspring's growth,development,nerves,endocrine,and reproductive system.CONCLUSION Data on BSG to rats maternal weight gain and food intake and to their offspring developmental landmarks,sexual maturation,or reflexes suggest that BSG gives no perinatal toxicity.
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Objective:To investigate the correlation between plexinC1 (PLXNC1) rs2272335 polymorphism and the family clus-tering genetic susceptibility to primary liver cancer (PLC) in Guangxi and the expression of PLXNC1. Methods:Genotype and alleles of rs2272335 were determined in 20 liver cancer family groups (79 cases) and 10 healthy normal control groups (40 cases) in Fusui County through Time of Flight Mass Spectrometer. Immunohistochemistry detected the PLEXNC1 protein expression. Results:For the alleles of PLXNC1 (rs2272335) site, the risk of hepatocellular carcinoma (HCC) for individuals with [C] allele was 4.16-fold (95%CI=0.37-47.3, P=0.032) compared with that for individuals with [T] allele among the members of the healthy normal control group. The fre-quencies of the [C] and [T] alleles were similar in the HCC patients and the core individuals of liver cancer families (P>0.05). For the genotype of the PLXNC1 (rs2272335) site, the differences in frequencies of TT, TC, and CC genotypes were not statistically significant among the PLC patients and the core individuals of the liver cancer families and normal controls. The PLXNC1 protein expression in HCC (3.12±1.12) was higher than in hepatocellular paracancerous tissues (1.54±0.67) and in benign hepatocellular lesions (1.23±0.87) (P<0.05). Conclusion:The [C] allele of PLXNC1 (rs2272335) site might be the risk gene for the occurrence of PLC family clustering in Guangxi. PLXNC1 protein overexpression was closely correlated with PLC oncogenesis.
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<p><b>OBJECTIVE</b>To study the radiobiological characteristic of human nasopharyngeal carcinoma cell lines CNE1 and CNE2 and the changes in expression MRN (Mre11-Rad50-Nbs1) complex in the cell lines exposed to irradiation.</p><p><b>METHODS</b>CNE1 and CNE2 were irradiated by a linear accelerator. Radiobiological characteristics were detected by colony assay and MTT assay. MRN complex expression were examined by Western blot.</p><p><b>RESULTS</b>Surviving fraction at 2 Gy (SF2), quasi-threshold Dose (Dq), and mean lethal dose (Do) of CNE1 were 0.56, 1.449 Gy and 1.480 Gy; SF2, Dq, and Do of CNE2 were 0.44, 0.776 Gy and 1.685 Gy, respectively. Survival fraction of CNE1 at the day 6 after 4 Gy irradiation was 0.59 and that of CNE2 was 0.79 when compared with control, with the up-regulated expressions of Rad50 in CNE1 and Mre11, Rad50 and Nbs1 in CNE2 (P < 0.05).</p><p><b>CONCLUSIONS</b>CNE1 and CNE2 were sensitive to radiation, but there were radioresistance cells in CNE2. The expressions of some components of MRN complex were up-regulated to repair DNA lesions induced by radiation.</p>
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Humans , Carcinoma , Cell Cycle Proteins , Metabolism , Cell Line, Tumor , Radiation Effects , DNA Repair , DNA Repair Enzymes , Metabolism , DNA-Binding Proteins , Metabolism , Gene Expression Regulation, Neoplastic , MRE11 Homologue Protein , Nasopharyngeal Neoplasms , Pathology , Radiotherapy , Nuclear Proteins , Metabolism , Radiation ToleranceABSTRACT
<p><b>OBJECTIVE</b>To investigate the regulatory effects of lanthanum chloride (LaCl3) on the activation of nuclear factor kappa B inhibitor (IκB) kinase beta (IKKβ) induced by tumor necrosis factor alpha (TNF-α).</p><p><b>METHODS</b>(1) Hela cells were cultured routinely in vitro. One portion of cells were collected and divided into TNF-α group (cultured with serum-free RMPI 1640 medium containing 20 ng/mL TNF-α for 30 min), low-dose LaCl3 + TNF-α group, moderate-dose LaCl3 + TNF-α group, high-dose LaCl3 + TNF-α group, LaCl3 group (cultured with serum-free RMPI 1640 medium containing 100 µmol/L LaCl3 for 30 min), and control group (cultured with serum-free RMPI 1640 medium for 30 min) according to the random number table. Cells in low-dose LaCl3 + TNF-α group, moderate-dose LaCl3 + TNF-α group, high-dose LaCl3 + TNF-α group were first cultured with serum-free RMPI 1640 medium containing 5, 25, 100 µmol/L LaCl3 for 4 h, and then stimulated with serum-free RMPI 1640 medium containing 20 ng/mL TNF-α for 30 min. There were 3 samples in each group. Cells were collected for detection of intracellular location of NF-κB/p65 protein by immunofluorescence staining. (2) Another portion of cells were collected and divided into TNF-α group, low-dose LaCl3 + TNF-α group, moderate-dose LaCl3 + TNF-α group, high-dose LaCl3 + TNF-α group, and control group with the same treatment as above. There were 3 samples in each group. The protein levels of NF-κB/p65 in nuclei, and the protein levels of IκBα, phosphorylated IκBα (p-IκBα) as well as IKKβ and phosphorylated IKKβ (p-IKKβ) in cytoplasm were determined by Western blotting. The binding activity between NF-κB/p65 in the nuclear and target gene was determined by NF-κB/p65 transcription factor kit (denoted as absorption value). Data were processed with analysis of variance or LSD-t test.</p><p><b>RESULTS</b>(1) High expression of NF-κB/p65 was observed in cytoplasm of control group. High expression of NF-κB/p65 was observed in nuclei of TNF-α group. The expression of NF-κB/p65 in cytoplasm of LaCl3 group was lower than that of control group. In groups treated with LaCl3 and TNF-α, NF-κB/p65 expression levels in nuclei and cytoplasm were decreased along with the increase in the concentration of LaCl3, which were all lower than those in TNF-α group. (2) There was certain amount of NF-κB/p65 protein expressed in nuclei of control group. The expression of NF-κB/p65 protein in nuclei of TNF-α group was higher than that of control group. In groups treated with LaCl3 and TNF-α, the expressions of NF-κB/p65 protein in nuclei were decreased along with an increase in the concentration of LaCl3. The level of IκBα in TNF-α group was significantly decreased but that of p-IκBα increased as compared with those in control group. Along with the increase in the concentration of LaCl3, the levels of IκBα gradually increased and the levels of p-IκBα gradually decreased in groups treated with LaCl3 and TNF-α. There were no statistical differences in expression levels of IKKβ among the 5 groups. The expression of p-IKKβ could be hardly observed in control group, but it was obviously increased in TNF-α group. The expression levels of p-IKKβ in groups treated with LaCl3 and TNF-α were gradually decreased along with the increase in the concentration of LaCl3. The absorption value in TNF-α group was 0.39 ± 0.03, which was higher than that in control group (0, t = -7.23, P<0.01). The absorption values in low-dose LaCl3 +TNF-α group, moderate-dose LaCl3 + TNF-α group, and high-dose LaCl3 +TNF-α group were respectively 0.17 ± 0.03, 0.15 ± 0.03, and 0, which were obviously lower than that in TNF-α group (with t values respectively -6.54, -5.92, -7.23, P values all below 0.01).</p><p><b>CONCLUSIONS</b>LaCl3 can block the activation of NF-κB signaling pathway by blocking the phosphorylation of IKKβ of Hela cells.</p>
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Humans , Culture Media , HeLa Cells , I-kappa B Kinase , Metabolism , I-kappa B Proteins , Metabolism , Lanthanum , Pharmacology , NF-KappaB Inhibitor alpha , Signal Transduction , Transcription Factor RelA , Metabolism , Tumor Necrosis Factor-alpha , PharmacologyABSTRACT
Aim To observe the effects of promethazine on the analgesia,hypnosis,amnesia and therapeutic index of isoflurane.Methods The experiments were designed to study promethazine on the analgesic effect of isoflurane by hot-plate test and writhing test,and to study the effect of promethazine on the sleeping time of isoflurane by the method of righting reflex,and the amnesia of isoflurane by Morris water maze,and the ED_(50),LD_(50) by sequential method in mice.Results The result of hot-plate test and writhing test indicated that promethazine could enhance the analgesic effect of isoflurane(P＜0.05 or P＜0.01);through the experiment of righting reflex, sleeping time of isoflurane in mice was extended by promethazine(P＜0.01);in Morris water maze experiment, the average latency in the combination of promethazine and isoflurane was longer than that of the promethazine group or isoflurane group(P＜0.05 or P＜0.01), while aiming to the residence time, the combination of the two was shorter than that in the third quadrant(P＜0.01 or P＜0.05),the TCPP of the group of isoflurance was more than that of the combination group;promethazine could decrease the ED_(50) of isoflurance(P＜0.01),but it did not obviously affect its LD_(50)(P＞0.05).Conclusion Promethazine can not only reinforce the effect of isoflurance on analgesia,hypnosis and amnesia, but also boost the therapeutic index of isoflurance.
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BACKGROUND:Up to date,inbred embryonic stem cells(ESCs) line mainly derived from 129 mouse strain and C57BL/6 strain,occasionally from BALB/c mouse strain,however,few reports concerning ESCs lines derived from Ch inese Kunmingmouse strain.OBJECTIVE:To isolate and culture mouse ESCs of kunming species,additionally,to identify its biological properties.DESIGN,TIME AND SETTING:The experiment with cells as observed subjects was conducted in the institute of Urology,First Affiliated Hospital of Nanchang University from May 2006 to June 2007.MATERIALS:Blastocysts of Kunmin mice with 3.5 days of embryonic age.METHODS:Inner cell mass from 3.5 days embryonic age were isolated from Kunming species mice,cultured on feeder layers of primary mice embryonic fibroblasts,and then the cells were isolated and subsequently cultured.MAIN OUTCOME MEASURES:Colony growth was observed and determined by alkaline phosphatase (AKP) staining;The expression of stage-specific embryonic antigen (SSEA)-1,and cell specific genes of c-Myc,Nanog,Oct3/4 and Sox2 were measured by immunofluorescence and RT-PCR;finally,the ability of differentiation in vitro and in vivo was identified.RESULTS:The ESCs-like colonies presented typical morphological characteristics of ESCs,which was positive to AKP and SSEA-1,and could express several cell specific genes,such as c-Myc,Nanog,Oct3/4 and Sox2,and differentiate into various cell types in vitro and in vivo.CONCLUSION:An ESCs line is successfully dedved from Kunming mice,which has typical biological characteristics of ESCs.
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BACKGROUND: Aromatic polyester can not be used alone as degradable material due to its poor biodegradation. In the School of Materials Science and Engineering, Nanchang University, aromatic/aliphatic copolyesters, a new kind of degradable biomaterial is synthesized by melt co-polycondensation and transesterification of terephthalyl chloride, bisphenol A, hexanediol as well as low lacticacid polymer, which has been patented. OBJECTIVE: To study the biocompatibility between aromatic/aliphatic copolyesters and bone marrow mesenchymal stem cells (BM-MSCs) in rats. DESIGN, TIME AND SETTING: An in vitro cellular-materials experiment. The experiment was performed at the Institute of Urology, the First Affiliated Hospital of Nanchang University between April 2006 and April 2007. MATERIALS: Five healthy, adult, male, Sprague Dawley rats were provided by the experimental animal center of Jiangxi University of Traditional Chinese Medicine. The bio-degradable polyester was aromatic/aliphatic copolyesters prepared by our group. METHODS: A new degradable biomaterial poly (4,4'-isopropylidenediphenyl terephthalate)-co-poly(hexylene terephthalate)-co-polylactide (PBHTL) was prepared into biomembrane by casting method. Simultaneously, polyvinyl chloride biomembrane was prepared with the same method. The leaching liquor of biomembrane was collected when the biomembranes were sterilized and immersed in culture medium. The BM-MSCs of rats with the 3~(rd) or 4(th) passage were incubated at the 96-well plates with density of 2×10~7/L and at 24-well plate with 1.3×10~5 per well. The experiment divided into 3 groups. In the negative control group, cells were cultured with DMEM. In the experimental group, 12.5%, 25%, 50%. 100% material leaching liquor were additional added based on DMEM. In the positive group, polyvinyl chloride leaching liquor was added except DMEM. MAIN OUTCOME MEASURES: The cytotoxicity of material was evaluated by neutral red uptake assay, basic fuchsin staining and MTT method. Growth of BM-MSCs on aromatic/aliphatic copolyesters membrane was estimated by electron microscope. RESULTS: The cell viability and metabolic capability were decreased 11%-16% in the positive control group at days 1, 3, 5, and 7 after culture. The absorbance value of the experimental group was significantly different from the positive control group (P < 0.001), which increased cell viability and metabolic capability with time prolonged (1%-4%). However, there was not obviouslydifference between the experimental group and negative group in absorbance value (P> 0.05). Acridine orange/ethylene dibromide staining showed that the BM-MSCs attached and grew in spindle-like manner on the surface of aromatic/aliphatic copolyesters biomembrane, which increased cell number with decreased apoptosis rate with time prolonged. CONCLUSION: The results demonstrated that aromatic/aliphatic copolyesters membrane has no eligible cytotoxicity to cell growth with good cell compatibility, which meets the requirements for applied biomaterials.
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<p><b>UNLABELLED</b>OBJECTIVE To explore the effects of human macrophage inflammatory protein-1 beta (hMIP-1beta) modification on the in vivo tumorigenicity and vaccine efficacy of tumor cells.</p><p><b>METHODS</b>Murine colorectal adenocarcinoma CT26 cells were transfected with a recombinant adenovirus carring the hMIP-1beta gene (AdhMIP-1beta). The efficacy of gene transfection was tested by X-gal staining. The hMIP-1beta level in the supernatant of hMIP-1beta gene-modified CT26 cells was assayed by ELISA, and the chemotactic activity for CD4+ T cells, CD8+ T cells, NK cells and immature dendritic cells (imDCs) were assayed by a transwell chamber. The changes of growth characteristics and in vivo tumorigenicity of hMIP-1beta gene-modified CT26 cells were also assessed. BALB/c mice were immunized with hMIP-1beta gene-modified CT26 tumor vaccine and the antitumor effect was evaluated.</p><p><b>RESULTS</b>hMIP-1beta gene could be transfected into CT26 cells by AdhMIP-1beta with an efficiency over 95%. The level of hMIP-1beta in the culture supernatant of hMIP-1beta gene-modified CT26 cells was 980 pg/ml and the supernatant displayed ramarkable chemotactic activity to CD4+ T cells, CD8+ T cells, NK cells and imDCs compared with LacZ gene-modified CT26 cells and control. When the hMIP-1beta gene-modifited CT26 cells were subcutaneously inoculated in BALB/c mice, the tumorigencity was delayed and suppressed, and overt necrosis and lymphocyte infiltration were observed in the tumor tissue, but not in those inoculated with LacZ gene-modified CT26 cells or parental CT26 cells. The mice immunized with hMIP-1beta gene-modified CT26 tumor vaccine could induce tumor specific CTL activity and nonspecific NK activity, and exhibited resistance to later challenge with wild-type CT26 cells.</p><p><b>CONCLUSION</b>hMIP-1beta gene-modified CT26 cells exhibit decreased tumorigenicity, and hMIP-1beta gene-modified tumor vaccine may induce a powerful specific and nonspecific antitumor response. The data suggested that hMIP-1beta gene-modified tumor vaccine may play a potent role in prevention of metastasis and recurrence of malignant tumors.</p>
Subject(s)
Animals , Female , Humans , Mice , Adenocarcinoma , Metabolism , Pathology , Adenoviridae , Genetics , CD4-Positive T-Lymphocytes , Allergy and Immunology , CD8-Positive T-Lymphocytes , Allergy and Immunology , Cancer Vaccines , Cell Line, Tumor , Chemokine CCL4 , Genetics , Metabolism , Chemotaxis, Leukocyte , Colonic Neoplasms , Metabolism , Pathology , Cytotoxicity, Immunologic , Dendritic Cells , Allergy and Immunology , Genetic Vectors , Killer Cells, Natural , Allergy and Immunology , Mice, Inbred BALB C , Neoplasm Transplantation , Random Allocation , Recombinant Proteins , Genetics , Metabolism , Transfection , Tumor BurdenABSTRACT
<p><b>OBJECTIVE</b>To construct a dendritic cell vaccine transduced by murine alpha-fetoprotein (mAFP) gene, and evaluate its immunoprotective effect on C57BL/6J mice during the induction of hepatocellular carcinoma by diethylnitrosamines, carbon tetrachloride and ethanol.</p><p><b>METHODS</b>Dendritic cells (DCs) were induced and augmented by murine IL-4 and GM-CSF, and transfected by recombinant adenovirus engineered with mAFP gene. Major MHC class I and II, B7.1 (CD80), B7.2 (CD86), CD18a, and CD54 molecules on DC were analyzed by FACS. 80 C57BL/6J male mice were randomly divided into 4 groups (20 mice per group): Simple DC inoculated group, pAdBM5-mAFP-DC inoculated group, pAdBM5-mAFP plasmid inoculated group, and PBS control group. They were immunized once with 5 x 10(5) DCs (0.1 ml)/mouse administered s. c. in the left flank or 100 mg pAdBMS-mAFP plasmid/mouse administered i. m. in the left tibialis anterior muscle. Inoculation was conducted once a week for 4 weeks after 3 times consecutive immunization initially. At the same time of immunization, DEN/CCl4/ethanol were given to induce hepatocellular carcinoma. Tumor incidence was assessed after 20 weeks.</p><p><b>RESULTS</b>A transgenic DC vaccine was successfully constructed and the mAFP transgenic DCs expressed high level molecules of major MHC class I and II , B7.1, B7.2, CD18a, and CD54. After the 20-week induction, the incidence of primary hepatocellular carcinoma (PLC) was 70.0% in simple DC inoculated group, 25.0% in pAdBMS-mAFP-DC inoculated group, 65.0% in pAdBM5-mAFP plasmid inoculated group, and 75.0% in PBS control group. There was a significant difference between group B and other groups (P < 0.05).</p><p><b>CONCLUSION</b>mAFP transgenic DC tumor vaccine inoculation may induce strong immunoprotection against liver carcinogenesis and tumor development and reduce PLC incidence induced by DEN/CCl4/ethanol.</p>
Subject(s)
Animals , Male , Mice , Adenoviridae , Genetics , B7-1 Antigen , Metabolism , Cancer Vaccines , Carbon Tetrachloride , Cells, Cultured , Dendritic Cells , Cell Biology , Allergy and Immunology , Metabolism , Diethylnitrosamine , Ethanol , Genetic Vectors , Histocompatibility Antigens Class I , Metabolism , Histocompatibility Antigens Class II , Metabolism , Intercellular Adhesion Molecule-1 , Metabolism , Liver Neoplasms, Experimental , Allergy and Immunology , Mice, Inbred C57BL , Random Allocation , Recombinant Proteins , Genetics , Metabolism , Transfection , alpha-Fetoproteins , Genetics , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To explore the influence of lanthanum chloride (LaCl3) on inducible nitric oxide synthase (iNOS) expression in RAW264.7 macrophages with lipopolysaccharide (LPS) induction, and to investigate its possible mechanisms.</p><p><b>METHODS</b>The RAW264.7 macrophages were randomly divided into four groups: i. e, control group (without treatment), LaCl3 group (with treatment of 2.5 micromol/L of LaCl3 for 24 hrs), LaCl3 + LPS group (with treatment of 2.5 micromol/L LaCl3 for 24h), and LPS group (with treatment of 1 mg/L LPS for 24 hrs). The iNOS protein expression was measured by immunofluorescence and Western blot. iNOS gene expression was assayed by reverse transcription-polymerase chain reaction (RT-PCR). NO production in culture supernatant was assayed by nitrate reductase method.</p><p><b>RESULTS</b>Immunofluorescence analysis showed that iNOS was located mainly in the cytoplasm. RAW264.7 cells with overexpression of iNOS accounted for 44.4%, which was obviously higher than that in LaCl3 + LPS group (11.8%, P < 0.05). There was a faint signal of FITC-labeled green tint in control group or LaCl3 group. The iNOS mRNA and protein expression, and the NO content in LPS group were significantly higher than those in control, LaCl3, and LaCl3 + LPS groups (P < 0.05).</p><p><b>CONCLUSION</b>LaCl3 can suppress LPS-induced iNOS overexpression at mRNA and protein level and reduce NO production, indicating that LaCl3 can antagonize the excessive activation of iNOS induced by LPS.</p>
Subject(s)
Animals , Mice , Cell Line , Lanthanum , Pharmacology , Lipopolysaccharides , Toxicity , Macrophages , Metabolism , Nitric Oxide , Nitric Oxide Synthase Type II , Metabolism , RNA, Messenger , Metabolism , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
<p><b>OBJECTIVE</b>To investigate the influence of lanthanum on lipopolysaccharide (LPS) induced NF-KB activation in murine peritoneal macrophage.</p><p><b>METHODS</b>Peritoneal macrophages were isolated and cultured by routine method, and randomly divided into 5 groups: i. e, control group, LPS group (with LPS stimulation for 30 min), La3+ group (with 2.5 micromol/L La3+ group for 30 min) , La3+ + LPS group( with 1 microg/ml LPS stimulation for 30 min after 30 min incubation with DMEM-F12 containing 2.5 microM of lanthanum.) ; La3+/LPS group (with 2.5 microM of lanthanum stimulation for 30 min, and then with 1 microg/ml of LPS for another 30 min after lanthanum was removed. The location of NF-kappaB p65 subunit (NF-kappaB/p65) in Mphi was detected by immunofluorescence and fluorescence microscope. The binding activity of NF-kappaB/p65 with DNA in nuclei was detected by TransAMTM NF-kappaB/p65 Transcription Factor assay kit. Meanwhile, the expression of NF-kappaB/p65 in nuclei, as well as IkappaBalpha in cytoplasm was measured by Western blotting. TNF-alpha content in culture supernatant were detected by ELISA.</p><p><b>RESULTS</b>(1) The green fluorescence in control, La3+, La3+ LPS and La+/LPS groups was mainly located in cytoplasm, while that in LPS group was located in nuclei. The fluorescent intensity in LPS group was (116 +/- 14), which was obviously higher than that in other 4 groups (42 +/-7,73 +/-30,48 +/- 11 and 67 +/- 19, respectively, P <0.01). (2) The IkappaBalpha protein level in cytoplasm in control (0.048 +/- 0.027), La3+ group (0.062 +/- 0.049), La3+ + LPS group (0.066 +/-0.031) and La3+/LPS group (0.108 +/- 0.017) was significantly lower than that in LPS group (0.435 +/-0.066, P <0.01). (3) The expression and activation of nucleus p65 protein in Mphi in LPS group was obviously higher than the other 4 groups, but changes in the IkappaBalpha expression between LPS group and other 4 groups was of controversy. (4) TNFalpha level in the culture supernatant in La3+ group was lower than that in control group ( P < 0.05) and below the detection limit (25 pg/ml). Moreover, it in La3+ + LPS group and La3*/LPS group was lower than that in LPS group (P <0.01), but higher than that in control group.</p><p><b>CONCLUSION</b>LPS can activate the nucleus translocation of NF-kappaB/p65 in Mphi of mice, increase NF-KB/p65 expression and activity, but reduce IkappaBalpha protein expression, which lead to increase of TNFalpha secretion. Lanthanum can inhibit lipopolysaccharide induced NF-kappaB activation.</p>