Objective To explore the cultural method of oral keratinocytes and make preparations for further investigation in using oral keratinocytes as a new choice of seed cells for the reconstruction of tissue-engineered urethra. Methods Oral keratinocytes of rabbits were isolated in vitro and seeded onto a culture dish with a feeder layer of 3T3 mouse fibroblasts inhibited by mitomycin(i3T3) or a culture dish without i3T3 respectively. Cell morphology was observed and cell growth was detected at intervals.Meanwhile,oral keratinocytes obtained from in vitro culture were performed immunofluorescence staining with broad-spectrum keratin antibody(AE1/AE3) and keratin 19 antibody(K19).The percentage of positive cells of passage 2 reactive to AE1/AE3 was assessed by flow cytometry. ResultsOral keratinocytes seeded onto a feeder layer of i3T3 exhibited finer morphous,better amplification capability,and could be passed for 7 or 8 generations.However,those cultured without i3T3 took on various morphous and could only be subcultured 2 generations before ageing.It was indicated by immunofluorescence staining that oral keratinocytes obtained from in vitro culture were positive for AE1/AE3 staining and 40% were positive for K19 staining.The result of flow cytometry revealed that the amout of positive keratinocytes reactive to AE1/AE3 was more than 95% of total cellular score. Conclusion Oral keratinocytes of rabbits can be cocultured with i3T3 in vitro and magnitude quantity can be attained,laying a favourable foundation for oral keratinocytes as a new choice of seed cells for urethral reconstruction with tissue engineering.

Objective To determine the frequency of polymorphism at exon 26 (C3435T) of muhidrug resistance 1 gene (MDR1) in epileptic patients in the southern Chinese and to study the association of this polymorphism with pharmacoresistance.Methods DNA samples were obtained from 134 patients,of whom 72 were resistant to antiepileptic drug treatment and 62 were responsive to the treatment. Genotypes of the C3435T polymorphism were determined by polymerase chain reaction (PCR) followed by restriction digestion and gel electrophoresis.Genotype and allele frequencies in the drug resistant group were compared to those in the response group by Chi-square analysis.Results Of all 134 patients,33 (24.6%) had CC genotype,72 (53.7%) had CT genotype,and 29 (21.6%) had TT genotype.The frequency of CC genotype was significantly higher in the pharmaeoresistance group (33.3%) than that in the responsive group (14.5%,P=0.012).The frequency of the C allele was also significantly higher in the pharmacoresistance group (57.6%) than that in the responsive group (44.4%,P=0.03).When patients were divided by types of seizure into three groups:generalized seizure group,partial seizure group,and undefined seizure group,the CC genotype and C allele were associated with pharmacoresistance in the partial seizure group.Conclusions In the southern Chinese,the CC genotype and C allele are associated with resistance to the antiepileptic treatment.This finding needs to be verified in studies with larger sample size.

Objective To build the experimental basement for the clinical use of cytokines induced killer(CIK)cells from the cord blood mononuclear cells(CBMNC)in tumor adoptive cellular immunotherapy, an effective protocol for their proliferation in vitro and cytotoxicity of CIK cells was established.Methods The lymphocytes from umbilical cord blood were isolated by density gradient centrifugation and suspended in medium with CD_3 mAb,rIL-2,rIL-1 and IFN-? as inducing agents to prepare CIK cells.At the same time, the lymphokine activated killer(LAK)and CBMNC were set as controls,which were only added IL-2 and not any cytokines during the whole culture.The changes of CIK cells before and after induction were observed with microscope and the phenotypes of the cells were analyzed by using flow cytometry.The proliferation of CIK cells were determined by trypan blue exclusion assay and the cytotoxic activity to lung cancer cell were tested with MTF method.Results According to the experiment,combining use of four types of cytokines could generate a great deal of CIK cells possessing highly cytotoxicity.From day 5 CIK cells became to prolif- erate and reached the peak at day 14.During the whole period,the relative percentage of CD_3~+ CD_(56)~+ cells in- creased significantly.Compared with LAK cells,which reached the proliferation peak at day 7 and then showed no evident proliferation.The control cells(CBMNC)showed no evident change of phenotypes and proliferation.CIK cells showed a higher antitumor activity on the tumor cells than LAK cells and CBMNC in vitro.Conclusion Umbilical cord blood can generate a great deal of CIK cells combining used with cy- tokines.Compared with classic LAK cells,umbilical cord blood CIK cells have the advantages of rapid prolif- eration speed and powerful cytotoxicity.CIK cells will be promising as a new strategy for the adoptive cellular immunotherapy of tumor.

In this paper, Liuwei Dihuang pill was used to study the identification of Chinese patent medicine by fluorescence sequencing typing technology. The DNA of Paeonia suffruticosa was used as template to amplify by five pair of FAM fluorescence labeling primers. Then, the amplified products were sequenced. The sequencing results were analyzed by GeneMarker V1.80 to screen the best fluorescence labeling primers. As a result, psbA-trnH fluorescence labeling primer was used to identify the raw materials of Liuwei Dihuang pill. The results showed that three kinds of raw plant medicinal materials in Liuwei Dihuang pill were able to be correctly identified by psbA-trnH fluorescence labeling primer. The fluorescence sequencing typing technology can stably and accurately distinguish raw medicinal materials in Chinese patent medicine.
DNA Primers ; chemistry ; genetics ; DNA, Plant ; chemistry ; genetics ; Drugs, Chinese Herbal ; chemistry ; standards ; Fluorescent Dyes ; chemistry ; Plants, Medicinal ; chemistry ; genetics ; Polymerase Chain Reaction ; instrumentation ; methods ; Quality Control ; Staining and Labeling

To simply and rapid authenticate Lonicera japanica. Rapid allele-specific PCR primer was designed base on trnL-trnF 625 G/T Single nucleotide polymorphism and the PCR reaction systems including annealing temperature was optimized; optimized results were performed to authenticate L. japanica and its 9 adulterants. When 100 x SYBR Green I was added in the PCR product of 87 degrees C initial denatured 1 min; 87 degrees C denatured 5 s, 68 degrees C annealing 5 s, 30 cycle; L. japanica visualize strong green fluorescence under 365 nm UV lamp whereas adulterants without. The results indicate rapid allele-specific PCR could authenticate L. japanica and its adulterants rapidly and simply.
Alleles ; DNA Primers ; genetics ; Drug Contamination ; prevention & control ; Lonicera ; classification ; genetics ; Polymerase Chain Reaction ; methods ; Quality Control

In order to confirm the existence of indoleamine 2, 3-dioxygenase (IDO) gene in swine, and to clone the novel gene followed by the molecule structure properties and expression pattern analysis, the porcine mRNA sequences homologous to human IDO were obtained from GenBank database by bioinformatics method. By using RT-PCR, the IDO gene was cloned from porcine endothelial cell line and the accuracy of the nucleic acid sequence was confirmed, and the expression pattern of the gene was detected. The three-dimensional structure model of porcine IDO was built referring to the tertiary structure of human IDO using biological sequence analysis software and database. The results showed that the porcine IDO was identified by sequencing. The nucleotide sequences were confirmed as a novel gene after submitted to Genbank. Porcine IDO was expressed in the lung, thymus, epididymis and anterior chamber with a basic level, however in peripheral blood mononuclear cells (PBMCs) the IDO gene was highly expressed. The three-dimensional structure model of porcine IDO was similar to that of human IDO. It was suggested that identification of the structure information of porcine IDO is essential to further investigate the immunologic function of the gene. Study of IDO on NK cells-mediated xenograft rejection will be a novel therapeutic target for the development of xenotransplantation.

Objective To discuss the jointly therapeutic effects of 6％ hydroxyethyl starch 130/0.4 (voluven) and furosemide on acute lung injury induced by paraquat (PQ) in swine.Methods The ALI/ ARDS models were established with 20％ PQ (20 mL) intraperitoneal injection in 18 healthy female piglets and randomly divided into three groups: voluven group (A), furosemide group (B) and voluven + furosemide group (C) (n =6 in each group).The heart rate (HR), mean arterial pressure (MAP), extravascular lung water index (ELWI), pulmonary vascular permeability index (PVPI), partial pressure of carbon dioxide (PaCO2) and oxygenation index (PaO2/FiO2) among the three groups were carefully measured by PICCO (pulse indicator continuous cardiac output) before modeling (baseline), just aftermodeling (t0), and 2 h (t2), 4 h (t4), 6 h (t6) and 8 h (t8) after trearment.Needle biopsies of lung tissue were made before modeling and at t0 and t8 and prepared for microscopy observation after Hematoxylineosin staining method (HE staning).Repeated measurement data were compared among repeated measures by the ANOVA and the difference between groups was compared by one-wey ANOVA.Results (1) The HR and MAP of all animals increased obviously while the oxygenation index declined significantly at modeling just made (t0) compared to those before modeling (all P ＜ 0.05).At the same time, the HR and MAP of A and B groups were significantly lower than those of C group after treatment (all P ＜ 0.05), while the oxygenation index of A and B were significantly higher (all P ＞ 0.05).(2) The ELWI and PVPI of all animals increased obviously at modeling just made (t0) compared to those before modeling (all P ＜ 0.05).The ELWI and PVPI were decreased after treatment.And at the same time, the ELWI and PVPI of A and B groups were significantly lower than those of C group after treatment (all P ＜ 0.05).(3) The alveolar tissue showed obviously injured changes at modeling just made (t0).The injury was relieved 8h after trearment, which was most markedly in group C.Conclusions With the combination of 6％ hydroxyethyl starch 130/0.4 and furosemide administered, the acute lung injury induced by paraquat poisoning can be effectively relieved, promoting gas exchange, and improve oxygenation.

This study was aimed to establish a rapid discrimination method of Pinellia ternataand its adulterants based on the odour fingerprints analysis.Typhonium flagelliforme and Arisaema Rhizome,which were the common adulterants of Pinellia ternata,were collected.The adulterants were mixed with Pinellia ternatain different proportions.E-nose technology was used to obtain the odour fingerprints of Pinellia ternataand its adulterants of different types and proportions.Chemometrics methods,such as the analysis of variance (ANOVA),principal component analysis (PCA) and discriminant factor analysis (DFA) were used in the analysis and discrimination on sensors response data collected by sensors.The results showed that there were obvious differences on the odour characteristics between Pinellia ternateand its adulterants.PCA can obviously discriminate Pinellia ternateand its adulterants.And the odour difference became obvious along with the increasing of the adulteration proportion.There was a linear relationship between e-nose signal and the proportion of Typhonium flagelliforme.The cumulative proportion in ANOVA of the DFA model was 100%.The correct recognition rate was not less than 97%.It was concluded that e-nose can be used for rapid discrimination of Pinellia ternataand its adulterants.This study provided new technology and method for the discrimination of adulterants of Chinese materia medica.

Objective To explore the clinical features, surgical techniques and treatment efficacy of blood blis?ter-like aneurysms (BBA) of the internal carotid artery (ICA). Method We retrospectively reviewed 45 BBAs of the ICA treated surgically at our department between Jan. 2006 and Jan. 2016. The data was analyzed. Results Direct clipping of BBA was performed in 38 patients, clipping over wrapping in 3 patients and trapping of ICA in 4 patients. Intraoperative rupture occurred in 12 patients (27%). Postoperative cerebral infarction developed in 10 cases (22%). Seven patients died (16%). During the follow-up time of 47 months (range, 6~126 months), Good results were achieved in 34 patients (76%) with a modified Rankin Scale (mRS) 0~2 and 4 patients were in mRS 3~5. Follow-up angiography revealed a re?currence of BBA in one patient, which was retreated with endovascular coiling. No rebleeding was found in all fol?lowed-up patients. Conclusions BBAs of the ICA are difficult to treat surgically with high risk and complications. Thoughtful pre-operative evaluation of BBA and application of various surgical strategies during operation can signifi?cantly reduce the surgical risk and achieve good outcomes.

Content of osteocyte in bone tissue is the most abundant, the most widely distributed, and embedding the cells in the mineralized bone matrix, the life can be close to the life of the body. Osteocyte formed by the cytoplasm dendritic communication network system between osteocyte and bone surface, is of great significance to maintaining the normal physiological function of bone tissue. Bone cells as the direct receptor of bone mechanical stress, through the release of bioactive factors such as PEG2, NO, ATP and classic Wnt/beta-catenin signaling pathway mechanical stress signal can be converted to bone formation and bone resorption of biochemical signals, and the biochemical signals were transfer to the other type cells of the tissue to regulate its function activities and cause bone reconstruction function. The microcracks surrounding osteocyte can disrupt the microtubule network system,cause surrounding osteocyte autophagy. In addition, osteocyte is very important for regulation of the body mineral balance, fat metabolism, and hematopoietic function.
Animals ; Autophagy ; Hematopoiesis ; Humans ; Minerals ; metabolism ; Osteocytes ; physiology ; Signal Transduction ; Stress, Mechanical