Objective To investigate the effects of different culture conditions(RPMI-1640,DMEM and DMEM/F12 medium)on the passage of MPM cells isolated from the tissues of Malignant pleural mesothelioma(MPM),and to study the effects of CDKN2B on the proliferation,invasion and apoptosis of MPM cells.Methods MPM cells were isolated from MPM tissues and cultured in RPMI-1640,DMEM and DMEM/F12 medium,respectively.Cell proliferation was examined by CCK-8,and the nuclei and chromosomes were observed by Wright-Giemsa staining.Fluorescence intensities of Calretinin,CD141,CK5,EMA and WT-1 were conducted by immunofluorescence assay.The mRNA and protein expression of CDKN2B were detected by RT-qPCR and Western blot,respectively.Transwell was used to detect cell invasion and flow cytometry was used to detect cell apoptosis.Results The established MPM cells showed good viability when passaged to the 10th generation in RPMI-1640,DMEM and DMEM/F12 cultures,and the MPM markers Calretinin,CD141,CK5,EMA and WT-1 were all expressed in the cells.The viability of MPM cells in RPMI-1640 culture medium was relatively stable.CDKN2B was downregulated in MPM cells(P<0.05),and overexpression of CDKN2B significantly suppressed the proliferation(P<0.05),invasion(P<0.05)and epithelial interstitial transformation of MPM cells(P<0.01),and promoted the apoptosis(P<0.01).Conclusion The established MPM cells were stably passaged in RPMI-1640 culture medium,and CDKN2B may be a potential target for the diagnosis and treatment of MPM.

H 1-antihistamines are widely used in the treatment of various allergic diseases, but there are still many challenges in the safe and rational use of H 1-antihistamines in pediatrics, and there is a lack of guidance on the prescription review of H 1-antihistamines for children.In this paper, suggestions are put forward from the indications, dosage, route of administration, pathophysiological characteristics of children with individual difference and drug interactions, so as to provide reference for clinicians and pharmacists.

Objective:To establish the optimal extraction technology of Xiaoer Yinqiao granules by orthogonal test. Methods:The effects of water amount,extraction duration and extraction times were investigated by orthogonal design using the contents of forsythia-side A and chlorogenic acid as the indices. Results: The optimum extraction process was as follows: adding 8-fold amount of water, and extracting 1. 5 h for the first time, and then adding 6-fold amount of water, extracting 1 h for the second and third time, respective-ly. Conclusion:The extraction technology is simple, reasonable and reliable.

Objective To evaluate the efficacy and safety of deoxyribonucleotide in intervention with wound healing.Methods A total of 144 patients above Type Ⅱ incision was included.All patients were received conventional therapy.And 72 cases in positive group were given thymopentin 1mg/d for 7 ～14 days,72 patients in treatment group were given deoxyribonucleotide 200mg/d for 7 ～ 14 days.Wounds were observed at 7,14,30 days,respectively.Results Postoperative infection rate(12.1％ vs 16.6％ vs 40％,P ＜ 0.05),pathogenic bacteria detection were decreased by both treatment and control groups.The 14 d complete healing rate (80％ vs 48.3 ％,P ＜ 0.05),weight gain [(2.1 ± 0.7) kg vs (0.6 ± 0.3) kg,P=0.016],clinical effect (89.3％ vs 61.3％,P =0.0018) of treatment group were higher than the control group.The 7 d wound area and complete healing time of treatment group were lower than the control group [(12.6 ±6.8)cm2 vs (18.7 ±7.1)cm2,P =0.018; (18.8 ± 11.2)d vs (21.4 ± 12.6)d,P =0.023].Deoxyribonucleotide can promote fibroblast proliferation and collagen production [(35.4 ± 7.6)ng/L vs (24.4 ± 6.9) ng/L,P =0.006].Conclusions Short-term application of deoxyribonucleotide and thymopentin can decrease postoperative infection through activating immune function.However,wound healing had no relationship with activating immune function seemly.Deoxyribonucleotide promotes wound healing through promoting fibroblast proliferation.

Objective To investigate effect of polymorphism of apolipoprotein E (APOE) gene on early apoptosis of astrocytes after hypoxic injury.Methods Astrocytes separated from APOE wild mice and APOE transgenic mice (ε3,ε4) were primarily cultured,and then purified and identified.Models of astrocyte hypoxic injury were set up by hypoxia.Morphological changes of astrocytes and mitochondria were observed by transmission electron microscope.Early apoptosis rate and changes of mitochondrial membrane potential were detected by flow cytometry.Results Cell foot process tumidness and mitochondria with irregular outline,vacuoles and irregular cristae were observed in each group by electron microscopy at six hours after hypoxia.There were no significant differences of cellular form changes among groups.Early apoptosis rate and decreasing degree of mitochondrial membrane potential in APOFε4 group were significantly higher than those in APOEε3 group and APOE wild group (P ＜ 0.05).Conclusion Compared with astrocytes from APOEε3 group and APOE wild group,mitochondrial membrane potential in astrocytes from APOEε4 group at early period after hypoxia declines more significantly,as may be one of causes for more astrocyte apoptosis.

Objective To investigate the effect of gene therapy which was mediated by electroporation on the expression of basic fibroblast growth factor (bFGF) duning mandible distraction.Methods Bilateral mandibular osteotomies were performed in New-Zealand rabbit.After a latency of 3 days,the mandibles were elongated using distractors with a rate of 0.8 mm/d for 7 days.The rabbits were randomly divided into 5 groups:2 μtg recombinant plasmids pIRES-hVEGF165-hBMP2,pIRES-hBMP2,pIRES-hVEGF165,pIRES and normal saline (NS) were injected into the distraction area of groups A,B,C,D and E,after completion of distraction,respectively.The lengthened mandibles were harvested and processed for immunohistochemical detection of bFGF,and the mean optic densities and integral optical density of bFGF positive cells were measured by computerized image analyzer.Results bFGF mainly located in fibroblasts,giant monocytes,polynuclear phagocytes,osetocytes,and osetoblasts in the connective tissue around bone tissue.The strongest expression was observed at the 7th day,and weakened at 14th day of consolidation stage,there were no significant difference among groups A,B and C,at the 7th day of consolidation.However,there were significant differences between gene therapy groups (A,B and C) and control groups (D and E) (P＜0.01).Conclusions Gene therapy can enhance and prolong the expression of bFGF in distraction gap,which promotes the cell differentiation and proliferation,extracellular matrix synthesis and new bone formation during distraction osteogenesis.This is probably one of the molecular mechanisms of the gene therapy promoting new bone formation in distraction gap.

ObjectiveTo establish a central nervous system axonal mechanical transection model in vitro and observe the axonal regeneration style and speed following the transection. MethodsThe cortical explants the mice were cultured in vitro,of which the axon and dendrite parts were marked using immunofluorescence.The model was built by mechanically transecting the axons and removing the severed axons.The axonal regeneration style was observed by tracing the undeveloped cellular bodies adhering to the residual axonal surface.The growth speed of the regenerated axons and normal axons were measured as well.Results ( 1 ) After mechauically transecting the axons of explants,the axonal regeneration was largely founded at the transected line.The undeveloped cellular bodies went over the transected line and spread to the distal area with axonai regeneration. (2) The axonal growth speed was (118 ± 32) μm/d and (72 ±41) μm/d in the transection group,but was (41 ± 17) μm/d and (32 ± 19) μm/d in the control group at 24 and 48 hours respectively. ConclusionThe regeneration style of the transected axons is the extension of the axonal stumps,rather than sprouting growth,and the growth speed is faster than that of the normal axons.

Objective To investigate the expression patterns of major bone growth factors and bone morphogenetic protein-2 (BMP2) in the distracted calluses following transfected gene during mandibular distraction osteogenesis in a rabbit model.Methods Bilateral mandibular osteotomies were performed in New-Zeland rabbits.After a latency of 3 days,the mandibles were elongated using distractors for 7 days.After the completion of distraction,the rabbits were randomly divided into 5 groups.Three animals each were sacrificed at the end of the delay phase,at 7,14,and 28 days after completion of distraction,respectively.The lengthened mandibles were harvested and processed for immunohistochemical detection of BMP2 expression,the mean optic densities and integral optical density of BMP2 positive cells were measured by computerized image analyzer.Results Elevated cellular expression of BMP2,in the distraction gap,was observed following mandibular distraction.BMP2 staining was mainly located in inflammatory cells,and the connective tissues arrounding the new bone.Their strongest expression was the 7th day,some of those growth factors expressed weakly or negatively.Conclusions Electroporation-mediated gene transfection can promote BMP2 expression effectively,which plays an important role in cell differentiation and proliferation during distraction osteo genesis.The BMP2 stimulates extracellular matrix synthesis,induces the proliferation and differentiation of fibroblasts and osteoblasts,which then promotes the new bone formation and repair.

Objective To establish a model fit for axonal regeneration research after its mechanical injury.Methods Cortical explants from mice were planted on culture dishes by microglass pipettes or routine glass pipettes.The cell body and dendrites in axonal area were detected by immunofluorescence and RT-PCR.Besides,purity of regenerated axons was also tested by immunofluorescence and RT-PCR after mechanical transection of axons under microscopy.Results Compared with explants planting by routine glass pipettes,in the outside 1/2 axons of explants planted by micro-glass pipettes,the immunofluorescence and RT-PCR showed negative nucleus and dendrites.In the regenerated axons following mechanical transection of explants planted by micro-glass pipette,the immunofluorescene and RT-PCR showed no regenerated axons nucleus mixed into the dendrites and nucleus.Conclusions Explants planted by micro-glass pipette obtains enough pure axons and regenerated axons.The establishment of models of axonal mechanical transection lays foundation for its molecular study after trauma.

Objective To investigate the influence of apolipoprotein E gene (APOE) polymorphism on the acute-phase brain electrical activity after mild/moderate traumatic brain injury. Methods The clinical data of 112 patients with mild/moderate traumatic brain injury were collected and the APOE genotypes were identified by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The brain electrical activity in every patient was recorded twice by using electroencephalogram within one week after injury. Qualitative and quantitative methods were used to determine the variations of brain electrical activity. Chi-square test, variance analysis and logistic regression analyses via SPSS version 11.5 were performed among APOE genotypes, electroencephalogram data and clinical data. Results The distributions of APOE genetypes and alleles matched Haldy-Weinberg Law in 112 patients. Of 22 patients with APOEε4, 12 patients (55%) presented with deteriorated electroencephalogram, which was significantly higher than those (16 of 90 patients, 18%) without APOEε4 (P ＜ 0. 01). Comparison of the first and second electroencephalograms demonstrated that the slow waves were increased significantly in patients with APOEε4 ( P ＜ 0. 01 ) but decreased in patients with APOEε2 and APOEε3 (P ＜0.05). The reduction of slow waves in APOEε2 carriers was more obvious than APOEε3 carriers (P ＜0.05). Univariate and multivariate logistic regression analyses showed that APOEε4 was a risk factor to electroencephalogram aggravation after traumatic brain injury. Conclusion APOEε4 is a risk factor to electroencephalogram aggravation during acute stage after mild/moderate traumatic brain injury. However,APOEε2 seems to be beneficial for recovery of brain electrical activity.