ObjectiveTo study the effect of testing amino acids levels in brain microdialysate by method of high performance liquid chromatography (HPLC) with α-aminobutyric acid (AABA) as the internal standard.MethodsReversed phase high performance liquid chromatography (RP-HPLC), orthophthaladehyde (OPA) precolumn derivatization method and fluorescence detector were used. The separation was performed using an ODS-C18 column. AABA, an isomeric compound of GABA, served as the internal standard, phosphate buffer solution and methanol as the mobile phase, gradient elution lasted in 35 min.Results7 kinds of amino acids and internal standard were separated completely. The mean recovery of amino acid was 88.2%—102.3%. The mean correlation coeffeicient of the linear relationship was 0.998±0.0015.Limits of detection for amino acids were 1.0—8.6 ppm respectively.ConclusionThe method mentioned above is simple and sensitive for operation, and can be used for determined levels of amino acids in microdialysate.

Objective To study the feature that murine cytomegalovirus(MCMV)infect macrophage strain RAW264.7 and the influence of virus infection on proliferation and apoptosis of RAW264.7 in vitro.Methods RAW was infected by MCMV Smith with multiplicity of infection(MOI)1,0.1 and 0.01,respectivelv.The cells and culture supernatant were collected at 6,12,24,36,48,72,96 and 120 h post-infection(P.i.).Cytopathic effect(CPE)was found with microscope.Virus particles and uhrastructural changes of RAW were observed by transmission electron microscope(TEM). Early antigen(EA)expression was assaved bv immunohistochemical method.The proliferation of MCMV was studied by plaque formation assay.The influence of virus infection on proliferation and apoptosis of RAW were measured by MTT method and flow cytometry.The mouse embryo fibroblast(MEF)susceptible to MCMV infection was positive contro1.Results RAW was swollen and desquamated on 24-48 h P.i..The full-grown virus particles and swollen organelles in RAW were displayed with TEM.Preliminary positive expression of EA was demonstra ted from 6 h(MOI=1 and 0.1)to 12 h(MOI=0.01)P.i..Virus titer in RAw supernatant increased obviouslv on 24 h p.i.and reached the peak on 96-120 h P.i..The proliferation of RAW could be obviously inhibited by MCMV on 72-120 h p.i..When infected by virus with MOI=0.1,necrotic cells of RAW increased on 72-120 h D.i.and the influence of MCMV infection on apoptosis of RAW was not obvious.Conclusion Macrophage strain RAW264.7 is susceptible to MCMV,and it emerges faster cytolytic and productive infection than MEF.MCMV can inhibit the proliferation of RAW but not influence the apoptosis of it.These results can provide a practical experimental model for studying immunological pathogenic mechanism of cytomegalovirus in vitro.

Objective To investigate the influence of murine cytomegalovirus(MCMV) infection on differentiation and differentiation gene expression of neural stem cells (NSCs) in vitro for studying the mechanisms of brain abnormalities calmed by congenital cytomegalovirns infection. Methods NSCs were separated from fetal BALB/c mouse and cultured and identified in vitro. The differentiation potency of NSCs was observed by immunnfluorescence. The NSCs infected by MCMV at dosage of multiplicity of infection (MOI) equaled to 5, I and 0. 1, respectively, were cultured in differentiation medium. The morphological changes of the cells were observed by inverted microscope. The ratios of NSCs and its differentiated cells were detected by flow cytometry. The expression changes of nestin, GFAP and NSE, markers of NSCs and its differentiated cells, were studied by immunofluorescence ( MOI = 1 ). The expression of early antigen (EA) of MCMV was detected to observe the infection process. Real-time RT-PCR method was employed to measure the expression levels of the key differentiation genes Wnt-3 and Wnt-7a in Wnt signal pathway of NSCs at early phage of differentiation culture. Results NSCs isolated from embryonic mouse brains could proliferate to form neurnspheres and strongly express Nestin and differentiate into NF-200 positive neurons or GFAP positive astrocytes. The NSCs of the infected groups couldn't adhere to the wall and appear differentia-tion growth, but showed swollen gradually after differentiation culture. The nostin expression of the infected groups downregulated slowly and was higher than that of the control groups ( P < 0.05 ). The GFAP and NSE expression of the infected groups were lower than that of the control groups (P <0.05). The EA of MCMV could be always detected in the cells of the infected groups. The ratios of nestin positive cells of the infected groups were higher than that of the control groups, but the ratios of GFAP and NSE positive cells of the for-mer were lower than that of the latter from 3rd to 9th day after differentiation culture ( P < 0.05 ). The levels of Wnt-3 mRNA and Wnt-7a mRNA of the infected groups were markedly lower than that of the control groups from 1st to 2nd clay and from 12th hour to 2nd day after differentiation culture respectively ( P < 0.05 ) . These changes of the infected groups became more obvious as MCMV MOI increased . Conclusion MCMV could inhibit significantly NSCs differentiate to neurons and astrocytes and lead to the decrease of dif-ferentiated cells. MCMV could inhibit or interfere with the gene expression of Wnt-3 and Wnt-7a in Wnt sig-nal pathway of NSCs. The effect that MCMV inhibited the differentiation and the differentiation gene expres-sion of NSCs showed dose-dependent with MCMV MOI. The inhibitory effect of MCMV on the differentiation of NSCs might be induced by interfering the differentiation gene expression of NSCs, which is possibly the one of primary causes of brain development disorders caused by congenital CMV infection.

Objective To investigate the biological function of M122 in pathogenesis of MCMV in developmental brain disorders and brain damage, screening for mouse brain cDNA library interacting with M122 was performed by a yeast two-hybrid system. Methods The reconstructed bait plasmid pGBKT7-M122 was transformed into yeast cells AH109 and screened on the nutrient deficiency medium SD/-Trp. After express of the bait protein in AH109 yeast strains was detected by Western blot analysis, yeast-two hybrid screening was performed by mating AH109 with Y187 containing mouse brain cDNA library plasmid. The diploid yeast cells were plated on the nutrient deficiency medium SD/-Trp/-Leu/-His/-Ade. The second screening was performed with SD/-Trp/-Leu/-His/-Ade containing X-α-gal. The plasmids in positive colonies were extracted and transformed into E. coli JM109 cells. After plasmid DNA in JM109 cells were extracted form positive colonies and sequenced, the results were analyzed by bioinformatic methods. The interactions between M122 protein and the protein obtained from positive colonies were further confirmed by repeating yeast-two hybrid. Then, autoactivations of the proteins obtained from positive colonies were detected.Results The reconstructed bait plasmid was transformed into yeast cells AH109 successfully. The bait protein expressed in the yeast cells AH109 stably. 24 proteins interacting with MCMV M122 were screened, including syntaxin 8 ( Stx8 ), phosphoglucomutase 2 ( Pgm2 ), potassium voltage-gated channel, shaker-related subfamily, beta member 1 ( Kcnab1 ), collagen, type ⅪⅩ, alpha 1 ( Col19a1 ), archain 1 ( Arcn1 ), cytidylate kinase( Cmpk), DnaJ(Hsp40) homolog, subfamily A, member 1 (Dnaja1), ATPase, Na+/K + transporting, beta 3 polypeptide( Atp1b3 ), SH3-domain GRB2-like ( endophilin ) interacting protein 1 ( Sgip1 ),ankyrin repeat domain 17 (Ankrd17), Smg-7 homolog, nonsense mediated mRNA decay factor(Smg7),sperm associated antigen 9 ( Spag9 ), FK506 binding protein 1a ( Fkbp1a), MYST histone acetyltransferase monocytic leukemia 4 ( Myst4), hyaluronan and proteoglycan link protein 1 ( Hapln1), autophagy-related 3 (Atg3), splicing factor, arginine/serine-rich 5 ( Sfrs5 ), zinc finger, C3HC-type containing 1 ( Zc3hc1 ),thioredoxin-related transmembrane protein 1 ( Txndc1 ), adaptor protein complex AP-1, gamma 1 subunit (Ap1g1), Cullin 1 ( Cul1 ), and so on. Three of them were formerly unknown proteins. M122 protein could interact with the proteins obtained from positive colonies in the yeast cells AH109. Ap1g1 and Cul1 were proved to have autoactivation. Conclusion A class of proteins in brain interacting with M122 has been obtained. It is presumed that these proteins are correlated with neuropathogenesis of the brain disorders caused by CMV, but the candidates still need further confirmation for the interaction.

Objective To investigate the risk factors of electrocoagulation syndrome after endoscopic submucosal dissection (ESD) in patients with colorectal lesions.Methods Clinical data of 145 patients with colorectal mucosal lesions undergoing ESD in People's Hospital of Wuhan University between September 2010 and September 2015 were retrospectively studied.Results Among 45 patients,post endoscopic submucosal dissection electrocoagulation syndrome (PEECS) was developed in 32 cases (22％).The median age in PEECS group was higher (t =-5.783,P =0.000),the median lesion size was larger(t =-5.590,P =0.000),the median length of hospital stay was longer (t =-6.841,P =0.000) than those in non-PEECS group.Univariate regression analysis showed PEECS was associated with the age,lesion size,lesion location,length of hospital stay,malignant tumor,polyps type,resection modality.Multivariable logistic regression analysis showed that the independent risk factors for the development of electrocoagulation syndrome were age ＞65 year (OR =1.123,95％ CI:1.013-1.244,P =0.027),lesion size ＞ 3.5 cm (OR =1.173,95％ CI:1.015-1.357,P =0.031),malignant tumor (OR =3.498,95 ％ CI:1.460-8.379,P =0.005),hospital stay ＞ 10 d (OR =2.480,95％ CI:1.346-4.569,P =0.004),non-rectal lesions (OR =12.612,95％ CI:3.446-46.157,P =0.000).Conclusion Attention should be paid for colorectal lesion patients with high risk of PEECS,when endoscopic submucosal dissection is performed.

Objective:To investigate the feasibility and safety of endovascular repair for traumatic thoracic aortic pseudoaneurysm.Methods:From Oct 2015 to Oct 2018, the clinical and followup data of 7 patients diagnosed as traumatic thoracic aortic pseudoaneurysm in Fuwai Hospital of Chinese Academy of Medical Sciences were analyzed retrospectively.Results:The patients average age was (51.2±11.0) years old. All patients underwent surgery in the hybrid operating room under general anesthesia. Two did thoracic endovascular aortic repair (TEVAR), three did TEVAR combined with chimney technique to reconstruct the left subclavian artery, and 1 had TEVAR combined with fenestration to reconstruct the left subclavian artery. One did TEVAR with left common carotid artery and left subclavian artery bypass. The mean operative time was (90.1±27.4) min, the mean postoperative hospital stay was (8.9±3.7) d, and the mean postoperative follow-up time was 42.4 months. All the patients received CTA reexamination of the aorta after 1, 6, 12 months and yearly thereafter. TypeⅠendoleak was found in one patient with chimney technique to reconstruct of left subclavian artery after operation. CT showed that the type Ⅰ endoleak disappeared 6 months after operation. There was no death, paraplegia or stroke during the perioperative period and follow-up period, and there was no aortic related reintervention.Conclusion:TEVAR is a safe and effective method for the treatment of traumatic pseudoaneurysm of thoracic aorta, and the early and mid-term results were satisfactory.

Objective:To evaluate the safety and feasibility of the in-situ needle fenestration combined with the in vitro physician modified fenestration technique to reconstruct supra-aortic branches during thoracic endovascular aortic repair (TEVAR) for aortic arch lesions requiring landing at Z0 and Z1.Methods:From Nov 2017 to Dec 2019, eighteen patients who underwent both the in-situ needle fenestration and the in vitro physician modified fenestration techniques to extend the proximal landing zone to Z0 and Z1 during TEVAR were included in our study.Results:Sixteen patients underwent in vitro physician modified fenestration ,two patients underwent in vitro physician modified fenestration to reconstruct both the left common carotid artery and the innominate artery. All eighteen patients received in-situ needle fenestration to preserve the left subclavian artery. Supra aortic branches were preserved in all patients (38/38, 100%). There was no Type Ⅰ endoleak. Type Ⅱ endoleak was found in four paitnets (4/18). Type Ⅲ endoleak occurred in one patient (1/18). Type Ⅳ endoleak in four patients (4/18). Type Ⅲ endoleak needed open aortic arch repair 6 months later. The median follow-up time was 12 months. One (1/18) died in 12 months and the other patients were doing well.Conclusions:The joint application of the in-situ needle fenestration and the in vitro physician modified fenestration to reconstruct supra-aortic branches during TEVAR for aortic arch pathologies requiring landing at Z0 and Z1 was satisfactory.

Objective To investigate the iodine nutritional status of targeted population in the high-risk areas of iodine deficiency disorders in Chongqing, so as to provide scientific evidence for establishing prevention and remedial measures. Methods Six towns were selected in Chengkou and Wuxi Counties to found suspected dementia patients born after first Jan, 1997. Two hundred children aged 8-10 years were investigated in every town. The thyroid volume, intelligence quotient(IQ) and urinary iodine of the children were examined. Forty women (pregnant and nursing women) were investigated in every town. The iodine content of salt from their home was measured. The thyroid volume was examined by palpation and B-uhrasound. IQ was measured by combined Raven Test in China(CRT-RC2). Urinary iodine was determined using the acid digest arsenic-cerium contacting method, and iodined salt was detected using direct titration method. Results Six suspected dementia patients were found in the local town hospital. Five eases were excluded. There was 1 case born in other place. The rates of goiter by palpation and B-ultrasound were 9.58%(92/960) and 8.89%(65/731), respectively. The median of urinary iodine of children and women was 319.15 μg/L and 248.42 μg/L, respectively. The mean of IQ of the children was 103.32. The coverage rate of iodine salt from residents was 98.82%(336/340). Conclusions The iodine nutrition of children was good and there is no newly occurred cretinism in Chengkou and Wuxi Counties. Goiter rate and median of urinary iodine aged 8-10 years and of women, coverage rate of iodine salt from resident has meet the standard set for basical elimination iodine deficiency disorders.

AIM: The influence of MCMV infection on differentiation and differentiation gene expression in neural stem cells ( NSCs) in vitro were investigated for studying the mechanisms of brain abnormalities caused by congenital cytomegalovirus infection. METHODS: NSCs were separated from fetal BALB/C mouse, and cultured and identified in vitro. The differentiation potency of NSCs was observed by immunofluorescence. The NSCs infected by MCMV at dosage of MOI( multiplicity of infection) equaled to 5, 1 and 0.1 .respectively, were cultured in differentiation medium. The morphological changes of infected cells were observed under inverted microscope. The ratios of NSCs and its differentiated cells were detected by flow cytometry. The expressions of nestin, GFAP and NSE, markers of NSCs and its differentiated cells, were studied by immunofluorescence( MOI = 1). The expression of early antigen ( EA ) of MCMV was detected to observe the infection process. Real - time RT - PCR method was employed to measure the expression levels of the key genes Neurog2, Myc and Ccnd1 in Wnt signal pathway of NSCs at early stage of differentiation culture. RESULTS: NSCs isolated from embryonic mouse brains proliferated to form neurospheres, strongly expressed nestin and differentiated into NF - 200 positive neurons or GFAP positive astrocytes. The infected NSCs did not adhere to the wall and appeared differentiation growths, but showed swollen gradually after differentiation culture. The nestin expression in the infected cells downregulated slowly and was higher than that in control groups ( P < 0.05). The GFAP and NSE expressions of the infected cells were lower than those in control groups ( P <0.05). The early antigen ( EA) of MCMV was always detected in the cells in infected groups. The ratios of nestin positive cells in infected groups were higher than those in control groups, but the ratios of GFAP and NSE positive cells of former were lower than that of the latter from 3rd to 9th d after differentiation culture(P < 0.05 ). The levels of Neurog2 mRNA and Myc mRNA in infected groups were markedly lower than those in normal control groups on 1st d and from 1st to 4th d after differentiation culture, respectively( P <0.05). The levels of Ccnd1 mRNA of infected groups were obviously lower than those in normal control groups from 12th h to 1st d( P <0.05 ). These changes in infected groups became more obvious as MCMV MOI increased. CONCLUSION: MCMV significantly inhibits differentiation of NSCs to neurons and astrocytes, and leads to the decrease in differentiated cells. MCMV inhibits or interferes with the gene expression of Newog2, Myc and Ccnd1 in Wnt signal pathway of NSCs. The effect that MCMV inhibits the expressions of differentiation and the differentiation genes in NSCs shows dose - dependent with MCMV MOI. The inhibitory effect of MCMV on the differentiation of NSCs might be induced by interfering with the expression of differentiation gene in NSCs, which is possibly the one of primary causes of brain development disorders induced by congenital CMV infection.

ObjectiveTo analyze the clinicopathologic features of hereditary cutaneomucosal venous malformation (VMCM) in a Chinese family.MethodsFamily history was investigated in a family with VMCM,and tissue specimens were obtained from the lesions of the proband and subjected to histopathological analysis.ResultsAmong 65 members from 5 generations of the family,19 were affected by VMCM,hinting an autosomal dominant inheritance.None of the family members experienced gastrointestinal bleeding,central nervous system disorders,or cardiac defects.Affected individuals usually presented with multiple irregularly sized,blue-violet,elevated and slightly indurated masses located in the oral mucosa and subcutaneous tissue of the extremities.Pathological analysis showed malformed veins with abnormally dilated cavities and irregularly thickened walls.Although small veins were abnormally proliferating and clustered,there was no endothelial discontinuity.The smooth muscle layer was thickned in a varying degree or absent.ConclusionA diagnosis of VMCM is made according to the inheritance manner,clinical manifestation and pathological findings.