Acellular embryoid bodiesin mice：preparation and effect of promoting differentiationofLewis lung carcinoma cells / 中国组织工程研究

BACKGROUND:Co-culture withembryonic stem cels or embryonic tissues can induce differentiation of carcinoma cels into normal epithelial cels or decreasemalignancyof carcinoma cels.Acelular embryoid bodies retain the structure and important cytokines of embryonic tissues. OBJECTIVE:To prepare acelular embryoid bodies from mouse embryonic stem cels and to investigate their effects on differentiation of mouse Lewis lung carcinoma cels at three-dimensional culturein vitro. METHODS:Mouse embryonic stem cels(D3)were dynamicaly cultured for 7 days to produce embryoid bodiesfolowedbydecelularization with 0.1% sodium dodecyl sulfate. Mouse Lewis lung carcinoma cels were co-cultured with acelular embryoid bodiesas test group or culturedinthree-dimensionalmatrigel mediumfor 7 days as control group, respectively. Cel proliferation and expression of E-cadherin were detected by immunohistochemical staining and western blot assay, respectively. In addition, mRNA expressions ofSlug and E-cadherin were observed using RT-PCR technology. RESULTSAND CONCLUSION:Uniform mouse embryoid bodieswere successfuly prepared, andwere completely decelularized with sodium dodecyl sulfate. After 7-day three-dimensionalmatrigelculture, in the control group,multicelular tumor spheroidswere formed,accompanied byahigherKi67positive rate;Lewis lung carcinoma cels in the test group were repopulated in the acelular embryoid bodies showing significantly lowerKi67positive rate. Compared with the control group, the absorbance ofPaxilin in the test group was significantly smaler, and the absorbance of E-cadherin was significantly higher (P< 0.05). Besides, mRNA expressions of Slug and E-cadherin were significantly decreased and increasedin the test group compared with the control group, respectively(P< 0.05). These findings indicate that the acelular embryoid bodies can promote differentiation of mouse Lewis lung carcinoma celsinthree-dimensional culturein vitro.

Analysis on common faults of clinical laboratory equipment on hospital ship / 医疗卫生装备

The clinical laboratory equipment on hospital ship was introduced, whose status and common faults were analyzed during Philippines humanitarian medical rescue mission and 3 times of Mission of Harmony. The experience was summarized for the maintenance of the clinical laboratory equipment. The problems and countermeasures were proposed for the clinical laboratory equipment.

A study of relationships between community acquired pneumonia patients with different syndrome patterns of traditional Chinese medicine and serum levels of C-reactive protein and mannose-binding lectin / 中国中西医结合急救杂志

Objective To study the regularity changes in serum levels of C-reactive protein(CRP)and mannose-binding lectin(MBL)in patients of community acquired pneumonia(CAP)with different syndrome patterns of traditional Chinese medicine(TCM),and to explore the new objective markers to differentiate the syndrome patterns of TCM. Methods According to The Guideline on TCM diagnosis and treatment of CAP(2011 edition),104 patients with CAP were selected and their syndromes were cassified into 3 classes and 8 patterns of syndrome:excessive class〔including following patterns:wind-heat invading lung(fengrexifei),exopathic cold and interior heat(waihanneire), accumulation of heat in lung(tanreyongfei),accumulation of phlegm-dampness in lung(tanshiyongfei)〕,deficient vital QI leading to lingering of pathogen class〔qi deficiency of lung and spleen(feipiqixu),both qi and yin deficiency (qiyinliangxu)〕,TCM critical class〔heat falling into pericardium(rexianxinbao),pathogen invasion and vital qi deterioration(xiexianzhengtuo)〕. In the same period,after physical examinations,100 healthy volunteers were chosen as healthy control group. The serum levels of CRP and MBL were detected before treatment and after treatment for 4 days and 7 days. Results Among the 104 CAP patients,the most popular class of syndrome was the excessive one(63.5%),followed by deficient vital QI leading to lingering of pathogen(19.2%)and TCM critical class(17.3%). The serum CRP level in CAP patients at each time point was higher than that in healthy control group,which had a different tendency to change over time in different syndrome patterns of TCM. With the prolongation of treatment time,the serum CRP levels in fengrexifei and waihanneire patterns returned to a normal level on the 7th day(mg/L:13.51±11.48,7.07±1.84 vs. 6.96±2.19,both P>0.05),in feipiqixu and qiyinliangxu patterns the CPR levels were higher,but its descent rate was relatively fast,and on the 7th day it was approximately normal in spite of being higher than the level in healthy control group(25.25±25.90,18.17±23.19 vs. 6.96±2.19,both P<0.05);in tanreyongfei and tanshiyongfei patterns,although the CPR levels were decreased,they still maintained at relatively high levels on the 7th day after treatment(51.70±27.33,49.28±30.57),and no downtrend of CPR was seen in rexianxinbao and xiexianzhengtuo patterns. Before treatment,the serum MBL levels in CAP patients with fengrexifei,waihanneire, tanreyongfei,tanshiyongfei,feipiqixu and qiyinliangxu patterns were higher than the level in healthy control group, and in rexianxinbao and xiexianzhengtuo patterns,the levels were lower than those in other patterns and kept being at relatively lower levels along with the prolongation of the therapy. Conclusion Serum CRP can be used as a reference marker for different syndrome patterns of TCM in patients with CAP,and low serum MBL level was a risk factor of severe syndrome patterns of TCM and a poor prognosis in CAP.

The role of hepatic stellate cells SSeCKS expression in liver fibrous / 中华内科杂志

Objective To investigate the change and effect of SSeCKS(src suppressed c kinase substrates)in the activation of hepatic stellate cells(HSCs).Methods HSCs were isolated from normal rats,the change of SSeCKS mRNA expression on HSCs culture in vitro was determined using real.time PCR.protein level was determined by Western blot and immunofluorescence methods.A rat model of liver fibrosis was established.The expression and location of SSeCKS and α-SMA(α-smooth muscle actin)in liver tissues were detected by immunofluorescence methods.Results SSeCKS mRNA expression WaS loW in freshly isolated HSCs cell and the expression increased in activated HSCs in vitro.In liver fibrosis tissue,the number of SSeCKS-positive cells was increased and these cells were distributed along the sinusoids which also contained α-SMA positive cells.Conclusion The expression of SSeCKS was increased in activated HSCs in vitro.Therefore.SSeCKS may be involved in the liver inflammation and fibrosis.

Observation of in Vitro Antibacterial Activity of Gymnema sylvestre Extractant / 中国中医药信息杂志

Objective To evaluate invitro antibacterial activity of Gymnema sylvestre extractant.Methods The antibacterial activity of Gymnema sylvestre extractant against staphyloccus aureus,bacillus coli,bacillus proteus,bacillus aeruginosus was detected by agar dilution method.Results The minimum inhibitory concentration were 0.65,1.25,1.25,2.5 mg/mL respectively.Conclusion Gymnema sylvestre extractant had strong antibacterial activity against various bacteria.

Construction of eukaryotic expression vector of human antimicrobial peptide LL-37 / 临床检验杂志

Objective To construct eukaryotic expression vector of human antimicrobial peptide LL-37 pPIC9-LL-37, and transform the plasmid pPIC9-LL-37 into P.pastoris GS115 to obtain the recombinant P.pastoris strains.Methods The full-length of antimicsobial peptide LL-37 gene was artificially synthesized by overlap extension method and was fused to pPIC9 and then the fused plasmid was transformed into E.coli DH5?.After analysis by PCR and sequencing,the plasmid pPIC9-LL-37 was transformed into P.pastoris.The colonies exhibiting the phenotype of His+Mut+ or His+Mut-were screened by means of MM and MD plates and the insertion was confirmed by PCR.Results The results of PCR and sequencing confirmed that the LL-37 gene was correctly inserted into pPIC9. The colonies of 10 His+Mut+ and 9 His+Mut-were obtained by means of MM and MD plates screening and were confirmed by PCR.Conclsion The recombinant P.pastoris strains containing LL-37 were successfully obtained.

Modification of 5′-UTR sequences of pPIC9 increases expression of antimicrobial peptide LL-37 / 第二军医大学学报

Objective:To study the influence of 5′-untranslated region modification of pPIC9 on expression of LL-37 in Pichia pastoris.Methods:The sequence GGATCCAA was deleted from 5′-UTR of pPIC9 and the modified product was trans- formed into E.coli DH5?to construct a modified eukaryotic vector pPIC9-EDIT.After PCR and sequencing,pPIC9-EDIT was ligated with LL-37 sequence coded by the biased codon of yeast,the product was then transformed into E.coli DH5?to con- struct the recombinant expression vector pPIC9-EDIT-LL-37,the latter was transformed into P.pastoris GS115 by spheroplas- ting and the insert was confirmed by PCR.The bacteriolytic activity to E.coli.DH5?was analyzed to screen the highest ex- pressing strain and to determine the best inducing time and concentration of methanol.The fermentation product was analyzed by Tricine-SDS-PAGE and Western blotting.The antibacterial activities of expression products of pPIC9-LL-37 and pPIC9-ED- IT-LL-37 were compared,and the changes of LL-37 protein expression were determined before and after modification.Results: pPIC9-EDIT and pPIC9-EDIT-LL-37 were successfully constructed.Expression of LL-37 gene was confirmed by PCR in P.pastoris after pPIC9-EDIT-LL-37 transformation.The highest expressing strain was identified;the best inducing time was 72 h and the best concentration of methanol was 0.5%.Tricine-SDS-PAGE and Western blotting analysis showed that the ex- pression product was LL-37.The expression level of LL-37 protein increased by 35 times after modification.Conclusion:Modifi- cation of pPIC9 5′-UTR can obviously improve expression of LL-37 protein in P.pastoris;it is worth to be used in the research of other heterogenous protein.