OBJECTIVEThe aim of this study is to establish a transgenic mouse model for cleft palate relevant gene thyroid transcription factor-2 (TTF-2), which can be used to study palatal shelf development when the expression pattern and regular activation of TTF-2 is altered.

METHODSThe C57BL/6J mouse TTF-2 gene was cloned through polymerase chain reaction (PCR) from the mouse genomic DNA. The TTF-2 gene was inserted into the expression vector pBROAD3-mcs to construct the recombinant expression vector pBROAD3-TTF-2. This expression vector was then microinjected into the male pronuclei of the fertilized mouse ovum. Thus, the TTF-2 transgenic mice model was established. The genotype of the transgenic mice was identified by PCR and Southern blot analysis. Immunohistochemistry identified the consistent expression of TTF-2 gene during its palatal shelf development.

RESULTSTTF-2 genes were microinjected into 982 fertilized ova. A total of 580 two-cell-stage embryos cultured and transplanted into the oviducts of 48 pseudopregnant female mice. Overall, 68 embryos were obtained for analysis. The genotype of the mice was determined through PCR and Southern blot analysis using genomic DNA extracted from tail biopsies of the transgenic fetus. A total of 13 TTF-2 transgenic mice were detected. The expression of TTF-2 gene during the palatal shelf development of the transgenic mice was consistently detected by immunohistochemistry.

CONCLUSIONThe recombinant expression vector pBROAD3-TTF-2 was integrated into mouse genome through microinjection. The transgenic mouse in the palatal shelf that consistently expressed TTF-2 was successfully established and displayed a cleft palate phenotype.

Animals ; Cleft Palate ; Disease Models, Animal ; Female ; Forkhead Transcription Factors ; Genotype ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Thyroid Gland

BACKGROUND:Half-ring sulcated external fixator is a bone external fixation device.Its structure is relatively simple,with multi-plane fixed,and small occupying space.So far,there are few experimental reports about construction of tibial defect model by it in large animals.OBJECTIVE:To validate the practicality and repeatability of half-ring sulcated external fixator in establishing tibial defect models in goats.DESIGN,TIME AND SETTING:The randomized controlled observation was performed at the Animal Laboratory of the Third Military Medical University from March 2005 to February 2007.MATERIALS:Nine healthy adult(Animal Center of Third Military Medical University);Half-ring sulcated external fixator,made by Professor Li of the Third Military Medical University;Osseous pin,ψ2.5 mm,Shanghai Medicai Instruments Co.,Ltd.METHODS:After animals were anesthetized,two needles were threaded in the same plane of cancellous bone about 1.5-2.0 cm above the superior articular surface of the tibia,named group 1.The angle between the two needles was 40°.One needle was threaded at the place of 3.0 to 5.0 cm under the group 1 and parallel with optional needle of the group 1,named group 2.Needles of group 4 were threaded at the cancellous bone that was 2.0 cm from the joint surface of inferior extremity of the tibia.One needle was threaded at the place of 3.0 to 5.0 cm from group 4 and parallel with optional needle of the group 4.The included angle between these two needles of group 2 and group 3 was 60°.There were totally 6 needles.Tibia was fixed by half-ring sulcated external fixator.According to the length of the tibia from X-ray,tibia and periosteum were amputated by wire saw between the two needles in the middle and inferior segment of the tibia to make segmental bone and periosteal defects of 20 percent of tibia.MAIN OUTCOME MEASURES:①Postoperative general state of the animals and effect of half-ring sulcated external fixator.②Self-repair of bone defects by X-ray and Lane-Sandhu scoring standards.RESULTS:Of the 9 selected goats,1 died of postoperative infection,and 1 developed pinhole infection and supplemented.All the others survived and were included in final analysis.The goats were awake within 2-6 hours after surgery and able to stand up to eat.The loosening nuts were screwed up.No external fixation failure or loosening was found.No skin was necrotic.Injured limb could touch the ground in 2 days,but could not bear load until 2 weeks.The goats could walk in three weeks with a slight limp,then moved freely without claudication in four weeks after the operation.At the 5th week after operation,the radiographs showed little periosteal reaction.At the 10th week after operation,sclerosis was found in the fracture ends,and medullary cavity started to close.Fifteen weeks after operation,the medullary cavity closed and periosteal reaction did not aggravate.Inaddition.Lane score was 0 at any time point.No bone formation was found in the histological examination at 5,10 and 15 weeks after operation,and Lane score was 0 at any time point.CONCLUSION:The goat model established in goat tibia with 20%defects by the half-ring sulcated external fixator shows no bone healing after 15 weeks by X-ray or histological exanimation,indicating this is a practical and repetitive method to establish animal models of large segmental defect for bone tissue engineering.

To investigate the epidemic and evolutionary trends of enterovirus (EV) in the external environment of Yunnan Province, China, molecular typing was performed on 4 EV strains that were isolated from environmental sewage in Yunnan. The VP1 region of isolates was amplified by RT-PCR using universal enterovirus primers, and the amplified VP1 region was sequenced for GenBank BLAST search and genotype analysis. The 4 EV strains were identified as ECHO7. Their nucleotide and amino acid homologies with the VP1 sequences of 68 ECHO7 strains retrieved from GenBank were measured by Mega software analysis. Our findings showed that ECHO7 strains from environmental sewage and population samples were in different evolutionary branches. These strains showed typical geographical and temporal differences; In addition, there were different transmission chains at the same time and in the same area. ECHO7 strains isolated from sewage water and patients with acute flaccid paralysis during the same period in Yunnan belonged to different clusters and evolved at different speeds. Special concerns are needed for this problem. Continuous molecular biological surveillance of human EV in the external environment of Yunnan will provide strong support for early warning of EV diseases.
China ; Databases, Genetic ; Enterovirus ; genetics ; isolation & purification ; Evolution, Molecular ; Humans ; Molecular Epidemiology ; Sequence Analysis ; Sewage ; virology

0.05).Seroquel showed high incidence rate of drowsiness,dizziness and weight gain(26.1%,23.9%,17.3%),chlorpromazine showed high incidence rate of extrapyramidal side effect,pyknocardia and erect collapse(60.9%,39.1%,32.6%).CONCLUSIONS:There was no significant difference of curative effect for the treat-ment of schizophrenia between seroquel-treated group and chlorpromazine-treated group,but seroque showed low side effect and high safety.

Objective To explore the effect of home-based cardiac rehabilitation training on ejection fraction, blood lipid and exercise endurance in patients with coronary heart disease. Methods Randomized controlled trials (RCTs) on the effect of home-based cardiac rehabilitation training on ejection fraction, blood lipid and exercise endurance in patients with coronary heart disease were searched via computer in PubMed, Web of Science, Embase, Cochrane Library database, Wanfang database, CNKI database, VIP database and CBMdisc. After literature screening, data extraction and quality evaluation, Revman5.3 software was used for meta-analysis. Results A total of 11 articles were included. Meta analysis showed that, compared with the conventional treatment group, home-based cardiac rehabilitation training could effectively improve patients' ejection fraction[MD=1.81,95%CI(0.68,2.95),P=0.002],exercise endurance[MD=21.97,95% CI(1.80, 42.14), P=0.03], and reduce cholesterol level[MD=-0.15,95%CI(-0.23,-0.07), P＜ 0.01]. However, there was no significant improvement on high-density lipoprotein, low-density lipoprotein, triglyceride (P＞0.05). Sensitivity analysis showed that the overall results of the study were relatively stable, and individual study had no significant influence on the overall results. Conclusions Home-based cardiac rehabilitation training can significantly improve patients' ejection fraction and exercise endurance and reduce cholesterol level, but in terms of improving patients' high density lipoprotein, low density lipoprotein and triglyceride, more large sample, multi-center long-term studies are needed to verify.

OBJECTIVETo study the major metabolites of antitumor lead compound T-OA (oleanolic acyl-3, 5, 6-trimethyl pyrazine-2-methyl ester) in rat urine, in order to preliminarily infer its metabolic mode in rats.

METHODRat urines of the blank group, the raw material group (ligustrazine TMP and oleanolic acid OA Moore equivalent) and the T-OA group were collected and freeze-dried; Solids were extracted by ethyl acetate; And then the extracts were re-dissolved with acetonitrile. HPLC-HRMS coupling technique was adopted to find the potential mass spectrum peak under ESI(+) (see symbol) ESI(-) modes. Metabolite-related information was obtained by comparing the three groups of spectra.

RESULTOne metabolite of OA and two metabolites of TMP were identified in the raw material group; none metabolite of T-OA but one phase II metabolite was detected in the T-OA group.

CONCLUSIONIt is the first time to identify one phase II metabolite of T-OA and one phase II metabolite of OA were identified in rat urine. On that basis, the researchers preliminarily inferred that T-OA does not show the efficacy in the form of raw material. The HPLC-HRMS method established could be used to identify metabolites of related derivative structures. This paper could also provide certain reference for designing pro-drugs based on oleanolic acid.

Animals ; Antineoplastic Agents ; chemistry ; metabolism ; urine ; Chromatography, High Pressure Liquid ; methods ; Drugs, Chinese Herbal ; chemistry ; metabolism ; Male ; Mass Spectrometry ; methods ; Molecular Structure ; Rats ; Rats, Sprague-Dawley

In order to explore the genotype distribution and molecular evolution of non-polio enterovirus (NPEVs)in Yunnan Province,the People's Republic of China, we sequenced and analyzed the partial VP1 coding region of 105 NPEVs isolated from acute flaccid paralysis (AFP) surveillance in Yunnan province during a 5- year study period from 2006 to 2010. The viral genomes of 105 NPEVs were translated to corresponding amino acid sequences and compared with those of the prototype strains, and the phylogenetic tree was constructed among these VP1 nucleotide sequences and other prototype strains from GenBank. Analysis showed that 18 isolates were classified into 7 serotypes of human enterovirus A species, while 77 isolates into 22 serotypes of B and 10 isolates into 4 serotypes of C species. However, we did not isolate any viruses which belonged to human enterovirus D species. Thus, under AFP surveillance, human enterovirus B species accounted for 73. 3% of the 105 isolates and was considered as the predominant one,followed by human enterovirus A(17. 1%) and human enterovirus C(9. 5%). Phylogenetic analysis showed that various serotypes of the virus and the corresponding prototype strains or other representative strains clustered into the same grooup, however, Yunnan strains and prototype strains were located in the different branches (except CA2,EV90 and EV76). The degree of variation was different even among the same genotype strains. This report showed that different genotype strains spread widely in Yunnan Province.
China ; epidemiology ; Enterovirus ; classification ; genetics ; isolation & purification ; Enterovirus Infections ; epidemiology ; virology ; Humans ; Molecular Sequence Data ; Molecular Typing ; Phylogeny

OBJECTIVETo investigate the relation between different extents of cleft malformation with the speech characteristics in patients with cleft palate.

METHODSThe formant frequency of vowel [i] of 46 incomplete cleft palate patients (ICCP group) and 56 complete cleft palate patients (CCP group) before and after cleft palate repair, as well as 30 normal people (C group), were measured and analyzed on spectrogram.

RESULTSThe comparison of F1 between C group and CCP, ICCP before surgery showed no difference. So did the comparison of F1 between C group and CCP, ICCP after surgery. The comparison of F2 between C group and CCP, ICCP before surgery showed significant difference. The value of the C group was the highest. The value of the ICCP was higher than that of CCP. So did the comparison of F2 between C group and CCP, ICCP after surgery. The comparison of F3 between C group and CCP, ICCP(including before and after surgery) was similar to the results of F2 between the three groups. The comparison of F1 between before and after surgery in ICCP group showed no difference. However, the same kind of comparison of F2 and F3 showed significant differences: Both the values after surgery were higher than those before surgery. The comparison of Fl, F2 and F3 between before and after surgery in CCP group was similar to that in ICCP group.

CONCLUSIONThe extent of the cleft malformation is closely related to the status of the speech in patients with cleft palate. With the malformation more severe, the tongue will move backward more obviously, the elevation of the soft palate after cleft palate repair will be less active. Two ways are recommended for those patients with CCP: (1) Early interceptive orthodontic treatment to reduce the extent of palate malformation; (2) The hard palate repair can be performed prior to the soft palate repair. Patients with severe cleft lip and palate can have hard palate repaired while accepting the early cleft lip repair.

Cleft Lip ; Cleft Palate ; Female ; Humans ; Male ; Palate, Hard ; Speech

OBJECTIVEThis report presented an overview on the epidemiology of enterovirus in Yunnan province, the People's Republic of China.

METHODSA total of 210 strains of non-polioviruses isolated under acute flaccid paralysis surveillance during a 5-year study period from 1997 to 2000 and 2004 were examined. Of the 210 non-polioviruses strains, a total of 12 strains of adenoviruses were serologically identified. The remaining 198 isolates were used for molecular typing, and the viral genomes of 195 nonpolio enteroviruses (NPEVs) were translated to corresponding amino acid sequences and compared with those of the prototype strains.

RESULTSBased on molecular typing, 5 isolates were classified into 5 serotypes of human enterovirus A species while 158 isolates into 34 serotypes of B and 32 isolates into 6 serotypes of C species. However, we did not isolate any viruses which belonged to human enterovirus D species. Thus, under acute flaccid paralysis surveillance, human enterovirus B species accounted for 75.2% of the 210 isolates and was considered as the predominant one, followed by human enterovirus C (12.2%), adenovirus (5.7%), and human enterovirus A (2.4%).

CONCLUSIONAlthough the epidemiological characteristics of NPEVs from Yunnan province remained "unknown", the molecular typing method had provided us a breakthrough to understand the epidemiology of these viruses.

China ; epidemiology ; Enterovirus ; classification ; genetics ; isolation & purification ; Enterovirus Infections ; epidemiology ; Genes, Viral ; Humans ; Molecular Epidemiology ; Serotyping

OBJECTIVETo observe the effect of dexamethasone on the proliferation and apoptosis of embryonic palatal mesenchymal (EPM) cells, and chose a proper concentration of dexamethasone which can effect the ordinary growth of embryonic palatal mesenchymal cells.

METHODSThe primary EPM cells were isolated and cultured in vitro, then we did biological assay. EPM cells were treated with different concentration dexamethasone (1 x 10(-9), 1 x 10(-8), 1 x 10(-7) and 1 x 10(-6) mol x L(-1)) respectively. The proliferation of EPM cells was evaluated using MTT method. Apoptosis was examined quantitatively with fluorescein stain.

RESULTSIn the condition of blood serum's concentration at 10%, optical density step down following the raise of dexamethasone's concentration. The effect of dexamethasone got to a summit at 3 days. Inhibition rate of dexamethasone at 1 x 10(-6) mol x L(-1) was the highest.

CONCLUSIONDexamethasone at 1 x 10(-6) mol x L(-1) can not only inhibit the growth of the EPM cells, but also will not lead to a large number of cells death. Therefore, this concentration can be used as a reference standard in future research. The most significant drug action time of dexamethason appears at the third day after administration, then the effect became weaken following the drug metabolism.

Animals ; Apoptosis ; Cell Proliferation ; Cells, Cultured ; Dexamethasone ; Humans ; In Vitro Techniques ; Mesenchymal Stromal Cells ; Mice