Objective To analyze the mutations in nucleotide sequences of transfusion transmitted virus(TTV) in neonatal infection.Methods Neonatal serum TTV-DNA was detected by a nested PCR technique.Fifteen Chinese neonates with positive TTV-DNA were diagnosed as TTV infection.ORF1 sequences of TTV-DNA from these neonates were determined.Results Homology of Chinese TTV(C01-C15) and Japanese TTV(N22)isolated ranged from 87.1%-97.7% at nucleotide level,but there were point mutations in Chinese TTV,such as GG→TT in locus 112 and 113,TTATC→CCTAT in locus 236-240.Conclusions Chinese and Japanese TTV isolated had the same genotype.Some gene mutations may increase the TTV pathogen,and result in neonatal hepatitis syndrome or hyperbilirubinemia.

Inheirted metabolic disodrers(IMD)involves in multiple substance dysbolism,which usually results in irreversible neurological lesions because of various categories and complicated clinical manifestations.In resent years,IMD became one of the hot spots in medical domain around the world,original diagnostic technique and management progressed unceasingly.This paper provides an overview of the traditio-nal detection and treatment about IMD,and reviews the new techniques such as gene analysis,gene chip,organ transplantation and enzyme replacement therapy at the same time.

The major goal of microbial ecology is to study the structure and function of complex micro-bial communities. New molecular biological techniques have been successfully applied to analyze mi-crobial community structure. However they do not provide information on the physiologic properties of the detected microorganisms. A new tool for structure-function analyses in microbial ecology, micro-autoradiography combined with fluorescence in situ hybridization (MAR-FISH) can be used to simul-taneously examine the phylogenetic identity and the specific activity of microorganisms within a com-plex microbial community at a single-cell level. This article reviews the principle, experimental steps of MAR-FISH technique. The application of this technique in study of the environmental microbial com-munity and function is also summarized.

Objective To compare the difference of early trauma experience between early-onset and late-onset obsessive-compulsive disorder(OCD),and the kinds of early trauma in the cause of OCD. Methods Three hundred and twenty-six patients with obsessive-compulsive disorder and 480 healthy con-trols were enrolled. Early Trauma Inventory-Short Form ( ETI-SF) was applied to assess the early trauma ex-perience,compare the difference of early trauma experience between early-onset ( n=138) and late-onset ( n=188) OCD,and the kinds of early trauma in the cause of OCD. Results There were significant differences in the early trauma experience between obsessive-compulsive disorder group and control group((3.90±3.61) vs (1.88±2.61), P<0.01).Scores of physical((1.12±1.38) vs (0.71±1.23), P=0.001),emotional((1.58± 1.71) vs (0.42±1.01), P<0.01),and sexual abuse((0.27±0.59) vs (0.09±0.36), P=0.001),and a gener-al traumatic experience((0.94±1.17) vs (0.66±1.09), P<0.01) were significant difference between patients and controls. Except sexual abuse((0.30±0.66) vs (0.24±0.67), P=0.42),there were significant differ-ences in the early trauma between early-onset and late-onset OCD((5.12±3.58) vs (3.01±3.38), P<0.01), especially emotional abuse((2.18±1.76) vs (1.12±1.53), P<0.01).247(75.8%) OCD patients and 79(57. 9%) healthy controls experienced early trauma(χ2=21.48, P<0.01). Experiencing one kind of early trau-ma,the prevalence of OCD was 39.1%,two kind was 61.0%,three kind of early trauma was 65.8%,and ex-periencing four or more kind of early trauma,the prevalence of OCD was up to 84.4%. Conclusion OCD patients have much more early trauma,especially early-onset OCD. Experiencing more kind of early trauma, and the prevalence of OCD higher may associated with the development of obsessive-compulsive disorder. It is important to consider the role of childhood trauma in the prevention and treatment of OCD.

Background Diabetes is one of the risk factors that leads to corneal neuropathy.Silent signal regulatory factor 1 (Sirt1) plays an important role in glucose metabolism,lipid metabolism,regulation of insulin secretion and is closely related to the nervous system disease.The relationship between Sirt1 and diabetic corneal neuropathy is not fully understood.Objective This study was to detect the expression of Sirtl in cornea and trigeminal ganglion with type 1 diabetes model mice and explore the association of Sirt1 expression with diabetic corneal neuropathy.Methods Eight C57BL/6-Ins2Akita/J male mice and eight wild-type C57BL/6 male mice in the same litter were selected as type 1 diabetes model group and control group,respectively.The mice of two groups were sacrificed in overdose anesthesia method at 12-month old.Histological examination of cornea and trigeminal ganglion was performed using hematoxylin and eosin staining.Expression and localization of Sift1 protein in cornea and trigeminal ganglion were detected using immunohistochemistry.Western blot assay and fluorescine quantitative PCR were respectively used to quantitatively analyze the expression of Sirt1 protein and Sirt1 mRNA.Results Trigeminal ganglion cells were uneven in size and shape with the loosened cellular arrangement and disorder neurofibrosis alignment,and the corneal epithelial cells were less in the C57BL/6-Ins2Akita/J mice,but the trigeminal ganglion cells and corneal epithelial cells were normal in wild-type C57BL/6 mice.Immunochemisty exhibited that Sirtl protein was expressed mainly in corneal epithelium and the expression of Sirtl protein was stronger in the C57BL/6 mice than that in C57BL/6-Ins2Akita/J mice.Fluorescine quantitative PCR assay showed that the gray scale value of Sirt1 mRNA in cornea in C57BL/6-Ins2Akita/J mice was lower than that of the wild-type C57BL/6 mice(0.56±0.03 vs.0.98±0.13) with significant difference (t =5.853,P =0.010).Western blot showed that the expression of Sirt1 protein in cornea was lower in C57BL/6-Ins2Akita/J mice than that of the wild-type C57BL/6 mice(0.78±0.017 vs.1.300±0.012) with significant difference(t =33.140,P =0.001).However,no significant differences were seen in the gray scale value of Sirt1 mRNA(2.45±0.18 vs.2.51±0.22) (t=0.587,P=0.599) and protein level(1.100±0.015 vs.1.110±0.017) (t =0.430,P=0.709) in trigeminal ganglion tissues between C57BL/6-Ins2Akita/J mice and wide-type C57BL/6 mice.Conclusions The corneal nerve and structure is abnormal in 12-month-old C57BL/6-Ins2Akita/J mouse.Sirt1 is involved in the pathogenesis of diabetic keratoneuropathy,suggesting that it may be a potential target.

Objective:To construct an eukaryotic expression vector of enhanced green fluorescence protein(EGFP) gene driven by telomerase catalytic subunit(hTERT) gene promoter and observe the specific expression of EGFP in lung cancer cell lines.Methods:The 1100bp promoter fragment was obtained by enzyme digestion from a recombinant plasmid of pGL3-hTERTp containing the hTERT promoter.The hTERT promoter was then subcloned into the upstream of the report gene EGFP of pEGFP-1 without promoter.The expression vector pEGFP-hTERTp was successfully constructed.The vector pEGFP-N1 containing cytomegalovirus(CMV) promoter was used as a positive control.The vector pEGFP-1 without promoter was used as a negative control.The vectors were transfected into human lung cancer cell lines 95D,NCI-H446,A2,A549,LTEP-a-2,YTMLC and normal MRC-5 through lipofectamine respectively.EGFP expression was detected under the fluorescence microscope.Results:pEGFP-hTERTp was confirmed by enzyme digestion with correct result.That the EGFP expression was detected in all of eight lung cancer cells transfected with pEGFP-hTERTp,but not in MRC cells.By contrast,high intensity EGFP expression was observed in both lung tumor cells and normal cells,which were transfected with pEGFP-N1.Conclusion:The EGFP controlled by hTERT promoter can be expressed specifically in lung cancer cell lines.hTERT promoter may be used as an excellent regulation element in tumor-targeting gene therapy.

Objective To highlight the characteristics of pulmonary sequestration (PS) with combination of practical experience and review of literature. Methods One patient with PS confirmed by biopsy was described and the relevant literatures of 279 cases were reviewed. Results PS can be divided into two types: intralobar type and extralobar type. The percentage of intralobar type is about 78.5%, intralobar type is 13.3%, other unreported is 8.2%. Clinically, PS was often combined by repeated pulmonary infection. The mis-diagnose rate is 72.8%. Etrograde arteriography or enhanced CT scan could find the abnormal artery, from which we can make correct diagnosis. The diagnose rate is 100%. Conclusion For patients with above clinical and X-ray symptoms, PS should be considered. Enhanced CT or retropgrade arteriography examination should be done as soon as possible. Timely diagnosis and surgery treatment are important for curing this kind of disease.

Objective To investigate the effect of MDR1 and MDR3 gene silence by shRNA of human breast carcinoma cell line MCF-7/Adr,and explore the role of MDR1 and MDR3 in adriamycin-resistance of breast carcinoma cells. Methods shRNA plasmid vector specifically targeting MDR1 and MDR3 gene was transfected into cells. The control group was transfected with empty vector. The concentration of adriamycin was detected by the flow cytometry (FCM). Cell apoptosis was analysed by FITC-Annexin-V/PI double staining. Cell viability and the IC50 of adriamycin on MCF-7/Adr cells were determined by MTT method. MDR1 and MDR3 mRNA were assessed by RT-PCR. P-gp expression was detectedby immunochemistry. Results After treatment with ABCB1 and ABCB4 shRNA plasmid vector, the apoptosis of MCF-7/Adr cells was (30.21±1.65)%and (22.07±2.17)% respectively. Compared with untransfecedgroup and empty vector transfection group the difference was significant(P＜0.01). MDR1 and MDR3 shRNAcould increase cellular adriamycin accumulation of MCF-7/Adr cells. MCF-7/Adr cells viability and the IC50were significantly decreased after transfection. Compared with untransfeced group and empty vector transfectiongroup, the mRNA level of MDR1 and MDR3 in MCF-7/Adr cells were decreased by (89.5±0.8)%and(85.1±1.2)%, the reduction of MDR1 and MDR3 mRNA was in a time-dependent manner. Immunochemistry proved that the expression of p-gp was significantly inhibited. Compared with untransfeced group and empty vector transfection group the difference was significant (P＜0.05). Conclusion The shRNA can effectively and specifically silence the expression of MDR1 and MDR3 gene, reverse the adriamycin-resistance mediated by P-gp in MCF-7/Adr cells. The reversal effect of adriamycin-resistance by shRNA of MDR1 is more effective than that of MDR3.

Objective To explore the application value of two-dimensional echocardiography with spatio-temporal im-age correlation (STIC) in fetal congenital heart disease (CHD) prenatal ultrasonography. Methods 11 036 hearts of fetus were inspected by severalviews order scanning method and for STIC volume database acquisition and off-line a-nalysis congenital heart disease fetus for autopsy after induced labor or contrast the follow-up results after birth. Re-sults 97 cases with spatio-temp-oral image correlation in 176 cases congenital heart and great vessels exception (dysrhythmias not including) with simple two-dimensional echocardiography,92 cases were accordant (one case with incorporative intracardiac malformation missed diagnosis);in screened congenital heart disease fetus,STIC (n=79) and routine ultrasonography (n=87) took (7.76±2.42) min and (9.68±2.13) min per case,respectively;in dif-ferent gestational weeks,the quality of the images derived from volume datasets were comparable to that directly ob-tained from 2D echocardiography. Conclusion STIC technology can be used as effective supplementary means of 2D echocardiography, and the combination can further improve the prenatal diagnosis of fetal congenital heart disease.

Objective To evaluate the effects of different doses of propofol or isoflurane on the cochlear blood flow (CBF) in guinea pigs.Methods Fifty-four adult male guinea pigs,aged 3 months,weighing 400-500 g,were randomly divided into 6 groups (n =9 each).In P1,P2 and P3 groups,propofol was infused for 115 min at 10,20 and 40 mg· kg-1 · h-1,respectively,after a loading dose of 5 mg/kg was injected over 5 min.In S1,S2 and S3 groups,isoflurane was inhaled for 120 min with end-tidal concentrations of 1％,2％ and 3 ％,respectively.Mean arterial pressure and CBF were recorded before administration (baseline,T1) and during the period of drug administration.Distortion product otoacoustic emission (DPOAE) was measured at T1,at the end of administration (T2) and 1 h after administration (T3).Five animals in each group were sacrificed and organs of Corti were harvested for observation of morphology of out hair cells by scanning electron microscopy.Results Propofol decreased MAP and increased CBF and DPOAE amplitude in a dose-dependent manner.Isoflurane decreased MAP and CBF in a dose-dependent manner.1％ isoflurane increased DPOAE amplitude,however,2％ and 3％ isoflurane decreased it and caused damage to out hair cells.Conclusion Propofol induces an increase in CBF in guinea pigs,while high concentration of isoflurane induces a decrease in CBF.Isoflurane inhibits CBF autoregulation,which makes CBF more sensitive to perfusion pressure,thus causing damage to hearing function.This is the reason why high concentration of isoflurane induces a decrease in CBF.