过表达人抗菌肽LL37腺病毒构建及转染阴道上皮细胞

BACKGROUND: LL37, the only antimicrobial peptide of the cathelicidin family identified from the human, not only promotes the proliferation of endothelial cells, but also plays an important role in angiogenesis and re-epithelialization. OBJECTIVE: To construct a recombinant adenovirus overexpressing human antimicrobial peptide LL37 gene, and to detect the expression and secretion of LL37 after transfected into the canine vaginal epithelial cells. METHODS: The cDNA encoding LL37 was amplified by PCR. Recombinant adenovirus expression plasmid encoding LL37 and green fluorescent protein (EGFP) was constructed, and identified using restriction endonuclease technology and DNA sequencing method. Adenovirus particles were generated by cotransfecting the 293-packaging cell line. The adenovirus were collected, amplified and concentrated, and viral titers were determined by end-point dilution assay by applying serial dilutions of the purified viruses to 293 cells. Primary cultured canine vaginal epithelial cells were transfected by the recombinant adenovirus. The transfection efficacy was observed by fluorescence microscope, and the cultured supernatant was collected to determine the expression of LL37 by ELISA method at 1, 2, 3, 5 and 7 days after transfection. RESULTS AND CONCLUSION:The adenovirus vector GV314-LL37 with the titer of 3×109 pfu/mL was successfully constructed and identified by DNA sequencing methods. Canine vaginal epithelial cells were successfully isolated and cultured and grew stably. After transfection, vaginal epithelial cells could express the EGFP and LL37 efficiently in a time-dependent manner detected by fluorescence microscope and ELISA method. The transfection efficacy of EGFP reached to 89% at 72 hours. The level of LL37 in the cell culture supernatant in the transfection group was significantly higher than that in the control group, the highest expression of LL37 was found at 3 days that lasting for 7 days. In conclusion, the recombinant adenovirus overexpressing human antimicrobial peptide LL37 gene is successfully constructed, which can express and secrete LL37 after transfected into canine vaginal epithelial cells, providing a foundation for constructing the tissue-engineered vagina possessing anti-infection and neovascularization. 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

Constructing the recombinant plasmid-pLXSN-SjYF-of Yolk Ferritin gene of Schistosoma japonicum

TO construct the recotabinant plasmid —— pLXSN-SjYF to prepare for expression and DNA vaccine of Schistosoma japonicum gene. Methods :Mnpliffing DNA fragment coding yolk fereltin from a female adult Sehistosoma japonicum DNAs by PCR. The fragment was inseted into pLXSN retrovirus vector by digesting with restrictive enzymes and linking reactions. The positive clone was screened on LB plates contmaining amplcillin asld identified by restrictive enzymes digestion and PCR amplification, Resttlts The specific DNA fragmem SjYF was amplified from the female adult SjDNAs. IaLXSN-SjYF was constructed successfully and the further research will be carried out.     

不明原因肺炎监测中SARS-CoV实时荧光RT-PCR检测方法的建立

Objective To develop and evaluate a real-time RT-PCR method for detecting SARS-CoV, based on TaqMan hybridization probe technology in order to provide a laboratory diagnosis for the examination of SARS-CoV infection during the surveillance of unexplained pneumonia. Methods Two pairs of primers and probes were designed to identify 1b and NP gene of SARS-CoV respectively. The two probes were 5'end labeled with FAM and 3'end labeled with TAMRA. The PCR reaction conditions were optimized according to the reaction conditions for detecting H5N1 which was set up by the National Influenza Center. Different concentration of plasmid DNA containing the target gene, 12 kinds of other respiratory viruses, mycoplasma pneumoniae and legionella pneumophilia were tested using this method to evaluate the specificity, sensitivity and reproducibility of the assay. Results All kinds of the viruses, mycoplasma pneumoniae and legionella pneumophilia tested were negative. The sensitivity of this assay for 1b gene was 10-9μg/ml DNA/reaction and for NP gene was 10 -7μg/ml DNA/reaction. The coefficients of variation (CV) value were 0.2% -0.9% during the reproducibility test. The whole process takes 2.5h including the extraction of RNA from the sample, and could be completed in the same machine, under the same condition with the detection of H5N1.Conclusion This real-time RT-PCR setting up based on TaqMan probe is a specific, rapid and sensitive method for detecting SARS-CoV. The establishment of this method will provide a strong support for quick examination of SARS-CoV infection during the unexplained pneumonia surveillance.     

人脐动脉平滑肌细胞培养及转染TFPI基因的实验研究

Objective The aim of the present study is to investigate the effect of tissue factor pathway inhibitor (TFPI) gene on cell cycle of human vascular smooth muscle cells.Methods Human vascular smooth muscle cells were separated from human umbilical artery and identified by immunohistochemical staining.The cells were transfected with various amount of pIRES-TFPI plasmid (1,2,and 3 μg/ml,respectively)and the TFPI expression in the cells were analyzed by RT-PCR.MTT assay was employed to detect the effect of TFPI gene on the proliferation of human vascular smooth muscle cells.Results The proliferation of vascular smooth muscle cells was inhibited in pIRES-TFPI group 5 and 7 days after gene transfection when compared with that of pIRES 1-neo transfection group.Conclusion The overexpression of TFPI gene in human umbilical artery vascular smooth muscle cells may contribute to the suppression of the proliferation of cells by gene transfection.     

Asymptomatic seminal infection of herpes simplex virus:impact on male infertility

In more than half of infertile men,the cause of their infertility is unknown.Several studies revealed the role of viral infections in male infertility.The aim of the present study was to determine the prevalence of herpes simplex virus-1 (HSV-1) and HSV-2 in semen from asymptomatic infertile male patients,and its association with altered semen parameters.A total of 70 semen samples were collected from infertile men who attended the Research and Clinical Center for Infertility in Yazd,Iran.Semen analysis and diagnostic real-time PCR using specific primers and probes for HSV-1 and HSV-2 DNA were performed.Comparison of semen parameters between virally infected and non-infected samples were performed with independent t-test and Mann-Whitney test.Semen analysis showed that infertile men fell into two groups,the male factor group and the unexplained group.HSV-1 and HSV-2 DNA was detected in 16 (22.9%) and 10 (14.3%) of 70 semen samples,respectively.All HSV-positive samples had abnormal semen parameters (the male factor group).Although HSV infection was not associated with sperm motility and morphological defects,it was correlated with lower sperm count in the seminal fluid.The findings suggest that asymptomatic seminal infection of HSV plays an important role in male infertility by adversely affecting sperm count.     

The immune response induced by hepatitis B virus principal antigens

Hepatitis B virus （HBV） infection occurs primarily in hepatocytes in the liver with release of infectious virions and non-infectious empty surface antigen particles into the bloodstream. HBV replication is non-cytopathic. Transient infections run a course of several months, and chronic infections are often life-long. Chronic infections can lead to liver failure with cirrhosis and hepatocellniar carcinoma. It is generally accepted that neutralizing anti-HBs antibodies plays a key role in recovery from HBV infection by containing the spread of infection in the infected host and facilitating the removal and destruction of viral particles. However, the immune response initiated by the T-cell response to viral antigens is also important for viral clearance and disease pathogenesis in HBV infection. The three structural forms of the viral proteins, the HBsAg, the particulate HBcAg, and the nonparticulate HBeAg, may preferentially elicit different Th cell subsets. The different IgG subclass profiles of anti-HBs, anti-HBc, and anti-HBe in different HBV infection status were revealed. Moreover, the different IgG subclass profiles in chronic carriers did not change with different ALT and AST levels and may reflect the difference between stimulating antigens, immune response, and the stages of viral disease and provide the basis for the use of vaccines and prophylactic treatments for individuals at high risk of human HBV infection. This review elucidates the detailed understanding of the immune responses induced during transient and persistent infection, and the development of immunotherapy and immunodiagnosis in patients with HBV infection, and possible means of reducing the liver damage.     

Production and secretion of biologically active human epidermal growth factor in Yarrowia lipolytica

The gene encoding human epidermal growth factor (hEGF) was expressed as a fusion protein with the leader peptide and pro I region of alkaline extracellular protease in the yeast Yarrowia lipolytica. hEGF was purified from culture supernatant by reverse-phase chromatography and analysed by Western-blot hybridisations. The biologically active hEGF in the purified sample was assayed using the radioreceptor assay and estimated to be 100 μg/l. However, the level of expression was found to be substantially low compared to the levels of homologous protein, alkaline extracellular protease (AEP), possibly due to degradation by secreted acid protease(s). A novel and sensitive bioassay was developed to determine the biological activity of hEGF produced at low levels and is based on the effect produced by hEGF in the regenerating tails of the wall lizard. Intramuscular injections of culture supernatant from the recombinant yeast and the standard hEGF led to a drastic reduction in tail regeneration confirming the biological activity of the recombinant hEGF. Received: 15 August / 19 September 1997     

Change of inflammatory cytokines after co-culture of inflamed cartilage blocks and chondrocytes transfected by interleukin-β1 receptor antagonist gene and transforming growth factor-β1 gene in vitro

Objective To evaluate the in vitro effects of recombinant human interluekin-β1 receptor antagonist(IL-1Ra) gene and transforming growth factor-β1 (TGF-β1) gene on rabbit osteoarthritis (OA).Methods Articular cartilages were extracted from mature New Zealand rabbits and by enzyme digestion,isolated for chondrocytes which were then identified with specific extracellular matrix collagen type Ⅱ stained immunocytochemistry.The chondrocytes were divided into IL-1Ra-transfected group (group A), TGF-β1?transfected group (group B) , combined IL-1Ra- and TGF-β1-transfected group (group C) , untransfected group (group D) and the blank control group (group E).LipofectamineTM 2000 Reagent was used as the vehicle for transfection among groups A, B and C.All the groups of chondrocytes were co-cultured with fragmented articular cartilages and added with 20 ng IL-β 1?expect for group E.The transgenic expression of chondrocytes was detected under fluorescence microscope at 12h,24h,2d,4d and 6 d after transfection and co-culture.In addition, radioimmunoassay (RIA) was used to determine the levels of IL-1βand TNF-α in each group at 2 d, 4 d and 6 d after transfection and co-culture.Results The chondrocytes were successfully isolated and cultured.Collagen type Ⅱ stained immunocytochemistry showed the brownish - yellow cytoplasm and unstained chromophobic nuclei.Under fluorescence microscope, the expression of enhanced green fluorescent protein was observed in groups A, BandC, which peaked at 24 hours after transfection (16.16±2.71)% vs (16.54±2.91)% vs (17.20±2.39)% and gradually declined 2 d later.At any time spots, the IL-1βevel was highest in group D, followed by group B, group A, group C, and group E.The level of TNF-a in each group was ordered by group D＞group A＞group B＞group C＞group E on days 2 and 6, and by group E＞group A＞group B＞group C＞group D on day 4.The level of TNF-α in group A was slightly higher than that of group B, but the difference was not statistical significance.There were statistical difference among the other groups.The expressions of IL-1βand TNF-α in groups A, B and C were significantly lower on day 6 than those on days 2 and 4.The level of IL-1βin groups D and E did not change with time, while the level of TNF -α was the lowest in group D and highest in group E on day 4.ConclusionsTransfection with IL-1Ra or TGF-β1 can reduce the level of inflammatory cytokines.Combined use of IL-1Ra and TGF-β1 genes may show control of inflammatory response and provide evidences for gene therapy of osteoarthritis.     

抗-HBc单项阳性患者中的隐匿性HBV感染

Objective This study was designed to explore the incidence rate of occuIt HBV infection in patients with anti-HBc positive alone and analyze the possible reasons of occuIt infection.Methods Sera of 183 patients carrying anti-HBc alone(A≤0.1) were collected and real-time PCR was used to select samples with HBV DNA positive.HBV pre-S/S amplification products were obtained by PCR,and clonal sequencing were then used for these samples with HBV DNA positive.Results DNA quantitative results of three samples were greater than 103 copies/ml in 183 samples,with a fraction of 1.6%.Pre-S/S sequencing results of two samples from these three samples were obtained.Point mutations within "a" determinant with Q129R/P mutations and co-existence of the mutant type and wild type were found in the two samples.Conclusions Occult HBV infection existed in samples with anti-HBc alone.Factors contributing to the loss of HBsAg detection by immunoassays include S gene mutations and low levels of cireulating antigen which are below the assay limit of detection.Occult HBV infection not only can lcad to a false clinical diagnosis,but also can result in hematological pollution due to such occult infection of blood donors.     

抗-HBc单项阳性患者中的隐匿性HBV感染

Objective This study was designed to explore the incidence rate of occuIt HBV infection in patients with anti-HBc positive alone and analyze the possible reasons of occuIt infection.Methods Sera of 183 patients carrying anti-HBc alone(A≤0.1) were collected and real-time PCR was used to select samples with HBV DNA positive.HBV pre-S/S amplification products were obtained by PCR,and clonal sequencing were then used for these samples with HBV DNA positive.Results DNA quantitative results of three samples were greater than 103 copies/ml in 183 samples,with a fraction of 1.6%.Pre-S/S sequencing results of two samples from these three samples were obtained.Point mutations within "a" determinant with Q129R/P mutations and co-existence of the mutant type and wild type were found in the two samples.Conclusions Occult HBV infection existed in samples with anti-HBc alone.Factors contributing to the loss of HBsAg detection by immunoassays include S gene mutations and low levels of cireulating antigen which are below the assay limit of detection.Occult HBV infection not only can lcad to a false clinical diagnosis,but also can result in hematological pollution due to such occult infection of blood donors.     

Level of functioning of siblings and parents of probands of varying degrees of retardation

A further analysis of already published data supports the position that retardates of low ability level less frequently have retarded siblings, retarded parents, and parents low in occupational level than do retardates higher in ability level. The analysis supports the position that there are two types of retarded individuals, persons retarded as a result of gene or chromosomal anomalies, brain injury, etc., who more frequently occur in the lower-level retardate group, and persons whose retardation represents polygenic segregation, who more frequently occur in the higher-level group.     

Modes of Inheritance of Errors of Refraction

Eighteen families in which both parents had refractions within the range of +4·0 D to −4·0 D and axial lengths seen in emmetropia (22·3-26·0 mm) showed coefficients of correlation of the order 0·5 indicative of polygenic inheritance. Such coefficients were seen for axial length (0·407) and for the cornea (0·487), but not for the lens (which is known to be yoked to the axial length). No such coefficients were seen in 19 families in which one of the parents had axial length outside the emmetropic range (nine families with long axes and 10 with short axes).

The pattern of polygenic inheritance for emmetropia (completely correlated optical components) and errors of refraction up to 4·0 D (inadequately correlated components: correlation ametropia) follows that seen in stature and other measurable characters. In contrast the high refractive errors with their abnormal axial lengths (component ametropia) are—like the extremes in stature—pathological anomalies with monofactorial inheritance.