"CapFinder" technology,which can be used to clone the full length of 5′ UTR sequence of mRNA,was described.This technology used the terminal transferase activity of certain MMLV RT variants that added 3-5 residues(predominantly dC) to the 3′end of the first-strand cDNA exhibited when MMLV RT reached the 5′cap structure of mRNA.In the reverse reaction system containing GGG oligo,the terminal transferase activity was harnessed by the GGG oligo whose terminal stretch of dG residues can anneal to the dC-rich cDNA tail and serve as an extended template for RT.After RT switch templates from the mRNA template to the GGG oligo,a complete cDNA copy of the original RNA was synthesized with the additional GGG oligo sequences at the end.5′UTR of mRNA can be amplified with GGG oligo as forward primer and a gene-specific reverse primer.5′UTR of Bt toxin receptor E-Cadherin gene in midgut of cotton bollworm was cloned.

Objective To establish embryonic stem cells (ESC) that can express green fluorescent protein (CFP) and differentiate them into neurons. It would provide tagging neurons for clinical transplantation to cure neural system diseases. Methods ESC (R1) was transfeeted with a plasmid containing the GFP by electroporation. A transgeuic cell line was obtained after selection with G418. The ESCs were characterized by AKP staining. Monolayer differentiation method was used to induce neural differentiation derived from GFP-ESC and immunofluorescence method was used to identify Tuj1 positive cells. Results There was no significant difference(X2=3.14,P0.05) in transfect rates between liposome and electroporation (65% vs 79%). The AKP staining of GFP-ESC was positive. GFP-ESC could be differentiated into neural cells. Conclusions These results show that ESC expressing GFP has been estabhshed, which can be differemiated into neurons.

Objective To investigate choroid thickness at horizontal meridian with optical coherence tomography (OCT) and compare the choroid thickness difference between first visit myopia children with those children who wear orthokeratology lens for more than 1 year.Methods This retrospective study enrolled 68 myopia children with low to moderate myopia (-1.00--6.00 D) who visited our hospital and took choroid images by OCT.The total subjects were divided into 2 groups.The subjects of 34 children in group 1 visited for myopia initially and wear spectacles.The other one group wear orthokeratology lens more than 1 year.Only the data of right eye were analyzed.Scans through the fovea at horizontal meridian were acquired with OCT.Choroid images were detected by custom software with 500 μm intervals up to 3.5 mm around fovea.Choroid thickness (CT) was calc~ated based on the average of the 7 zones.Statistical analysis was performed to evaluate choroid thickness at each zone.ANOVA was used to compare choroid thickness differences between various zones in each group.Paired t test was used to compare choroid thickness difference at the same zone between two groups.Results The mean age of OK lens group was (12.3 ± 1.8) years old,and the spectacles group was (11.8 ± 1.4) years old,there was no statistical difference.From temple to nasal choroid,the mean CT of the orthokeratology lens group were (296.7 ± 61.8) μm (T3),(290.7 ± 58.9) μm (T2),(285.7 ± 57.4) μm (T1),(278.5 ±57.7) μm (M),(262.2 ±57.9) μm (N1),(239.8 ±59.7) μm (N2),(214.7 ±59.0) μm (N3);And the mean CT of the spectacles group were (294.2 ± 45.4) μm (T3),(292.0±44.0) μm (T2),(283.6 ±45.5) μm (T1),(272.0 ±51.6) μm (M),(255.2 ± 56.3) μm (N1),(236.5 ±58.1)μm (N2),(212.8 ±57.7) μm (N3),respectively.The thicknesses were significantly thicker in temple zones than that in nasal zones in each group (all P ＜ 0.05),but the CT was not significantly different between the two groups in each zone (all P ＞ 0.05).Conclusion The choroid thickness has regional deference in myopia children,the thickest is in the temple and the thinnest in the nasal region.There is no significant difference between the children who initially corrected by spectacles and those who wear OK lens more than 1 year.

Objective To explore the expression and regulatory mechanism of hypoxia inducible factor-1?(HIF-1?)during fracture healing. Methods Mouse models of tibia fracture healing were established,and callus samples were collected 1,3,7,14,21 and 28 days after fracture.The development of callus and new bone formation were evaluated with roentgenology,Micro-CT and tetracycline double labeling method,and the expression of HIF-1?,vascular endothelial growth factor(VEGF),Runx2 and ALP in callus were detected with RT-PCR,Western blotting and immunohistochemistry.The relationship between HIF-1? and fracture healing was analysed. Results The expression of HIF-1? was detected in cells in the fracture sites as well as in evolved osteoblasts,chondrocytes and osteocytes in early callus under hypoxia.The highest expression rate of HIF-1? achieved on the 7th day after fracture,lasted for about 7 days,then decreased gradually,and returned to intact level on the 28th day after fracture.The expression tendency of VEGF resembled that of HIF-1?.Bone formation activity was more active in early callus,and the callus volume peaked on the 14th day after fracture and decreased gradually.The mineralization of callus mainly took place in the late healing period(14th to 28th day after fracture).Conclusion Cells involved in fracture healing are hypoxia-responsive cells,which express HIF-1?.HIF-1? can regulate cell state and function,and can promote angiogenesis so as to play a crucial role in fracture healing.

OBJECTIVETo explore the diagnosis and treatment of a case of neonatal alloimmune thrombocytopenia purpura (NAITP) caused by anti HPA-3a antibody.

METHODSThe platelet counts and purpuric symptom in the newborn were clinical examined. The HPA-1-21bw genotypes of the newborn and his parents were detected by multiple DNA-PCR, gene sequencing and genotyping. The HPA specificity antibody in the sera of newborn and his mother were detected by flow cytometry (FCM), and the HPA specificity antibody was identified by monoclonal antibody-specific immobilization of platelet antigens (MAIPA).

RESULTSThe newborn had the typical symptom of NAITP, multiple subcutaneous petechia, hematuria and coffee-like vomitus. The HPA genotype of the newborn was HPA-3ab, while that of his mother and his father were HPA-3bb and HPA-3aa, respectively. The sera of newborn and his mother existed antibody against the platelet of newborn's father. The HPA antibody of the newborn and his mother were identified as anti HPA-3a. The newborn was approved a patient of NAITP caused by anti HPA-3a antibody.

CONCLUSIONThe diagnosis and treatment for NAITP newborn caused by anti HPA-3a antibody in this study was the first domestic report. It could provide successful experiences and references for the similar cases.

Antibody Specificity ; immunology ; Antigens, Human Platelet ; immunology ; Genotype ; Humans ; Infant, Newborn ; Isoantibodies ; adverse effects ; immunology ; Male ; Thrombocytopenia, Neonatal Alloimmune ; etiology ; immunology

OBJECTIVETo study the role of extracellular matrix (ECM) in neural differentiation of mouse embryonic stem cells (ESCs).

METHODSMouse ESCs were incubated in the ESC conditioned medium, and the formation of embryonic bodies (EBs) were induced in bacteriological dishes using high-concentration all-trans retinoic acid (RA). The EBs were seeded on different matrixes (gelatin, fibronectin, and laminin/poly-L-ornithine) to test their impact on neural differentiation of the ESCs using immunofluorescence assay. The effect of laminin/poly-L-ornithine on the growth of neurites was evaluated with fluorescence microscopy.

RESULTSHigh-concentration RA activated and accelerated the differentiation of ESCs toward nestin-positive neural progenitor cells. Fibronectin supplement in the matrix dose-dependently promoted ESC differentiation into neural progenitor cells, while laminin/poly-L-ornithine increased the growth of the neurites and induced the maturation of the differentiated neural cells.

CONCLUSIONECM plays an important role in neural differentiation of mouse ESCs, and application of FN produces the most conspicuous effect during the differentiation of the ESCs into the neural progenitor cells;laminin/poly-L-ornithine is the most effective during their differentiation into neural cells.

Animals ; Cell Differentiation ; drug effects ; Cells, Cultured ; Culture Media ; Embryonic Stem Cells ; cytology ; drug effects ; Extracellular Matrix ; physiology ; Fibronectins ; pharmacology ; Laminin ; pharmacology ; Mice ; Neurons ; cytology ; drug effects ; Peptides ; pharmacology ; Tretinoin ; pharmacology

·AIM: To observe the therapeutic effect of visual perception training combined with total nutrition meal sequential therapy in the treatment of myopic amblyopia.·METHODS:Totally 73 children ( 135 eyes ) with myopic amblyopia were divided into control group ( 36 cases, 67 eyes) and treatment group (37 cases, 68 eyes) according to random number table. The control group were treated with traditional spectaculars and grating covering combined with fine eyesight training;the treatment group were treated with visual perception training combined with total nutrient meal sequential therapy. The visual acuity, diopter and average diopter of two groups were compared before and after treatment at 3, 6mo and 1a. The curative effect of two groups of children was compared after 1a treatment. And the adverse reactions were recorded in two groups during the treatment period. The recurrence rate of amblyopia in 1a follow-up was compared between two groups.·RESULTS: The difference of visual acuity between two groups was not significant at 3mo (P>0. 05). The visual acuity of the treatment group was significantly higher than that of the control group at 6mo and 1a (P<0. 05). There was no significant difference in diopter between the two groups after 3, 6mo and 1a (P>0. 05), but the average annual refractive changes in the treatment group were significantly lower than that in the control group ( P<0. 05). The basic cure rate and total effective rate of the treatment group were significantly higher than that of the control group (P<0. 05). There were no severe adverse reactions occurred between two groups during the treatment period. The recurrence rate of amblyopia in the treatment group was significantly lower than that in the control group (P<0. 05) after 1a follow-up.· CONCLUSION: Visual perception training combined with total nutrition meal sequential therapy in the treatment of myopic amblyopia in preschool children can significantly improve patients' visual acuity, reduce the average annual diopter changes, improve the therapeutic effect, reduce the recurrence rate of amblyopia.

Objective To establish a real time PCR assay with a novel fluorescence quencher for identification of mutation of clarithromycin -resistance gene of Helicobacter pylori.Methods Two mutations of 23S rDNA gene in Helicobacter pylori,No.2142 and 2143,were chosen as targets for detection,and then the primers and the probe with a novel fluorescence quencher were designed.The genome DNA of Helicobacter pylori was extracted,and then detected by real time PCR reported here.Meanwhile,the specificity,reproducibility and sensitivity of the assay were evaluated.Finally,the real time PCR described here,the real time PCR based on TaqMan,and a sequencing assay were applied to detect 55 Helicobacter pylori strains isolated from clinical specimens,respectively.The results from three assays were compared with each other in order to further evaluate the applicability of this assay in clinic.Results It indicated that the mutation points related to clarithromycin -resistance,A2142G and A2143G,were identified by real time PCR with a novel fluorescence quencher rapidly and accurately.Moreover the coefficient of variation was less than 5%.The limit of detection was 100 copies/reaction.While this assay was applied directly to detect 55 Helicobacter pylori strains,the results were in accordance with those obtained from a TaqMan real time PCR and a sequencing assay,respectively.Conclusion The real time PCR described here was a simple,reliable and accurate approach and substituted for the TaqMan real time PCR for identification of two mutation points of clarithromycin -resistance,A2142G and A2143G in Helicobacter pylori.Thus,a novel tool for diagnosis of gene mutation was provided and the results might be regarded as a substantial evidence for clinical individual therapy.

A significant-source of allergens come from house dust that contain particles derived from arthropods, molds, and pet dander. This study evaluated mite and booklouse fauna from vacuumed dust samples in Beijing China (a temperate zone). Our survey was carried out in Beijing in the homes of mite allergic patients who visited our Allergy Department. In total, 38 homes were selected for the collection of dust samples by vacuuming, from December 2008 to January 2010. The flotation method was used to isolate mites from house dust. Permanent slides were prepared for mite specimens and mites were identified and counted under a microscope. In total, 1,798 separate mite and insect specimens were found in 345 dust samples taken from 38 homes. A total of 95 individual Dermatophagoides (D) siboney were detected in 35 dust samples from 19 homes (representing 5.3% of all mite and insect species found in house dust); in addition, this mite was found to co-exist with D. farinae (Hughes, 1961) in 33 dust samples. Our results demonstrated the presence D. siboney that co-existed with D. farinae in house dust in Beijing China (a temperate zone).
Allergens ; Arthropods ; China ; Dander ; Dermatophagoides farinae ; Dust* ; Fungi ; Humans ; Hypersensitivity ; Insects ; Mites* ; Pyroglyphidae ; Vacuum*

OBJECTIVETo study the regulation of hypoxia inducible factor-1alpha (HIF-1alpha) on osteoblast function in postmenopausal osteoporosis.

METHODSFrom October 2004 to May 2006, Cre-Loxp recombinase was used to create mice which the HIF-1alpha gene in osteoblasts was conditional knock-out, 24 female wild-type (WT) mice and 24 female conditional knock-out (CKO) mice of 3 months old were operated on ovariotomy. At 0,4,8 weeks after operation, bone histomorphometry parameters were measured with computer image analysis in HE stain sections and in tetracycline bone double labeling fluorescence sections; Bone density and the trabecular bone architecture parameters were measured by Micro-CT; The mRNA expression of vascular endothelial growth factors (VEGF), RunX2, OC, ALP were detected with quantitative RT-PCR; The protein expression of VEGF and RunX2 were detected with Western-blotting.

RESULTSIn CKO mice, the trabecular number, volume, thickness, bone density, mineral apposition rate (MAR), the expression of VEGF, RunX2, OC, ALP on mRNA level and the expression of VEGF, RunX2 on protein level decreased significantly compared with WT mice especially in 8 weeks after operation.

CONCLUSIONSThe bone formation ability of osteoblasts in CKO mice was reduced compared with WT mice after ovariotomy. HIF-1alpha can regulate the bone formation ability of osteoblasts in postmenopausal osteoporosis.

Animals ; Blotting, Western ; Core Binding Factor Alpha 1 Subunit ; genetics ; metabolism ; Disease Models, Animal ; Female ; Femur ; metabolism ; pathology ; physiopathology ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; genetics ; physiology ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Osteoblasts ; metabolism ; physiology ; Osteoporosis, Postmenopausal ; genetics ; metabolism ; physiopathology ; Ovariectomy ; Random Allocation ; Reverse Transcriptase Polymerase Chain Reaction ; Vascular Endothelial Growth Factor A ; genetics ; metabolism