China ; Humans ; Nanostructures ; toxicity ; Nanotechnology

Objective:To study the expression level of peripheral blood mircoRNA-155 and the variation characteristics of CD4+T cell percentage and to expound the clinical significance of mircoRNA-155 quantitative test and CD 4+T cell percentage cytological test.Methods:Fluorescent quantitative PCR had been used to detect the expression level of mircoRNA -155 in the peripheral blood.Flow cytometry had been used to detect the cell percentage of the peripheral blood T lymphocyte subset CD 3+T cell,CD4+T cell and CD8+T cell.The mircoRNA-155 expression levels and the cell percentages of CD 4+T were tested respectively in the selected 40 patients with atopic dermatitis ,30 patients with skin eczema and 30 healthy controls.Finally,the differences of the above groups were counted and analyzed .Results:The relative expression levels of mircoRNA-155 of the atopic dermatitis group were higher than those of the eczema group and the normal control group ( P<0.05 ).The CD4+T cell percentage of the atopic dermatitis group was higher than those of the eczema group and the normal control group ( P<0.05 ) .Conclusion: The expression level of the peripheral blood mircoRNA-155 in the patients with atopic dermatitis is increased significantly and CD 4+T cell percentage in the patients is raised as well.Consequently ,the clinical detection of miRNA-155 and CD4+T cell shall be of great significance .

Objective:To study the cytotoxic effects on tumor cell lines Hela,BGC823,MCF-7,L342,H9101,D6 induced by Fas ligand and anti-human DR5 monoclonal antibodies(Anti-DR5 mAb) and the underlying mechanism.Methods:Fas/DR5 mRNA were detected by RT-PCR. Cytotoxicity exerted by FasL/Anti-DR5 mAb on tumor cell lines was measured by MTT colorimetry and the induced apoptosis was determined by agarose gel electrophoresis.Results:The expression of DR5 on BGC823 and Hela cells were higher and DR5 didn’t express in D6. The expression of Fas on H9101 and L342 were higher and Fas didn’t express in D6. Cell line H9101 and L342 were sensitive to Anti-DR5 mAb and FasL in a dose dependent manner; Cell line MCF-7 and BGC823 were sensitive to FasL but were partially sensitive to Anti-DR5 mAb; Cell line Hela was partially sensitive to FasL but was sensitive to Anti-DR5 mAb; Cell line D6 was insensitive to two apoptosis inductions.Conclusion:Apoptosis induced by Fas ligand and Anti-DR5 mAb vary among tumor cell lines. The underlying mechanism may be relevant to Fas/DR5 mRNA expression,the release of Caspase-8 and Bcl-2.

Objective:To investigate the correlation between single nucleotide polymorphism ( SNP ) of rs7517847 and rs10489629 on IL-23R gene and susceptibility of ankylosing spondylitis in Jilin.Methods:IL-23R gene polymorphisms of 188 cases on ankylosing spondylitis were detected by polymerase chain reaction-restriction fragment length polymorphism ( PCR-RFLP ) , compared with those of 100 cases of healthy control group.Results:The frequency distribution differences ,between AS group and comparison group of separate genotype and the allele on two SNPs (rs7517847 and rs10489629) both showed statistical significance (P<0.05).Meanwhile, by the assumed genetic way , compared homozygous mutant GG of rs 7517847 with TG+TT, compared homozygous mutant AA of rs10489629 with GA+GG,such frequency distribution difference between the above two groups also showed statistical significance ( P<0.05).Conclusion:Polymorphisms about IL-23R gene of the rs7517847 and rs10489629 are both associated with susceptibility of AS in Jilin.The risk of AS will be increased if the person both with G/A allele and GG/AA genotype , which may be one of susceptive factors by AS.

Objective:To characterize the CD4+CD45RA+,CD4+CD45RO+,CD8+CD45RA+ and CD8+CD45RO+T lymphocyte subsets in the peripheral blood of the patients with chronic hepatitis B and to explore their relations with the disease state.Methods:The peripheral blood was collected from 104 patients with chronic hepatitis B and 30 healthy individuals,then CD4+CD45RA+,CD4+CD45RO+,CD8+CD45RA+ and CD8+CD45RO+T lymphocyte subsets were detected by flow cytometry with three-color fluorescence technology.Results:To compare with the control,the percent of CD8+CD45RA+T lymphocyte in patients with mild and severe grad CHB decreased markedly,the percent of CD8+CD45RO+T lymphocyte increased markedly,CD4+,CD8+,CD4+CD45RA+ and CD4+CD45RO+T lymphocyte did not change obviously. Compared with the mild grade CHB,the percent of CD8+CD45RA+T lymphocyte in patients with severe grade CHB decreased obviously(P

Objective: To construct the expressing vector of the extracellular domain of death receptor 5 (DR5), express it E.coli, identify the purified DR5 protein, and study its biological activity. Methods: The extracellular domain of DR5(eDR5) was assembled by overlapping PCR. The expression vector pET-22b(+)/ DR5 was constructed and transformed into E.coli BL21(DE3). The expression of eDR5 protein was induced by IPTG and purified by Ni 2+ -affinity chromatographic column. The purity and specificities were detected by SDS-PAGE and ELISA, respectively. The blocking effects of purified eDR5 on FMU1.5-induced apoptosis of U343, U373 cells were observed. Results: The extracellular domain of DR5 was obtained by overlapping PCR. The eDR5 protein was expressed in both supernatants and inclusion bodies with a yield more than 30% of total bacterial proteins. The purity of eDR5 was more than 95% and the yield reached 9 mg/ml. The result of ELISA showed the purified protein was eDR5. Purified eDR5 partially blocked the apoptosis of U343 cells induced by FMU1.5 and TRAIL. Conclusion: The successful construction, expression, and purification of the extracellular domain of DR5 protein lays a foundation for further study of DR5 function.

Gene knockout by ZFNs (zinc-finger nucleases) is efficient and specific, and successfully applied in more than 10 organisms. Currently, it is unclear whether this technology can be used for knocking-out enhanced green fluorescent protein (EGFP) gene in transgenic goats. Here we constructed and used ZFN-coding plasmids to produce genetic knockouts in the cells of cloned fetus produced from donor cells by microinjection of EGFP gene. Following introduced plasmids into caprine primary cultured fetus fibroblasts by electroporation, targeting of a transgene resulted in sequence mutation. Using the flow cytometric analysis, we confirmed the disappearance of EGFP expression in treated cells. Sequence from PCR products corresponding to targeted site showed that insertion of a G into the exon of EGFP resulted in frame shift mutation. These results suggest that ZFN-mediated gene targeting can apply to caprine fetus fibroblasts, which may open a unique avenue toward the creation of gene knockout goats combining with somatic cell nuclear transfer.
Animals ; Base Sequence ; Cloning, Organism ; Electrophoresis ; Endonucleases ; genetics ; metabolism ; Fetus ; Fibroblasts ; metabolism ; Gene Knockout Techniques ; Gene Targeting ; methods ; Goats ; Green Fluorescent Proteins ; genetics ; Molecular Sequence Data ; Mutation ; Zinc Fingers

To knock out β-lactoglobulin (BLG) gene and insert human lactoferrin (hLF) coding sequence at BLG locus of goat, the transcription activator-like effector nucleases (TALEN) mediated recombination was used to edit the BLG gene of goat fetal fibroblast, then as donor cells for somatic cell nuclear transfer. We designed a pair of specific plasmid TALEN-3-L/R for goat BLG exon III recognition sites, and BLC14-TK vector containing a negative selection gene HSV-TK, was used for the knock in of hLF gene. TALENs plasmids were transfected into the goat fetal fibroblast cells, and the cells were screened three days by 2 μg/mL puromycin. DNA cleavage activities of cells were verified by PCR amplification and DNA production sequencing. Then, targeting vector BLC14-TK and plasmids TALEN-3-L/R were co-transfected into goat fetal fibroblasts, both 700 μg/mL G418 and 2 μg/mL GCV were simultaneously used to screen G418-resistant cells. Detections of integration and recombination were implemented to obtain cells with hLF gene site-specific integration. We chose targeting cells as donor cells for somatic cell nuclear transfer. The mutagenicity of TALEN-3-L/R was between 25% and 30%. A total of 335 reconstructed embryos with 6 BLG-/hLF+ targeting cell lines were transferred into 16 recipient goats. There were 9 pregnancies confirmed by ultrasound on day 30 to 35 (pregnancy rate of 39.1%), and one of 50-day-old fetus with BLG-/hLF+ was achieved. These results provide the basis for hLF gene knock-in at BLG locus of goat and cultivating transgenic goat of low allergens and rich hLF in the milk.
Animals ; Animals, Genetically Modified ; genetics ; Female ; Fibroblasts ; Gene Knock-In Techniques ; Gene Knockout Techniques ; Goats ; genetics ; Humans ; Lactoferrin ; genetics ; Lactoglobulins ; genetics ; Milk ; chemistry ; Nuclear Transfer Techniques ; Plasmids ; Pregnancy ; Transfection

Objective:To explore the feasibility and application value of three dimensional (3D) printing technology in creating models of abnormal fetal aortic arch and its branches.Methods:Eleven cases of abnormal fetuses confirmed fetal aortic arch and its branches anomalies from March 2019 to July 2020 in Renmin Hospital of Wuhan University were prospectively enrolled. All the fetuses underwent two dimensional(2D) echocardiography and spatio-temporal image correlation (STIC) technology examination. The 3D volume images of fetal heart were post-processed by Mimics software to create images of the great vessels and their branches in standard tessellation language format (STL) file. The STL file was output to the 3D printer and the 3D printing models of fetal great vessels and their branches were obtained. Compared with conventional ultrasound, the characteristics and application value of 3D printed models of abnormal fetal aortic arch and its branches were analyzed.Results:Eleven fetuses were successfully modeled and printed out large blood vessels and their branch models. The 3D printing model had its own advantages in displaying large blood vessels and their branch abnormalities. It could provide high quality imaging anatomical details and visualize great vessels origin, branch and position and can better display vascular ring spatial relations.Conclusions:It is feasible to use 3D printing technology to make the fetal aortic arch and its branch abnormal model. The 3D printing model can directly display its characteristic changes and provide a certain reference basis for accurately determining the type of vascular ring in the prenatal stage.

Objective To develop and validate a method for detecting factor 8 gene (F8) mutations in hemophilia A patients by Ion Torrent semiconductor sequencing .Methods Intron 22 and intron 1 inversions of F8 gene were identified by long distance PCR (LD-PCR), other mutations in the F8 gene were identified by Ion Torrent sequencing.Candidate variants were validated by Sanger sequencing .Sanger sequencing was applied to screen HA carriers from 11 female family members in the 8 pedigrees.One pregnant woman was offered prenatal diagnosis via analyzing the fetal DNA obtained through amniocentesis . Results Four missense mutations ( c.1331A >C, 1648C >T, c.6506G >A, c.6544C >T), two frameshift mutations ( c.2393 _2394insT, c.6320delG), one splicing mutation ( IVS5 +5G >A), one nonsense mutation (c.43C >T) and one Inv22 mutation were identified in all nine probands respectively . Among 11 female family members, 10 females were identified to be HA carriers, and one didn′t carry the maternal pathogenic mutation.Prenatal diagnosis result showed that the fetus inherited the wild -type maternal allele and was predicted to be unaffected by HA .Conclusion The targeted Ion Torrent sequencing is a reliable and efficient method to detect F8 mutations in patients with Hemophilia A disease .