Animals ; Bone Marrow Cells ; cytology ; Cell Hypoxia ; Cells, Cultured ; Female ; Male ; Mesenchymal Stromal Cells ; cytology ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Rabbits ; Vascular Endothelial Growth Factor A ; genetics ; metabolism

Asphyxia Neonatorum ; blood ; drug therapy ; Creatine Kinase, MB Form ; blood ; Female ; Humans ; Infant, Newborn ; Male ; Naltrexone ; analogs & derivatives ; therapeutic use ; Natriuretic Peptide, Brain ; blood ; Peptide Fragments ; blood

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 relationship between insulin resistance and other metabolic disorders in patients with essential hypertension (EH).Methods Glucose metabolism rate (GMR) was measured by euglycemic insulin clamp technique,and salt sensitivity was tested by increase in blood pressure after salt load and its decreases alter depletion of sodium in 26 healthy subjects and 84 patients with EH.Results GMR lowered significantly in patients with EH than that in healthy subjects,P

Objective To investigate whether erythropoietin(Epo) is potentially beneficial in protecting cultured retinal neurocytes.Methods Primary isolated retinal nerve cells were cultured.Expressions of Epo and EpoR protein in cultured retinal neurocytes were decected by immunohistochemical analysis.Survival of cultured neurocytes that were incubated in the presence of Epo or glutamate in the presence or absence of Epo were estimated by determining the activity of their mitochondrial dehydrogenases using the MTT assay.Results Epo and EpoR protein were expressed on the cultured retinal neurocytes.The presence of different concentrations of Epo did not improve the survival of retinal neurocytes,and Epo could prevent glutamateinduced toxicity. Conclusion Epo is beneficial in protecting mixed cultured retinal neurocytes from glutamate-induced cytotoxicity.

HER2, a member of epidermal growth factor receptor family proteins, is overexpressed in about 30% of human breast cancer. Increased levels of HER2 are associated with poor patient prognosis and enhanced metastasis. RNA interference (RNAi) is developed recently as a new technique which can inhibit gene expression specifically in mammalian cells. On the basis of previous study,in which two target sequences with favorable RNAi effect on HER2 were identified, a series of dual promoter siRNA-expressing vectors containing two opposing U6 and H1 promoters were constructed. After transfection of HER2-overexpressing SKBR3 breast cancer cells with the siRNA-expressing vectors, downregulation of HER2 was identified quantitatively. Subsequently, the siRNA-expressing cassettes were subcloned into lentiviral vectors by LR recombination reaction and lentivirus was prepared successfully. The results from infection of SKBR3 cells with siRNA-expressing lentivirus demonstrated that lentiviral-mediated RNAi could downregulate HER2 expression efficiently through fluorescent quantitative PCR (FQ-PCR), western blot, and FACS analysis. Furthermore, cell growth was inhibited in cell proliferation assay after treatment with siRNA lentivirus.A new tool for clarifying the function of HER2 in cancer metastasis and developing the gene therapy drug was offered.

Objective To analyze the electrocardiography(ECG)data of pressure overload-induced cardiac hy pertrophy rats.Methods Pressure overload cardiac hypertrophy was induced by abdominal aorta constriction. Echocardiogram and heart weight measurement demonstrated the occurrence of cardiac hypertrophy.Standardized ECG parameters of limb and chest were measured and statistically analyzed.Results Two weeks after hypertrophy models were established,echocardiogram showed greater LVPWTd,IVSTd,LVDd.ECG showed that left axis deviation and higher R waves in V_A,V_B,V_C(P

The biological!activities i.e. antineoplastic activities and antiviral activity of the two novel kinds of interferons: hIFN-?1 and hIFN-? were studied and compared. First the fusion expression vector: pcDNA3.1A-hIFN-?1-His and pcDNA3.1A-hIFN-?-His by PCR was constructed,then the two kinds of plasmids were transfected into WI-38 (human embryonic lung cells) with liposome. And cytopathic effect (CPE) suppression test was used to study and compare the antiviral activities of rhIFN-?1-His and rhIFN-?-His, meanwhile MTT assay was used to detect their antineoplastic activities.It was found that, antiproliferative activity and MxA protein induction shown by rhIFN-?1-His is more powerful than of rhIFN-?-His. The antiviral molecular mechanisms of both hIFN-?1 and hIFN-? are related to MxA.The foundation for further study on the bioactivities and mechanism of action of hIFN-?1 and hIFN-? was established.

The known members of inhibitor of growth (ING) gene family are considered as candidate tumor suppressor genes. ING4, a novel member of ING family, is recently reported to regulate brain tumour angiogenesis through transcriptional repression of NF-?B-responsive genes, induce G2/M arrest by the increased p21 expression in a p53-dependent manner, suppress the loss of contact inhibition and represses activation of the hypoxia inducible factor, which plays an important role in the progression of tumorigenesis. However, seldom studies about ING4 inducing tumor cells apoptosis were reported.The C6 cells (mouse glioma cells) were infected respectively with the blank adenovirus carrying GFP (Ad) and the recombinated Ad-hING4-His, then RT-PCR assay was used to detect the transcriptions of hING4, as well Western-blotting assay was ued to detect the expressions of hING4. The effects of hING4 expression upon C6 cells were observed, and the growth curve was drawed and tumor control rates were calculated. The C6 cells, which were affected by blank Ad and Ad-hING4-His, were respectively observed by LSCM (laser scan confocal microscope) and transmission electron microscope (TEM), detected by flow cytometry; and the genomic DNA of both groups were extracted and electrophoresised in agarose gel to examinate the DNA fragments. The results showed hING4 can significantly inhibit the growth of C6 cells by promoting the cell’s apoptosis, which probably is the first one to prove this property of ING4.The experimental and theoretical foundation for gene therapy for gliomas with ING4 in the future was established.

Objective To improve the coverage rate of medical equipment metrological verification to ensure medical equipment safety and efficiency.Methods PDCA cycle was used to analyze the main problems in medical equipment metrological verification from the aspects of personnel,institution,management and flow,and the solutions were put forward accordingly.A certain of verification devices were sampled after verification,and the coverage rate of medical equipment metrological verification in the hospital was estimated based on the corresponding value of the sampled devices.Results Statistical analysis on the verification devices in 3 a showed the coverage rate of verification was enhanced from 45.5％ to 97.5％.Conclusion PDCA cycle contributes to increasing the coverage rate of medical equipment metrological verification.