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OBJECTIVE@#To transinfect SD adipose tissue-derived stem cell (ADSC) in vitro with a recombinant adenoviral vector containing human B-domain-deleted FVIII (BDDhFⅧ), so as to lay the foundation for the treatment of hemophilia A by using ADSC combined with BDDhFⅧ gene.@*METHODS@#ADSCs were isolated from the inguinal adipose tissue of SD rats and passed to third passage for identification. Third passage ADSCs were transfected in vitro with recombinant adenovirus vector Ad-BDDhFⅧ-GFP. The experiments were divided into Ad-BDDhFⅧ-GFP-transfected ADSCs group (A), Ad-GFP-transfected ADSC group (B), and untransfected ADSC group (C). CCK-8 method was used to detect the proliferation of transfected cells in three groups, and the expression level of hFⅧ antigen in cell supernatant was detected by ELISA. RT-PCR and Western blot respectively were used to detect the mRNA and protein expression of BDDhFⅧ in the three groups after transfection.@*RESULTS@#The growth curve of third passage cells isolated and cultured showed an inverted "S" shap; the flow cytometry detection showed the positive expression of CD29, CD90, CD44, and the negative expression of CD45 in third passage cells. After the adipogenic and osteogenic induction, the cells could transformed to adipogenic and osteogenic directions. CCK-8 detection showed that the proliferation of cells in 3 groups not was influenced. ELISA showed that the expression of hFⅧAg in group A was significantly higher than that in group B and C (P＜0.05). RT-PCR showed that compared with group A, there was no target band in B and C groups, and BDDhFⅧ gene was not expressed. The results in group A were consistent with the length of amplified fragments, and BDDhFⅧ target gene was expressed. Western blot analysis showed that the expression of hFⅧ protein in group A was significantly higher than that in group B and C. (P＜0.05).@*CONCLUSION@#Recombinant adenovirus Ad-BDDhFⅧ-GFP can effectively transfect rat ADSC in vitro, which lays an experimental foundation for gene therapy of hemophilia A.
Subject(s)
Animals , Humans , Rats , Adenoviridae , Adipose Tissue , Cell Differentiation , Cells, Cultured , Factor VIII , Rats, Sprague-Dawley , Stem Cells , TransfectionABSTRACT
Objective:To explore the potential relationship between nutritional risk evaluated by NRS 2002 and the indexes of physical and biochemical parameters.Methods:From January 2015 to December 2015,218 hospitalized patients with chronic obstructive pulmonary disease (COPD) were randomly selected to assess the nutrition risk by using NRS 2002,and the physical examination indexes and biochemical indicators were also collected.The potential correlations between NRS 2002 score and the physical and biochemical parameters were analyzed.Results:The incidence of nutritional risk (the score of NRS 2002 ≥ 3) was 31.7％ in 218 COPD inpatients.Both age and pulmonary function was significantly related to the incidence of nutritional risk (P ＜ 0.05).The relevance also was found between NRS 2002 score and 2 physical indexes (BMI and FFMI) and 2 biochemical indicators (ALB and PA).Conclusion:The frequency of nutritional risk is very high among hospitalized patients with COPD,especially in old patients.NRS 2002 is a suitable nutritional risk screening tool in COPD patients,but a comprehensive nutrition assessment should combine proper nutritional risk screening tool with physical examination indexes and biochemical indicators in clinical applications.
ABSTRACT
<p><b>OBJECTIVE</b>To establish a highly sensitive and specific dual-color fluorescence in situ hybridization (D-FISH) method used for chromosomal localization of foreign genes in double transgenic mice.</p><p><b>METHODS</b>Two strains of double transgenic mice were used in this experiment, one was integrated with the herpes simplex virus thymidine kinase (HSV-tk) and the enhanced green fluorescence protein (eGFP), the other was with the short hairpin RNA interference(RNAi) and beta(654). Splenic cells cultured in vitro were arrested in metaphase by colchicine and hybridized with digoxigenin-labeled and biotinylated DNA probes, then detected by rhodamine-conjugated avidin and FITC-conjugated anti-digoxigenin.</p><p><b>RESULTS</b>Dual-color fluorescence signals were detected on the same metaphase in both transgenic mice strains. In HSV-tk/eGFP double transgenic mice, strong green fluorescence for HSV-tk and red for eGFP were observed and localized at 2E5-G3 and 8A2-A4 respectively. In beta(654)/RNAi mice, beta(654) was detected as red fluorescence on chromosome 7D3-E2, and RNAi showed random integration on chromosomes. It was detected as green fluorescence on chromosome 12B1 in one mouse, while on 1E2.3-1F and 3A3 in the other.</p><p><b>CONCLUSION</b>Highly sensitive and specific D-FISH method was established using the self-prepared DNA probes, and chromosomal localization of the foreign genes was also performed in combination with G-banding in double transgenic mice. This technology will facilitate the researches in transgenic animals and gene therapy models.</p>