| Objectives To observe the expression of human coagulation factor ?(hF?)gene mediated by adenovirus in adipose-derived stem cells(ADSCs)of mice,and to explore whether ADSCs have the ability to carry and transport hF? in gene therapy of hemophilia B and the possibility of ADSCs as a cell vector for gene therapy of hemophilia B.Methods 1)Isolation,Culture and Identification of Cells Four male SPF-grade C57BL/6J mice aged 3-4 weeks were killed by cervical dislocation.Adipose tissue was taken from both sides of the groin under sterile condition.After digestion with 1% collagenase,primary mouse adipose stem cells were cultured and subcultured,and to observe the shapes of cells.Growth curve was studied by using CCK-8 method.Cells surface markers were identified through flow cytometry,and the diferentiation ability were identified by adipogenic and osteogenic induction.2)Cell grouping and transfection The third generation cells with good growth were randomly divided into three groups: blank group(DMEM was used instead of adenovirus to transfect ADSCs),blank group(Ad-GFP was used to transfect ADSCs),and experimental group(Ad-hF? was used to transfect ADSCs).To observe morphological changes and the growth curve after adenovirus containing hF? gene transfected to ADSCs.3)Cell detection after transfection The expression of hF? gene was detected by RT-PCR.The expression of hF? protein in ADSCs and in culture supernatant was detected by Western blot?The hF? protein amount in the transfected ADSCs was measured by ELISA,the coagulation activity in culture supernatant was measured by one-stage method.Results 1)Cell morphology and growth curve ADSCs of primary isolated mice showed small spherical shape,strong refractive property,and began to adhere to the wall at 4-6 hours.After 72 hours,the cells showed long spindle-shaped fibrous and swirling arrangement.The growth rate of ADSCs of the third generation was slow in 1-2 days,increased exponentially in 3-5 days,and slowed down gradually in 6-7 days,and the growth curve was S-shaped.2)Induction of adipogenic osteogenesis Oil red O staining and inverted microscope observation showed red lipid droplets form.After alizarin red staining,orange-red calcified bone nodes were observed under inverted phase contrast microscopy.3)Phenotypic identification of flow cytometry CD29(99.91%)and CD90(99.02%)were highly expressed in the third generation of ADSCs,but almost no CD45(0.94%)was expressed.4)Viral transfection When MOI = 300 was taken,the intensity of GFP expression in cells 24,48 and 72 hours after virus transfection was the strongest,and the transfection efficiency could reach more than 80%.The cell morphology after transfection were not significantly different from those before transfection.5)mRNA expression RT-PCR proved hF? gene could be expressed in ADSCs.6)Expression of hF? protein in cells and supernatants Western blot confirmed hF? protein could be expressed in ADSCs and in culture supernatant.7)F?:Ag testing F?:Ag in cell supernatant was determined by ELISA as 21.33 ±3.93 ng/(106 cells.24h)on the first day,12.63±0.86 ng/(106 cells.24h)on the third day and 12.63±2.36 ng /(106 cells.24h)on the ninth day.8)F?:C testing F?:C in culture supernatant was 8.5% by one-stage assay.It is concluded that adenovirus can successfully transfect mouse ADSCs to secrete hF? protein,which lays a foundation for subsequent cell therapy for hemophilia B.Conclusions 1)Adenovirus carrying hF? gene can effectively transfect ADSCs.2)ADSCs modified by hF? gene can secrete hF? protein with coagulation activity.Figure 10;Table 4;Reference 68. |
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