Research On ADSCs Transfected With Adenovirus Containing BMP-9

Objective:1.To obtain purification ADSCs and set up a ADSCs cultural method stably and high efficiently.2.To explore an empirical method about ADSCs transfected with BMP-9, confirm ADSCs with BMP-9 can differentiate to the osteoblast and gain gene-modified seed cells which can be used for bone tissue engineering.Methods:1.Separation,cultivation and identification of ADSCs: Obtained primary ADSCs was with the I-collagenase digestion. Following amplification and purification of ADSCs, morphology was observed by microscope, proliferation in vitro was detected by MTT and cell count, and expression of surface antigen CD29 and CD44 was detected by immunohistochemistry.2.Transfection into ADSCs with BMP-9: adenovirus containing BMP-9 infected ADSCs in P3 period, effect of infection was observed by fluorescence microscope and infection efficiency was determined by flow cytometry. Expression of BMP-9 mRNA in ADSCs infected was detected by RT-PCR. Osteoblastic activity of ADSCs infected was detected by semi-quantity kit of alkaline phosphatase, alkaline phosphatase and alizarin red staining of calcium nodules.Results:1.Separation,cultivation and identification of ADSCs(:1)Morphology of primary ADSCs were spindle-shaped or angular, similar to fibroblasts, cluster-like growth. At the time of 10 days 90% cells were fusions.(2)ADSCs with passage and purification were fibroblast-like, distribution,size, consistent with cells close and swirl-like growth. At the time of 5 days 90% cells were fusions, which showing activity proliferation.(3)Growth curve showed: day 1 to 3 was lag phase of serial subcultivation, day 4 to 6 was logarithmic growth phase, and day 7 to 9 was growth inhibiting phase, in which cells growth slowed down.(4)Cumulative multiplication curve showed: population doubling time was about 55 hours, proliferation rate of cells in P3 period was fastest of all, and after P8 period, proliferation rate of cells slowed down and cells showed aging sign.(5)Immunohistochemistry showed: surface antigen CD29 and CD44 of ADSCs were both positive.2.Transfection into ADSCs with BMP-9:(1)Adenovirus containing BMP-9 could infect ADSCs successfully.(2)Fluorescent was observed at the time of 24h and the fluorescence intensity over time gradually after ADSCs infected by adenovirus containing BMP-9. After 3 days, infect rate could reach to more than 80%(.3)Lag phase of ADSCs with adenovirus was slightly extended, quantity reduced slightly and doubling time was slightly extended, which didn't affect cell proliferation.(4)BMP-9 mRNA in cells with adenovirus was positive and the expression was sustained. There was no expression in cells without adenovirus.(5)Alkaline phosphatase and alizarin red staining of calcium nodules were positive in ADSCs infected. The activity of alkaline phosphatase was growing trend and infected group was significantly higher than non-infected.Conclusions:1.ADSCs, which were separated from adipose tissue of rabbit, cultivated and purified, had obvious characteristics of stem cells. The characteristics were high growth in vitro, amplified rapidly and maintained stable reproductive activity after passage of long-term. It will offer basis for ADSCs infected with BMP-9 osteoinduction further.2.Adenovirus containing BMP-9 could be infected ADSCs safely and efficiently. BMP-9 gene infected had higher expression and a significant role in osteogenesis. Therefore, ADSCs infected with adenovirus containing the length of human BMP-9 could be a reliable method to research on BMP-9 inducting ADSCs osteogenesis.