碳納米纖維支架與兔脂肪干細(xì)胞的體外相容性研究.pdf

1、PartⅠ：Characteristics of Isolation and Culture of Rabbit Adipose-Derived Stem Cells in vitro Objectives：To establish the method for isolation and culture of rabbit adipose-derived stem cells (ADSCs)in vitro，identifying &

2、 analyzing its biological characteristics，and exploring the optimal condition for culture of adipose-derived stem cells in vitro． Methods：Three female New Zealand rabbits were selected，the adipose-derived stem cells in i

3、nguinal region were harvested by digestion of collagenase I enzymatic hydrolysis．After the differential adherent culture conducted to the selected passage cells，the cells extracted and purified．The flow cytometry is adop

4、ted to indentify adipose-derived stem cells using monoclonal antibodies of mouse-anti-rabbit CD44 and CD166．The proliferation and growth curve of the 4th generation of ADSCs were detected to investigate the morphological

5、 features and proliferation states of the primary and passage adipose-derived stem cells (ADSCs)，while adipogenic and osteogenic inductions and identification were also implemented．ADSCs were induced for differentiation

6、in osteogenic medium for 3 w and in adipogenic medium for 2 w．Finally，the target genes were detected once a week using RT-PCRtechnique． Results：The growth morphology of primary and passaged ADSCs：the morphology of primar

7、y cell was smaller and grows adherently in the shape of long-spindle or polygon; the shape of passage cell achieved by differential adherent culture was expanded in cobble-stone-like distribution，and the growth rate of t

8、he passage cell that is in active growth and migration was found significantly faster than primary cell． The result of flow cytometry shows that the positive rate of surface markers CD44 and CD166 of rabbit adipose-deriv

9、ed stem cells is 61％ and 62％，respectively． The growth curve of rabbit ADSCs presents the shape S，the growth residence phase persists for two days followed by logarithmic phase at day 3 and platform phase probably at day

10、7． ADSCs can be induced to differentiation：During osteogenic differentiation，Runx2 gene of the experiment group have stronger expression in Week 1，then the band intensity gradually decreased; the expressions of COLL-1 an

11、d OC genes both increased along with time，until Week 3 when the relevant band was the brightest．During adipogenic differentiation，the band intensities of experiment group PPARγ and LPL genes increased along with time，and

12、 no expression was detected in leptin gene until Week 2．All the target genes of the control group cells had no positive expression． Conclusions：ADSCs were isolated and cultured successfully from subcutaneous adipose tiss

13、ue of New Zealand rabbit in the experiment and a set of simple and effective methods for isolation，culture and identification of ADSCs were established．Rabbit ADSCs are expected to be an option as the seed cells of tissu

14、e engineering due to its excellent capacities of growth，proliferation and differentiation in vitro.
　　 PartⅡ：Biocompatibility of Carbon Nanofibrous Scaffold and Rabbit Adipose-Derived Stem Cells in vitro． Objectives：

15、To co-culture the adipose-derived stem cells (ADSCs)and carbon nanofibrous scaffold，observe the biological performance of ADSCs on carbon nanofibrous materials，investigate the effect of carbon nanofibrous materials on pr

16、oliferation，adhesion and differentiation of ADSCs and to evaluate the biocompatibility of materials of carbon nanofibrous scaffold． Methods：PLLA/PCL/F-MWNTs complex solution is used in synthesis of carbon nanofibrous sca

17、ffold with electrospinning technology．The 4th generation of well-growing ADSCs that we got from part one of our experiment，is selected to be cultured with carbon nanofibrous materials．The effect of carbon nanofibrous sca

18、ffold on the proliferation of rabbit adipose-derived stem cells is measured by MTT assay，and the adhesion rate of cells on materials is calculated．Finally，the osteogenic and adipogenic differentiation are performed on ca

19、rbon nanofibrous scaffold materials and the markers，Runx2，COLL-1 and OC for osteogenesis and PPARγ，LPL and leptin for adipogenesis are detected using RT-PCRtechnique． Results：The cells grow well after co-culture of ADSCs

20、 and carbon nanofibrous scaffold; there is no significant difference in absorbance value measured by MTT method between experiment group (cells with carbon nanofibrous scaffold)and control group (cells without scaffold)a

21、nd the absorbance value in experiment group is greater than that of control group．The cellular adhesion measured with-carbon-nanofibrous-scaffold group is more significant than that without-carbon nanofibrous-scaffold gr

22、oup．After inducing ADSCs toward osteogenic and adipogenic differentiation on the surface of carbon nanofibrous scaffold，the gene band appears in both experiment group and control group detected by RT-PCRtechnique while t

23、he brightness in experiment group is slightly weaker than that in control group． Conclusions：The rabbit adipose-derived stem cells present normal growth，proliferation and adhesion with carbon nanofibrous scaffold in vitr