The Study Of Exogenous TGF-β₁ And IGF-1 Induce Adipose Derived Stem Cells To Differentiate To Chondrocyte

ObjectiveTo investigate the methods of isolation, cultivation and amplification of Adipose derived stem cells (ADSCs) and the feasibility of inducting ADSCs to differentiate to chondrocyte in vivo by monolayer culture with exogenous Transforming growth factor-β₁ (TGF-β₁)and Insulin-like growth factor-1(IGF-1).Materials and methodsThe adipose tissue was obtained from rats' epiploica and fatty capsule of kidney, the isolation and cultivation of ADSCs is processed by enzyme digesting ;the expression of CD29 and CD44 were detected by immunofluorescence; The passaged cells were exposed to four kinds of defined mediums for chondrogenic phenotype differentiation. After two weeks of induction, the identification of chondrogenic phenotype were pursued by RT-PCR,Western blot and immunofluorescence.Results(1)The primary cultured ADSCs could adhere to the culture flask within the first 24h ,and it may form the colony in about 5 days and get 90% confluence within 7 days. The offspring cells grew more rapidly and was basically fibroblast-shaped when cultured in vitro;(2)the result of immunofluorescence shows that surface protein CD29 and CD44 were positively expressed on ADSCs;(3) After chondrogenic induction, the growth velocity of each group was slower than before especially in the control group. The appearance of each group except control group became polygonal ,we can see pseudopodia and black particles around the nucleus when observed in microscope . After 14 days chondrogenic induction , the cells became buninoid and aggregate into cell nodules , the morphology changes were more obviously in the TGF-β₁ group and T+Igroup;(4)the detection of Collaggen II by immunofluorescence after chondrogenic induction shows the following findings: the blank group shows negative expression but weakly positive,positive and powerful positive expression in the IGF-1 group,TGF-β₁group and TGF-β₁+IGF-lgroup;(5) Reverse- Transcription-Polymerase Chain Reaction(RT-PCR) Analysis: Collagen II mRNA was positively expressed in other groups but blank group was negative. the amount of the Collagen II mRNA expression was augmented in the 4 groups. Significant statistical difference was found among each group (P<0.01) ; IGF-1group,TGF-β₁group and T+Igroup shows a augmented expression of Aggrecan mRNA while blank group and control group show negative results. there's no significant statistical difference between the blank group and control group (P>0.05) but significant difference between the negative 2 groups and positive 3 groups and among the positive 3 groups (P<0.01) ;(6)the result of Western blot show that Collagen II protein was positively expressed in other 4 groups but negatively in the blank group, Significant statistical difference was found among each group (P<0.01)Conclusion(1)ADSCs can be isolated from rat's adipose tissue and and their biological characteristics are stable; (2) The surface protein CD29 and CD44 were positively expressed on ADSCs (3)Either exogenous IGF-1 or TGF-β₁ can induce ADSCs to express chondrogenic phenotype and it can seen a cooperativity when they act together...