Experimental Study Of Rat's BMSCs Differentiation To Chondrocytes Transfected By TGF-β1 And IGF-I Gene Alone And Together

PurposeInvestigate the methods of isolation,cultivation and the detection of surface maker of rat bone marrow mesenchymal stem cells(MSCs).To provide a new method for tissue- engineering cartilage seed cells,rat's bone marrow stormal cells(BMSCs)were transfected with TGF-β1 and IGF-1 gene alone and together,then observaed and compared the destined gene secretion and the differentiation of transfeced BMSCs into chondrocytes.Methodswe amplified and extracted the plamid of pcDNA3.1-IGF-1,pcDNA3.1-TGF-β₁, then degisted it by enzymes,electrophoresed and measured its sequence.BMSCs of Wister rat were separated and purificated by the density gradient centrifuge assay combined with pastes the wall parting method,inverted phase contrast microscope was used to observe the morphology change of primary and passaged cells.Five groups were classified, according to the transfect situation:the non-transfected group(Group A),the transfected by vector group(Group B),the transfected by TGF-β₁ group(Group C),the transfected by IGF-1 group(Group D)and the transfected by TGF-β₁ and IGF-1 group (Group E)..Immunofluorescence was used to assay cell surface markers.Rat's BMSCs were ransfected with TGF-β1 and IGF-â… gene alone and together,than we selected the cells with tansgenes,MTT was also used to test the multiplication activities of selected cells,meanwhile,RT-PCR,Western blot were used to analyze the expression leve- s of the selected cells.ResultsThe result of electrophoresis demonstrated that two bands of the target gene,IGF-1 and TGF-β₁,meanwhile its sequence was identical with the sequence of GenBank cDNA. The primary cells of rat's BMSCs had few processing cells after 24h cultivation,typicall amplification cluster was formed on days four or five,and 80～90%fusion was reached on days nine or ten.The shape of the cells became similar after passaging.Few cells died after 24h of transfection,cell clone was formed after three weeks of selection,and the cells could be passaged at the four weeks,most cells became polygon,some cells had obscure boundary,round,nucleus dissymmetry with obvious particles around the nucleus obviously.The cell surface markers detected by Immune fluorescence CD29 and CD44 were positively expressed,whereas CD34 and CD45 were negatively expressed. The cells' A value tested by MTT at the wavelength of 490nm,group A:0.432±0.038,group B:0.42 8±0.041,group C:0.664±0.086,group D:0.655±0.045 and group E: 0.833±0.103.The differences between Group A,B and group C,D,E is not statistically significant(P＜0.01),and the difference between group A and B or between C and D and E also is not statistical significance(P＞0.05).RT-PCR and Western blot was used to detect the expression of target gene and protein,the expressin of TGF-β1 was most in group C,0.925 0±0.022 0,124.341 7±2.982 0,followed by group E,0.771 7±0.012 0,101.766 7±1.241 0(P＜0.01);The expression of IGF-1 was most in group E,1.020 0±0.026 0,111.045 0±6.248 0,followed by group D,0.465 0±0.042 0,11-1.045 0±6.248 0(P＜0.01),whereas collagenâ…¡,most in group E,0.980 0±0.034 0,120.355 0±12.550 0.followed by group C,0.720 0±0.026 0,72.246 7±7.364 0(P＜0.01)ConclusionsAfter TGF-β₁ and IGF-1 gene was introduced into mesenchymal stem cells(MSCs), TGF-β₁,IGF-1 and collagenâ…¡expression were higher than control group,so did proliferative capacity.All these results indicate that MSCs can be used as the seed cells of tissue-engineering cartilage...