The Study Of RAd5-BMP2 And RAd5-TGFβ3 Vector Transfected And Induced BMSCs From Diannan Small-ear Pig Into Chondrogenic Differentiation

Part 1 The culture and identification of BMSCs from Diannan Small-ear Pig in vitro[Objective】To find amplification method of bone marrow mesenchymal stem cells from Diannan Small-ear Pig,and get a better amplification method for seed cells of cartilage tissue engineering.[Method】Bone marrow mesenchymal stem cells were harvested from Diannan Small-ear Pig,then cultured with centrifugation and adherent method.The CD29, CD44, CD45 markers of BMSCs in the primary.the first and the second generation were examined with flow cytometry. And the second generation of BMSCs were induced into bone and chondrogenic differentiation.the ability of osteoblast differentiation was examined with alizarin red, Von kossa staining.the ability of chondrogenic differentiation was examined with Western blot.[Results】(1)Cells shape were spindle and fibroblast-like.(2) Flow cytometry results: P₀CD29 positive rate was 73.10%,P₀CD44 positive rate was 69.74%,P₀CD45 negative rate was 99.68%.P₁CD29 positive rate was 99.44%,P₁CD44 positive rate was 99.96%,P₁CD45 negative rate was 95.81%.P2CD29 positive rate was 99.49%,P2CD44 positive rate was 99.52%,P₂CD45 negative rate was 99.93%.(3)alizarin red staining,Von kossa staining showed positive after BMSCs were induced 3 weeks.(4) Collagen II Western blot showed positive after BMSCs were induced 3 weeks. [Conclusion](1)Centrifugation and adherent method could culture BMSCs from Diannan Small-ear Pig,it was an efficient method of amplification. (2) The second generation of BMSCs from Diannan Small-ear Pig with high purity and could be used as seed cells of tissue engineering. (3)The second generation of BMSCs from Diannan Small-ear Pig with multiple differentiation potential, could differentiate to chondrogenic,and could be used as seed cells of tissue engineering.Part 2 Construction of rAd5-BMP2-EGFP and rAd5-TGFp3 vectors[Objective]To construct rAd5-BMP2-EGFP and rAd5-TGFβ3 transfection vectors[Method]BMP2 gene was amplified with PCR;then PCR products were connected to the T vectors and were sent to sequence;BMP2 T vectors were identified with BamHI,EcoRI double digestion;BMP2 were subcloned into the shuttle vector PEC3.1-GIE,BMP2 expression cassettes would be transferred to pGSadeno adenovirus vectors through LR in vitro homologous recombination,and package adenovirus.rAd5-BMP2-EGFP vectors were successfully constructed through fluorescence microscopy observation, and determined virus titer. TGFβ3 gene was amplified with PCR;then PCR products were connected to the T vectors and were sent to sequence;TGFβ3 T vectors were identified with BamHI,EcoRI double digestion;TGFβ3 were subcloned into the shuttle vector PEC3.1,TGFβ3 expression cassettes will be transferred to pGSadeno adenovirus vectors through LR in vitro homologous recombination,and package adenovirus.rAd5-TGFβ3 vectors were successfully constructed with PCR identification, and determined virus titer.[Results](1)By PCR amplification,the size of BMP2 gene was 1.2k.(2) PCR products were connected to the T vectors and the sequence result was no base with mutation or loss or increased.(3)The size of BamHI,EcoRI double digestion BMP2 T vector was 1.2k.(4) PCR identification of BMP2 pGSadeno adenovirus vectors bands could be seen in 1.2k size.(5)The third round of the virus titer>109pfu/ml, and could see the obvious green fluorescence.(6)By PCR amplification,the size of TGFP3 gene was 930bp.(7) PCR products were connected to the T vectors and the sequence result was no base with mutation or loss or increased. (8) The size of BamHI,EcoRI double digestion TGFβ3 T vector was 930bp.(9)PCR identification of TGF(33 pGSadeno adenovirus vectors bands could be seen in 930bp size.(10) The third round of the virus titer>109pfu/ml,and PCR identification of virus could be seen in 514bp size.[Conclusion] We successfully constructed rAd5-BMP2-EGFP and rAd5-TGFβ3 vectorsPart 3 rAd5-BMP2 and rAd5-TGFp3 induced BMSCs from Diannan Small-ear Pig to chondrogenic differentiation[Objective] rAd5-BMP2 and rAd5-TGFβ3 were transfected into BMSCs from Diannan Small-ear Pig,then BMP2 and TGF(33 expression were examined in BMSCs, changes were observed in the chondrogenic differentiation.[Method] rAd5-BMP2-EGFP were alone transfected,rAd5-BMP2-EGFP and rAd5-TGF(33 were together transfected into BMSCs from Diannan Small-ear Pig, the rate of fluorescence infection on 1,2,3,4 days were examined by flow cytometry. rAd5-BMP2 and rAd5-TGFβ3 were together transfected,HK were alone transfected into BMSCs from Diannan Small-ear Pig,cultured with normal cells at the same time,then cells morphology were observed, BMP2 and TGFβ3 were examined with Western blot in BMSCs after 7,21 days,collagen II were examined with Western blot after three weeks.[Results]The positive rate of fluorescence on 1,2,3,4days by flow cytometry in transfected rAd5-BMP2-EGFP group was 28.15%,77.84%,81.57%,84.86%,in transfected rAd5-BMP2-EGFP and rAd5-TGFβ3 group was 19.80%,35.26%,78.62%, 77.27%.The Western blot examination of BMP2 and TGFβ3 on 7,21 days in BMSCs was positive.The Western blot examination of collagen II in BMSCs after three weeks was positive. 【Conclusion](1)BMP2 and TGFβ3 could be simultaneously transfected and effectively expessed in BMSCs of Diannan Small-ear Pig.the best transfection time was at 72 hours.(2) BMP2 and TGF(33 could induced BMSCs of Diannan Small-ear Pig into chondrogenic differentiatation.