The Differentiation Of Human Mensenchymal Stem Cells Deriving From Bone Marrow Transfected With Ectogenic TGF-β₁ Gene By Liposome-mediated Ex Vivo

Objective: To study the differentiation of human mensen- chymal stem cells driving from bone marrow(hBMSCs) transfected with TGF-β1 gene through liposome-mediated ex vivo. Method: 1. The isolation and culture of hBMSCs. ⑴ Bone marrow of 10 ml were sucked in greater trochanter of male adult femur, then Percoll cells isolation liquid(1.073g/ml) was used to dissociate hBMSCs and make primary culture. ⑵ Replaced DMEM medium contained 10%FBS every two or three days and observed cellular morphologic change and growth behaviors. ⑶ When hBMSCs were propagated to the fourth generation, we taked parts of cells to be cultured with conditional medium (complete medium with dexaiafrma 10-8mol/L, ascorbic acid 50mg/L, beta-sodium glycerophosphate 100mmol/L) and haved alkaline phosphatase stain and economycin stain respectively in the given time. 2. The construction and amplification of pACCMV-hTGF-β1 gene. ⑴ Recombinant plasmids were contructed by the second medical university of shanghai. ⑵Amplification of recombinant plasmids. ①the E.coli JM109 was Proliferated in the plate culture medium with the method of painting under 37℃ to stay overnight. ②Then, under 37℃, we picked a mono-colony and put it into the liquid culture medium, shaking it to OD260=0.3~0.4 . ③The germs were made into the sensitive state with the method of CaCL2. ④The plasmids contained the purpose gene were added into them. Thus, we could transfect the plasmids cDNA into the thallus by 42℃ heat-shock . ⑤Kept them staying overnight under 37℃ in the liquid culture medium. ⑥The bacterio-liquid was spreaded on the plate culture medium contained the Aminobenzylpenicillin,the X-gal and IPTG. ⑦Then,we picked out the needed germs by drug-resistance screening and the blue-white spots test. ⑧After it, we continued to culture the bacterio-liquid overnight. ⑨The plasmids were purifyed by Centrifugation, and measured the density and the purity by spectrophotography (the concentration and the purity of plasmid cDNA that drawed out is 0.067μg/μL and 1.782 respectively). ⑩ Finally, we could evaluate plasmids cDNA extracted with mono-restriction endonuclease enzyme and the agar gel electrophoresis. ⒊ The transfection of hBMSCs. ⑴ We divided hBMSCs into the transfected cells group and the untransfected cells group. One day before transfceting, we digested the fourth generation hBMSCs with 0.25%trypsin and 0.2% EDTA(the ratio of volum is 1:1), and we inoculated it into petri dishes whose diameter is 35mm with the concentration of 5.0×104/ml and laid previously four pieces of cover glasses in petri dish. ⑵ Dispensed transfecting fluid referring to the introduction of cationic liposomes transfection reagent (TransFastTM Reagent), preservated at -20℃ for use. ⑶ The plasmids contained GFP gene were transfected into hBMSCs to detect and optimize transfection efficiency. ⑷ Transfected pACCMV-hTGF-β1 gene into hBMSCs at optimized transfection efficiency. ⑸Finally, we could make immunocyto- chemical fluorescence stain about TGF-β1, typeⅠ and typeⅡ collagen to observe the differentiation of transfected cells. Result: 1. 48 hours after inoculating hBMSCs, we could see cells and cellular colonies distributing rarefied with invert microscope. Fusiform cells showed typical fibroblast-like appearance. About two weeks later, cells fused on the whole, meanwhile, the morphologic change of cells colonies shows radiating or whirlpool-like. Detached primary cells showed alive 100% staining with 4% trypan blue. Digested cells appeared to be shrinked but the ratio of alive cells reached to 93%~98%. 2. When propagating to about 50%, the fourth generation hBMSCs were cultured with conditional culture medium. After a week, there had brownish particles in celluar plasm with alkaline phosphatase stain. About two weeks later, hBMSCs formed Calcified nods, which were dyed into golden color with economycin when observed with confocal microscopy. 3. Immunocytochemical fluorescence staining indicated, after transfecting 48 hours, there were parts of c...