目的 比较3种成软骨诱导培养基对单层培养和微球培养的人脂肪干细胞（human adipose mesenchymal stem cells，hASCs）体外成软骨分化的效果。 方法 从人脂肪组织中提取hASCs，分别采用单层培养和微球培养，加入3种不同成分的成软骨诱导培养基，A组：胎牛血清（FBS）+ 10 ng/ml转化生长因子3（TGF-β3）；B组：胰岛素-转铁蛋白-亚硒酸钠（ITS）+ 1 ng/ml TGF-β3；C组：ITS+10 ng/mL TGF-β3。3周后提取单层培养的细胞蛋白进行成软骨相关标志物检测，并对细胞微球进行阿尔新蓝、甲苯胺蓝、天狼星红染色，比较成软骨效果。 结果 3周后单层培养体系C组的软骨标志物表达最高，微球培养阿尔新蓝、甲苯胺蓝、天狼星红染色C组着色最深。 结论 作为培养基添加物，ITS在诱导hASCs成软骨分化时的效果优于FBS，而作为刺激hASCs成软骨的重要生长因子TGF-β3，10 ng/ml的浓度比1 ng/ml作用更强。
Objective To compare the chondrogenic differentiation of human adipose mesenchymal stem cells (hASCs) in vitro by three components of chondrogenic induction medium. Methods hASCs were extracted from human adipose tissues and then performed in monolayer culture and microsphere culture respectively. Three kinds of different components of chondrogenic induction medium (group A: Fetal bovine serum (FBS) + high concentration TGF-β3, group B: Insulin, Transferrin, Selenium Solution (ITS) + low concentration TGF-β3, group C: ITS + high concentration TGF-β3) were added. After 3 weeks, the cell proteins in monolayer culture were extracted for detection of cartilage-related markers, and the cell microspheres were stained with Toluidine blue, Alcian blue, Sirius red to compare the effect of cartilage formation. Results After 3 weeks, the expression of cartilage markers was the highest in the group C of monolayer culture system, and the staining of microspheres was also the deepest in the group C. Conclusions As a medium additive, ITS has a better effect than FBS in inducing chondrogenic differentiation of hASCs. As an important growth factor stimulating chondrogenesis of hASCs, the concentration of 10 ng/ml TGF-β3 has a stronger effect than the concentration of 1 ng/ml.
[1] Han Y, Li XZ, Zhang YB, et al. Mesenchymal stem cells for regenerative medicine[J]. Cells, 2019, 8(8): 886. DOI: 10.3390/cells8080886.
[2] Brittberg M, Lindahl A, Nilsson A, et al. Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation[J]. N Engl J Med, 1994, 331(14): 889-895. DOI: 10.1056/NEJM199410063311401.
[3] Saris D, Price A, Widuchowski W, et al. Matrix-applied characterized autologous cultured chondrocytes versus microfracture: two-year follow-up of a prospective randomized trial[J]. Am J Sports Med, 2014, 42(6): 1384-1394. DOI: 10.1177/0363546514528093.
[4] Lynch TS, Patel RM, Benedick A, et al. Systematic review of autogenous osteochondral transplant outcomes[J]. Arthroscopy, 2015, 31(4): 746-754. DOI: 10.1016/j.arthro.2014.11.018.
[5] Shaikh N, Seah MKT, Khan WS. Systematic review on the use of autologous matrix-induced chondrogenesis for the repair of articular cartilage defects in patients[J]. World J Orthop, 2017, 8(7): 588-601. DOI: 10.5312/wjo.v8.i7.588.
[6] 李永津, 伍慧媚, 林涌鹏, 等. HuR诱导骨髓间充质干细胞成软骨分化的作用探析[J]. 中国临床解剖学杂志, 2018, 36(3): 304-307. DOI: 10.13418/j.issn.1001-165x.2018.03.014.
[7] Lyssiotis CA, Lairson LL, Boitano AE, et al. Chemical control of stem cell fate and developmental potential[J]. Angew Chem Int Ed Engl, 2011, 50(1): 200-242. DOI: 10.1002/anie.201004284.
[8] Langer R, Vacanti JP. Tissue engineering[J]. Science, 1993, 260(5110): 920-926. DOI: 10.1126/science.8493529
[9] Alfaqeh HH, Hui CK, Saim AB, et al. Growth medium with low serum and transforming growth factor beta 3 promotes better chondrogenesis of bone marrow-derived stem cells in vitro and in vivo[J]. Saudi Med J, 2011, 32(6): 640-641. PMID: 21666950
[10] Endo K, Fujita N, Nakagawa T, et al. Effect of fibroblast growth factor-2 and serum on canine mesenchymal stem cell chondrogenesis[J]. Tissue Eng Part A, 2019, 25(11-12): 901-910. DOI: 10.1089/ten.TEA.2018.0177.
[11] Ho STB, Yang Z, Hui HPJ, et al. A serum free approach towards the conservation of chondrogenic phenotype during in vitro cell expansion[J]. Growth Factors, 2009, 27(5): 321-333. DOI: 10.1080/08977190903137595.
[12]Cho H, Lee A, Kim K. The effect of serum types on Chondrogenic differentiation of adipose-derived stem cells[J]. Biomater Res, 2018, 22: 6. DOI: 10.1186/s40824-018-0116-z.
[13]Li YY, Lam KL, Chen AD, et al. Collagen microencapsulation recapitulates mesenchymal condensation and potentiates chondrogenesis of human mesenchymal stem cells-a matrix-driven in vitro model of early skeletogenesis[J]. Biomaterials, 2019, 213: 119210. DOI: 10.1016/j.biomaterials.2019.05.021.
[14] Jooybar E, Abdekhodaie MJ, Alvi M, et al. An injectable platelet lysate-hyaluronic acid hydrogel supports cellular activities and induces chondrogenesis of encapsulated mesenchymal stem cells[J]. Acta Biomater, 2019, 83: 233-244. DOI: 10.1016/j.actbio.2018.10.031.
[15] Han JW, Yang TT, Gao J, et al. Specific microRNA expression during chondrogenesis of human mesenchymal stem cells[J]. Int J Mol Med, 2010, 25(3): 377-384. DOI: 10.3892/ijmm_00000355.
京ICP备08002354号-3
