膀胱平滑肌细胞条件培养液诱导脐带MSCs向平滑肌细胞分化的实验研究_《中国修复重建外科杂志》

作者：

员海超 , 柳良仁 , 郑硕 , 刘振华 , 杨璐 , 蒲春晓 , 李金洪 , 龙丹 , 魏强 , 韩平

四川大学华西医院泌尿外科（成都，610041）;

关键词：

脐带MSCs 膀胱平滑肌细胞条件培养液平滑肌细胞诱导分化

DOI：

10.7507/1002-1892.20130330

视频：

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摘要 全文 图表 视频 参考文献 施引文献 补充材料

目的探讨膀胱平滑肌细胞（bladder smooth muscle cells， BSMCs）条件培养液能否诱导脐带MSCs（umbilical cord MSCs，UCMSCs）向平滑肌细胞（smooth muscle cells，SMCs）分化，为组织工程技术应用于泌尿系统修复重建寻找可供选择的种子细胞。方法取足月新生儿脐带和行膀胱全切术患者捐赠的正常膀胱组织，分别分离培养UCMSCs和BSMCs。收集第1～5代BSMCs的培养液，与完全培养基以1∶1比例配制成BSMCs条件培养液。取第3代UCMSCs作为诱导细胞，使用BSMCs条件培养液培养为诱导组（A组），完全培养基培养为对照组（B组），倒置显微镜观察两组细胞形态变化；另设单纯BSMCs为阳性对照组（C组）。培养7、14 d，采用免疫荧光染色和Western blot检测各组细胞中α-平滑肌肌动蛋白（α-smooth muscle actin，α-SMA）、Calponin、平滑肌肌球蛋白重链（smooth muscle myosin heavy chain，SM-MHC）的表达情况。结果诱导培养后，A组细胞逐渐变长，由短棒状、多个突起逐渐转变为长梭形，与BSMCs形状相似；B组细胞形态未见明显变化。免疫荧光染色示，C组BSMCs中α-SMA、Calponin和SM-MHC均呈阳性表达。培养7 d，A、B组可见α-SMA呈阳性表达；14 d时，A组α-SMA阳性表达逐渐增多，B组无明显变化。培养7 d，A组可见Calponin阳性表达，14 d时阳性表达明显增多；B组各时间点均未见Calponin阳性表达。各时间点A、B组均未见SM-MHC阳性表达。Western blot检测示各组细胞α-SMA、Calponin和SM-MHC蛋白表达情况与免疫荧光染色结果基本一致。结论BSMCs条件培养液能诱导UCMSCs向SMCs分化，UCMSCs有望成为泌尿系统修复重建可供选择的种子细胞之一。

引用本文： 员海超,柳良仁,郑硕,刘振华,杨璐,蒲春晓,李金洪,龙丹,魏强,韩平. 膀胱平滑肌细胞条件培养液诱导脐带MSCs向平滑肌细胞分化的实验研究. 中国修复重建外科杂志, 2013, 27(12): 1505-1511. doi: 10.7507/1002-1892.20130330 复制

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29.	Karahuseyinoglu S, Cinar O, Kilic E, et al. Biology of stem cells in human umbilical cord stroma: in situ and in vitro surveys. Stem Cells, 2007, 25(2): 319-331.
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33.	Zhang R, Jack GS, Rao N, et al. Nuclear fusion-independent smooth muscle differentiation of human adipose-derived stem cells induced by a smooth muscle environment. Stem Cells, 2012, 30(3): 481-490.

1. Gage FH. Cell therapy. Nature, 1998, 392(6679 Suppl): 18-24.
2. Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science, 1997, 276(5309): 71-74.
3. Zuk PA, Zhu M, Ashjian P, et al. Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell, 2002, 13(12): 4279-4295.
4. Seale P, Asakura A, Rudnicki MA. The potential of muscle stem cells. Dev Cell, 2001, 1(3): 333-342.
5. Hutson EL, Boyer S, Genever PG. Rapid isolation, expansion, and differentiation of osteoprogenitors from full-term umbilical cord blood. Tissue Eng, 2005, 11(9-10): 1407-1420.
6. RusCiucǎ D, Soritǎu O, Suşman S, et al. Isolation and characterization of chorionic mesenchyal stem cells from the placenta. Rom J Morphol Embryol, 2011, 52(3): 803-808.
7. Rao MS, Mattson MP. Stem ceils and aging: expanding the possibilities. Mech Ageing Dev, 2001, 122(7): 713-734.
8. Kern S, Eichler H, Stoeve J, et al. Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue. Stem Cells, 2006, 24(5): 1294-1301.
9. Sinha S, Hoofnagle MH, Kingston PA, et al. Transforming growth factor-beta1 signaling contributes to development of smooth muscle cells from embryonic stem cells. Am J Physiol Cell Physiol, 2004, 287(6): C1560-1568.
10. Tian H, Bharadwaj S, Liu Y, et al. Differentiation of human bone marrow mesenchymal stem cells into bladder cells: potential for urological tissue engineering. Tissue Eng Part A, 2010, 16(5): 1769-1779.
11. Gonzalez R, Griparic L, Umana M, et al. An efficient approach to isolation and characterization of pre- and postnatal umbilical cord lining stem cells for clinical applications. Cell Transplant, 2010, 19(11): 1439-1449.
12. Oh SH, Muzzonigro TM, Bae SH, et al. Adult Bone marrow derived cells trans-differentiating in to insulin-producing cells for the treatment of type I diabetes. Lab Invest, 2004, 84(5): 607-617.
13. Ende N, Chen R, Reddi AS. Effect of human umbilical cord blood cells on glycemia and insulitis in type 1 diabetic mice. Biochem Biophys Res Commun, 2004, 325(3): 665-669.
14. Lim JH, Byeon YE, Ryu HH, et al. Transplantation of canine umbilical cord blood-derived mesenchymal stem cells in experimentally induced spinal cord injured dogs. J Vet Sci, 2007, 8(3): 275-282.
15. Coutts M, Keirstead HS. Stem cells for the treatment of spinal cord injury. Exp Neurol, 2008, 209(2): 368-377.
16. Pereira WC, Khushnooma I, Madkaikar M, et al. Reproducible methodology for the isolation of mesenchymal stem cells from human umbilical cord and its potential for cardiomyocyte generation. J Tissue Eng Regen Med, 2008, 2(7): 394-399.
17. Ichim TE, Alexandrescu DT, Solano F, et al. Mesenchymal stem cells as anti-inflammatories: implications for treatment of Duch-ennemuscular dystrophy. Cell Immunol, 2010, 260(2): 75-82.
18. Corcos J, Loutochin O, Campeau L, et al. Bone marrow mesenchymal stromal cell therapy for external urethral sphincter restoration in a rat model of stress urinary incontinence. Neurourol Urody, 2011, 30(3): 447-455.
19. Zhao W, Zhang C, Jin C, et al. Periurethral injection of autologous adipose-derived stem cells with controlled-release nerve growth factor for the treatment of stress urinary incontinence in a rat model. Eur Urol, 2011, 59(1): 155-163.
20. Fu Q, Cao YL. Tissue engineering and stem cell application of urethroplasty: from bench to bedside. Urology, 2012, 79(2): 246-253.
21. Sharma AK, Bury MI, Marks AJ, et al. A nonhuman primate model for urinary bladder regeneration using autologous sources of bone marrow-derived mesenchymal stem cells. Stem Cells, 2011, 29(2): 241-250.
22. Mitchell KE, Weiss ML, Mitchell BM, et al. Matrix cells from Wharton’s jelly form neurons and glia. Stem cells, 2003, 21(1): 50-60.
23. Wang L, Tran I, Seshareddy K, et al. A comparison of human bone marrow-derived mesenchymal stem cells and human umbilical cord-derived mesenchymal stromal cells for cartilage tissue engineering. Tissue Eng Part A, 2009, 15(8): 2259-2266.
24. Wang M, Yang Y, Yang D, et al. The immunomodulatory activity of human umbilical cord blood-derived mesenchymal stem cells in vitro. Immunology, 2008, 126(2): 220-232.
25. Ma L, Feng XY, Cui BL, et al. Human umbilical cord Wharton’s Jelly-derived mesenchymal stem cells differentiation into nerve-like cells. Chin Med J (Engl), 2005, 118(23): 1987-1993.
26. Shapiro AM, Lakey JR, Ryan EA, et al. Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med, 2000, 343(4): 230-238.
27. Campard D, Lysy PA, Najimi M, et al. Native umbilical cord matrix stem cells express hepatic markers and differentiate into hepatocyte-like cells. Gastroenterology, 2008, 134(3): 833-848.
28. Zhang YN, Lie PC, Wei X. Differentiation of mesenchymal stromal cells derived from umbilical cord Wharton’s jelly into hepatocyte-like cells. Cytotherapy, 11(5): 548-558.
29. Karahuseyinoglu S, Cinar O, Kilic E, et al. Biology of stem cells in human umbilical cord stroma: in situ and in vitro surveys. Stem Cells, 2007, 25(2): 319-331.
30. Schneider RK, Puellen A, Kramann R, et al. The osteogenic differentiation of adult bone marrow and perinatal umbilical mesenchymal stem cells and matrix remodelling in three-dimensional collagen scaffolds. Biomaterials, 2010, 31(3): 467-480.
31. 刘晓芳, 王延洲, 徐惠成, 等. 细胞间接触诱导骨髓间充质干细胞向平滑肌细胞分化的实验研究. 第三军医大学学报, 2010, 32(6): 516-519.
32. Mohana Kumar B, Maeng GH, Lee YM, et al. 212 porcine bone marrow-derived mesenchymal stem cells differentiate in vitro into smooth muscle cells. Reprod Fertil Dev, 2011, 24(1): 218.
33. Zhang R, Jack GS, Rao N, et al. Nuclear fusion-independent smooth muscle differentiation of human adipose-derived stem cells induced by a smooth muscle environment. Stem Cells, 2012, 30(3): 481-490.

《中国修复重建外科杂志》

膀胱平滑肌细胞条件培养液诱导脐带MSCs向平滑肌细胞分化的实验研究

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《中国修复重建外科杂志》

膀胱平滑肌细胞条件培养液诱导脐带MSCs向平滑肌细胞分化的实验研究

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