膀胱平滑肌細胞條件培養液誘導臍帶MSCs向平滑肌細胞分化的實驗研究_《中國修復重建外科雜志》

作者：

員海超 , 柳良仁 , 鄭碩 , 劉振華 , 楊璐 , 蒲春曉 , 李金洪 , 龍丹 , 魏強 , 韓平

四川大學華西醫院泌尿外科（成都，610041）;

關鍵詞：

臍帶MSCs 膀胱平滑肌細胞條件培養液平滑肌細胞誘導分化

DOI：

10.7507/1002-1892.20130330

視頻：

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摘要 全文 圖表 視頻 參考文獻 施引文獻 補充材料

目的探討膀胱平滑肌細胞（bladder smooth muscle cells， BSMCs）條件培養液能否誘導臍帶MSCs（umbilical cord MSCs，UCMSCs）向平滑肌細胞（smooth muscle cells，SMCs）分化，為組織工程技術應用于泌尿系統修復重建尋找可供選擇的種子細胞。方法取足月新生兒臍帶和行膀胱全切術患者捐贈的正常膀胱組織，分別分離培養UCMSCs和BSMCs。收集第1～5代BSMCs的培養液，與完全培養基以1∶1比例配制成BSMCs條件培養液。取第3代UCMSCs作為誘導細胞，使用BSMCs條件培養液培養為誘導組（A組），完全培養基培養為對照組（B組），倒置顯微鏡觀察兩組細胞形態變化；另設單純BSMCs為陽性對照組（C組）。培養7、14 d，采用免疫熒光染色和Western blot檢測各組細胞中α-平滑肌肌動蛋白（α-smooth muscle actin，α-SMA）、Calponin、平滑肌肌球蛋白重鏈（smooth muscle myosin heavy chain，SM-MHC）的表達情況。結果誘導培養后，A組細胞逐漸變長，由短棒狀、多個突起逐漸轉變為長梭形，與BSMCs形狀相似；B組細胞形態未見明顯變化。免疫熒光染色示，C組BSMCs中α-SMA、Calponin和SM-MHC均呈陽性表達。培養7 d，A、B組可見α-SMA呈陽性表達；14 d時，A組α-SMA陽性表達逐漸增多，B組無明顯變化。培養7 d，A組可見Calponin陽性表達，14 d時陽性表達明顯增多；B組各時間點均未見Calponin陽性表達。各時間點A、B組均未見SM-MHC陽性表達。Western blot檢測示各組細胞α-SMA、Calponin和SM-MHC蛋白表達情況與免疫熒光染色結果基本一致。結論BSMCs條件培養液能誘導UCMSCs向SMCs分化，UCMSCs有望成為泌尿系統修復重建可供選擇的種子細胞之一。

引用本文： 員海超,柳良仁,鄭碩,劉振華,楊璐,蒲春曉,李金洪,龍丹,魏強,韓平. 膀胱平滑肌細胞條件培養液誘導臍帶MSCs向平滑肌細胞分化的實驗研究. 中國修復重建外科雜志, 2013, 27(12): 1505-1511. doi: 10.7507/1002-1892.20130330 復制

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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.	劉曉芳, 王延洲, 徐惠成, 等. 細胞間接觸誘導骨髓間充質干細胞向平滑肌細胞分化的實驗研究. 第三軍醫大學學報, 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.

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, Su?man 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. 劉曉芳, 王延洲, 徐惠成, 等. 細胞間接觸誘導骨髓間充質干細胞向平滑肌細胞分化的實驗研究. 第三軍醫大學學報, 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.

《中國修復重建外科雜志》

膀胱平滑肌細胞條件培養液誘導臍帶MSCs向平滑肌細胞分化的實驗研究

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《中國修復重建外科雜志》

膀胱平滑肌細胞條件培養液誘導臍帶MSCs向平滑肌細胞分化的實驗研究

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