Transforming growth factor beta1 gene silenced adipose-derived stem cell transplantation reduces thrombocytopenic purpura

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21 1 2017 01 08 Chinese Journal of Tissue Engineering Research January 8, 2017 Vol.21, No.1 β1 ( 050031). β1 [J]. 2017 21(1):60-65. DOI: 10.3969/j.issn.2095-4344.2017.01.011 ORCID:0000-0002-3236-5704() β1 β1 mrna sirna 3 1979 2006 050031 :R394.2 :B :2095-4344 (2017)01-00060-06 2016-10-17 β1 β1-sirna β1 (GP-APS) 4 20 µl β1 2 10 6 L 1 CM-Dil β1 20 µl 5 mg/kg 14 d EILSA 3 β1 CM-Dil PAIgG β1 (P < 0.05) β1 β1 PAIgG β1 β1 (20130276) Transforming growth factor beta1 gene silenced adipose-derived stem cell transplantation reduces thrombocytopenic purpura Yin Wan-yi, Shen Yang, Liu Qing-chi, Jia Xiao-hui, Zhang Li-hong (Department of Hematology, First Hospital of Hebei Medical University, Shijiazhuang 050031, Hebei Province, China) Abstract BACKGROUND: Some studies have shown that stem cell therapy offers new promise for the treatment of thrombocytopenic purpura, but there are still difficulties in the perfect treatment for thrombocytopenic purpura. Yin Wan-yi, Master, Attending physician, Department of Hematology, First Hospital of Hebei Medical University, Shijiazhuang 050031, Hebei Province, China Corresponding author: Yin Wan-yi, Department of Hematology, First Hospital of Hebei Medical University, Shijiazhuang 050031, Hebei Province, China 60 P.O. Box 10002, Shenyang 110180

. β1 OBJECTIVE: To investigate the effect of transplantation of transforming growth factor beta1 (TGF-β1) gene silenced mouse adipose-derived stem cells (ADSCs) in mice with thrombocytopenic purpura. METHODS: The mouse ADSCs were cultured in vitro and transfected by the sirna expression plasmids containing TGF-β1 (TGF-β1-siRNA). Mouse model of thrombocytopenic purpura were made by guinea pig anti-mouse platelet serum intraperitoneally injected. Then, model mice were randomly assigned into model, ADSCs, TGF-β1 silenced ADSCs, or prednisone group, followed by intravenous injection of 20 µl normal saline, 2 10 6 /L CM-Dil-labeled ADSCs, 20 µl TGF-β1 silenced ADSCs suspension via the tail vein, or intragastrical administration of 5 mg/kg prednisone, respectively. The mice in each group were sacrificed after 14 days of treatment. RESULTS AND CONCLUSION: Compared with the other three groups, the TGF-β1 silenced ADSCs group showed a significant increase in the number of CM-Dil positive cells, blood platelet count, and proportion of thromocytogenic megakaryocytes, while a marked decrease in the total number of megakaryocytes, PAIgG values and TGF-β1 level (P < 0.05). To conclude, TGF-β1 gene silenced ADSCs transplantation can effectively reduce the TGF-β1 and PAIgG values in ITP mice, and promote the differentiation and maturation of megakaryocytes in the bone marrow, which can effectively improve the number of platelets, playing a role in the treatment of ITP. Subject headings: Stem Cells; Tissue Engineering; Transforming Growth Factor beta; Blood Platelets Funding: the Medical Research Project of Hebei Health Department, No. 20130276 Cite this article: Yin WY, Shen Y, Liu QC, Jia XH, Zhang LH. Transforming growth factor beta1 gene silenced adipose-derived stem cell transplantation reduces thrombocytopenic purpura. Zhongguo Zuzhi Gongcheng Yanjiu. 2017;21(1):60-65. 0 Introduction 2 β1 [1-3] (GP-APS) β1 1Materials and methods 1.1 1.2 2015 4 2016 7 1.3 8 Balb/cSPF 18 22 g 080373 SCXK( )2013-1007 SYXK( )2013-0005 + 1.4 1.4.1 37 1 min (SCW-CJ ) 1 10 L-DMEM 37 5%CO 2 (3111Forma ) 2 d 85% 3 g/l 5 min 10% (L-530 ) 1 500 r/min5 min 4 (P4 ) CM-Dil 5 µl 1.5 ml EP 1 ml CM-Dil 90% PBS 340 µl/cm 2 37 5%CO 2 20 min 37 5 ml 10 min 2 24 h ISSN 2095-4344 CN 21-1581/R CODEN: ZLKHAH 61

. β1 ( Nikon )CM-Dil 5CM-Dil =(/ ) 100% PBS 3 1 10 10 L 1 1.4.2 P4 [4] β1 β1-sirna β1 sirna 5 -GAT CCG GGC AGC TGT ACA TTG ACT TTT CAA GAG AAA GTC AAT GTA CAG CTG CCC TTT TTT G-3 5 -AAT TGA AAA AAG GGC AGC TGT ACA TTG ACT TTC TCT TGA AAA GTC AAT GTA CAG CTG CCC G-3 573 bp β1 8 h PBS 2 72 h 1 mg/l 3 ( ) ( ) β1-sirna( β1-sirna) 1.4.3 Western blot3 14 β1 3 14 5 10 8 L 1 1 ml/ 24 h 12 h BCA 50 µg 10% 1 h β1 GAPDH 4 1 hecl 3Quantity one ( ) GAPDH 1.4.4 CM-Dil 5 µl CM-Dil 1 ml 1.5 ml EP CM-Dil90% PBS3 20 min 5 ml 10 min 230 d 62 1.4.5 GP-APS (GP-APS) [2-4] BALB/C EDTA-Na2( ) (FICA) (FCA) FCA 0 4 FICA 1 2 45 1 500 r/min 10 min GP-APS20 [2-3] 80 Balb/c GP-APS 56 30 min 1 4GP-APS 1 3 5 7 9 11 13 d100 µl GP-APS 80 4 20 20 µl 2 10 6 L 1 CM-Dil 20 µl β1 2 10 6 L 1 CM-Dilβ1 20 µl 5 mg/kg1 /d 14 d 1.4.6 EDTA-Na2 14 d EDTA-Na2 BC-3000 Plus ( ) 14 d PBS ( ) 25 µl 1.4.7 PAIgG β1 (14 d ) 20 ELISA PAIgG β1 (ELISA Kit for Mouse PAIgG ELISA Kit for Mouse P.O. Box 10002, Shenyang 110180

. β1 β1) 37 120 min 5 3 min ( PAIgG)100 µl 37 60 min 100 µl 37 30 min 100 µl ( Bio-Rad ) PAIgG 1.5 CM-Dil PAIgG β1 1.6 x _ ±s SPSS 17.0 P < 0.05 2Results 2.1 80 2.2 2 β1 2.3 P4 M-Dil 3 1ACM-Dil 98% 1B 2.4 β1 Western blot TGF-β 3 d β1 β1 14β1 ( 2) 2.5 CM-Dil CM-Dil CM-Dil [(16.69±3.17)%] β1[(22.98± 4.06)%] (P < 0.05 3) 2.6 β1 β1 (P < 0.05) β1 ( 1) A B β1-sirna 3 d β1 14 d β1 1 CM-Dil ( 200) Figure 1 Morphology of CM-Dil-labeled adipose-derived stem cells (immunofluorescent staining, 200) A B CM-Dil (CM-Dil ) GAPDH 2 3 14 d β1 Figure 2 Expression of transforming growth factor β1 in each group at 3 and 14 days after cell transfection A B C D 3 CD-Dil ( 200) Figure 3 Morphology of CM-Dil-labeled adipose-derived stem cells after transfection (immunofluorescent staining, 200) A CM-Dil B CM-Dil C CM-Dil D β1 CM-Dil ISSN 2095-4344 CN 21-1581/R CODEN: ZLKHAH 63

. β1 1 (x _ ±s n=20) Table 1 Number and proportion of megakaryocytes in each group ( 10 9 L -1 ) (n) (%) 310.42±75.62 67.72±5.01 6.67±1.46 335.33±81.40 ab 59.14±4.35 ab 7.81±1.56 ab 354.92±88.16 ab 55.37±4.38 ab 8.22±1.39 ab β1 406.41±102.11 a 48.26±3.99 a 8.85±1.49 a a P < 0.05 β1 b P < 0.05 2 (x _ ±s n=20) Table 2 Classification of megakaryocytes in each group 32.20±5.90 58.90±5.49 5.20±1.48 29.30±3.95 a 60.80±4.05 6.40±1.26 ab 25.10±4.46 a 62.90±3.96 a 6.80±1.69 ab β1 25.50±4.84 ac 62.60±6.20 a 7.10±1.79 a a P < 0.05 ADSCs b P < 0.05 c P < 0.05 3 PAIgG β1 (x _ ±s n=20) Table 3 Comparison of serum PAIgG values and transforming growth factor β1 levels among groups PAIgG (µg/l) β1 (ng/l) 24.56±7.86 359.22±40.24 23.34±6.12 ab 353.45±33.30 ab 23.17±5.82 ab 342.63±46.14 ab β1 22.24±4.83 a 265.08±38.63 a a P < 0.05 β1 b P < 0.05 β1 β1 ( β1 < <)(P < 0.05) β1 (β1> >)(P < 0.05)1 2 β1 2.7 PAIgGβ1 3 PAIgG β1 (P < 0.05) > > β1(p < 0.05) β1 (P > 0.05) β1 β1 (P < 0.05) 3Discussion [5-7] Zuk [8] 2001 [9-10] Balb/c CD29 CD4495%5% CD34 β1adsc Western blot β1β1 β1 β1 β1 PAIgG ELISA PAIgG β1 β1 β1 PAIgG [11] [12] ( 3 d) ( ) [13] 14 d (72 h) 2 2 β1 PAIgG 64 P.O. Box 10002, Shenyang 110180

. β1 (GP) Fab [14] [15-16] PAIgG β1 PAIgG [17-18] β1 β1 β1 β1 PAIgG 2009 Ethical issues in animal experimentation, CNKI 3 ( ) 3.0 4 References [1],,,.IL-10 TGF-β1 [J].,2016,31(12):2450-2452. [2],,. [J].,2005,23(5):928. [3],,,. [J].,2010,14(24):4462-4468. [4],,. [J].,2009,32(5):403-406. [5] Docheva D, Popov C, Mutschler W, et al. Human mesenchymal stem cells in contact with their environment: surface characteristics and the integrin system. J Cell Mol Med. 2007;11(1):21-38. [6] Lee HL, Yu B, Deng P, et al. Transforming growth factor-β-induced KDW4B promotes chondrogenic differentiation of human mesenchymal stem cells. Stem Cells. 2016;34(3):711-719. [7] Kang WC, Oh PC, Lee K, et al. Increasing injection frequency enhances the survival of injected bone marrow derived mesenchymal stem cells in a critical limb ischemia animal model. Korean J Physiol Pharmacol. 2016;20(6):657-667. [8] Zuk PA, Zhu M, Asijian P, et al. Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell. 2002;13(12): 4279-4295. [9] Bodle JC, Hanson AD, Loboa EG. Adipose-derived stem cells in functional bone tissue engineering: lessons from bone mechanobiology. Tissue Eng Part B Rev. 2011;17(3):195-211. [10] Cheng NC, Estes BT, Awad HA. Chondrogenic differentiation of adipose-derived adult stem cells by a porous scaffold derived from native articular cartilage extracellular matrix. Tissue Eng Part A. 2009;15(2):231-241. [11] Tejinder SB, Sonam S, Mridu M, et al. Changes in megakaryocytes in case of thrombocytopenia: bone marrow aspiration and biopsy analysis. J Clin Diagn Res. 2013; 7(7): 473-479. [12],,,. [J].,2014,34(2):231-233. [13],,,. [J].,2004,10(2):30-32. [14] Warrier R, Chauhan A. Management of immune thrombocytopenic purpura: an update. Ochsner J. 2012; 12(3): 221-227. [15] Chang M, Nakagawa PA, Williams SA, et al. Immune thrombocytopenic purpura (ITP) plasma and purified ITP monoclonal autoantibodies inhibit megakaryocytopoiesis in vitro. Blood. 2003;102(3):887-895. [16] McMillan R, Wang L, Tomer A, et al. Suppression of in vitro megakaryocyte production by antiplatelet autoantibodies from adult patients with chronic ITP. Blood. 2004;103(4):1364-1369. [17],,. [J]., 2003,32(4):348-350. [18] Houwerzijil EJ, Blom NR, vail der Want JJ, et al. Ultrastructural study shows morphologic features of apoptosis and para-apoptosis in megakaryocytes from patients with idiopathic thromboeytopenic purpura. Blood. 2004;103(2): 500-506. ISSN 2095-4344 CN 21-1581/R CODEN: ZLKHAH 65

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