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

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1 Chinese Journal of Tissue Engineering Research January 8, 2017 Vol.21, No.1 β1 ( ). β1 [J] (1): DOI: /j.issn ORCID: () β1 β1 mrna sirna :R394.2 :B : (2017) β1 β1-sirna β1 (GP-APS) 4 20 µl β 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 ( ) 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 , 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 , Hebei Province, China Corresponding author: Yin Wan-yi, Department of Hematology, First Hospital of Hebei Medical University, Shijiazhuang , Hebei Province, China 60 P.O. Box 10002, Shenyang

2 . 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, /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 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): Introduction 2 β1 [1-3] (GP-APS) β1 1Materials and methods Balb/cSPF g SCXK( ) SYXK( ) min (SCW-CJ ) 1 10 L-DMEM 37 5%CO 2 (3111Forma ) 2 d 85% 3 g/l 5 min 10% (L-530 ) r/min5 min 4 (P4 ) CM-Dil 5 µl 1.5 ml EP 1 ml CM-Dil 90% PBS 340 µl/cm %CO 2 20 min 37 5 ml 10 min 2 24 h ISSN CN /R CODEN: ZLKHAH 61

3 . β1 ( Nikon )CM-Dil 5CM-Dil =(/ ) 100% PBS L 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 bp β1 8 h PBS 2 72 h 1 mg/l 3 ( ) ( ) β1-sirna( β1-sirna) Western blot3 14 β L 1 1 ml/ 24 h 12 h BCA 50 µg 10% 1 h β1 GAPDH 4 1 hecl 3Quantity one ( ) GAPDH CM-Dil 5 µl CM-Dil 1 ml 1.5 ml EP CM-Dil90% PBS3 20 min 5 ml 10 min 230 d GP-APS (GP-APS) [2-4] BALB/C EDTA-Na2( ) (FICA) (FCA) FCA 0 4 FICA r/min 10 min GP-APS20 [2-3] 80 Balb/c GP-APS min 1 4GP-APS d100 µl GP-APS µl L 1 CM-Dil 20 µl β L 1 CM-Dilβ1 20 µl 5 mg/kg1 /d 14 d EDTA-Na2 14 d EDTA-Na2 BC-3000 Plus ( ) 14 d PBS ( ) 25 µl PAIgG β1 (14 d ) 20 ELISA PAIgG β1 (ELISA Kit for Mouse PAIgG ELISA Kit for Mouse P.O. Box 10002, Shenyang

4 . β1 β1) min 5 3 min ( PAIgG)100 µl min 100 µl min 100 µl ( Bio-Rad ) PAIgG 1.5 CM-Dil PAIgG β1 1.6 x _ ±s SPSS 17.0 P < Results 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 < ) 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 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 CN /R CODEN: ZLKHAH 63

5 . β1 1 (x _ ±s n=20) Table 1 Number and proportion of megakaryocytes in each group ( 10 9 L -1 ) (n) (%) ± ± ± ±81.40 ab 59.14±4.35 ab 7.81±1.56 ab ±88.16 ab 55.37±4.38 ab 8.22±1.39 ab β ± a 48.26±3.99 a 8.85±1.49 a a P < 0.05 β1 b P < (x _ ±s n=20) Table 2 Classification of megakaryocytes in each group 32.20± ± ± ±3.95 a 60.80± ±1.26 ab 25.10±4.46 a 62.90±3.96 a 6.80±1.69 ab β ±4.84 ac 62.60±6.20 a 7.10±1.79 a a P < 0.05 ADSCs b P < 0.05 c P < 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± ± ±6.12 ab ±33.30 ab 23.17±5.82 ab ±46.14 ab β ±4.83 a ±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

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