人脐带间充质干细胞移植治疗大鼠深静脉血栓
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摘要
背景:人脐带间充质干细胞具有归巢至缺血部位并在局部促进血管新生的能力,而新血管生成对深静脉血栓的机化及再通至关重要,但是将人脐带间充质干细胞用于血栓的研究尚未见相关报道。目的:探讨人脐带间充质干细胞移植治疗对深静脉血栓溶解、机化及再通的影响。方法:用"狭窄法"建立下腔静脉血栓模型大鼠,制备P3代人脐带间充质干细胞悬液,细胞浓度2×109 L-1,将建模后大鼠随机分为4组。对照组:建模后第1,2,3天尾静脉注射PBS1mL;实验组1:术后第1天尾静脉注射细胞悬液1 mL,第2,3天注射PBS 1 mL;实验组2:术后第1,2天注射细胞悬液1 mL,第3天注射PBS 1 mL;实验组3:术后第1,2,3天注射细胞悬液1 mL。实验方案经南通大学动物实验伦理委员会批准,批准号为20180315-001,批准时间2018-04-01。于术后第7天取出下腔静脉内血栓,通过苏木精-伊红染色及免疫组织化学染色观察比较各组血栓内新生血管数及溶解率。结果与结论:①与对照组相比,实验组2,3血栓内血管密度显著增高(P <0.01);②所有实验组的溶解率较对照组增高,但差异无显著性意义(P> 0.05);③结果表明,一定剂量人脐带间充质干细胞移植能显著增加血栓内的小血管数量,有助于血栓的机化及再通。
BACKGROUND: Human umbilical cord mesenchymal stem cells can home to ischemic sites and form functional blood vessels locally.Neovascularization is critical for the organization and recanalization of deep vein thrombosis. However, transplantation of human umbilical cord mesenchymal stem cells for the treatment of thrombosis has not been reported.OBJECTIVE: To investigate the dissolution, organization and recanalization of deep vein thrombosis after the transplantation of human umbilical cord mesenchymal stem cells.METHODS: Deep vein thrombosis model was established by "narrow method" in rats. Suspension of the third generation human umbilical cord mesenchymal stem cells at a density of 2×109 cells/L was prepared. The model rats were randomly divided into four groups. In control group, 1 mL of PBS was injected into the tail vein on days 1, 2, and 3 after modeling. In experimental group 1, 1 mL of cell suspension was injected into the tail vein on day 1 after modeling, and 1 mL of PBS was injected on days 2 and 3. In experimental group 2, 1 mL of cell suspension was injected on days 1 and 2 after modeling, and 1 mL of PBS was injected on day 3. In experimental group 3, 1 mL of cell suspension was injected on days 1, 2, and 3 after modeling. The experimental protocol was approved by the Animal Experimental Ethics Committee of Nantong University with the approval No. 20180315-001 on April 1, 2018. Samples from deep vein thrombosis rats were taken on day 7 after modeling. Number of new blood vessels and dissolution rate in each group were detected by hematoxylin-eosin staining and immunohistochemical staining.RESULTS AND CONCLUSION: Compared with the control group, the capillary density of the experimental groups 2 and 3 was significantly increased(P < 0.01). The dissolution rate of all the experimental groups was higher than that of the control group, but the difference was not statistically significant(P > 0.05). All these results reveal that a certain amount of human umbilical cord mesenchymal stem cells transplanted can increase the number of microvessels in the thrombosis, and accelerate organization and recanalization of the thrombosis.
BACKGROUND: Human umbilical cord mesenchymal stem cells can home to ischemic sites and form functional blood vessels locally.Neovascularization is critical for the organization and recanalization of deep vein thrombosis. However, transplantation of human umbilical cord mesenchymal stem cells for the treatment of thrombosis has not been reported.OBJECTIVE: To investigate the dissolution, organization and recanalization of deep vein thrombosis after the transplantation of human umbilical cord mesenchymal stem cells.METHODS: Deep vein thrombosis model was established by "narrow method" in rats. Suspension of the third generation human umbilical cord mesenchymal stem cells at a density of 2×109 cells/L was prepared. The model rats were randomly divided into four groups. In control group, 1 mL of PBS was injected into the tail vein on days 1, 2, and 3 after modeling. In experimental group 1, 1 mL of cell suspension was injected into the tail vein on day 1 after modeling, and 1 mL of PBS was injected on days 2 and 3. In experimental group 2, 1 mL of cell suspension was injected on days 1 and 2 after modeling, and 1 mL of PBS was injected on day 3. In experimental group 3, 1 mL of cell suspension was injected on days 1, 2, and 3 after modeling. The experimental protocol was approved by the Animal Experimental Ethics Committee of Nantong University with the approval No. 20180315-001 on April 1, 2018. Samples from deep vein thrombosis rats were taken on day 7 after modeling. Number of new blood vessels and dissolution rate in each group were detected by hematoxylin-eosin staining and immunohistochemical staining.RESULTS AND CONCLUSION: Compared with the control group, the capillary density of the experimental groups 2 and 3 was significantly increased(P < 0.01). The dissolution rate of all the experimental groups was higher than that of the control group, but the difference was not statistically significant(P > 0.05). All these results reveal that a certain amount of human umbilical cord mesenchymal stem cells transplanted can increase the number of microvessels in the thrombosis, and accelerate organization and recanalization of the thrombosis.
引文
[1]Vedovetto V, Dalla Valle F, Milan M, et al. Residual vein thrombosis and trans-popliteal reflux in patients with and without the post-thrombotic syndrome. Thromb Haemost.2013;110(4):854-855.
[2]Sarolo L, Turatti G, Milan M, et al. Trans-popliteal reflux in limbs with and without deep-vein thrombosis of the same subject:cross-sectional study. Thromb Res. 2017;154:53-54.
[3]Altmann J, Sharma S, Lang IM. Advances in our understanding of mechanisms of venous thrombus resolution.Expert Rev Hematol. 2016;9(1):69-78.
[4]Mukhopadhyay S, Johnson TA, Sarkar R, et al. Serpins in venous thrombosis and venous thrombus resolution. Methods Mol Biol. 2018;1826:197-211.
[5]Schonfelder T, Brandt M, Kossmann S,et al. Lack of T-bet reduces monocytic interleukin-12 formation and accelerates thrombus resolution in deep vein thrombosis. Sci Rep. 2018;8(1):3013.
[6]McGuinness CL, Humphries J, Waltham M, et al. Recruitment of labelled monocytes by experimental venous thrombi.Thromb Haemost. 2001;85(6):1018-1024.
[7]Modarai B, Burnand KG, Humphries J, et al. The role of neovascularisation in the resolution of venous thrombus.Thromb Haemost. 2005;93(5):801-809.
[8]Li WD, Li XQ. Endothelial progenitor cells accelerate the resolution of deep vein thrombosis. Vascul Pharmacol. 2016;83:10-16.
[9]Evans CE, Grover SP, Humphries J, et al. Antiangiogenic therapy inhibits venous thrombus resolution. Arterioscler Thromb Vasc Biol. 2014;34(3):565-570.
[10]Choi M, Lee HS, Naidansaren P, et al. Proangiogenic features of Wharton's jelly-derived mesenchymal stromal/stem cells and their ability to form functional vessels. Int J Biochem Cell Biol. Mar 2013;45(3):560-570.
[11]Gorkun AA, Shpichka AI, Zurina IM, et al. Angiogenic potential of spheroids from umbilical cord and adipose-derived multipotent mesenchymal stromal cells within fibrin gel. Biomed Mater. 22 2018;13(4):044108.
[12]Boroujeni ME, Gardaneh M. Umbilical cord:an unlimited source of cells differentiable towards dopaminergic neurons.Neural Regen Res. 2017;12(7):1186-1192.
[13]Bullard JD, Lei J, Lim JJ, et al. Evaluation of dehydrated human umbilical cord biological properties for wound care and soft tissue healing. J Biomed Mater Res B Appl Biomater.2018. doi:10.1002/jbm.b.34196.
[14]Rabbani S, Soleimani M, Sahebjam M, et al. Simultaneous delivery of wharton's jelly mesenchymal stem cells and insulin-like growth factor-1 in acute myocardial infarction. Iran J Pharm Res. 2018;17(2):426-441.
[15]Yousefifard M, Nasirinezhad F, Shardi Manaheji H, et al.Human bone marrow-derived and umbilical cord-derived mesenchymal stem cells for alleviating neuropathic pain in a spinal cord injury model. Stem Cell Res Ther. 2016;7:36.
[16]徐海环,董化江,商崇智,等.脐带间充质干细胞移植急性脑创伤大鼠损伤区域微血管密度的变化[J].中国组织工程研究,2017,21(33):5320-5324.
[17]Yaghoobi K.Neural regeneration after spinal cord injury treatment by lavandula angustifolia and human umbilical mesanchymal stem cell transplantation.Neural Regen Res.2017;12(1):68-69.
[18]Li XY, Zheng ZH, Li XY, et al. Treatment of foot disease in patients with type 2 diabetes mellitus using human umbilical cord blood mesenchymal stem cells:response and correction of immunological anomalies. Curr Pharm Des. 2013;19(27):4893-4899.
[19]李茂,游成东,黄文.人脐带间充质干细胞移植治疗小鼠皮肤溃疡[J].中国组织工程研究,2018,22(33):5315-5320.
[20]杨晓清,张沐,杨兵,等.人脐带华通氏胶间充质干细胞的分离、培养、鉴定及冻存、复苏[J].南通大学学报(医学版),2010,30(6):413-415, 419.
[21]白云城,赵学凌,周如丹,等.大鼠下腔静脉血栓模型的建立及手术技巧[J].南京医科大学学报(自然科学版),2014,34(3):394-399.
[22]Alias S, Redwan B, Panzenboeck A, et al. Defective angiogenesis delays thrombus resolution:a potential pathogenetic mechanism underlying chronic thromboembolic pulmonary hypertension. Arterioscler Thromb Vasc Biol. Apr2014;34(4):810-819.
[23]Wakefield TW, Linn MJ, Henke PK, et al. Neovascularization during venous thrombosis organization:a preliminary study. J Vasc Surg. 1999;30(5):885-892.
[24]Chabasse C, Siefert SA, Chaudry M, et al. Recanalization and flow regulate venous thrombus resolution and matrix metalloproteinase expression in vivo. J Vasc Surg Venous Lymphat Disord. 2015;3(1):64-74.
[25]付健,唐博,陈以宽,等.新型大鼠下腔静脉血栓模型的建立及血栓溶解演变过程研究[J].解放军医学杂志,2015,40(8):610-615.
[26]Aguilera V, Briceno L, Contreras H, et al. Endothelium trans differentiated from Wharton's jelly mesenchymal cells promote tissue regeneration:potential role of soluble pro-angiogenic factors. PLoS One. 2014;9(11):e111025.
[27]Kupcova Skalnikova H. Proteomic techniques for characterisation of mesenchymal stem cell secretome.Biochimie. 2013;95(12):2196-2211.
[28]Au P, Tam J, Fukumura D, et al. Bone marrow-derived mesenchymal stem cells facilitate engineering of long-lasting functional vasculature. Blood. 2008;111(9):4551-4558.
[29]Edwards SS, Zavala G, Prieto CP, et al. Functional analysis reveals angiogenic potential of human mesenchymal stem cells from Wharton's jelly in dermal regeneration.Angiogenesis. 2014;17(4):851-866.
[30]Chen J, Liu Z, Hong MM, et al. Proangiogenic compositions of microvesicles derived from human umbilical cord mesenchymal stem cells. PLoS One. 2014;9(12):e115316.
[31]Arutyunyan I, Fatkhudinov T, Kananykhina E, et al. Role of VEGF-A in angiogenesis promoted by umbilical cord-derived mesenchymal stromal/stem cells:in vitro study. Stem Cell Res Ther. 2016;7:46.
[32]Zhu J, Liu Q, Jiang Y, et al. Enhanced angiogenesis promoted by human umbilical mesenchymal stem cell transplantation in stroked mouse is Notch1 signaling associated. Neuroscience.2015;290:288-299.
[33]Kamolz LP, Keck M, Kasper C. Wharton's jelly mesenchymal stem cells promote wound healing and tissue regeneration.Stem Cell Res Ther. 2014;5(3):62.
[34]Francois S, Eder V, Belmokhtar K, et al. Synergistic effect of human bone morphogenic protein-2 and mesenchymal stromal cells on chronic wounds through hypoxia-inducible factor-1 alpha induction. Sci Rep. 2017;7(1):4272.
[35]Mahmoudian-Sani MR, Rafeei F, Amini R, et al. The effect of mesenchymal stem cells combined with platelet-rich plasma on skin wound healing. J Cosmet Dermatol. 2018;17(5):650-659.
[2]Sarolo L, Turatti G, Milan M, et al. Trans-popliteal reflux in limbs with and without deep-vein thrombosis of the same subject:cross-sectional study. Thromb Res. 2017;154:53-54.
[3]Altmann J, Sharma S, Lang IM. Advances in our understanding of mechanisms of venous thrombus resolution.Expert Rev Hematol. 2016;9(1):69-78.
[4]Mukhopadhyay S, Johnson TA, Sarkar R, et al. Serpins in venous thrombosis and venous thrombus resolution. Methods Mol Biol. 2018;1826:197-211.
[5]Schonfelder T, Brandt M, Kossmann S,et al. Lack of T-bet reduces monocytic interleukin-12 formation and accelerates thrombus resolution in deep vein thrombosis. Sci Rep. 2018;8(1):3013.
[6]McGuinness CL, Humphries J, Waltham M, et al. Recruitment of labelled monocytes by experimental venous thrombi.Thromb Haemost. 2001;85(6):1018-1024.
[7]Modarai B, Burnand KG, Humphries J, et al. The role of neovascularisation in the resolution of venous thrombus.Thromb Haemost. 2005;93(5):801-809.
[8]Li WD, Li XQ. Endothelial progenitor cells accelerate the resolution of deep vein thrombosis. Vascul Pharmacol. 2016;83:10-16.
[9]Evans CE, Grover SP, Humphries J, et al. Antiangiogenic therapy inhibits venous thrombus resolution. Arterioscler Thromb Vasc Biol. 2014;34(3):565-570.
[10]Choi M, Lee HS, Naidansaren P, et al. Proangiogenic features of Wharton's jelly-derived mesenchymal stromal/stem cells and their ability to form functional vessels. Int J Biochem Cell Biol. Mar 2013;45(3):560-570.
[11]Gorkun AA, Shpichka AI, Zurina IM, et al. Angiogenic potential of spheroids from umbilical cord and adipose-derived multipotent mesenchymal stromal cells within fibrin gel. Biomed Mater. 22 2018;13(4):044108.
[12]Boroujeni ME, Gardaneh M. Umbilical cord:an unlimited source of cells differentiable towards dopaminergic neurons.Neural Regen Res. 2017;12(7):1186-1192.
[13]Bullard JD, Lei J, Lim JJ, et al. Evaluation of dehydrated human umbilical cord biological properties for wound care and soft tissue healing. J Biomed Mater Res B Appl Biomater.2018. doi:10.1002/jbm.b.34196.
[14]Rabbani S, Soleimani M, Sahebjam M, et al. Simultaneous delivery of wharton's jelly mesenchymal stem cells and insulin-like growth factor-1 in acute myocardial infarction. Iran J Pharm Res. 2018;17(2):426-441.
[15]Yousefifard M, Nasirinezhad F, Shardi Manaheji H, et al.Human bone marrow-derived and umbilical cord-derived mesenchymal stem cells for alleviating neuropathic pain in a spinal cord injury model. Stem Cell Res Ther. 2016;7:36.
[16]徐海环,董化江,商崇智,等.脐带间充质干细胞移植急性脑创伤大鼠损伤区域微血管密度的变化[J].中国组织工程研究,2017,21(33):5320-5324.
[17]Yaghoobi K.Neural regeneration after spinal cord injury treatment by lavandula angustifolia and human umbilical mesanchymal stem cell transplantation.Neural Regen Res.2017;12(1):68-69.
[18]Li XY, Zheng ZH, Li XY, et al. Treatment of foot disease in patients with type 2 diabetes mellitus using human umbilical cord blood mesenchymal stem cells:response and correction of immunological anomalies. Curr Pharm Des. 2013;19(27):4893-4899.
[19]李茂,游成东,黄文.人脐带间充质干细胞移植治疗小鼠皮肤溃疡[J].中国组织工程研究,2018,22(33):5315-5320.
[20]杨晓清,张沐,杨兵,等.人脐带华通氏胶间充质干细胞的分离、培养、鉴定及冻存、复苏[J].南通大学学报(医学版),2010,30(6):413-415, 419.
[21]白云城,赵学凌,周如丹,等.大鼠下腔静脉血栓模型的建立及手术技巧[J].南京医科大学学报(自然科学版),2014,34(3):394-399.
[22]Alias S, Redwan B, Panzenboeck A, et al. Defective angiogenesis delays thrombus resolution:a potential pathogenetic mechanism underlying chronic thromboembolic pulmonary hypertension. Arterioscler Thromb Vasc Biol. Apr2014;34(4):810-819.
[23]Wakefield TW, Linn MJ, Henke PK, et al. Neovascularization during venous thrombosis organization:a preliminary study. J Vasc Surg. 1999;30(5):885-892.
[24]Chabasse C, Siefert SA, Chaudry M, et al. Recanalization and flow regulate venous thrombus resolution and matrix metalloproteinase expression in vivo. J Vasc Surg Venous Lymphat Disord. 2015;3(1):64-74.
[25]付健,唐博,陈以宽,等.新型大鼠下腔静脉血栓模型的建立及血栓溶解演变过程研究[J].解放军医学杂志,2015,40(8):610-615.
[26]Aguilera V, Briceno L, Contreras H, et al. Endothelium trans differentiated from Wharton's jelly mesenchymal cells promote tissue regeneration:potential role of soluble pro-angiogenic factors. PLoS One. 2014;9(11):e111025.
[27]Kupcova Skalnikova H. Proteomic techniques for characterisation of mesenchymal stem cell secretome.Biochimie. 2013;95(12):2196-2211.
[28]Au P, Tam J, Fukumura D, et al. Bone marrow-derived mesenchymal stem cells facilitate engineering of long-lasting functional vasculature. Blood. 2008;111(9):4551-4558.
[29]Edwards SS, Zavala G, Prieto CP, et al. Functional analysis reveals angiogenic potential of human mesenchymal stem cells from Wharton's jelly in dermal regeneration.Angiogenesis. 2014;17(4):851-866.
[30]Chen J, Liu Z, Hong MM, et al. Proangiogenic compositions of microvesicles derived from human umbilical cord mesenchymal stem cells. PLoS One. 2014;9(12):e115316.
[31]Arutyunyan I, Fatkhudinov T, Kananykhina E, et al. Role of VEGF-A in angiogenesis promoted by umbilical cord-derived mesenchymal stromal/stem cells:in vitro study. Stem Cell Res Ther. 2016;7:46.
[32]Zhu J, Liu Q, Jiang Y, et al. Enhanced angiogenesis promoted by human umbilical mesenchymal stem cell transplantation in stroked mouse is Notch1 signaling associated. Neuroscience.2015;290:288-299.
[33]Kamolz LP, Keck M, Kasper C. Wharton's jelly mesenchymal stem cells promote wound healing and tissue regeneration.Stem Cell Res Ther. 2014;5(3):62.
[34]Francois S, Eder V, Belmokhtar K, et al. Synergistic effect of human bone morphogenic protein-2 and mesenchymal stromal cells on chronic wounds through hypoxia-inducible factor-1 alpha induction. Sci Rep. 2017;7(1):4272.
[35]Mahmoudian-Sani MR, Rafeei F, Amini R, et al. The effect of mesenchymal stem cells combined with platelet-rich plasma on skin wound healing. J Cosmet Dermatol. 2018;17(5):650-659.