CD34~+干细胞种植对人工血管内膜形成的影响
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摘要
目的：
1.探讨CD34+干细胞种植膨体聚四氟乙烯(expanded polytera fluoroethylene, ePTFE)和涤纶人工血管对移植人工血管内皮形成和中远期通畅率的影响。
2.检测人工血管移植后血清6-k-PGF1α和TXB2的变化,探讨其变化与骨髓CD34+细胞种植人工血管内皮细胞增殖、内膜形成的关系
方法：
1.CD34+干细胞分离纯化：选取济南地区家犬20只,无菌条件抽取犬骨髓,梯度离心提取单个核细胞,经免疫磁珠标记方法分离并提纯CD34+干细胞,经流式细胞检测验证。
2.人工血管CD34+干细胞种植及移植：CD34+干细胞种植于ePTFE和涤纶人工血管,把犬分为ePTFE血管实验组和涤纶血管实验组及ePTFE对照组和涤纶对照组,各组中分别把同一种人工血管同时植入同一条犬的下腔静脉和腹主动脉
3. PLT、PGF1α、TXB2测定分析：在术前、术后3天、7天、14天、30天、60天时股静脉采血血细胞计数仪测定血小板,分离血清EIA方法测定血清PGF1α、TXB2浓度,计算PGF1α/TXB2的比值。
4.移植血管内膜检测：在术后10、30、60、100天解剖获取标本,观察种植组和对照组移植后的通畅率；免疫组化法标记血管内皮细胞,通过大体观察和光镜观察新生内膜厚度；扫描电镜观察人工血管内皮增殖及分布,比较动脉和静脉系统骨髓CD34+细胞种植人工血管后内皮化及通畅率
结果：
1.免疫磁珠标记方法分离的犬骨髓干细胞经流式细胞仪鉴定为CD34+细胞。经台盼兰染色后细胞计数板计数活细胞为(2.6±0.3)×107／ml。
2.血清PGF1α、TXB2、PLT变化：实验组血小板浓度先上升后下降并维持在一定水平,而对照组明显上升后维持在一个较高水平；实验组血清PGF1α先降低后升高,而对照组明显降低后维持在一个较低水平；实验组浓度先上升后下降并维持在一定水平,而对照组明显上升后维持在一个较高水平；实验组血清P/T的比值先降低后升高,而对照组明显降低后维持在一个较低水平。
3.人工血管通畅率：CD34+干细胞预种植人工血管植入动脉闭塞率0,ePTFE组狭窄率25%；涤纶组狭窄率25%。CD34+干细胞预种植人工血管植入静脉ePTFE组闭塞率12.5%,狭窄率37.5%；涤纶组闭塞率0,狭窄率37.5%。对照组植入动脉闭塞率0,狭窄率100%；对照组植入静脉闭塞率100%。
4.人工血管内膜增殖情况：光镜下观察HE染色试验组涤纶血管新生内膜贴附腔面紧密且完整,ePTFE血管新生内膜在HE染色后与血管腔面有部分分离。实验组两种人工血管新内膜厚度小于对照组新内膜厚度,实验组两种血管新内膜厚度存在差异(p<0.05)
5.人工血管内皮细胞增殖及分布：扫描电镜观察可见实验组30天人工血管自吻合口向人工血管中间方向的腔面新生内膜内皮细胞密度逐渐减少；100天人工血管腔面内膜内皮细胞排列均匀完整。涤纶人工血管的新内膜覆以较完整内皮细胞层,细胞排列紧密,走行与血管长轴一致。ePTFE血管新内膜较同期涤纶血管新生内膜欠完整,少量区域内皮层缺乏表面附着纤维素和血细胞,其间见坏死和衰老的内皮细胞对照组人工血管内膜表面无内皮细胞覆盖,仅见纤维组织排列。
结论：
1.免疫磁珠标记方法分离CD34+细胞经纯化后细胞数量多增殖能力强,细胞的数量能满足20-30cm长,直径6-8mm人工血管腔面的内皮化种植。
2.经纯化的CD34+细胞种植于ePTFE和涤纶人工血管,均能保持理想的内皮化和通畅率。涤纶人工血管CD34+干细胞种植后贴附腔面程度和内皮化效果优于ePTFE人工血管。
3.动脉系统骨髓CD34+细胞种植人工血管后内皮化程度和通畅率优于静脉系统骨髓CD34+细胞种植人工血管后内皮化程度和通畅率。
4.骨髓CD34+干细胞种植人工血管应用于血管置换后能抑制血小板的粘附聚集和活化,抑制生长因子、细胞因子、粘附分子、趋化因子等生物活性物质的产生。
5.骨髓CD34+干细胞种植人工血管应用于血管置换后使人工血管管腔内较早的产生内皮细胞,而血管内皮细胞可产生前列环素腺苷、一氧化氮、VEGF等生物活性物质,从而有效抑制PGF1α和P/T比值的过度降低、TXB2和血小板的过度升高,进一步抑制内膜的过度增生和血栓形成。
Objective:To investigate endothelialization and medium-long term patency rate of ePTFE (expanded polytera fluoro ethylene) and Dacron artificail blood vessel planted by canine bone marrow CD34+ stem cells. And study the relationship between the 6-Keto-PGF1a, TXB2 and the endotheliosis and thrombosis of the new endomembrance on the prostheses seeded by CD34+ stem cells.
Methods:CD34+ stem cells were isolated from canine bone marrow and purified by immunomagnetic beads system, which were seeded on the surface of ePTFE and Dacron blood vessel prostheses. Dogs were separate to ePTFE and Dacron experimental group and control groups. Implant the same sort of artificial blood vessel into the abdominal aorta artery(AAA) and inferior vena cava(IVC) of the same dog. Blood was collected in preoperative and 3,7,14,30,60 days postoperative from femoral vein. PGFla and TXB2 concentrations were detected by EIA assay. Obtaining the specimens of the experimental groups and the control groups to compare the patency rate in the 10,30,60,100 days after transplantation. Compare the patency rate of the experimental group and control group. Blade the specimens, identify the endothelial cell by immunohistochemistry method. Observe the characteristic of tunica intima yasorum and intima endothelial of related time section by optical and scanning electronic microscope, observe the new born intima thickness, and compare the endothelialization of the artery and venous system of those implanted artificial blood vessel described above.
Results:(1) The isolated cells from canine bone marrow were identified to be CD34+ stem cells by flow cytometry. Those CD34+ cells were isolated and purified by immunomagnetic beads system, the concentration of cells were measured by trypan blue dyeing method, is 2.6(±0.3)×10'/ml.
(2) The PLT concentration in the experimental group increased at first, then decreased and maintained at a certain level, while the control group, after a marked increase, remained at a high level;The serum PGF1αin experimental group reduced firstly and then increased while the control group decreased significantly and then maintained at a lower level; TXB2 concentration in the experimental group increased at first, then decreased and maintained at a certain level, while the control group, after a marked increase, remained at a high level; P/T ratio in experimental group reduced firstly and then increased while the control group decreased significantly and then maintained at a lower level.
(3) For the experimental group:in the artery case, the occlusion rate is O, for ePTFE group the stenosis rate is 25%, for the Dacron group the stenosis rate is 25% too. In the vein case, for ePTFE group, the occlusion rate is 12.5%, the stenosis rate is 37.5%, and occlusion rate and stenosis rate is 0 and 37.5% for the Dacron group. For the control group, the stenosis rate is 0 and 100% for the artery and vein implanted blood vessels.
(4)Observe by the optical microscope, the new born inner membrane is dense and complete for the HE staining Dacron blood vessel, while the intima of the ePTFE staining vessel is partly separately. The new intima thickness of those two experimental group is smaller than the control group, while there are some difference between those two experimental group.
(5)Observe by SEM:for the 30 days specimens of the experimental group, the endothelial cell density decrease gradually from the stoma to the middle of the artificial blood vessel; for the 100 days specimens, the endothelial cells ranked regularly and completely. For the Dacron artificial blood vessel, the endothelial cells form a complete inner membrane which follow the long axis of the original artificial blood vessel. For the ePTFE blood vessel, the new intima is less complete, there are some area lack of endothelial cell, while some blood cell and cellulose attach instead. There are some necrotic and senile endothelial cell between. For the control group, the situation is simple, there are only some cellulose, no endothelial cell is found to cover the inner wall.
Conclusion:(1)The CD34+ stem cells isolated by immuno-magnetic beads system can be purified, and can have an appropriate increment capability, the quantity of the cells is enough to implant in one artificial blood vessel with length 20-30cm and diameter 6-8mm.
(2) Purified CD34+ cells were implanted into ePTFE and Dacron artificial blood vessels, the intima reach expected endothelialization and patency rate. Such a method is better for Dacron than ePTFE artificial blood vessel.
(3) The endothelialization and patency rate of the implanted artery blood vessels is better than those implanted venous blood vessels.
(4) It can inhibit the adhesion, aggregation and activation of platelet, also inhibit the production of bioactivators through displacing blood vessel with prostheses seeded with CD34+ stem cell.
(5)It makes endothelial cell grow faster, which can produce prostacyclin, adenosine, nitric oxide, VEGF and other bioactivators through displacing blood vessel with prostheses seeded with CD34+ stem cell. And then they inhibit PGF1αand P/ T ratio of over-reduced effectively, restrain TXB2 and platelet excessive rise, finnally prevent intimal hyperplasia and thrombosis
1.探讨CD34+干细胞种植膨体聚四氟乙烯(expanded polytera fluoroethylene, ePTFE)和涤纶人工血管对移植人工血管内皮形成和中远期通畅率的影响。
2.检测人工血管移植后血清6-k-PGF1α和TXB2的变化,探讨其变化与骨髓CD34+细胞种植人工血管内皮细胞增殖、内膜形成的关系
方法：
1.CD34+干细胞分离纯化：选取济南地区家犬20只,无菌条件抽取犬骨髓,梯度离心提取单个核细胞,经免疫磁珠标记方法分离并提纯CD34+干细胞,经流式细胞检测验证。
2.人工血管CD34+干细胞种植及移植：CD34+干细胞种植于ePTFE和涤纶人工血管,把犬分为ePTFE血管实验组和涤纶血管实验组及ePTFE对照组和涤纶对照组,各组中分别把同一种人工血管同时植入同一条犬的下腔静脉和腹主动脉
3. PLT、PGF1α、TXB2测定分析：在术前、术后3天、7天、14天、30天、60天时股静脉采血血细胞计数仪测定血小板,分离血清EIA方法测定血清PGF1α、TXB2浓度,计算PGF1α/TXB2的比值。
4.移植血管内膜检测：在术后10、30、60、100天解剖获取标本,观察种植组和对照组移植后的通畅率；免疫组化法标记血管内皮细胞,通过大体观察和光镜观察新生内膜厚度；扫描电镜观察人工血管内皮增殖及分布,比较动脉和静脉系统骨髓CD34+细胞种植人工血管后内皮化及通畅率
结果：
1.免疫磁珠标记方法分离的犬骨髓干细胞经流式细胞仪鉴定为CD34+细胞。经台盼兰染色后细胞计数板计数活细胞为(2.6±0.3)×107／ml。
2.血清PGF1α、TXB2、PLT变化：实验组血小板浓度先上升后下降并维持在一定水平,而对照组明显上升后维持在一个较高水平；实验组血清PGF1α先降低后升高,而对照组明显降低后维持在一个较低水平；实验组浓度先上升后下降并维持在一定水平,而对照组明显上升后维持在一个较高水平；实验组血清P/T的比值先降低后升高,而对照组明显降低后维持在一个较低水平。
3.人工血管通畅率：CD34+干细胞预种植人工血管植入动脉闭塞率0,ePTFE组狭窄率25%；涤纶组狭窄率25%。CD34+干细胞预种植人工血管植入静脉ePTFE组闭塞率12.5%,狭窄率37.5%；涤纶组闭塞率0,狭窄率37.5%。对照组植入动脉闭塞率0,狭窄率100%；对照组植入静脉闭塞率100%。
4.人工血管内膜增殖情况：光镜下观察HE染色试验组涤纶血管新生内膜贴附腔面紧密且完整,ePTFE血管新生内膜在HE染色后与血管腔面有部分分离。实验组两种人工血管新内膜厚度小于对照组新内膜厚度,实验组两种血管新内膜厚度存在差异(p<0.05)
5.人工血管内皮细胞增殖及分布：扫描电镜观察可见实验组30天人工血管自吻合口向人工血管中间方向的腔面新生内膜内皮细胞密度逐渐减少；100天人工血管腔面内膜内皮细胞排列均匀完整。涤纶人工血管的新内膜覆以较完整内皮细胞层,细胞排列紧密,走行与血管长轴一致。ePTFE血管新内膜较同期涤纶血管新生内膜欠完整,少量区域内皮层缺乏表面附着纤维素和血细胞,其间见坏死和衰老的内皮细胞对照组人工血管内膜表面无内皮细胞覆盖,仅见纤维组织排列。
结论：
1.免疫磁珠标记方法分离CD34+细胞经纯化后细胞数量多增殖能力强,细胞的数量能满足20-30cm长,直径6-8mm人工血管腔面的内皮化种植。
2.经纯化的CD34+细胞种植于ePTFE和涤纶人工血管,均能保持理想的内皮化和通畅率。涤纶人工血管CD34+干细胞种植后贴附腔面程度和内皮化效果优于ePTFE人工血管。
3.动脉系统骨髓CD34+细胞种植人工血管后内皮化程度和通畅率优于静脉系统骨髓CD34+细胞种植人工血管后内皮化程度和通畅率。
4.骨髓CD34+干细胞种植人工血管应用于血管置换后能抑制血小板的粘附聚集和活化,抑制生长因子、细胞因子、粘附分子、趋化因子等生物活性物质的产生。
5.骨髓CD34+干细胞种植人工血管应用于血管置换后使人工血管管腔内较早的产生内皮细胞,而血管内皮细胞可产生前列环素腺苷、一氧化氮、VEGF等生物活性物质,从而有效抑制PGF1α和P/T比值的过度降低、TXB2和血小板的过度升高,进一步抑制内膜的过度增生和血栓形成。
Objective:To investigate endothelialization and medium-long term patency rate of ePTFE (expanded polytera fluoro ethylene) and Dacron artificail blood vessel planted by canine bone marrow CD34+ stem cells. And study the relationship between the 6-Keto-PGF1a, TXB2 and the endotheliosis and thrombosis of the new endomembrance on the prostheses seeded by CD34+ stem cells.
Methods:CD34+ stem cells were isolated from canine bone marrow and purified by immunomagnetic beads system, which were seeded on the surface of ePTFE and Dacron blood vessel prostheses. Dogs were separate to ePTFE and Dacron experimental group and control groups. Implant the same sort of artificial blood vessel into the abdominal aorta artery(AAA) and inferior vena cava(IVC) of the same dog. Blood was collected in preoperative and 3,7,14,30,60 days postoperative from femoral vein. PGFla and TXB2 concentrations were detected by EIA assay. Obtaining the specimens of the experimental groups and the control groups to compare the patency rate in the 10,30,60,100 days after transplantation. Compare the patency rate of the experimental group and control group. Blade the specimens, identify the endothelial cell by immunohistochemistry method. Observe the characteristic of tunica intima yasorum and intima endothelial of related time section by optical and scanning electronic microscope, observe the new born intima thickness, and compare the endothelialization of the artery and venous system of those implanted artificial blood vessel described above.
Results:(1) The isolated cells from canine bone marrow were identified to be CD34+ stem cells by flow cytometry. Those CD34+ cells were isolated and purified by immunomagnetic beads system, the concentration of cells were measured by trypan blue dyeing method, is 2.6(±0.3)×10'/ml.
(2) The PLT concentration in the experimental group increased at first, then decreased and maintained at a certain level, while the control group, after a marked increase, remained at a high level;The serum PGF1αin experimental group reduced firstly and then increased while the control group decreased significantly and then maintained at a lower level; TXB2 concentration in the experimental group increased at first, then decreased and maintained at a certain level, while the control group, after a marked increase, remained at a high level; P/T ratio in experimental group reduced firstly and then increased while the control group decreased significantly and then maintained at a lower level.
(3) For the experimental group:in the artery case, the occlusion rate is O, for ePTFE group the stenosis rate is 25%, for the Dacron group the stenosis rate is 25% too. In the vein case, for ePTFE group, the occlusion rate is 12.5%, the stenosis rate is 37.5%, and occlusion rate and stenosis rate is 0 and 37.5% for the Dacron group. For the control group, the stenosis rate is 0 and 100% for the artery and vein implanted blood vessels.
(4)Observe by the optical microscope, the new born inner membrane is dense and complete for the HE staining Dacron blood vessel, while the intima of the ePTFE staining vessel is partly separately. The new intima thickness of those two experimental group is smaller than the control group, while there are some difference between those two experimental group.
(5)Observe by SEM:for the 30 days specimens of the experimental group, the endothelial cell density decrease gradually from the stoma to the middle of the artificial blood vessel; for the 100 days specimens, the endothelial cells ranked regularly and completely. For the Dacron artificial blood vessel, the endothelial cells form a complete inner membrane which follow the long axis of the original artificial blood vessel. For the ePTFE blood vessel, the new intima is less complete, there are some area lack of endothelial cell, while some blood cell and cellulose attach instead. There are some necrotic and senile endothelial cell between. For the control group, the situation is simple, there are only some cellulose, no endothelial cell is found to cover the inner wall.
Conclusion:(1)The CD34+ stem cells isolated by immuno-magnetic beads system can be purified, and can have an appropriate increment capability, the quantity of the cells is enough to implant in one artificial blood vessel with length 20-30cm and diameter 6-8mm.
(2) Purified CD34+ cells were implanted into ePTFE and Dacron artificial blood vessels, the intima reach expected endothelialization and patency rate. Such a method is better for Dacron than ePTFE artificial blood vessel.
(3) The endothelialization and patency rate of the implanted artery blood vessels is better than those implanted venous blood vessels.
(4) It can inhibit the adhesion, aggregation and activation of platelet, also inhibit the production of bioactivators through displacing blood vessel with prostheses seeded with CD34+ stem cell.
(5)It makes endothelial cell grow faster, which can produce prostacyclin, adenosine, nitric oxide, VEGF and other bioactivators through displacing blood vessel with prostheses seeded with CD34+ stem cell. And then they inhibit PGF1αand P/ T ratio of over-reduced effectively, restrain TXB2 and platelet excessive rise, finnally prevent intimal hyperplasia and thrombosis
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23. Niklason LE, Gao J, Abbott W, et al.Functional arteries grown in virto. [J] Science,1999,284:489 493.
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28. 刘冰,李哈莉,张小明.骨髓干细胞在血管外科的基础研究与临床应用.[J]中华普通外科杂志,2003,18(4)：255-256.
29. Shi Q, Wu MH-D, Hayashida N, et al. Proof of fallout endothelialization of impervious Dacron grafts in the aorta and inferior vena cava of the dog. [J] J Vasc Surg.1994;20: 546-556.
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32. Shi Q, Wu MH-D, Fujita Y, et al. Genetic tracing of arterial flow surface endothelialization in allogeneic marrow transplanted dogs. [J] Cardiovasc Surg.1999;7:98-105.
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36.柏树令,赵丹.CD34抗原的生物学特性及其临床应用.[J]解剖科学进展,2005,11(1)：54-56.
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41.张小明,李伟,李清乐.骨髓种植人工血管在静脉系统的实验研究.[J]中华普通外科杂志,2004,19：46-49.
42.Falk J, Townsend L E, Vogel L M, et al. Improved adherence of genetically modified endothelial cells to small- diameter expanded polytetrafluoroethylene grafts in a canine mode. J
43. Bowlin GL, Rittgers SE. Electrostatic endothelial cell seeding technique for small-diameter(<6mm) vascular prostheses: feasibility testing. [J] Cell Transplant.1997,6(6):623
44. Carr HM, Vohra R, Sharnna H, et al. [J] Endothelial cell seeding kinetics under chronic flow in prosthetic grafts. Ann Vasc Surg,1996,10(5):469
45. Bhat VD, Klitzman B, Koger K, et al. Improving endothelial cell adhesion to vasculargraftsurfaces:clinical need and strategies. [J] J Biomater Sci Polym Ed,1998,9(11):11-17;
46.Dardik A, Liu A, Ballermann BJ. Chronic in vitro shear stress stimulates endothelial cell retention on prosthetic vascular grafts and reduces subsequent in vivo neointimal thickness. [J] J Vase Surg,1999,29(1):157
47. Shan- hui Hsu, Hsinang-jung Tseng, Meng- shou Wu. Comparative in vitro evaluation of different preparations diameter polyurethane vascular grafts. [J] Artifical Organs, 2000,24:119.
48. Jan M S, Lee Y J, Shin S J,et al.Oxidied lowdensity lipoprotein stimulates endothelin-1 and mRNA expression from rat mesangial cells. [J] J-Lab-Clin-Med,1997,129 (2):224-230.
49. Lerman A, Edwards B S, et al. Circulation and tissue endothlin immunoreacyivity in advance atherosclerosis. [J] N Engl J Med,1991,325: 997-1001.
50.宋良文,王雪青,张文静,等.氧化低密度脂蛋白于内皮素在促进血管平滑肌细胞增生中的内在联系[J].中国动脉硬化杂志,1994,2(2-3)：84-86.
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53. Dustiag G J,Monesdes, Vane J R前列腺素及其中间体和前体：心血管效应及其正常和一场循环血流中的调节作用.[J]国外医学心血管分册,1980,2：65-278.
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