骨髓CD34~+干细胞种植人工血管应用于血管置换后内皮化和通畅率的研究

目的:探讨骨髓CD34+细胞分别种植于常用膨体聚四氟乙烯(expanded polytera fluoroethylone,ePTFE)人工血管和涤纶人工血管的内皮化程度和通畅率.方法:选杂种犬16条,依人工血管不同分为ePTFE血管实验组(6条)和涤纶皿管实验组(6条)及ePTFE对照组(2条)和涤纶对照组(2条),实验犬采自体骨髓,提取CD34+细胞种植覆膜人工血管,对照犬采用单纯自体血预凝人工血管,将ePTFE或涤纶人工血管分别植入所有实验犬的下腔静脉和腹主动脉.术后第30、60、100天取标本,观察通畅率,并分别用光学显微镜、电子显微镜和免疫组织化学方法观察新生内膜表面内皮化情况.结果:对照组静脉全部阻塞.实验组第30天人工血管腔面新生内膜内皮细胞密度自吻合口向中间方向逐渐减少,第60天的人工血管内皮基本覆盖管壁,第100天人工血管腔面内膜内皮细胞排列均匀完整,而对照组内膜表面无内皮细胞覆盖.结论:经纯化的CD34+细胞种植于ePTFE和涤纶人工血管较未种植的人工血管中远期有较好的内皮化和通畅率.     

犬骨髓CD34＋细胞种植人工血管置换后短期内皮化的实验研究

目的：探讨骨髓CD34＋细胞种植于常用膨体聚四氟乙烯（expanded polytera fluoroethylene，ePTFE）人工血管和涤纶人工血管后二者的内皮化程度。方法：选杂种犬8条，依支架覆膜不同分为ePTFE组和涤纶组，每组实验犬2条，对照犬2条。实验犬采自体骨髓，提取CD34＋细胞种植覆膜支架，对照犬采用单纯自体血预凝覆膜支架。将人工血管植入犬的下腔静脉和腹主动脉。在术后第10、30天观察植入的人工血管通畅情况，采用免疫组织化学方法鉴定内膜细胞来源，在光镜和电镜下观察人工血管新生内膜表面内皮化情况。结果：术后第10天实验组与对照组差异无统计学意义；术后第30天腔面新生内膜内皮细胞自人工血管吻合口向中间逐渐减少（P〈0．05）；而对照组内膜表面第10、30天均无内皮细胞覆盖。结论：经纯化的CD34＋细胞种植于ePTFE和涤纶人工血管，均获得理想的内皮化。     

术前放疗对犬腹主动脉和下腔静脉人工血管置换术后人工血管内膜的影响

目的 观察术前放疗对犬腹主动脉、下腔静脉人工血管置换术后人工血管内膜的影响.方法 杂种犬18只,随机分为放疗组和对照组各9只.放疗组对标记的拟行人工血管移植的血管区行分割放疗,6周后于肾动脉下腹主动脉、下腔静脉行ePTFE人工血管置换术,4周后取标本.对照组除不放疗外各项操作同放疗组.观察两组动物人工血管的通畅率,并行HE染色和免疫组化PCNA和CD34染色.结果 放疗组有2犬术后当天死亡,对照组全部存活.放疗组术后3条静脉人工血管完全堵塞,对照组2条完全堵塞,两者差异无统计学意义(P>0.05).两组动脉人工血管均通畅,内膜完整,但内皮细胞覆盖不完全.放疗组的吻合口近、远端处CD34阳性细胞数差异无统计学意义,而在人工血管中部放疗组低于对照组(P<0.05);放疗组吻合口近、远端及人工血管中部内膜厚度均较对照组薄(P<0.05);而放疗组3个点PCNA阳性细胞表达较对照组为低(P<0.05).结论 术前放疗对人工血管置换术后4周内膜的增生有抑制作用,不会降低其通畅率.     

血小板中cAMP与cGMP及血清LDL-ch对犬下腔静脉移植物内膜增生的影响

为探讨静脉系移植物通畅率的影响因素，对自体静脉碎片种植Ｄａｃｒｏｎ后植入下腔静脉（ＩＶＣ）的１３只犬及全血预凝Ｄａｃｒｏｎ后植入ＩＶＣ作对照的８只犬血清脂蛋白胆固醇、血小板环－磷酸腺苷（ｃＡＭＰ）、环－磷酸鸟苷（ｃＧＭＰ）及移植物内膜厚度进行了测定。结果：种植组通畅率（６１．５％）高于对照组（２５．０％），Ｄａｃｒｏｎ腔面术后２周完全内皮化；对照阻塞组与种植阻塞组血小板ｃＡＭＰ低于对照通畅组与种植通畅组，血清低密度脂蛋白胆固醇（ＬＤＬ－ｃｈ）高于对照通畅组与种植通畅组，前二组内膜厚度大于后二组。结果提示：内皮层不完整所致腔面前列环素及血小板ｃＡＭＰ减少，可能是静脉系移植物内膜增生的主要原因，高ＬＤＬ－ｃｈ血症可能对其有促进作用；人工血管内皮化，抗血小板与降血脂处理对预防内膜增生及提高通畅率可能有帮助。     

血小板中cAMP与cGMP及血清LDL—ch对犬下腔静脉移植内膜增？…

为探讨静脉系移植物通畅率的影响因素，对自体静脉碎片种植Ｄａｃｒｏｎ后植入下腔静脉的１３只犬及全血预凝Ｄａｃｒｏｎ后植入ＩＶＣ作对照的８只犬血甭脂蛋白胆固醇，血小板环－磷酸腺苷，环－磷酸鸟苷及移植物内膜厚度进行了测定。结果：种植组通畅率高于对照组，Ｄａｃｒｏｎ腔面术后２周内皮化；对照阻塞组与种植阻塞组血小板ｃＡＭＰ低于对照通畅组与种植通畅组，血清低密度脂蛋白胆固醇高于对照通畅组与种植通畅组，前二组内     

犬自体静脉与腹膜碎片镶嵌种植人工血管的研究

目的:探讨加速人工血管内皮化,提高人工血管移植后通畅率的途径.方法:犬自体静脉与腹膜碎片镶嵌种植人工血管,行股动脉移植.结果与结论:细胞镶嵌种植后的人工血管在短期内形成了管腔内皮化,提高了人工血管移植通畅率.光、电镜及免疫组化观察,证实了移植血管新生内膜的细胞组成.内膜厚度测定,种植组与对照组间差异显著(P<0.01 ),内皮化后抑制了内膜的增生.腹膜间皮细胞与内皮细胞在形态及功能上的相似性,使其同样具有很好的应用前景.镶嵌种植技术为一快速、简便的人工血管内皮化方法,具有较高的临床应用价值.     

小口径脱细胞基质人工血管移植的研究

目的 探讨脱细胞基质( DCM)人工血管用于小口径血管移植的可行性.方法 40条雄性杂种犬随机分为DCM、膨体聚四氟乙烯(ePTFE)人工血管及自体颈外静脉3组行右颈总动脉置换术,彩超监测移植物通畅率.术后4、8周活体取材,标本行苏木素-伊红(HE)、免疫组织化学染色及扫描电镜检查.结果 3组移植物1周通畅率(75.0％、64.3％、100.0％)差异无统计学意义(P＞0.05);自体颈外静脉组4、8周通畅率( 100.0％、88.9％)优于DCM组(56.3％、26.7％)及ePTFE组(57.1％、23.1％,P＜0.05),后两组差异无统计学意义(P＞0.05).DCM人工血管4、8周血栓形成面积小于ePTFE人工血管,吻合口内膜内皮化程度高于后者.结论 小口径DCM人工血管在抑制血栓形成及加快内皮化方面优于ePTFE人工血管.     

骨髓种植人工血管在静脉系统的实验研究

目的研究骨髓种植的人工血管在静脉系统的应用 ,旨在探索一种新的、更理想的静脉代用品。方法选北京地区杂种犬 8条 ,实验组和对照组各 4条。实验组采用自体骨髓种植的涤纶双绒人工血管置换肾下下腔静脉 ,对照组则采用单纯自体血浆预凝人工血管。术后 10d获取人工血管标本 ,观察其通畅率 ,行光镜、电镜检查 ,比较新生内膜厚度、新生内膜表面内皮化情况 ,并通过检测6 keto PGE1α和TXB2水平 ,比较分析实验组和对照组抗血栓能力。结果实验组全部移植血管均通畅 ,对照组 2 / 4条通畅 ,光镜下实验组的内膜厚度明显薄于对照组 (P <0 0 1) ,电镜发现实验组人工血管达到完全内皮化 ,而对照组表面则无成片内皮细胞存在。实验组 6 keto PGF1α水平明显高于对照组 (P <0 0 1) ,而TXB2水平实验组低于对照组 (P <0 0 5 )。结论骨髓种植的人工血管在静脉系统 10d时能实现人工血管腔面的快速完全内皮化 ,新生内皮细胞具有抑制新生内膜增生和抗血栓形成的能力。     

微泡造影剂结合超声介导一氧化氮合成酶基因转染防治移植静脉再狭窄

目的 探讨微泡造影剂及超声辐照介导内皮型一氧化氮合成酶(eNOS)基因转染防治移植静脉血管桥再狭窄的可行性.方法 重组真核表达载体pcDNA3.1-eNOS经微泡造影剂及超声辐照介导体外转染大鼠颈外静脉,再将静脉段间置植入颈总动脉,建立SD大鼠颈外静脉-颈总动脉移植模型.于术后4周取移植静脉标本分别进行苏木素-伊红(HE)染色,免疫组织化学染色及Western blot法分析,观察eNOS基因在静脉桥中的功能表达和静脉桥新生内膜的增生.结果 Western blot法分析表明微泡造影剂及超声辐照介导eNOS基因转染组的移植血管桥内eNOS表达最明显;增殖细胞核抗原( PCNA)检测阳性率为(21.04±3.51)％,细胞凋亡指数为(12.11±1.23)％,新生内膜厚度(25.0±3.5) μm,内膜/中膜比值为0.43,均明显低于其他组(P＜0.05).结论 微泡造影剂及超声辐照介导eNOS基因转染可以有效抑制移植静脉桥新生内膜的增生.     

体外扩增恒河猴自体内皮细胞人工血管移植研究（二）

目的：为自体内皮细胞移植的临床应用奠定理论基础。方法：将１５只恒河猴随机分为两组。实验组１０例，用衬里有自体静脉内皮细胞的人工血管置换髂总动脉；对照组５例，用未衬里内皮细胞塞外，其余８条人工血管术后四周均保持通畅，内膜下组织碍度为８０±１２μｍ；对照组５条人工血管全部阻塞。结论自体内皮移植可有效地南昌市人工血管的通畅率。     

Suppression of ICAM-1 in human venous endothelial cells by small interfering RNAs

Objective: Cardiopulmonary bypass-mediated release of proinflammatory cytokines promotes the transendothelial migration of leukocytes. Among others, intercellular adhesion molecule (ICAM) is essential for this migratory process within the venous bypass graft, which finally contributes to a diminished early patency rate by thickening of the intima. Small interfering ribonucleic acids (siRNAs) are efficient and specific modulators of endogenous gene expression. This study describes the application of siRNAs to suppress ICAM-1 expression on the surface of human venous endothelial cells. Methods: Primary cultures of human venous endothelial cells were either transfected with ICAM-1 siRNA, with a scrambled control siRNA or cultured without transfection. ICAM-1 expression was analyzed with or without TNF- stimulation by flow cytometry. Results: Upon TNF- stimulation, cells transfected with ICAM-1 siRNA showed a six- to seven-fold decreased ICAM-1 expression compared to untransfected cells or cells transfected with the scrambled control siRNA. Conclusions: This is the first report that ICAM-1 expression can be effectively silenced by siRNAs on endothelial cells from human saphenous veins. This new technology may render novel therapeutic concepts to reduce early graft failure by protecting venous bypass grafts against early intra- or postoperative leukocyte infiltration.     

The effect of preformed confluent endothelial cell monolayers on the patency and thrombogenicity of small calibre vascular grafts

Endothelial cell seeding has been proposed as a method to improve the patency rates in small calibre prosthetic vascular grafts. The seeding methods used at present leave much of the graft luminal surface devoid of endothelial cells and thus still significantly thrombogenic. We have developed a method to preform confluent endothelial cell monolayers, on the grafts prior to implantation, and this study investigates the effect of these monolayers on the early thrombogenicity and patency of polytetrafluoroethylene (PTFE) grafts. Small diameter PTFE grafts were seeded with canine endothelial cells obtained from the external jugular vein. Each of five dogs then received a graft seeded with its own cells and a contralateral, non-seeded control graft. At 1 and 10 weeks after graft implantation graft thrombogenicity was assessed by the use of Indium labelled platelets. The thrombogenicity index (TI) of each graft was determined from counts of gamma activity recorded over a period of 7 days. Grafts were subsequently removed at 12 weeks. At 1 week the mean TI for the seeded grafts was 0.123 (SD 0.019) and that for the controls 0.183 (SD 0.017) (p = 0.005). At 10 weeks only the seeded grafts could be assessed because all of the control grafts had occluded. At this point in time the seeded grafts had a mean TI of 0.159 (SD 0.011) (p = 0.047 vs. seeded at 1 week). By the time of removal at 12 weeks, all control grafts were occluded but only one of the seeded grafts had occluded (p = 0.025). In conclusion, the use of preformed, confluent endothelial cell monolayers for seeding prosthetic grafts significantly reduces the early graft thrombogenicity and improves graft patency. It does not, however, completely halt the increase in thrombogenicity which occurs during the early post-implantation period.     

Prostacyclin production from seeded prosthetic vascular grafts.

Endothelial cell seeding has been proposed as a method of improving patency rates in small-calibre prosthetic vascular grafts. In vivo, endothelial cells normally produce prostacyclin (PGI2), a potent antiplatelet agent. The aim of this study was to determine whether seeded grafts show significant PGI2 production after in vivo implantation. Grafts were seeded with either autologous canine venous endothelial cells or autologous microvascular endothelial cells. After 12 weeks, PGI2 production was assessed under basal and stimulated conditions. Seeded grafts were compared with non-seeded controls and the corresponding aorta. The overall patency rate in seeded grafts was 80 per cent compared with 10 per cent in non-seeded grafts (P < 0.01). Grafts seeded with cells from either source produced significantly more PGI2 than unseeded grafts in both basal and stimulated states (P < 0.05). The aorta produced significantly more PGI2 than seeded grafts under both conditions (P < 0.01). Endothelial cell seeding produces a functional graft and leads to an improved patency rate.     

内皮型一氧化氮合酶基因体外转染对犬内皮祖细胞功能的影响

目的探讨人内皮型一氧化氮合酶基因(heNOS)转染对犬骨髓源内皮祖细胞(EPC)功能的影响。方法用携带heNOS基因的5型腺病毒作为载体(Ad5-heNOS),对体外定向培养扩增的骨髓源犬EPC进行转染,以未转染的犬EPC作为对照。用酶联免疫吸附试验(ELISA)和硝酸还原酶法检测转染后的EPC中heNOS蛋白的表达和NO的产量,并检测转染后EPC的增殖、黏附、迁移、抗衰老和成血管能力等功能。结果 Ad5-heNOS转染48 h后,转染组EPC的eNOS蛋白表达量和NO产量均明显高于未转染组[(2091.67±172.489)pg/mL vs.(158.00±30.914)pg/mL;(49.5±5.16)μmol/Lvs.(39.7±7.24)μmol/L](均P<0.01);转染组EPC的细胞增殖数、黏附数、迁移数均明显高于未转染组(0.52±0.03 vs.0.31±0.02;28.00±1.41 vs.11.83±1.45;109.67±6.95 vs.72.67±6.29)(均P<0.01),而衰老细胞百分数明显低于未转染组(0.22±0.02 vs.0.32±0.01)(P<0.01);转染heN...     

Small-diameter blood vessels engineered with bone marrow-derived cells

OBJECTIVE: The objective of this study is to investigate if bone marrow-derived cells (BMCs) regenerate vascular tissues and improve patency in tissue-engineered small-diameter (internal diameter = 3 mm) vascular grafts. SUMMARY BACKGROUND DATA: BMCs have demonstrated the ability to differentiate into endothelial-like cells and vascular smooth muscle-like cells and may offer an alternative cell source for vascular tissue engineering. Thus, we tissue-engineered small-diameter vascular grafts with BMCs and decellularized arteries. METHODS: Canine BMCs were differentiated in vitro into smooth muscle alpha-actin/smooth muscle myosin heavy-chain-positive cells and von Willebrand factor/CD31-positive cells and seeded onto decellularized canine carotid arteries (internal diameter = 3 mm). The seeded grafts were implanted in cell donor dogs. The vascular-tissue regeneration and graft patency were investigated with immunohistochemistry and angiography, respectively. RESULTS: The vascular grafts seeded with BMCs remained patent for up to 8 weeks in the canine carotid artery interposition model, whereas nonseeded grafts occluded within 2 weeks. Within 8 weeks after implantation, the vascular grafts showed regeneration of the 3 elements of artery (endothelium, media, and adventitia). BMCs labeled with a fluorescent dye prior to implantation were detected in the retrieved vascular grafts, indicating that the BMCs participated in the vascular tissue regeneration. CONCLUSIONS: Here we show that BMCs have the potential to regenerate vascular tissues and improve patency in tissue-engineered small-diameter vascular grafts. This is the first report of a small-diameter neovessel engineered with BMCs as a cell source.     

Effects of gene transfection of human bcl-2 on concordant cardiac xenografts in hamster to rat model

OBJECTIVES: Concordant cardiac xenografts are known for delayed vascular rejection. Therapy combining with FK506 and cobra venom factor prolongs graft survival. The proposed underlying mechanism holds that cytoprotective proteins such as Bcl-2 play a role here. We studied the effects of gene transfection of human-bcl-2 on graft survival and coronary artery lesions in concordant cardiac xenografts, and discuss the role of cytoprotective genes in vascular xenograft rejection. METHODS: Golden-Syrian-hamster hearts were heterotopically transplanted into Lewis rats given FK506 (1 mg/kg daily) and cobra venom factor (0.2 mg/kg; day 0 and 1) intramuscularly. They were divided into 2 groups--grafts transfected vector with the human-bcl-2 gene (Group-B(+)) and vector without the gene (Group-B(-)) using the HVJ liposome method; 4 or 5 grafts from each group were explanted 1, 2, 3, or 4 weeks and more than 1 month after transplantation and evaluated by H-E, Elastic-Van-Gieson and immunohistochemical staining of Bcl-2. Coronary arterial lesions were examined using a scoring method. RESULTS: Bcl-2 expression in endothelial cells in Group-B(+) was confirmed within 2 weeks after transplantation but not thereafter. The coronary score in Group-B(+) was significantly lower than that in Group-B(-) within 2 weeks after transplantation but not thereafter. CONCLUSIONS: In this hamster-to-rat cardiac xenograft model, the bcl-2 gene was successfully transfected to the coronary endothelium and lasted 2 weeks. During Bcl-2 expression, coronary vascular lesions were suppressed more than in the untransfected group.     

In vivo h-VEGF165 gene transfer improves early endothelialisation and patency in synthetic vascular grafts.

OBJECTIVES: Small-diameter synthetic vascular graft performance is inferior to autologous vein grafts. This study tested the hypotheses that local in vivo administration of plasmids encoding for human vascular endothelial growth factor (VEGF), or co-administration of plasmids encoding for human vascular endothelial growth factor/plasmids encoding for fibroblast growth factor-2 in the tissues surrounding a porous synthetic vascular graft would enhance graft endothelialisation and, consecutively, graft patency. METHODS: First, optimal gene for small-diameter synthetic graft endothelialisation was studied in rat abdominal aorta model (n=132): plasmids encoding for human vascular endothelial growth factor; co-administration of plasmids encoding for human vascular endothelial growth factor/plasmids encoding for fibroblast growth factor-2; or control plasmids were injected around 60 microm ePTFE graft. Second, optimal small-diameter synthetic graft design for endothelialisation was explored in rabbit abdominal aorta model (n=90). Various ePTFE grafts or pre-clotted polyester grafts were used with/without plasmids encoding for human vascular endothelial growth factor. Third, clinically used medium-size synthetic grafts were investigated with/without plasmids encoding for human vascular endothelial growth factor in dog carotid (n=20) and femoral arteries (n=15). Endothelialisation was assessed in midgraft area with scanning electron microscopy. RESULTS: In rats, plasmids encoding for human vascular endothelial growth factor enhanced endothelialisation; whereas co-administration of plasmids encoding for human vascular endothelial growth factor/plasmids encoding for fibroblast growth factor-2 had worst outcome at 1 week (NS), 2 weeks (P=0.01) and 4 weeks (P=0.02). In rabbits, pre-clotted polyester grafts had a trend for faster endothelialisation than ePTFE grafts (P=0.08); whereas plasmids encoding for human vascular endothelial growth factor enhanced endothelialisation compared to controls at 2 weeks (P=0.06), however, the effect reversed at 4 weeks (P=0.03). In dogs, synthetic graft patency was improved by plasmids encoding for human vascular endothelial growth factor in femoral position (P=0.103); whereas all carotid grafts were patent at 6 weeks. CONCLUSIONS: Thus, these data suggested that endothelialisation was fastest in pre-clotted polyester grafts; and that local application of plasmids encoding for human vascular endothelial growth factor had a potential to improve early endothelialisation and patency in synthetic vascular grafts.     

兔颈动脉移植后血管内皮型一氧化氮合酶基因表达的变化及意义

目的探讨兔颈动脉移植后移植血管段内皮型一氧化氮合酶信使核糖核酸(eNOSmRNA)表达的变化及其意义。方法建立大白兔颈动脉移植模型。将30只兔按随机数字表法分为4组。A组(n=3)自体血管移植;B组(n=9)新鲜异体血管移植;C组(n=9)同种异体血管经青霉素和链霉素处理常温保存后移植;D组(n=9)同种异体血管经青霉素和链霉素处理冷冻保存后移植。观察比较移植后1～3周移植血管段组织形态学变化和eNOS mRNA的表达。结果光学显微镜下观察A组结构正常,仅有炎性细胞浸润;其余各组术后1周内膜开始增生,2周内膜明显增厚,3周内膜、中膜增厚最明显,同时伴有血栓形成。其中B组改变最严重,B组血管移植后eNOS mRNA表达明显低于其他三组(P<0.05)。结论同种异体动脉血管移植后,血管eNOS基因表达明显下调,可造成移植后血管内膜增生和血栓形成。