Seeding of autogenous endothelial cells to inner surface of small-caliber dacron vascular prostheses. An experimental study on canines

We presented the experimental results of endothelial cells (EC) seeding in small caliber interpositional dacron grafts of canine carotid arteries. Six weeks after surgery, 9 (56.25%) seeded, and 6 (37.5%) control grafts remained patent. The mean thrombus-free surface area was 83.4% in the seeded and 62% in the controls. The mean volume of thrombus was 74 mg and 218 mg respectively (P less than 0.05). Morphologically, the endothelial layer extension on the graft from the host artery was less than 1 cm. Scanning electron microscopy revealed that a thin luminal layer was composed of flat polygonal cells at the seeded midgrafts, and that fibrous tissues were covered with numerous platelets, fibrin, and blood cells at the unseeded midgrafts. Transmission electron microscopy demonstrated that cells on the seeded midgrafts were characterized by endothelial cells. We conclude that endothelial cells seeding could improve the performance of small-caliber dacron prostheses.     

降血脂剂对犬人工血管内膜增生的影响

OBJECTIVE: To investigate the effects of the lipid-lowering drugs in alleviating endothelial hyperplasia in the inferior vena cava (IVC) grafts in dogs. METHODS: The Dacron grafts seeded with autologous venous fragments were implanted into the IVC of 20 dogs, including 12 dogs receiving oral lipid-lowering drugs serving as the treatment group and the other 8 without medication as the control group. The levels of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-ch) and high-density lipoprotein cholesterol (HDL-ch) in the serum were measured regularly, and all the grafts harvested to measure the thickness of the endothelium. RESULTS: The total patency rate of the IVC were higher in the treatment group (75%) than in the control group (37.5%), and new endothelial lining was formed two weeks after the operation. Compared with the control group, the endothelial thickness of the grafts at the proximal (P<0.01), middle (P<0.05) and distal segments (P<0.05) of the IVC were all smaller in the treatment group, which also had lower serum LDL-ch and TC levels (both P<0.05) but with comparable HDL-ch levels (P>0.05). CONCLUSION: Administration of lipid-lowering drugs may reduce the level of serum LDL-ch and TC and the endothelial thickness of the grafts to improve the patency rate of the vessels.     

生物血管异种移植的初步研究

目的为了寻求一种新的小口径血管代用品,建立异种移植的动物实验模型,以观察异种移植物的安全性、可靠性、通畅性及组织学改变。方法共采用17只杂种雌性犬,实验组10只,植入经环氧化物处理的猪血管移植物;对照组7只,植入人造血管。手术方法为右侧股动静脉瘘。术后通过超声和血管造影方法来观察移植血管的通畅性,并在术后3月将移植物取出,进行病理学检查,观察移植前后移植物的组织学改变。结果术后第一周、二周行Doppler超声检查结果,两组动静脉瘘均通畅,2周内血管通畅率为100%。术后3个月动脉造影检查后,生物血管组(PG)通畅5只,通畅率62.5%,e-PTFE组通畅4只,通畅率66.7%。两组数据统计学处理,差异无显著性(P>0.05)。术后3月对移植物取材,进行光镜及扫描电镜病理学检查,通畅的生物血管吻合口无狭窄,吻合部位有新的内膜覆盖,周围组织无钙化,有新生的内皮细胞覆盖。结论经环氧化物处理的猪的血管移植物(PG)生物血管作为异种移植物,生物相容性好,具有一定的可行性。     

降血脂剂对犬人工血管内膜增生的影响

目的探讨降血脂剂对减轻静脉系人工血管移植物内膜增生的作用。方法将自体静脉碎片种植的Dacron植入20只犬下腔静脉(IVC),除对照组(n=8)外,实验组(n=12)术后给降血脂剂口服,两组动物定期处死,测定比较两组血中总胆固醇(TC)、低密度脂蛋白胆固醇(LDL-C)、高密度脂蛋白胆固醇(HDL-C)含量以及人工血管内膜厚度。结果实验组血管通畅率(75%)高于对照组(37.5%),Dacron腔面术后2周完全内皮化。与对照组比较,实验组移近心端(P<0.01)、中部(P<0.05)及远心端(P<0.05)的植物内膜厚度均显著薄于对照组。动物处死时,实验组血清TC与LDL-C含量均显著低于对照组(P<0.05),HDL-C含量无组间差异(P>0.05)。结论降血脂剂可显著降低犬血脂,对减轻人工血管内膜增生、提高通畅率有一定作用。     

Patency rate and complications of polytetrafluoroethylene grafts compared with polyurethane grafts for hemodialysis access

Background

The survival of hemodialysis patients requiring dialysis depends on the long-term functioning and patency of the vascular access. Prosthetic vascular grafts are inevitably used for patients whose vessels are unsuitable for an autogenous arteriovenous (AV) fistula. The purpose of this study was to compare the patency rate and associated complications using different types of grafts.

Methods

This prospective study was conducted on patients who did not have an appropriate vein for arteriovenous fistula from January 2004 through July 2006. They were divided into two groups, sex, age, and basic data matched. Polytetrafluoroethylene (PTFE) and polyurethane (PVAG) were the two types of grafts used in this study. The functionality of the graft was assessed immediately 1 day and 2 weeks after operation. The clinical follow-up was performed each 3 months until 24 months.

Results

One-year patency rate was reported to be 64% and 52% in the PTFE and PVAG groups, respectively. There was no significant difference in 1-year (64% versus 52%) and 2-year (49% versus 41%) patency rate of the PTFE and PVAG grafts used as vascular access. There was also no difference between the numbers of complications reported in the two groups.

Conclusion

It could be concluded that either PTFE or PVAG grafts can be used with the same expected outcomes.     

新型组织工程化血管移植物桥接腹主动脉缺损

目的将硅胶管置入大鼠腹腔内预制出腹膜组织管构建新型的自体血管移植物。方法将预制的硅胶棒(直径1.5mm,长20mm)褥式缝合在大鼠的腹膜下,4周后硅胶棒被管状壁层腹膜组织包裹;应用形成的壁层腹膜组织管作为血管移植物桥接大鼠腹主动脉缺损;术后4周取材,观察其通畅率并进行光镜电镜观察。结果25只大鼠中血管通畅21只,血栓4只,通畅率为89.1%(21/25)。血管移植物内表面可见新生的内皮细胞覆盖,内皮细胞下有少量肌样细胞,管壁最外层富含胶原纤维。结论在体内预制的大鼠壁层腹膜组织管可以作为血管移植物桥接大鼠腹主动脉缺损,这种非血管组织构建的血管移植物在血流作用下,其管壁被新生的内皮细胞和肌样细胞覆盖,证明了细胞对周围环境的适应性。     

Patency rate and complications of polytetrafluoroethylene grafts compared with polyurethane grafts for hemodialysis access

Abstract

Background. The survival of hemodialysis patients requiring dialysis depends on the long-term functioning and patency of the vascular access. Prosthetic vascular grafts are inevitably used for patients whose vessels are unsuitable for an autogenous arteriovenous (AV) fistula. The purpose of this study was to compare the patency rate and associated complications using different types of grafts.

Methods. This prospective study was conducted on patients who did not have an appropriate vein for arteriovenous fistula from January 2004 through July 2006. They were divided into two groups, sex, age, and basic data matched. Polytetrafluoroethylene (PTFE) and polyurethane (PVAG) were the two types of grafts used in this study. The functionality of the graft was assessed immediately 1 day and 2 weeks after operation. The clinical follow-up was performed each 3 months until 24 months.

Results. One-year patency rate was reported to be 64% and 52% in the PTFE and PVAG groups, respectively. There was no significant difference in 1-year (64% versus 52%) and 2-year (49% versus 41%) patency rate of the PTFE and PVAG grafts used as vascular access. There was also no difference between the numbers of complications reported in the two groups.

Conclusion. It could be concluded that either PTFE or PVAG grafts can be used with the same expected outcomes.     

携载VEGF基因的聚四氟乙烯血管材料对内皮细胞生长的促进作用

目的:研究聚四氟乙烯(PTFE)血管材料携载血管内皮生长因子(VEGF)基因并促进内皮细胞生长的可行性. 方法:将pCDI-hVEGF121/pCDI质粒溶液处理过的PTFE人工血管修剪成圆片,置于生理盐水中并于0.5, 1, 2, 4, 8, 16,24, 48和72 h测定溶液的吸光度,计算出溶液的DNA浓度,绘出体外释放曲线. 分离和培养人脐静脉内皮细胞,将内皮细胞种植到携带基因质粒的PTFE材料表面,在6, 24, 72和120 h检测细胞增殖情况并用ELISA法检测培养液中VEGF蛋白的表达量. 结果:体外释放曲线提示在0.5～4 h的这段时间内,质粒释放较快,随后进入缓慢增长期,直至72 h仍有质粒的释放. 在24, 72和120 h时间点,VEGF质粒组PTFE材料上的内皮细胞数和增长倍率都明显高于空质粒组材料(P<0.05),在6, 24, 72和120 h时间点,VEGF质粒组培养液的VEGF蛋白水平和增长倍率也明显高于空质粒组(P<0.01). 结论:证实了PTFE材料携带VEGF基因转染内皮细胞并具有促进其生长的可能性.     

自体骨髓间充质干细胞复合膀胱去细胞基质修复犬膀胱缺损的实验研究

目的 探讨利用自体骨髓间充质干细胞(BMSCs)复合膀胱去细胞基质(BAMG)进行犬膀胱缺损修复重建的可行性。方法 25只杂种犬,5只用于制备BAMG,20只随机分为两组进行膀胱部分切除术。A组用单纯BAMG修复膀胱缺损,B组用复合自体BMSCs的BAMG修复膀胱缺损。分别在术后1、2、4、8、12周取材进行组织学和免疫组织化学检测评价膀胱组织再生情况。结果 所有动物术后均存活。组织学检测结果表明B组术后2周时已有尿路上皮形成,术后8周和12周时在修复区再生出与正常膀胱组织相似的膀胱壁结构。A组术后4周观察到尿路上皮再生,术后12周时仍然缺乏良好的平滑肌组织再生。结论 自体BMSCs复合BAMG比单纯BAMG更能促进膀胱缺损区修复和组织再生。自体BMSCs是组织工程膀胱可供选择的种子细胞来源。     

肱动脉-头臂静脉内瘘——慢性肾功能衰竭成功的次选血管通路

目的　对肱动脉 头臂静脉内瘘及前臂袢型PTFE人造血管的生存率及并发症进行分析比较。方法　对 1997年 10月～ 2 0 0 0年 10月间 34例共 35次肱动脉 头臂静脉内瘘手术及 2 9例共 36次PTFE人造血管术进行回顾性分析。所有患者均为以前桡动脉 头臂静脉内瘘失败或丧失功能者。结果　肱动脉 头臂静脉内瘘及PTFE人造血管 1年、2年、3年的生存率分别为 96 .43%和 5 4.2 3%、96 .43%和 48.2 2 %、48.2 2 %和 39.19%(P <0 .0 1)。PTFE人造血管术后并发症次数及最终血管通路丧失功能的数目均明显多于肱动脉 头臂静脉内瘘术 (P <0 .0 1)。PTFE术后并发症以血栓形成最常见 ,其他并发症包括感染、假性动脉瘤、人造血管外露和血清瘤等 ,均需外科手术干预。结论　上臂肱动脉 头臂静脉使用寿命长 ,并发症少 ,作为桡动脉 头臂静脉内瘘失败后的次选血管通路较PTFE人造血管更为理想     

局部缓释紫杉醇抑制犬人工血管移植术后吻合口内膜增生的初步研究

目的 探索预防周围动脉人工血管旁路移植术（VGB）后吻合口内膜增生（IH）性再狭窄的新方法。方法 取大型杂种犬5只,全麻后行双侧髂股动脉VGB,并结扎、切断人工血管桥之间的自体动脉。同一犬随机选取一侧人工血管（VG）及远近端吻合口喷紫杉醇．生物蛋白胶（FG）混合物,另一侧喷FG作为对照。4周后取出两侧包括远近端吻合口在内的VG,对吻合口进行肉眼、病理组织学及电镜检查,测量内膜厚度及内膜面积,并进行免疫组化检查。结果 5只实验犬两侧VG均通畅,远近端吻合口可见新生内膜。实验侧吻合口新生内膜厚度和内膜面积均显著小于对照侧（P〈0．05）。扫描电镜检查示：实验侧吻合口可见覆盖一层完整的内皮细胞（EC）,EC排列规则整齐,其表面有较少血液成分附着。对照侧EC排列不规则,并见大量血液成分附着。吻合口新生内膜透射电镜检查示：对照侧细胞丰富,细胞类型单一,主要为典型的平滑肌细胞（VSMC）;基质为规则密集排列的胶原纤维。实验侧可见多种类型的细胞;基质中胶原纤维稀疏,并可见大量异物颗粒。结论以FG作为载体,在VG吻合口外局部应用小剂量紫杉醇可以起到抑制吻合口IH的作用。紫杉醇能够从吻合口外渗透至吻合口内,并能在内膜中停留至少4周以上。近期安全有效。     

Human vascular smooth muscle cells and endothelial cells cocultured on polyglycolic acid (70/30) scaffold in tissue engineered vascular graft

Background Current prosthetic, small diameter vascular grafts showing poor long term patency rates have led to the pursuit of other biological materials. Biomaterials that successfully integrate into surrounding tissue should match not only the mechanical properties of tissues, but also topography. Polyglycolic acid (70/30) has been used as synthetic grafts to determine whether human vascular smooth muscle cells and endothelial cells attach, survive and secrete endothelin and 6-keto-prostaglandin F1α (6-keto-PGF1α).Methods Endothelial cells and smooth muscle cells were isolated from adult human great saphenous vein. They were seeded on polyglycolic acid scaffold in vitro separately to grow vascular patch (Groups A and B respectively) and cocultured in vitro to grow into vascular patch (Group C). Smooth muscle cells and endothelial cells were identified by immunohistochemical analysis and growth of cells on polyglycolic acid was investigated using scanning electron microscopy. The levels of endothelin and 6-keto-PGF1α in the culturing solutions were examined by radioimmunology to measure endothelial function. Results Seed smooth muscle cells adhered to polyglycolic acid scaffold and over 28 days grew in the interstices to form a uniform cell distribution throughout the scaffold. Then seed endothelial cells formed a complete endothelial layer on the smooth muscle cells. The levels of endothelin and 6-keto-prostaglandin F1 alpha in the culturing solution were (234±29) pg/ml and (428±98) pg/ml respectively in Group C and (196±30) pg/ml and (346±120) pg/ml in Group B; both significantly higher than in Groups A and D (blank control group, all P&lt;0.05 ). Conclusions Cells could be grown successfully on polyglycolic acid and retain functions of secretion. Our next step is to use human saphenous vein smooth muscle cells and endothelial cells to grow tubular vascular grafts in vitro.     

体外扩增恒河猴自体内皮细胞人工血管移植研究

目的为自体内皮细胞移植的临床应用奠定理论基础。方法将恒河猴表浅静脉内皮细胞在体外培养１３．９±１．４天;扩增培养的自体细胞衬里于用纤维蛋白胶预衬的膨体聚四氟乙烯（ｅＰＴＦＥ）人工血管,继续培养９天。１５只恒河猴随机分为两组。实验组（１０只）用衬有自体静脉内皮细胞的人工血管置换骼总动脉;对照组（５只）用未衬里内皮细胞的人工血管置换。结果细胞数增加１４８±８９倍。所培养的细胞为二倍体细胞,纯度９９％。原代及传代细胞培养上清液中６－酮－前列腺素Ｆ１ａ（６－ｋｅｔｏ－ＰＧＦ１ａ）和ＶｏｎＷｉｌｅｂｒａｎｄ因子（ｖＷＦ）含量差异无显著意义。细胞种植后２小时和９天,人工血管腔面见一层均匀的基质,基质表面有一层连续的内皮细胞单层。内皮细胞紧密排列。呈梭状,形态饱满。实验组除１只于术后１２小时因近端吻合口破裂腹腔内出血死亡,１只人工血管于术后４周阻塞外,其余８只恒河猴人工血管术后４周均保持通畅,内膜下组织厚度为８０±１２μｍ;对照组５条人工血管全部阻塞。结论自体内皮细胞移植可有效地提高人工血管的通畅率。     

CD34+干细胞的分化及其在人工血管内皮化中的应用

目的:探讨外周血、骨髓和脐血中CD34 细胞作为人工血管内皮化的种子细胞来源问题.方法:采集犬外周血和骨髓及人脐血,经免疫磁珠分离出内皮细胞祖细胞,内皮细胞生长因子(VEGF)诱导分化为内皮细胞并扩增,光镜、扫描电镜和免疫细胞化学鉴定;将培养细胞种植于人工血管,扫描电镜观察.结果:经流式细胞仪测定,分离后的细胞中CD34 细胞:外周血(26.30±2.42)% 、骨髓(41.84±3.65)%、脐血(74.62±4.46)%,骨髓和脐血中CD34 细胞数量明显多于外周血,且增殖、分化能力强;CD34 细胞培养2周细胞长满瓶底并达到增殖高峰,细胞呈"铺路石"状排列.Ⅷ因子,CD31免疫细胞化学染色均为阳性,透射电镜细胞胞浆内可见W-P小体;扫描电镜下,内皮细胞平铺于人工血管表面,成单层排列,细胞排列无轴向性.结论:外周血、骨髓和脐血经免疫磁珠分离系统可分离出CD34 细胞,CD34 细胞经VEGF诱导可定向分化为内皮细胞.骨髓和脐血CD34 细胞可作为人工血管内皮化的种子细胞来源.     

冻干辐照脐动脉移植后组织结构的变化

用冻干辐照脐动脉（ＬＩＵＡ）建立狗颈动脉移植模型，观察在体内ＬＩＵＡ组织结构的变化。７条阌以侧颈动脉间置移植ＬＩＵＡ，术后２－１０周取材进行组织学观察。１４条中８条通畅（５７．１％）。术后８周ＬＩＵＡ管腔完全再内皮化，中膜出现纤维母细胞物滋养血管，胶原纤维增生；至术后１０周中膜出现平滑肌细胞。结果提示机体能和ＬＩＵＡ的纤维框架重建正常动脉的组织结构。     

静脉人工血管桥内膜增生的形态学特征及洛伐他汀的干预作用

目的:探讨静脉人工血管桥内膜增生特点及洛伐他汀可能的干预作用。方法:实验犬12只,随机分为实验组和对照组,建立颈静脉人工血管搭桥模型,以洛伐他汀作为实验干预药物,4周后取出人工血管标本,观察人工血管通畅情况,观察吻合口内膜增生形态、厚度以及新生小血管情况等,并比较两组间的差异。结果:术后4周,实验组与对照组均有3条人工血管完全闭塞,全部吻合口均可见明显内膜增生,实验组近端、远端内膜增生厚度低于对照组(P值分别为0.045,0.040),增生内膜表面可见内皮细胞覆盖,增生内膜中可见新生小血管形成,实验组近、远端内膜中新生小血管的数量均多于对照组(P值分别为0.041,0.031)。结论:4周时,吻合口内膜增生明显,同时伴有新生小血管形成,洛伐他汀可以减少内膜增生的厚度,并能够促进新生小血管的形成。     

骨髓间质干细胞和脱细胞基质构建组织工程血管的动物实验

目的以骨髓细胞作为种子细胞,血管脱细胞组织基质作为支架、构建组织工程血管。方法分离小猪骨髓单个核细胞,分别置于内皮细胞培养液和平滑肌细胞培养液中培养,观察分化细胞的抗原标志;多步骤法制备犬血管脱细胞组织基质,将未经分化的骨髓单个核细胞种植于脱细胞基质构建组织工程血管,移植至骨髓供体小猪,代替部分肺动脉血管。结果小猪骨髓细胞经内皮细胞培养液或平滑肌细胞培养液培养后,分别出现成熟内皮细胞或平滑肌细胞的形态学改变,并分别表达内皮细胞或平滑肌细胞的特异性抗原;组织工程血管移植100d后管腔通畅,无血栓、钙化或动脉瘤形成,内皮细胞和平滑肌细胞分化、增殖良好;移植血管的最大负载为2．76N,最大伸长度为20．31mm。结论骨髓单个核细胞复合脱细胞基质可成功构建组织工程血管。     

局部应用肝细胞生长因子对静脉移植物内膜增生的影响

目的:探讨局部应用肝细胞生长因子静脉移植物内膜增生的影响。方法:取犬一侧颈外静脉,对半剪成两段后分别吻合在双侧股动脉上,其中一侧静脉移植物在移植前用肝细胞生长因子处理(实验组),另一侧为单纯的静脉移植(对照组)。术后2周、4周分别切除移植静脉,计算机图像分析系统测量和计算内膜厚度、内膜面积,进行扫描电镜检查,用免疫组织化学方法检测静脉移植物平滑肌细胞增殖细胞核抗(PCNA)的表达。结果:实验组静脉移植物术后2周、4周内膜厚度和内膜截面积明显低于对照组(P<0.01)。实验组静脉移植物术后2周、4周的PCNA指数也明显低于对照组(P<0.01;P<0.05)。扫描电镜检查显示实验组静脉移植物内皮层破坏程度轻于对照组。结论:局部应用肝细胞生长因子能抑制静脉移植物内膜的增生。     

新型小口径生物型人造血管的光镜和扫描电镜观察

目的 观察小口径生物型人造血管移植后6月内不同时期的血管材料腔内面内膜层的组织病理学和扫描电镜变化,探讨研制一种新型小口径生物型人造血管.方法 19只健康杂种犬作为实验对象,手术切除3 cm颈总动脉,以口径4mm的小口径新型人造血管进行桥接.其中19只犬的19侧颈部随机分为1、8、12、24周四组,各含4、4、6、5条动脉.四组的近、远心端均行端-端吻合术.以彩色多普勒B超监测移植血管2周内通畅情况,并于取材前行颈总动脉DSA造影术,观察人造血管不同时间的通畅率.术后1、8、12、24周分期切取标本,标本经处理后分别作光镜和扫描电镜检查.结果 四组19条人造血管除12周组和24周组各1条血管阻塞外,其余血管均通畅.组织病理学及扫描电镜检查显示:植入后1周,血管吻合口处有少量的纤维组织增生混杂有薄层少量红色血栓形成,尚未见内皮细胞生长.移植后8周,宿主组织通过人造血管孔隙长入血管腔内参与移植血管新内膜的形成,新内膜形成较均匀,其中有丰富的胶原纤维组织增生,但仍未见内皮细胞生长.移植后12周,镜下于吻合口处,可见新内膜表面有不连续的内皮细胞生长,且内皮细胞形态成熟,其下的胶原纤维增生更致密;而于人造血管中段,可见不成熟的内皮细胞呈岛状生长,新内膜层结构较吻合口处疏松,其间尚可见炎性细胞生长.移植后24周,通畅的人造血管整段管腔内面均可见内皮细胞生长,吻合口附近新内膜表面可见连续的内皮细胞生长.结论 以新型生物型人造血管修复犬颈总动脉缺损是可行的,新内膜形成早和完整,24周内自然内皮化相对满意,是一种很有发展前途的新型人造血管.     

人脐静脉内皮细胞用于构建组织工程心脏瓣膜的实验研究

目的：以人脐静脉血管内皮细胞(HUVECs)为种子细胞，体外构建组织工程心脏瓣膜(TEHV)，并对其分泌纤溶物质——组织型纤溶酶原激活物(t-PA)及其抑制物(PAI-1)的作用进行观察。方法：获取并扩增HUVECs，种植在猪脱细胞主动脉瓣叶上，体外静态构建TEHV。观察内皮细胞的形态学变化和生长状况。收集瓣膜培养液，采用发色底物法检测瓣膜内皮细胞分泌的t-PA和PAI-1活性。结果：以猪脱细胞主动脉瓣叶作支架，HUVECs做种子细胞，体外成功构建TEHV，种植的HUVECs在瓣膜表面长成一层连续的细胞层，生长状态良好。瓣膜表面的内皮细胞能够分泌纤溶物质t-PA和PAI-1。结论：HUVECs种植在猪脱细胞瓣膜支架上可以构建TEHV，且瓣膜内皮细胞能够分泌t-PA和PAI-1。