转基因内皮细胞在小口径人工血管上种植及表达的体外实验研究
摘要
转基因内皮细胞在小口径人工血管上种植及表达的体外实验研究
小口径人工血管内皮化的研究已有二十余年，但至今仍没有真正意义上的临床应用，主要原因在于种有EC的小口径人工血管植入早期只是部分内皮化，其血管内壁并没形成一层完整的EC层，所以早期易形成血栓而造成血管堵塞。我们认为这种早期血栓形成的原因与EC本身的生物活性，EC的种植方法和植入早期的抗凝治疗有关。本研究总结过去经验，从细胞水平研究EC的种植过程，对适合于种植用的EC进行了较全面系统地研究，首次提出了“加压旋转种植法”，还首次用腺病毒做载体将人pro-UK cDNA转入种植在人工血管上的EC内，并在体外测得其产物的表达，为小口径人工血管能早日进入临床应用提供了理论基础和实验依据。
在本研究的第一部分中，我们主要在细胞水平上从四个方面对适用于种植用的EC的进行了较全面地研究：
1．Collagenase与Trypsin消化EC的比较：取6根脐带，分成2组，每组各3根。Collagenase组用0.1％Collagenase消化EC。Trypsin组用0.25％Trypsin消化EC。结果显示，Collagenase组EC从接种到融合用6.8±0.5天，而Trypsin组EC从接种到融合用11.5±0.8(p＜0.05)。从融合后细胞数上来看，Collagenase组平均为(106.7±16.4)×10~4／瓶，Trypsin组平均为(64.7±26.8)×10~4／瓶(p＜0.05)。光镜下观察用Trypsin消化EC，细胞生长缓慢，不易融
There is still no successful clinical use of small diameter vascular graft so far after the first report of ECs seeded on vascular prostheses in canine by Herring in 1978. The main limitaiton is early thrombosis after implanting caused by endothelialization only in part. These are relating to not only the biological characteristics of ECs, but also the method of ECs seeding and thrombolytic therapy. In this research, we studied the ECs specially for seeding in cellular level, created 'compressed-rotated seeding' method and finally transferred the human pro-UK cDNA into the ECs seeded on small diameter vascular graft throuth adenovirus vector. The gene expression was detected by Western blot in vitro.
Our purpose of the first part of this study was to study what kind of ECs are better for seeding on vascular grafts. First, we digested cells from the intima of human umbilical vein vessel. These cells were confirmed to be endothelial cells by specific factor Ⅷ related antigen stained with immunofluorescence. And then the studies were taken as follows:
1. Comparing collagenase with trypsin: 6 umbilical cords were divided into 2 groups. Collagenase group (3 cords) were digested using 0.1% collagenase. Trypsin group were digested using 0.25% trypsin. The results revealed that the days of collagenase group from seeding to
小口径人工血管内皮化的研究已有二十余年，但至今仍没有真正意义上的临床应用，主要原因在于种有EC的小口径人工血管植入早期只是部分内皮化，其血管内壁并没形成一层完整的EC层，所以早期易形成血栓而造成血管堵塞。我们认为这种早期血栓形成的原因与EC本身的生物活性，EC的种植方法和植入早期的抗凝治疗有关。本研究总结过去经验，从细胞水平研究EC的种植过程，对适合于种植用的EC进行了较全面系统地研究，首次提出了“加压旋转种植法”，还首次用腺病毒做载体将人pro-UK cDNA转入种植在人工血管上的EC内，并在体外测得其产物的表达，为小口径人工血管能早日进入临床应用提供了理论基础和实验依据。
在本研究的第一部分中，我们主要在细胞水平上从四个方面对适用于种植用的EC的进行了较全面地研究：
1．Collagenase与Trypsin消化EC的比较：取6根脐带，分成2组，每组各3根。Collagenase组用0.1％Collagenase消化EC。Trypsin组用0.25％Trypsin消化EC。结果显示，Collagenase组EC从接种到融合用6.8±0.5天，而Trypsin组EC从接种到融合用11.5±0.8(p＜0.05)。从融合后细胞数上来看，Collagenase组平均为(106.7±16.4)×10~4／瓶，Trypsin组平均为(64.7±26.8)×10~4／瓶(p＜0.05)。光镜下观察用Trypsin消化EC，细胞生长缓慢，不易融
There is still no successful clinical use of small diameter vascular graft so far after the first report of ECs seeded on vascular prostheses in canine by Herring in 1978. The main limitaiton is early thrombosis after implanting caused by endothelialization only in part. These are relating to not only the biological characteristics of ECs, but also the method of ECs seeding and thrombolytic therapy. In this research, we studied the ECs specially for seeding in cellular level, created 'compressed-rotated seeding' method and finally transferred the human pro-UK cDNA into the ECs seeded on small diameter vascular graft throuth adenovirus vector. The gene expression was detected by Western blot in vitro.
Our purpose of the first part of this study was to study what kind of ECs are better for seeding on vascular grafts. First, we digested cells from the intima of human umbilical vein vessel. These cells were confirmed to be endothelial cells by specific factor Ⅷ related antigen stained with immunofluorescence. And then the studies were taken as follows:
1. Comparing collagenase with trypsin: 6 umbilical cords were divided into 2 groups. Collagenase group (3 cords) were digested using 0.1% collagenase. Trypsin group were digested using 0.25% trypsin. The results revealed that the days of collagenase group from seeding to
引文
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