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VeröffentlichungsnummerCN100443064 C
PublikationstypErteilung
AnmeldenummerCN 200510032124
Veröffentlichungsdatum17. Dez. 2008
Eingetragen8. Sept. 2005
Prioritätsdatum8. Sept. 2005
Auch veröffentlicht unterCN1762311A
Veröffentlichungsnummer200510032124.9, CN 100443064 C, CN 100443064C, CN 200510032124, CN-C-100443064, CN100443064 C, CN100443064C, CN200510032124, CN200510032124.9
Erfinder吴忠仕, 胡建国
Antragsteller吴忠仕;胡建国
Zitat exportierenBiBTeX, EndNote, RefMan
Externe Links:  SIPO, Espacenet
Preparation process of biological valve-possessed duct for pulmonary artery vessel restoration or reconstruction
CN 100443064 C
Zusammenfassung  übersetzt aus folgender Sprache: Chinesisch
本发明涉及到制备用于心血管系统组织修复或重建的生物带瓣管道的方法,特别是取材来源于异种生物组织的方法。 The present invention relates to a method for preparing biological tissue repair or reconstruction of the cardiovascular system of the valved conduit, especially biological tissue is taken from heterogeneous approach. 本发明提供的用于肺动脉血管修复或重建的生物带瓣管道的制备方法,包括如下步骤:1)获取牛颈静脉,并漂洗,消毒;2)采用染料介导的光氧化反应使牛颈静脉基质交联;3)消毒并保存;其特征在于在第2步之前先用去细胞剂处理第1步获得的牛颈静脉。 Preparation for the repair or reconstruction of the pulmonary artery biological valved conduit of the present invention provides, comprises the following steps: 1) Get the bovine jugular vein, and rinsing, disinfect; 2) the use of light-mediated oxidation dye of bovine jugular vein crosslinked matrix; 3) disinfection and preservation; wherein prior to step 2 cells were first treated with de obtained in Step 1 of bovine jugular vein. 本发明提供的生物带瓣管道的制备方法,能获得具有良好的生物物理性能,抗钙化性能强,免疫原性低,无细胞毒性,有利于宿主细胞生长的生物带瓣管道。 Preparation of biological valved conduit of the present invention provides access to excellent biophysical properties, anti-calcification properties, low immunogenicity, non-cytotoxic, biological valved conduit in favor of the host cells.
Ansprüche(9)  übersetzt aus folgender Sprache: Chinesisch
1、用于肺动脉血管修复或重建的生物带瓣管道的制备方法,包括如下步骤:1)获取牛颈静脉,并漂洗,消毒;2)采用染料介导的光氧化反应使颈静脉基质交联;3)消毒并保存;其特征在于在第2步之前先用去细胞剂处理第1步获得的牛颈静脉;所采用的去细胞剂是采用非离子型的去污剂,还联合采用消化酶或/和核酸酶;消化酶为胰蛋白酶;核酸酶为脱氧核糖核酸酶和核糖核酸酶。 1. A method for the preparation of the pulmonary vascular repair or reconstruction of biological valved conduit, comprising the following steps: 1) Get the bovine jugular vein, and rinsing, disinfect; 2) the use of light-mediated oxidation dye so jugular vein matrix crosslinking ; 3) disinfection and preservation; wherein prior to step 2 cells were first treated with de obtained in Step 1 of bovine jugular vein; the use of acellular agent is non-ionic detergents, also jointly adopt digest enzymes or / and nuclease; digestive enzyme trypsin; nuclease enzymes for DNA and RNA enzymes.
2、 根据权利要求1所述的用于肺动脉血管修复或重建的生物带瓣管道的制备方法,其特征在于所用的去污剂为曲那通ioo。 2. The method according to claim preparation pulmonary vascular repair or reconstruction of biological valved conduit for 1, characterized in that the detergent used for the song that pass ioo.
3 、 根据权利要求2所述的用于肺动脉血管修复或重建的生物带瓣管道的制备方法,其特征在于去污剂的浓度为0. 25—1%;当消化酶为胰蛋白酶时浓度为0. 025〜0. 05%胰蛋白酶;当核酸酶为脱氧核糖核酸酶和核糖核酸酶时,其浓度分别为10—40 unit/ ml及0. l—O. 4mg/ml。 3. Preparation of pulmonary vascular repair or reconstruction of biological valved conduit according to claim 2, wherein the concentration of detergent is 0. 25-1%; when the digestive enzymes trypsin concentration 0. 05% trypsin 025~0; when the nucleic acid enzyme DNase and RNase was, in concentrations of 10-40 unit / ml and 0. l-O 4mg / ml.
4、 根据权利要求3所述的用于肺动脉血管修复或重建的生物带瓣管道的制备方法,其特征在于所用的去污剂处理时间为24--48小时,胰蛋白酶处理时间为30—120分钟,脱氧核糖核酸酶和核糖核酸酶混合后处理时间为24—72小时。 4. The method according to claim preparation pulmonary vascular repair or reconstruction of biological valved conduit for 3, wherein the detergent is used in the processing time 24--48 hours, trypsin processing time 30-120 minutes, and the treatment time DNase RNase was 24-72 hours after mixing.
5、 根据权利要求4所述的用于肺动脉血管修复或重建的生物带瓣管道的制备方法,其特征在于曲那通100浓度为0. 5%,处理时间为48小时, 消化酶为胰蛋白酶,其浓度为0.05%,处理时间为30分钟;以及脱氧核糖核酸酶和核糖核酸酶,其浓度分别为20u/ml及0.2mg/ml,处理时间为24小时。 5. The method according to claim preparation pulmonary vascular repair or reconstruction of biological valved conduit for 4, characterized in that song that pass 100 for the 0.5% concentration, the processing time is 48 hours, digestive enzymes trypsin at a concentration of 0.05%, the processing time was 30 minutes; and DNase and RNase, the concentrations of 20u / ml and 0.2mg / ml, the processing time was 24 hours.
6、 根据权利要求1至5之一所述的用于肺动脉血管修复或重建的生物带瓣管道的制备方法,其特征在于光氧化反应中采用亚甲基蓝,并在pH:7. 6;0sm: 320mosm条件下,150〜500W白炽灯悬空照射20〜48小时。 6. A method for preparing biological valved conduit for pulmonary vascular repair or reconstruction of 1 to 5 of the preceding claims, characterized in that the photo-oxidation reaction using methylene blue, and at pH: 7 6; 0sm:. 320mosm Under the conditions, 150~500W vacant incandescent illumination 20~48 hours.
7、 根据权利要求6所述的用于肺动脉血管修复或重建的生物带瓣管道的制备方法,其特征在于在亚甲基蓝溶液中,pH:7.6;0sm: 320mosm条件下浸泡2—4小时。 7. Preparation of pulmonary vascular repair or reconstruction of biological valved conduit according to claim 6, wherein the methylene blue solution, pH: 7.6; 0sm: Soak 2-4 hours 320mosm conditions.
8、 根据权利要求6所述的用于肺动脉血管修复或重建的生物带瓣管道的制备方法,其特征在于在进行光氧化反应时,生物带瓣管道处于不高于25'C温度条件下。 8. A method for preparing repair or reconstruction of the pulmonary artery biological valved conduit according to claim 6, wherein during photo-oxidation, biological valved conduit at a temperature of not higher than 25'C.
9、 一种用于肺动脉血管修复或重建的生物带瓣管道,其特征在于它是根据权利要求l一8之一的方法制备的。 Biological valved conduit 9, for repair or reconstruction of the pulmonary artery, characterized in that it is one method for preparing a 8 l of claim.
Beschreibung  übersetzt aus folgender Sprache: Chinesisch

用于肺动脉血管修复或重建的生物带瓣管道及制备方法 Biological valved conduit and preparation methods for the repair or reconstruction of the pulmonary artery

技术领域 Technical Field

本发明涉及到制备用于心血管系统组织修复或重建的生物带瓣管道的方法,特别是取材来源于异种生物组织的方法。 The present invention relates to a method for preparing biological tissue repair or reconstruction of the cardiovascular system of the valved conduit, especially biological tissue is taken from heterogeneous approach. 技术背景 BACKGROUND

在右室与肺动脉连接发育不良的复杂先天性心脏病如共同动脉干,肺动脉闭锁,右室双出口,矫正性大动脉转位等的外科治疗需要应用生物带瓣 In the right ventricle and pulmonary artery connection dysplasia complex congenital heart disease, such as common truncus arteriosus, pulmonary atresia, double outlet right ventricle, corrected transposition of great arteries and other applications that require surgical treatment of biological valved

管道。 Pipeline. 没有带瓣管道,这类疾病治疗效果极差。 No valved conduit, poor treatment of such diseases. .上世纪60年代以来,国内外采用同种带瓣主动脉或肺动脉管道,并取得了良好效果。 . On since the 1960s, at home and abroad using the same aorta or pulmonary valved conduit, and achieved good results. 但同种带瓣管道来源困难,尤其适合于婴幼儿手术的更少,并且,存在远期钙化,耐久性不佳等问题。 But valved conduit sources difficult, especially for infants less surgery, and the presence of long-term calcification, poor durability issues. 80年代国外研制出猪肺动脉或主动脉带瓣管道,但临床应用效果欠佳,远期钙化严重,失功衰败,耐久性差。 80 years abroad, developed a porcine pulmonary artery or aortic valved conduit, but poor clinical effect, long-term calcification serious dysfunction decline, poor durability. 上世纪90年始国外开展牛颈静脉带瓣管道研究并应用于临床。 90 years of the last century began to carry out foreign bovine jugular vein conduit research and clinical application. 牛颈静脉带瓣管道具有取材方便, 天然具有的瓣膜类似于人的肺动脉瓣结构,抗反流性能强,血管直径跨度范围大,更适合于临床婴幼儿病例。 Bovine jugular vein conduit having easily obtained, having a valve similar to the natural human pulmonary valve structure, strong anti-flow performance, the vessel diameter large span, more suitable for clinical cases of infants and young children. 目前国际上,唯一牛颈静脉带瓣管道产品为Medtronic公司生产的商品名为Tongtegra,。 At present the international level, the only bovine jugular vein conduit products for Medtronic, the trade name Tongtegra ,. 该产品系采用常规生物组织交联剂戊二醛固定制备而成。 The product is a biological tissue using conventional fixed with glutaraldehyde prepared from. 该产品在欧美等国应用,近期和中期临床效果报道结果不一。 The products in Europe and the United States to use the immediate and mid-term clinical results reported mixed results. 效果不佳的原因主要为组织转化、吻合口狭窄及血栓形成等。 The main reason for ineffective organizational transformation, anastomotic stenosis and thrombosis. 而这些原因主要与制备技术相关。 These primarily related to the preparation of technical reasons. 戊二醛交联处理生物组织存在下列缺陷:l)细胞毒性作用,交联后的组织中长期残留的戊二醛对细胞有毒性作用,宿主细胞不能在组织中生长。 Glutaraldehyde cross-linked biological tissue following defects exist: l) cytotoxicity organization long after crosslinking residual glutaraldehyde cell toxic effects on the host cell can not grow in the organization. 2)醛基的存在等原因导致交联后组织易钙化,钙化是带瓣管道衰败的重要原因之一。 2) the presence of an aldehyde group of other causes tissue after cross-linking is easy to calcification, calcification is a major reason for the decline of the valved conduit. 3)免疫原性消除不完全,是钙化的原因之一,也是导致炎症反应机制之一。 3) does not entirely eliminate the immunogenicity, calcification is one of the reasons, is also one of the inflammatory response mechanisms. 4)残留的细胞及碎屑是组织钙化的主要位点之一,也是免疫原性的重要来源。 4) cells and debris remaining one of the major sites of calcified tissue, but also an important source of immunogenic. 图1是用传统戊二醛方法处理获得的带瓣管道的样品图(A),以及及植入犬体内一年后改变(B);图2戊二醛处理的牛颈静脉瓣膜(A)及植入犬体内一年后改变(B)。 FIG. 1 is a conventional method for processing glutaraldehyde obtained valved conduit sample diagram (A), and implanted in dogs and changed after one year (B); bovine jugular vein valve 2 glutaraldehyde (A) and implanted in dogs after one year change (B).

改进处理制备技术是提高牛颈静脉带瓣管道性能的重要环节。 Improved handling of preparation technology is an important part of bovine jugular vein valved conduit properties. 染料介导的光氧化是异种生物组织交联的一种非掺入性技术。 Light oxidation dye mediated crosslinking is heterogeneous biological tissue incorporation of a non-technical. 是通过光化学反应使得组织胶原交联,无细胞毒性。 By photochemical reaction makes collagen cross-linking, no cytotoxicity. 对于不同的组织,光氧化固定方法在一些细节上有差异,但是大体方法一致,即把要固定的材料浸入溶有染料的溶液中,进行光辐射。 For different organizations, photo-oxidation method fixed differences in some detail, but generally consistent method, namely the material to be fixed is immersed in a solution of the dye solution, the light radiation. 所用的染料有如下几种:亚甲蓝、亚甲绿、孟加拉玫瑰红、核黄素、原黄素、荧光素、五磷酸吡眵醛、伊红等。 The dyes used are summarized as follows: methylene blue, methylene green, rose bengal, riboflavin, proflavine, fluorescein, five phosphate topiramate gum aldehyde, eosin, etc. 其中应用最多的是亚甲蓝,染料的使用浓度为0. 01%〜0.1%。 Among the most widely used is methylene blue, a dye used in a concentration of 0.01% ~0.1%. 使用的溶液可以是低离子强度缓冲液、高离子强度缓冲液、磷酸缓冲盐溶液或组织缓冲液。 The solution may be the use of a low ionic strength buffer, a high ionic strength buffer, phosphate buffered saline or tissue buffer. 溶液的pH值6. 8〜8. 6,渗透压为300mmo1/ kg左右,溶液温度(TC〜25'C,最佳是10 'C左右,可以避免温度过高引起组织蛋白的变性。根据所固定的材料的不同,光辐射的强度和时间不一样, 一般是用1〜2只150〜500W的灯泡, 放置在离样本2〜12cm高度:光照的时间从几分钟到160h,光照的强度和时间有一个综合的指标一流明小时,一般100〜20000流明小时。 PH of the solution 6. 8~8. 6, an osmotic pressure of about 300mmo1 / kg, solution temperature (TC~25'C, most preferably 10 'C or so, to avoid overheating caused by degeneration of tissue proteins. Depending on different fixation materials, intensity and duration of light radiation is not the same, is generally used only 1 to 2 150~500W bulb placed in height from sample 2~12cm: illumination from minutes to 160h, light intensity and Time has a comprehensive index class next hour, general 100~20000 lumen hours.

按现有技术中公开的染料介导的光氧化交联技术没有用来处理牛颈静脉带瓣管道的方法。 According to the prior art disclosed in the dye-mediated photo-oxidation crosslinking technology is not the method used to process bovine jugular vein valved conduit. 本申请人用该技术来处理牛颈静脉带瓣管道,其结果仍然是不理想。 The applicant used the technology to deal with bovine jugular vein conduit, the result is still not ideal.

另一方面,去细胞技术在组织工程学领域已广泛应用。 On the other hand, to the cell technology has been widely applied in the field of tissue engineering. 去细胞后保留的细胞外基质,是一种天然的有利于宿主细胞附着、生长和迁移的支架结构。 After the decellularized extracellular matrix reservations, it is in favor of a natural host cell attachment, growth and migration of the stent structure. 细胞成分的充分去除,降低了免疫原性,也消除了细胞残核这个可能 Full removal of cellular components, reduced immunogenicity, but also eliminates the cell nucleus residues that may

的钙化位点,提高了抗钙化性能。 Calcification sites and improve the anti-calcification properties. 文献报道去细胞的方法有两种:(1)去垢剂脱细胞:去垢剂是一类可溶于水的脂类,它有亲水部分和疏水部分,因此能裂解脂膜,溶解抗原,清除免疫复合物。 The method has been reported to cells in two ways: (1) acellular detergents: detergent is a kind of water-soluble lipids, it has a hydrophilic portion and a hydrophobic portion, thus cracking lipid film was dissolved antigen , clearance of immune complexes. 去垢剂分为:①离子型:带电荷头部(+或-),如十二烷基硫酸钠(SDS)、胆酸钠、脱氧胆酸钠等。 Detergents divided into: ① Ionic: charged head (+ or -), such as sodium dodecyl sulfate (SDS), sodium cholate, sodium deoxycholate. 其缺点是使蛋白质高度变性,并使蛋白质以单体形式分离。 The disadvantage is highly protein denaturation and protein separation in monomeric form. ②非离子型:带非极性头部,如Triton X-100 、 Triton-X114 、辛葡糖苷、Tween20等。 ② nonionic: with nonpolar head, such as Triton X-100, Triton-X114, octyl glucoside, Tween20 like. 其对蛋白质和蛋白质间的相互作用干扰较弱,对蛋白质变性的作用亦较弱。 It interfere with protein and protein interaction between the weak role of protein denaturation was also weak. ③两性型:带有+和-电荷头部,如CHAPS、 Zwitterget等。 ③ Gender type: with + and - charges the head, such as CHAPS, Zwitterget like. (2)消化酶脱细胞:采用胰蛋白酶、脱氧核糖核酸酶和核糖核酸酶作为脱细胞剂。 (2) Acellular digestive enzymes: trypsin, DNase and RNase as acellular agent. 这两种脱细胞液,根据不同组织和临床应用情况可自由选择。 Both acellular solution, depending on the organizational and clinical application of the freedom to choose. 一般采用联合使用方法。 Commonly used method for combination use. 发明内容 DISCLOSURE

本发明为提供用于肺动脉血管修复或重建的生物带瓣管道,采用去细胞技术与染料介导的光氧化交联技术处理牛颈静脉带瓣管道。 Biological valved conduit of the present invention to provide for the repair or reconstruction of the pulmonary artery, using dye to the cell technology and light-mediated oxidative cross-linking technology bovine jugular vein conduit.

本发明提供的用于肺动脉血管修复或重建的生物带瓣管道的制备方法, 包括如下步骤:1)获取牛颈静脉,并漂洗,消毒;2)采用染料介导的光氧化反应使牛颈静脉基质交联;3)消毒并保存;其特征在于在第2步之前先用去细胞剂处理第1步获得的牛颈静脉。 Preparation for the repair or reconstruction of the pulmonary artery biological valved conduit of the present invention provides, comprises the following steps: 1) Get the bovine jugular vein, and rinsing, disinfect; 2) the use of light-mediated oxidation dye of bovine jugular vein crosslinked matrix; 3) disinfection and preservation; wherein prior to step 2 cells were first treated with de obtained in Step 1 of bovine jugular vein.

本发明的关键在于选择合适的去细胞方法并且釆用非戊二醛的蛋白质交联方法。 The key to the present invention is to select the appropriate method to cells and preclude non-glutaraldehyde cross-linked protein method.

釆用去细胞技术与染料介导的光氧化交联技术联合处理牛颈静脉带瓣管道,可以利用两者的优点,使制备的带瓣管道达到很好的性能。 Bian spent cell technology and dye-mediated photo-oxidation crosslinking techniques combined treatment of bovine jugular vein conduit, you can use the advantages of both, so valved conduit prepared to achieve good performance. 作为去细胞技术,它的作用是将牛颈静脉组织细胞中的细胞成分如细胞核等充分去除,从而达到消除钙化位点,降低免疫原性的目的。 As to cell technology, its role is to sufficiently remove bovine jugular vein tissue cells such as the nucleus and other cellular components, so as to eliminate calcification sites, reduce immunogenicity purposes. 而染料介导的光氧化技术的应用是使组织中的蛋白质充分交联,提供一个稳定的骨架结构。 The application of the dye mediated photo-oxidation technology is to make the organization fully cross-linked proteins, providing a stable framework structure. 牛颈静脉采自本地黄牛及青海牦牛,体重为100〜600公斤,健康,检疫合格。 Bovine jugular vein were collected from local cattle and Yak, 100~600 kg body weight, health, quarantine. 牛颈静脉通常拥有多组瓣膜,每组瓣膜通常为三叶或两叶,少部分为四叶。 Bovine jugular vein usually have multiple sets of valves, each valve is generally clover leaf or two, a small four leaves. 瓣叶发育程度不等,约有20-30%的牛颈静脉具有发育及抗反流性能良好的瓣膜。 Leaf lobes varying degrees, about 20-30% of bovine jugular vein with good development and performance of the anti-reflux valve. 瓣叶菲薄,类似于人肺动脉。 Leaflets thin, similar to human pulmonary artery. 瓣窦处血管壁薄,修剪时易损伤。 Valve sinus blood vessel wall is thin, easy to damage when pruning. 同一牛颈静脉瓣膜组间距离统计结果为6.4士2.6cm。 Among the same group of bovine jugular vein valve 6.4 from the statistical results disabilities 2.6cm. 牛颈静脉通常有多支小血管分支开口,制备时需结扎。 Bovine jugular vein is usually more support small blood vessels branch opening, the preparation required ligation. 牛颈静脉外膜纤维结缔组织丰富,与中膜分界不清。 Bovine jugular vein adventitia rich fibrous connective tissue, and in the film boundaries unclear. 不同牛系之间颈静脉的瓣膜发育无明显区别。 Among different bovine jugular vein valve system development no significant difference. 血管直径为8〜22mm,在无菌条件下去除筋膜组织,检测瓣膜发育状态及抗反流性能。 Vessel diameter of 8~22mm, fascia tissue removed under sterile conditions, the detection valve development status and anti-reflux properties. 瓣膜发育及抗反流性能良好的血管浸入O. 1%新洁尔灭30min, PBS 漂洗。 Good development and anti-reflux valve performance vessels immersed O. 1% benzalkonium bromide 30min, PBS rinse. 根据现有技术,用于去细胞的去污剂有多种,包括离子型、非离子型以及两性型。 According to prior art, a detergent used to a variety of cells, including ionic, nonionic and amphoteric. 本发明选用非离子型去污剂,例如曲那通IOO。 In the invention, a non-ionic detergent, such as song that pass IOO. 本发明在去细胞的步骤中,采用多步骤的手段。 The present invention in steps to the cells, the use of means of multi-step. 除采用非离子型去污剂外,还联合采用消化酶或/和核酸酶,所用的消化酶为胰蛋白酶,核酸酶为脱氧核糖核酸酶和核糖核酸酶。 In addition to the use of non-ionic detergents, but also combined the use of digestive enzymes or / and a nuclease, an enzyme is used trypsin digestion, nuclease is RNase and DNase. 因此,本发明进一步提供去细胞处理的方法是联合采用非离子型的去污剂和消化酶或/和核酸酶处理的方法;去污剂的浓度为0.25—1%,消化酶为胰蛋白酶;核酸酶为脱氧核糖核酸酶和核糖核酸酶,当消化酶为胰蛋白酶时浓度为0.025〜0.05%胰蛋白酶;当消化酶为脱氧核糖核酸酶和核糖核酸酶时,其浓度分别为10—40 unit/ ml及0.1~~0.4mg/ml。 Accordingly, the present invention further provides a method to cell treatment using a combination of non-ionic detergent and digestive enzymes and / or nuclease treatment methods; detergent concentration of 0.25-1%, digestive enzyme trypsin; nuclease DNase and RNase is when digestive enzymes trypsin concentration 0.025~0.05% trypsin; when digestive enzymes are DNase and RNase, its concentrations were 10-40 unit / ml and 0.1 ~~ 0.4mg / ml. 按现有技术中公开的方法,在用胰蛋白酶去细胞时,溶液中还有一定浓度的EDTA。 By the method disclosed in the prior art, in the cells with trypsin to the solution there is a certain concentration of EDTA. 上述各种去细胞剂的处理时间也是一个很重要的参数。 The processing time of the various acellular agent is also a very important parameter. 本发明中优选去污剂处理时间为24—48小时,胰蛋白酶处理时间为30-120分钟,脱氧核糖核酸酶和核糖核酸酶混合后处理时间为24—72小时。 In the present invention, detergent treatment time is preferably 24-48 hours, trypsin treatment time of 30 to 120 minutes, DNase and RNase treatment after mixing time was 24-72 hours. 最优选曲那通100浓度为0. 5%,处理时间为48小时,消化酶为胰蛋白酶,其浓度为0. 025~0.125%,处理时间为30—60分钟;以及脱氧核糖核酸酶和核糖核酸酶,其浓度分别为20 unit/ ml及0. 2mg/ml,处理时间为24小时。 Optimal selections 100 that pass 0.5% concentration, the processing time was 48 hours, digestive enzymes trypsin at a concentration of 0.025 ~ 0.125%, the processing time is 30-60 minutes; and deoxyribonuclease and ribose nuclease, in concentrations of 20 unit / ml and 0. 2mg / ml, the processing time was 24 hours. 对于光氧化反应,本发明中具体选用了亚甲基蓝,并在pH:7.6;0sm: 320mosm条件下,150〜500W白炽灯悬空照射20〜48 h。 For the photo-oxidation, the present invention specifically chose methylene blue, and pH: 7.6; 0sm: under 320mosm conditions, 150~500W vacant incandescent illumination 20~48 h. 优选在亚甲基蓝溶液中,PBS(pH:7.6;0sm: 320mosm)条件下浸泡2—4 小时。 Preferably methylene blue solution, PBS (pH: 7.6; 0sm: 320mosm) 2-4 hours soaking conditions. 在进行光氧化反应时,生物带瓣管道处于不高于25'C温度条件下。 During photo-oxidation, biological valved conduit at a temperature of not higher than 25'C. 例如可以用水浴的方法控制被处理的生物带瓣管道的温度不高于25'C。 For example, a method of controlling the temperature of the water bath biological valved conduit being processed is not higher than 25'C. 本发明提供的用于肺动脉血管修复或重建的生物带瓣管道的制备方法,能获得具有良好的生物物理性能,抗钙化性能强,免疫原性低,无细胞毒性,有利于宿主细胞生长的生物带瓣管道。 The present invention provides a method for preparing biological for repair or reconstruction of the pulmonary artery valved conduit can be obtained with good biophysical properties, anti-calcification properties, low immunogenicity, non-cytotoxic, is conducive to the growth of the host cell organism valved conduit. 附图说明图1是戊二醛处理的牛颈静脉带瓣管道(A)及植入犬体内一年后改变(B);图2是戊二醛处理的牛颈静脉瓣膜(A)及植入犬体内一年后改变(B);其中箭头指示瓣膜结构;图3用本发明的方法处理后,牛颈静脉血管(A)及瓣膜(B)组织形态变化(HE染色X20);图4用本发明的方法处理后,牛颈静脉带瓣管道(A)及植入犬体内一年后改变(B),其中箭头指示瓣膜位置;图5用本发明的方法处理后,牛颈静脉瓣膜(A)及植入犬体内一年后改变(B),其中箭头指示瓣膜位置;图6用本发明的方法处理后,牛颈静脉植入犬体内一年后血管组织内皮样细胞再生图(HE染色(X 100 )箭头示内皮样细胞);图7不同技术处理制备的牛颈静脉组织蛋白提取试验结果,其中F:新鲜组织,P:光氧化处理组织,GA:戊二醛处理组织,DE:单纯去细胞组织,DP:去细胞结合光氧化处理组织,DGA:去细胞结合戊二醛处理组织,M: marker。 Brief Description of the bovine jugular vein conduit 1 is glutaraldehyde (A) and implanted in dogs after one year change (B); bovine jugular vein valve (A) in FIG. 2 is a glutaraldehyde-treated and planting A year later changed into dogs (B); wherein the arrows indicate the valve structure; Figure 3 after the processing method of the present invention, bovine jugular vein (A) and the valve (B) Histopathological changes (HE staining X20); Figure 4 After the processing method of the present invention, bovine jugular vein conduit (A) and implanted in dogs after one year change (B), wherein arrows indicate valve position; Figure 5 processing method of the present invention, bovine jugular vein valve (A) and implanted in dogs after one year change (B), in which the arrows indicate valve position; Figure 6 processing method of the present invention, bovine jugular vein dogs one year after implantation of vascular endothelial-like cells regenerate tissue images ( HE staining (X 100) arrows endothelial-like cells); 7 bovine jugular vein tissue protein extract prepared different technical processing test results, where F: fresh tissue, P: photo-oxidation process organization, GA: glutaraldehyde tissue, DE: simple to cells and tissues, DP: cell binding to photo-oxidation process organization, DGA: binding to cells treated with glutaraldehyde tissue, M: marker. 12, 24, 36及48为光氧化处理时间;图8利用Western blotting技术检测不同方法处理制备的BJVC的免疫原性结果,其中F:新鲜组,GA:戊二醛组,P:光氧化组,DP:去细胞结合光氧化组;图9人脐静脉内皮细胞在去细胞结合光氧化技术制备的牛颈静脉血管片上种植后电镜结果(SEM一x1200.箭头示人脐静脉内皮细胞)。 12, 24, 36 and 48 for the photo-oxidation processing time; 8 use Western blotting technique to detect different approach to the preparation of BJVC immunogenicity results, where F: Fresh Group, GA: glutaraldehyde group, P: photo-oxidation group , DP: to cell binding photooxidation group; 9 human umbilical vein endothelial cells to cell binding electron microscope after planting the bovine jugular vein sheet photo-oxidation technology to prepare (SEM a x1200 arrows in human umbilical vein endothelial cells). 具体实施例实施例11)自屠宰场获取牛颈静脉,牛种属可为本地黄牛,体重为100〜600公斤, 健康,检疫合格。 Specific Examples Example 11) from slaughterhouse Get bovine jugular vein, bovine species for local cattle, 100~600 kg body weight, health, quarantine. 血管直径为8〜22rnm,在无菌条件下去除筋膜组织,检测瓣膜发育状态及抗反流性能。 Vessel diameter of 8~22rnm, fascia tissue removed under sterile conditions, the detection valve development status and anti-reflux properties. 瓣膜发育及抗反流性能良好的血管浸入0.1%新洁尔灭30min, PBS漂洗。 Good development and anti-reflux valve performance dipped 0.1% benzalkonium bromide vascular 30min, PBS rinse. 2) 去细胞处理:按照表1随机分十组。 2) to cell treatment: according to Table 1 were randomly divided into ten groups. 采用不同浓度、不同作用时间、不同去细胞剂按下列方法组合处理血管组织。 With different concentrations, different time, different cell agents to deal with the following combination of methods vascular tissue. 观察不同脱细胞方法处理后的牛颈静脉血管片的大体形态、脱细胞的完整程度、胶原弹力纤维保存的完整性、热皱縮温度的变化和最大抗张强度下降的程度。 Gross morphology was observed after different treatment methods acellular bovine jugular vein sheet, the degree of completeness of acellular collagen and elastic fibers to save the integrity of the heat shrinkage temperature changes and maximum tensile strength decreased. ①采用去垢剂曲那通(Triton) X-100的PBS液,浓度小于1%, 合适浓度为0. 25〜0. 596, 37° C下处理24〜48小时,PBS漂洗;② 0.025〜0.059G胰蛋白酶/0.0296EDTA的PBS在37° C下处理30〜 120min,PBS漂洗,30〜60 min更为合适;③加入20u/ml DNase-1及0. 2mg/ml RNase-A的PBS在37° C下处理24—48小时,PBS漂洗, 置于PBS中。 ① song that through the use of detergents (Triton) PBS was X-100, the concentration of less than 1%, a suitable concentration for dealing 24~48 hours 0. 25~0 596, 37 ° C lower, PBS rinse;. ② 0.025~ 0.059G /0.0296EDTA trypsin in PBS at 37 ° C for processing 30~ 120min, PBS rinse, 30~60 min more appropriate; ③ join 20u / ml DNase-1 and 0. 2mg / ml RNase-A of PBS in Processing at 37 ° C for 24-48 hours, PBS rinsed and placed in PBS. 3) 管道置于高渗PBS液(pH:7.6 ;600〜680 mosm)中浸泡2—4h ,再置于含0.01〜0.1%亚甲基蓝PBS (pH:7.6;Osm: 320mosm)中平衡2〜4h 后,150〜500W白炽灯悬空照射20〜48 h。 3) pipe placed hypertonic PBS solution (pH: 7.6; 600~680 mosm) soaked 2-4h, then placed in containing 0.01~0.1% methylene blue PBS (pH: 7.6; Osm: 320mosm) after the balance 2~4h , 150~500W vacant incandescent illumination 20~48 h. 反应体系置于0〜25'C恒温水浴内,照射过程中连续搅拌,吹入空气,并控制pH值。 0~25'C placed in the reaction system constant temperature water bath, continuous stirring during irradiation, air is blown, and control the pH. 处理完后充分漂洗脱色。 After sufficient rinsing bleaching process. 4) 置于复合碘剂中浸泡3天灭菌处理或0.1%新洁尔灭中30〜60min,再置于50〜60%酒精中保存。 4) in complex iodine soak 3 days, or 0.1% benzalkonium bromide sterilized in 30~60min, then placed in 50 ~ 60% alcohol for storage. 复合碘剂配方及处理方法参照1995年美国专利(专利号为5437287)进行。 Iodine complex formulations and treatment methods reference 1995 US patent (No. 5,437,287) is. 第4步处理完毕后,组织釆用涤纶无菌袋包装,保存液为DMEM液,1096DMSO及l(^FBS。组织逐步降温至-80 ° C,在冷冻状态下,采用y射线消毒(剂量为25〜40kGy),消毒后,组织低温或常温保存。表l,<table>table see original document page 11</column></row> <table>结果:所有脱细胞处理后的各组牛颈静脉血管片中除了m组的瓣膜结构有破坏外,其他组的血管壁和瓣膜结构完好,和新鲜组相似;各组的热铍縮温度和新鲜对照组比较无显著性差异。过高浓度的胰蛋白酶(0.125%) 或过长的胰蛋白酶消化时间(24h)可破坏瓣膜结构和牛颈静脉血管中胶原弹力纤维,使得最大抗张强度比新鲜对照组下降20%以上;单纯的曲那通乂-100(0.25%)加低浓度的胰蛋白酶(0.025%)脱细胞处理的牛颈静脉血管片残留部分细胞结构;温和的多步骤去垢剂-酶消化法(0.25%或0.5。/。曲那通X-100, 0.025%或0.05%胰蛋白酶和0.02%EDTA, 20u/mlDNase-I和0.2mg/mlRNase-A)脱细胞处理后的带瓣牛颈静脉血管片比较理想:血管壁和瓣膜大体形态和处理前相似,光镜和电镜观察显示细胞成分去除完全, 胶原弹力纤维保存完整见图3。结论:多步骤去垢剂-酶消化法(曲那通X-100 ,胰蛋白酶和核酸酶) 脱细胞处理后的带瓣牛颈静脉脱细胞基质是比较理想的组织工程血管支架。实施例2去细胞光氧化处理的牛颈静脉血管抗张强度研究。方法:采集本地牛颈静脉20根,每根血管分成五段,随机分成新鲜组、 去细胞组、光氧化组、去细胞光氧化组、GA组。每组n-20。戊二醛组采用0.625%戊二醛固定6小时,0.3%戊二醛保存;光氧化组采用0.1%亚甲基蓝作为介导剂,光照时间为48小时,光照强度为4000流明小时,介导剂温度为12-18'C,复合碘剂消毒,60%酒精保存;去细胞结合光氧化组采用0.5。/。曲那通X-100处理48小时,0.05%胰蛋白酶和0.02%EDTA, 30min 及20u/ mlDNase-I和0. 2mg/ml RNase-A , 24h。各步间PBS液漂洗多次(即表1中VII组方案)。采用与光氧化组相同技术进一步染料介导光氧化处理,复合碘剂消毒,60%酒精保存。新鲜组血管采用60%酒精保存。自所有实验用牛颈静脉距离瓣窦lcm以上处横行裁剪长4cm,宽0.5cm的血管条。 Step 4 after treatment is completed, the organization preclude the use of polyester aseptic bags, storage solution is DMEM liquid, 1096DMSO and l (^ FBS. Tissue gradually cooling down to -80 ° C, in a frozen state, the use of y radiation sterilization (dose of 25~40kGy), after disinfection, cold or room temperature preservation organization table l, <table> table see original document page 11 </ column> </ row> <table> Result: Each group of bovine jugular vein after all acellular vascular structure of the film in addition to valvular m group destructive, the other group of the vessel wall and the valve structure intact, and similar to the fresh group; no significant difference between the groups of beryllium heat shrink temperature and high concentration of fresh pancreatic control group. Protease (0.125%) or too long trypsin digestion time (24h) can damage the valve structure and bovine jugular vein collagen and elastic fibers, so that the maximum tensile strength than fresh fall more than 20% in the control group; a simple song that Tong Yi in - 100 (0.25%) plus low concentration of trypsin (0.025%) of acellular bovine jugular vein residual sheet portion cellular structure; multi-step mild detergent - enzymatic digestion (0.25% or 0.5 / song that. through the X-100, 0.025% or 0.05% trypsin and 0.02% EDTA, 20u / mlDNase-I and 0.2mg / mlRNase-A) acellular treated valved bovine jugular vein sheet ideal: the vessel wall and the valve substantially former shape and handling similar to light and electron microscopy showed that the cell component completely removed, collagen and elastic fibers intact Figure 3. Conclusions: Multiple-step detergent - enzymatic digestion (song that through X-100, trypsin and nuclease ) acellular treated valved bovine jugular vein acellular matrix is ideal for tissue engineering vascular stent tensile strength of Bovine jugular vein 2 to cell light anti-oxidation treatment method embodiment: the acquisition of local bovine jugular vein 20 , each vessel is divided into five sections, were randomly divided into the fresh group, decellularized group, group photo-oxidation, photo-oxidation to the cell group, GA groups. Each group n-20. glutaraldehyde group was 0.625% glutaraldehyde six hours, 0.3% glutaraldehyde preservation; photooxidation group with 0.1% methylene blue as a mediating agent, illumination time of 48 hours, the light intensity of 4,000 lumens hour, mediated coolant temperature is 12-18'C, compound iodine disinfection, 60% Alcohol saved; to cell binding photooxidation group with 0.5 / song that through X-100 for 48 hours and 0.05% trypsin and 0.02% EDTA, 30min and 20u / mlDNase-I and 0. 2mg / ml RNase-A,.. 24h. between each step of rinsing several times with PBS (ie, group program VII Table 1). using the same set of photo-oxidation technology to further dye-mediated photo-oxidation process, complex iodine disinfection, save 60% alcohol. fresh group of vessels with 60 % alcohol save since all experiments using bovine jugular vein valve sinus lcm distance above the transverse cut at length 4cm, 0.5cm wide strip of blood vessels. 采用STRON生物拉力仪测定牛颈静脉血管条的生物力学性能。 Rally measured using STRON mechanical properties of biological organisms bovine jugular vein strip. 结果:各组的最大应力如下表2。 RESULTS: The maximum stress shown in Table 2. 去细胞光氧化处理组的最大应力显著强于新鲜组。 The maximum stress to cell light oxidation was significantly stronger than the fresh group. 去细胞处理对应力无明显影响。 To stress it had no effect on cell treatment. 但去细胞光氧化处理的血管应力显著低于戊二醛交联组。 But go light oxidation treatment of vascular cell stress was significantly lower than glutaraldehyde cross-linked groups. 表2,不同技术处理的牛颈静脉血管片的最大应力比较 <table>table see original document page 13</column></row> <table> Table 2, the maximum stress different technical processing of bovine jugular vein sheet of relatively <table> table see original document page 13 </ column> </ row> <table>

结论:去细胞结合光氧化处理制备牛颈静脉提高了组织的抗张强度,但不及戊二醛处理的材料的抗张强度大。 Conclusion: Combining the preparation of acellular bovine jugular vein light oxidation increases the tensile strength of the organization, but the material is not as glutaraldehyde tensile strength. 实施例3不同技术处理制备的牛颈静脉组织蛋白提取试验方法:采集本地牛颈静脉,采用戊二醛交联、光氧化交联及去细胞光氧化交联制备牛颈静脉(方法同实施例2)。 Bovine jugular vein tissue protein extraction technology Standard Test Method for 3 different examples of the preparation process: collecting local bovine jugular vein, using glutaraldehyde cross-linked, photo-oxidation crosslinking and acellular light oxidative crosslinking preparation of bovine jugular vein (the same way as Example 2). 不同技术制备的牛颈静脉及新鲜的牛颈静脉和去细胞处理的牛颈静脉捣碎,加入组织提取液,取20ixl提取液作聚丙烯酰胺凝胶电泳蛋白分析。 Bovine jugular bovine jugular vein and various technical preparation of fresh bovine jugular vein and to cells treated with mashed, adding tissue extracts, take 20ixl extract as polyacrylamide gel electrophoresis protein analysis. 结果:见图7,与新鲜管道相比,戊二醛处理组和光氧化反应组处理的管道组织蛋白提取量都明显减少,这说明光氧化反应与戊二醛一样对牛颈静脉起到固定作用,组织稳定性显著增加,且随着光氧化反应作用时间的增加,组织蛋白提取量进行性减少。 Results: Figure 7, compared with fresh piping, piping tissue protein extraction amount glutaraldehyde-treated group and the photo-oxidation treatment group were significantly reduced, indicating that photo-oxidation as glutaraldehyde fixed bovine jugular vein role play , organizational stability increased significantly, and with the increase of light oxidation treatment time, amount of tissue protein extraction decreases. 去细胞处理对组织稳定性无明显影响。 Go cell treatment had no significant effect on the stability of the organization. 实施例4不同交联技术制备的牛颈静脉免疫原性比较研究方法:采集本地牛颈静脉,采用戊二醛交联、光氧化交联及去细胞光氧化交联制备牛颈静脉(具体方案同实施例2)。 A Comparative Study of the immunogenicity of bovine jugular vein Preparation Method 4 different crosslinking Example: the collection of local bovine jugular vein, using glutaraldehyde cross-linked, photo-oxidation crosslinking and acellular light oxidative crosslinking preparation of bovine jugular vein (specific programs in Example 2). 不同技术制备的牛颈静脉及新鲜的牛颈静脉分别匀浆提取抗原,再与完全弗氏佐剂混合制成复合乳剂抗原后免疫新西兰大白兔,双向免疫扩散法测定兔血清抗体滴度。 Bovine jugular vein preparation of different technologies and fresh bovine jugular vein tissue extract antigens were measured immune rabbit serum antibody titers rabbits, double immunodiffusion after another with complete Freund's adjuvant emulsion blend complex antigen. 取新鲜牛颈静脉免疫抗血清,采用免疫印迹法(Western blotting)体外检测交联后的牛颈静脉带瓣管道免疫原性。 Fresh bovine jugular vein immune antisera, using immunoblotting (Western blotting) bovine jugular vein conduit immunogenicity after crosslinking in vitro assay. Western blotting方法:(l)SDS-PAGE: 取浓縮胶浓度为4.0%,分离胶浓度为10%的垂直平板电泳,每板依次加入新鲜牛颈静脉、PC、 PH 、 GA交联处理的牛颈静脉织抗原。 Western blotting method: (l) SDS-PAGE: harvested, concentrated gel concentration was 4.0% separating gel concentration of 10% of the vertical slab electrophoresis, are then added to each plate of fresh bovine jugular vein, cattle PC, PH, GA crosslinking treatment jugular vein tissue antigens. 组织抗原在加样前均与样品缓冲液以i : i对比稀释并加热处理(100'C,4min)。 Before loading tissue antigens were associated with sample buffer with i: i compare dilution and heat treatment (100'C, 4min). 电泳结束后,凝胶用考马斯亮蓝染色或进行电转膜。 After electrophoresis, the gel was stained with Coomassie blue staining or electrical transfer film. (2)电转膜:将蛋白条带从凝胶转移至硝酸纤维素膜(NC膜),转移电压为恒压45 V,转移时间为3 h ,电转移后的凝胶用考马斯亮蓝染色以观察蛋白转移效率。 (2) electricity transfer film: The protein bands were transferred from the gel to a nitrocellulose membrane (NC film), the transfer voltage of the constant voltage 45 V, the transfer time of 3 h, gel electrophoresis, transferred after staining with Coomassie Brilliant Blue protein transfer efficiency was observed. (3)免疫印迹反应:用含1%BSA的Tween 80/Tris盐缓冲液(Tween 80,TBS)封闭NC膜,然后将NC膜先后与1 : 40血清及1 : 12000AP羊抗兔IgG反应,最后以BCIP/NBT作为底物进行显色反应,条带出现而背景较淡时以蒸馏水冲洗终止反应。 (3) immunoblot reaction: containing 1% BSA in Tween 80 / Tris-buffered saline (Tween 80, TBS) closed NC film, then the film has with NC 1: Reaction 12000AP goat anti-rabbit IgG,: 40 serum and 1 Finally, BCIP / NBT as substrate color reaction, while the background bands appear lighter distilled water to stop the reaction. 结果:抗体稀释至1:1000后,新鲜牛颈静脉组织抗血清仍有明显条带显示,说明抗体的效价至少可达1:1000 ,完全能满足实验的需要。 Results: The antibodies were diluted to 1: 1000, and fresh bovine jugular vein tissue antiserum strip show there are still significant, indicating that the antibody titer of up to at least 1: 1000, can meet the needs of the experiment. 新鲜牛颈静脉组织有特异性重复出现的条带,至少包括6个不同分子量的抗原,说明新鲜牛颈静脉组织有抗原性且抗原分布有特异性,经光氧化及去细胞结合光氧化处理的牛颈静脉基本未出现明显条带,5组经GA交联处理的牛颈静脉有1组出现条带,但无特异性重复出现的条带,说明光氧化处理的牛颈静脉组织抗原性显著降低,经去细胞结合光氧化处理的牛颈静脉的抗原性更低(见图8)。 Fresh bovine jugular vein organizations with recurring item-specific, include at least six different molecular weight antigens, indicating that fresh bovine jugular vein tissue antigenicity and antigen-specific distribution by light oxidation and to photo-oxidation process of cell binding Basic obviously bovine jugular vein strip does not appear, there is a group of five groups banding by GA crosslinked bovine jugular vein, but no specific recurring bands, indicating that bovine jugular vein tissue antigens of the photo-oxidation process significantly reduced antigenic cells by binding to light oxidation bovine jugular vein of less (see Figure 8). 实施例5去细胞光氧化处理的牛颈静脉血管抗钙化性能研究方法:采集本地牛颈静脉,采用戊二醛交联、光氧化交联及去细胞光氧化交联处理牛颈静脉(具体方案同实施例2)。 Anti-calcification properties of Example 5 to cell light oxidation bovine jugular vein implementation: collecting local bovine jugular vein, using glutaraldehyde cross-linked, photo-oxidation and crosslinking to cell photo-oxidation crosslinking treatment of bovine jugular vein (specific programs in Example 2). 裁剪成lxl.5cm2带瓣叶的试片。 Lxl.5cm2 cut into test pieces with leaflets. 选择离乳的雄性SD大白鼠(由中南大学湘雅医学院实验动物中心提供,均符合国家实验动物规定),皮下包埋,喂养60天,获取标本,原子吸收分光光度仪测定组织钙含量。 Choose from the milk of male SD rats (provided by the Central South University Xiangya School of Medicine Laboratory Animal Center, are in line with the provisions of the national animal), subcutaneous embedding, feeding 60 days, obtain specimens, atomic absorption spectrophotometer tissue calcium content. 结果:管壁及瓣膜钙含量结果分别为253土37.6, 170.6 ±29. 5, 121.7 ±21.8Mg/mg; 25. 6 ±3. 8, 21. 7 ±2. 9, 12. 8 ± 2. 6 mg/mg。 Results: The wall and valvular calcium content results were 253 soil 37.6, 170.6 ± 29 5, 121.7 ± 21.8Mg / mg; 25. 6 ± 3 8, 21. 7 ± 2 9, 12. 8 ± 2.... 6 mg / mg. 组间比较三组之间均有显著性差异。 Between groups were statistically significant differences between the three groups. 结论:去细胞结合染料介导光氧化处理制备的牛颈静脉显著提高了抗钙化性能。 Conclusion: go light-mediated cell binding dye oxidation bovine jugular vein preparation significantly improves the performance of the anti-calcification. 实施例6去细胞结合光氧化处理的牛颈静脉管道重建犬肺动脉与右室连接方法:采集牛颈静脉带瓣管道,去细胞处理结合光氧化技术制备。 6 cell binding to light oxidation bovine jugular vein conduit pulmonary artery reconstruction embodiment of right ventricular connection methods: Acquisition of bovine jugular vein conduit, prepared photo-oxidation process to cell binding. 应用带瓣管道作本地家犬右室流出道重建,结扎自身肺动脉,重建前后直接测定肺动脉压、右室压及体循环压,术后七天内超声检测,术后一年超声及心导管检测血流动力学。 Applications valved conduit for local dog right ventricular outflow tract reconstruction, ligation itself pulmonary artery, pulmonary artery pressure was measured before and after reconstruction directly, right ventricular pressure and circulation pressure, within seven days after the ultrasound, ultrasound and cardiac catheterization year after detection of blood flow kinetics. 结果:六只犬重建后均长期存活。 Results: After six dogs were long-term survivors rebuild. 重建右室流出道后,管道内瓣膜远端血压为22/14〜37/10 mm Hg。 Reconstruction of the right ventricular outflow tract, the distal end of the pipe valve blood pressure 22 / 14~37 / 10 mm Hg. 与重建前肺动脉压比较(包括收縮压、 舒张压及平均压)差异无显著性(PX).05),但管道内舒张压显著高于右室舒张压(pO.Ol)。 Comparison with the reconstruction of the anterior pulmonary artery pressure (including systolic, diastolic and mean pressure) difference was not statistically significant (PX) .05), but the pipeline right ventricular diastolic pressure was significantly higher than diastolic blood pressure (pO.Ol). 重建后右室血压为26/5〜41/0 mm Hg,与重建前比较, 右室舒张压无明显变化(p>0.05)。 After the reconstruction of the right ventricular blood pressure of 26 / 5~41 / 0 mm Hg, compared with before reconstruction, right ventricular diastolic pressure was no significant change (p> 0.05). 但收縮压增加有显著性(p〈0.05),术后超声检测管道内瓣膜纤细,瓣膜启闭良好,多无返流或极轻度返流。 But there was a significant increase in systolic blood pressure (p <0.05), postoperative ultrasound pipeline valve slim, good heart valves, and more without regurgitation or very mild regurgitation. 跨瓣压差小。 Pressure difference across the valve. 术后一年及一年半后处杀动物获取牛颈静脉标本检测,牛颈静脉管道通畅,管壁仍具有较好弹性,外形完整(图4)瓣叶无明显增厚,活动好(图5),管壁及瓣叶内膜有一层内皮样细胞生长(图6)。 After a year and a year and a half later kill the animals get bovine jugular vein specimens, bovine jugular vein conduit patency, the wall still has good elasticity, shape intact (Fig. 4) leaflets no thickening, a good activity (Fig. 5), wall and leaflets intima layer of endothelial-like cell growth (Figure 6). 结论:从近期和远期血流动力学观察看,自制牛颈静脉带瓣管道可以作为右室流出道重建的良好材料。 Conclusion: From the short and long term hemodynamic observation, the homemade bovine jugular vein conduit can be used as the right ventricular outflow tract reconstruction of good material. 实施例7 Example 7

人脐静脉内皮细胞在去细胞结合光氧化处理的牛颈静脉片上的种植方法:采集牛颈静脉,去细胞处理结合染料介导光氧化技术处理牛颈静脉。 Human umbilical vein endothelial cells grown in cell binding to bovine jugular vein segments on the photo-oxidation process: collecting bovine jugular vein, to the cell processing combined with dye mediated photo-oxidation technology bovine jugular vein. y射线低温灭菌处理。 y ray low-temperature sterilization treatment. 获取人脐静脉内皮细胞,传代培养,在血管内皮细胞生长因子等诱导下培养成内皮细胞。 Get in human umbilical vein endothelial cells, subculture, cultured endothelial cells into vascular endothelial growth factor induced. 在牛颈静脉片上种植培养。 On bovine jugular vein of crop cultivation.

结果:内皮细胞在牛颈静脉片上种植生长并长期存活(图9),细胞密集生长,排列有序,贴壁牢靠。 Results: Endothelial cells on bovine jugular vein of crop growth and long-term survival (Figure 9), cell density growth, orderly, adherent solid.

结论:去细胞结合光氧化处理的牛颈静脉适合人内皮细胞生长。 Conclusion: to cell binding bovine jugular vein photo-oxidation process for the human endothelial cell growth.

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