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Preparation process of biological valve-possessed duct for pulmonary artery vessel restoration or reconstruction

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CN100443064C
CN100443064C CN 200510032124 CN200510032124A CN100443064C CN 100443064 C CN100443064 C CN 100443064C CN 200510032124 CN200510032124 CN 200510032124 CN 200510032124 A CN200510032124 A CN 200510032124A CN 100443064 C CN100443064 C CN 100443064C
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artery
process
restoration
biological
valve
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CN 200510032124
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Chinese (zh)
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CN1762311A (en )
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吴忠仕
胡建国
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吴忠仕;胡建国
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Abstract

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 of preparing a biological conduit with valves for tissue repair or reconstruction of the cardiovascular system, in particular a method drawn from heterogeneous biological tissue. 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。 A method for the preparation of the pulmonary artery repair or reconstruction of a biological conduit with valves of the present invention is provided, comprising the following steps: 1) Get bovine jugular vein, and rinsed, disinfected; 2) reactive dye mediated photooxidation of bovine jugular vein crosslinked matrix; 3) sterilized and stored; wherein prior to step 2 first de-treated bovine jugular vein cells obtained in the first step. 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。 The method of preparing a biological conduit with valves of the present invention provides can be obtained with good biophysical properties, anti-calcification properties, low immunogenicity, non-cytotoxic, conducive to biological valved conduit host cells are grown.

Description

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 conduit with valves and preparation methods for the pulmonary vascular repair or rebuild

技术领域 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 of preparing a biological conduit with valves for tissue repair or reconstruction of the cardiovascular system, in particular a method drawn from heterogeneous biological tissue. 技术背景 technical 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 right ventricular and pulmonary artery is connected dysplasia complex congenital heart disease such as truncus arteriosus, pulmonary atresia, double outlet right ventricle, transposition of other corrective surgical treatment requires application 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。 . Since the 60s of the last century, the use of domestic and foreign valved homograft aortic or pulmonary 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 the difficulty valved homograft conduit source, especially for infants less surgery, and the presence of long-term calcification, poor durability and other 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。 Foreign-developed 80 years 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。 To carry out the 90 years before the pipeline study abroad bovine jugular vein 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 native human pulmonary valve structure, strong anti-reflux properties, span a large vessel diameter, and 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,。 The current international, the only bovine jugular vein conduit products for Medtronic, Inc. under 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 prepared by a conventional fixed biological tissue with glutaraldehyde. 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 product applications in Europe and other countries, and the recent 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 the poor effect metaplasia, anastomotic stenosis and thrombosis. 而这些原因主要与制备技术相关。 These reasons primarily related to preparation techniques. 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 crosslinked biological tissue is present the following drawbacks: l) the cytotoxic effect, long-term tissue remaining after crosslinking glutaraldehyde toxic to cells, the host cells can not grow in the tissue. 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 or the like causes the post-crosslinking is easy to tissue calcification, calcification is an important 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) not completely 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) residual cells and tissue debris is one of the main sites of calcification, is 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 sample treated by conventional methods glutaraldehyde FIG valved conduit obtained in (A), and changes, and (B) one year after implantation in dogs; bovine jugular venous glutaraldehyde FIG 2 (A), and change (B) one year after implantation in dogs.

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 processing techniques to improve the preparation of bovine jugular vein valved conduit important link performance. 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。 Dye-mediated photo-oxidation heterogeneous biological tissue is a non-crosslinked incorporated technique. 是通过光化学反应使得组织胶原交联,无细胞毒性。 By photochemical reaction so that the crosslinking of collagen, non-cytotoxic. 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 tissues, photooxidation fixing method has differences in some detail, but the same general method, i.e. the material to be immobilized is immersed in a solution of the dye dissolved, light irradiation. 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 the following: methylene blue, methylene green, rose bengal, riboflavin, proflavin, fluorescein, five phosphate pyrazole aldehyde gum, eosin and the like. 其中应用最多的是亚甲蓝,染料的使用浓度为0. 01%〜0.1%。 Wherein the most widely used is methylene blue, the dye concentration is 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 used 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 about 10 'C, to avoid overheating caused by degeneration of tissue proteins. Depending on the fixed different materials, the light intensity of the radiation and the time is not the same, is generally used only 1 to 2 150~500W bulb, placed at a height from the sample 2~12cm: illumination from a few minutes to 160H, and the light intensity time has a comprehensive indicator of the Ming-class hours, usually 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。 Dye mediated photooxidation crosslinking technology disclosed by the prior art no method for bovine jugular vein valved conduit process. 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 technique bovine jugular vein conduit, the result is still unsatisfactory.

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。 The decellularized extracellular matrix of retention, a natural host facilitates 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 Sufficiently remove cellular components, reduced immunogenicity, but also eliminates the possibility that the cell nucleus residues

的钙化位点,提高了抗钙化性能。 Calcification sites, 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 reported in the literature to cells in two ways: (1) detergent decellularization: detergent water is a class of lipid-soluble, it has a hydrophilic portion and a hydrophobic portion, so that cleaves the lipid membrane, the antigen is dissolved , 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: a head charged (+ or -), such as sodium dodecyl sulfate (SDS), sodium cholate, sodium deoxycholate and the like. 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 that the height of the denatured proteins, which were isolated 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。 Which is weak interaction between proteins and protein interference, on protein denaturation also weak. ③两性型:带有+和-电荷头部,如CHAPS、 Zwitterget等。 ③ amphoteric: with + and - charge 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) digestive enzyme acellular: 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 decellularization solution, depending on the clinical application and the organization may be freely selected. 一般采用联合使用方法。 Method for combination use is generally used. 发明内容 SUMMARY

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。 The present invention provides for the pulmonary artery to repair or reconstruction of biological valved conduit, using the technology of the dye to cells mediated photooxidation crosslinking techniques 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。 A method for the preparation of the pulmonary artery repair or reconstruction of a biological conduit with valves of the present invention is provided, comprising the following steps: 1) Get bovine jugular vein, and rinsed, disinfected; 2) reactive dye mediated photooxidation of bovine jugular vein crosslinked matrix; 3) sterilized and stored; wherein prior to step 2 first de-treated bovine jugular vein cells obtained in the first step.

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 of the present invention is to select the appropriate method of cell protein and the non-crosslinked glutaraldehyde method Bian.

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 technology combined treatment of bovine jugular vein conduit, can take advantage of both of the valved conduit made 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 the cell technology, its role is the cellular component of bovine jugular vein tissue cell such as the nucleus and the like sufficiently removed, so as to eliminate calcification sites, the purpose of reducing immunogenicity. 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。 And the application of light dye oxidation mediated tissue proteins are sufficiently crosslinked to provide 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 Qinghai, 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 usually two or clover leaf, small part of the four-leaf. 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。 The degree of leaf lobes ranging from 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。 Sinus node at the vascular wall is thin, easy to damage during trimming. 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 statistical results from 2.6cm to 6.4 persons. 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 generally small blood vessels branch aperture multivessel required ligation preparation. 牛颈静脉外膜纤维结缔组织丰富,与中膜分界不清。 Bovine jugular vein rich outer membrane of connective tissue fibers, and medial boundaries unclear. 不同牛系之间颈静脉的瓣膜发育无明显区别。 Bovine jugular vein between different valve system developed 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。 Vascular diameter 8~22mm, fascia tissue removed under sterile conditions, developmental states, and detecting valvular 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。 Development and good anti-reflux valve performance vessel was immersed in 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 the prior art, for a variety of detergents to the cells, including the ionic, nonionic and amphoteric. 本发明选用非离子型去污剂,例如曲那通IOO。 The present invention, use of non-ionic detergents, for example, through the song that IOO. 本发明在去细胞的步骤中,采用多步骤的手段。 In the present invention, the step to the cell, means for using 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 non-ionic detergents, but also employed in conjunction digestive enzymes and / or nuclease enzyme is used trypsin digestion, the nuclease is DNase and RNase. 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 cells treated using a combination of non-ionic detergent and digestive enzymes and / or enzyme-treated nucleic acid; 0.25-1% detergent concentration, digestive enzyme is trypsin; nuclease is a deoxyribonuclease and ribonuclease, when the digestive enzyme is trypsin concentration 0.025~0.05% trypsin; when digestive enzymes are DNase and RNase at a concentration of 10-40 unit respectively / 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。 According to the method disclosed in the prior art, when the cells were trypsinized to the solution as well as 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 agents to the cells 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。 The present invention, detergent treatment time is preferably 24-48 hours, trypsin treatment time of 30 to 120 minutes after mixing DNase and RNase treatment time of 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, treatment time of 48 hours, the digestive enzyme is trypsin, at a concentration of 0.025 ~ 0.125%, the processing time is 30-60 minutes; and deoxyribonuclease and ribose nuclease that concentrations of 20 unit / ml and 0. 2mg / ml, a treatment time of 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 photooxidation, in the present invention is specifically chosen methylene blue, and pH: 7.6; 0sm: 320mosm under conditions, 150~500W suspended 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 in methylene blue solution, PBS (pH: 7.6; 0sm: 320mosm) under soaking conditions for 2-4 hours. 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。 Carrying out photo-oxidation, biological valved conduit at a temperature 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。 The method may be, for example, a water bath to control temperature of the biological conduit with valves to be 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 preparation method for the biological repair or reconstruction of pulmonary artery conduit with valves, can be obtained with good biophysical properties, anti-calcification properties, low immunogenicity, non-cytotoxic, host cells are grown is conducive to biological 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 BRIEF DESCRIPTION OF bovine jugular vein conduit of FIG. 1 is a glutaraldehyde-treated (A) and the change (B) one year after implantation in dogs; bovine jugular venous (A) of FIG. 2 is a glutaraldehyde-treated explants and a year later changed into dogs (B); wherein the arrows indicate the valve structure; FIG. 3 after treatment with the process of the present invention, bovine jugular vein (a) and the valve (B) Histopathological changes (HE staining X20); FIG. 4 after treatment by the process of the present invention, bovine jugular vein conduit (a) and one year after the change implantation in dogs (B), wherein the arrows indicate the position of the valve; Figure 5 with the treatment method of the present invention, bovine jugular vein valve (a) and one year after the change implantation in dogs (B), wherein the arrows indicate the position of the valve; Figure 6 treated by the method of the present invention, bovine jugular vein year after implantation in dogs vascular endothelial tissue regeneration FIG like cells ( HE staining (X 100) arrows endothelial-like cells); FIG. 7 bovine jugular vein tissue protein extract prepared with different technical processing test results, wherein F: fresh tissue, P: photooxidation tissue, GA: glutaraldehyde treated tissue, DE: simple decellularized tissue, DP: decellularized tissue binding photooxidation process, DGA: cell binding to 合戊二醛处理组织,M: marker。 Together glutaraldehyde treated 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 photooxidation process time; FIG. 8 immunogenic processing result BJVC prepared using different methods to detect the Western blotting, wherein F: Group fresh, GA: glutaraldehyde group, P: Group photooxidation , DP: to cell binding photooxidation group; FIG. 9 human umbilical vein endothelial cells bind after planting electron microscope of a bovine jugular vein sheet prepared by photooxidation technique acellular (SEM a x1200 arrows 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 to obtain bovine jugular vein, beef cattle species can be local, 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。 Vascular diameter 8~22rnm, fascia tissue removed under sterile conditions, developmental states, and detecting valvular 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。 Development and good anti-reflux valve performance vessel was immersed in 0.1% benzalkonium bromide 30min, PBS rinse. 2) 去细胞处理:按照表1随机分十组。 2) Cells were treated to: in ten randomly set in Table 1. 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。 Different concentrations, different time, different cells to vascular tissue treated by the combination of the following methods. 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。 General morphology was observed different method of acellular bovine jugular vein of the sheet, the degree of completeness of decellularization, the integrity of the stored elastic force of the collagen fibers, the temperature change of the thermal shrinkage and a maximum tensile strength is reduced. 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 using detergents that through curved (Triton) X-100 in PBS solution, a concentration of less than 1%, a concentration of the appropriate treatment 24~48 hours at 0. 25~0 596, 37 ° C, PBS rinse;. ② 0.025~ 0.059G trypsin /0.0296EDTA treated with PBS at 37 ° C 30~ 120min, PBS rinse, 30~60 min more appropriate; ③ PBS was added 20u / ml DNase-1 and 0. 2mg / ml RNase-a in treatment at 37 ° C 24-48 hours, rinsed with PBS, 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。 After 320 mOsm) equilibrated 2~4h: 3) tubing is placed hypertonic PBS solution (pH: 7.6;:; 600~680 mosm) soaked 2-4h, then placed 0.01~0.1% methylene blue-containing PBS (pH Osm 7.6 , 150~500W suspended 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 was placed in the reaction system constant temperature water bath, continuous stirring during irradiation, air blowing, and pH control. 处理完后充分漂洗脱色。 It has been dealt with thoroughly rinsed bleaching. 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) iodine complex was placed in soaking 3 days in sterilized or 30~60min 0.1% benzalkonium bromide, 50 to 60% alcohol and then placed in 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 formula and approach the reference year 1995 US patent (Patent No. 5,437,287) were. 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%E After Step 4 has been processed, it precludes the use of polyester tissue sterile bag, storage solution with DMEM solution, 1096DMSO and l (^ FBS. Tissue gradually cooled to -80 ° C, in a frozen state, using y radiation sterilization (dose 25~40kGy), after disinfection, storage at room temperature or cryogenic tissue table l, <table> table see original document page 11 </ column> </ row> <table>: after all of acellular bovine jugular vein of each group in addition to vascular valve structure film has damaging m group, the other groups of the vascular wall and the valve structure intact, fresh and similar groups; no significant difference in the thermal shrinkage temperature of each group of beryllium fresh control group and the high concentration of trypsin. protease (0.125%) or too long trypsin digestion time (24h) breakable valve structures and bovine jugular vein collagen elastic fibers, so that the maximum tensile strength decreased by 20% compared to the fresh control group; pure song that pass qe - 100 (0.25%) plus low concentrations of trypsin (0.025%) of the cellular structure of acellular bovine jugular vein remaining sheet; mild multi-step detergent - enzymatic digestion (0.25% or song that 0.5./ through X-100, 0.025% or 0.05% trypsin and 0.02% E DTA, 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. DTA, 20u / mlDNase-I and 0.2mg / mlRNase-A) after acellular bovine jugular vein valved sheet over the comparison: a vessel wall and the valve and the pre-treatment gross morphology similar light and electron microscopy showed that the cell component is removed complete, intact collagen elastic fibers shown in Figure 3. Conclusion: step detergent - enzymatic digestion (song that pass X-100, trypsin and nucleases) bovine jugular vein valved off after the cells were treated acellular matrix is . ideal tissue engineered vascular stent embodiment 2 of bovine jugular vein decellularization photooxidation process according tensile strength anti method: collecting local bovine jugular vein 20, each vessel is divided into five sections, were randomly divided into the fresh group, decellularized group, photooxidation group decellularized group photooxidation, GA n-20 groups each group using glutaraldehyde 0.625% glutaraldehyde for 6 hours and 0.3% glutaraldehyde preservation;. photooxidation group with 0.1% methylene blue as mediators, illumination time of 48 hours, light intensity of 4000 lumens hours, mediators temperature of 12-18'C, disinfecting iodine complex, save 60% alcohol; cell binding to photooxidation group with 0.5./ song. that X-100, 48 hours, 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。 0.05% trypsin and 0.02% EDTA, 30min and 20u / mlDNase-I and 0. 2mg / ml RNase-A, 24h. Step PBS solution among rinsed multiple times (i.e., program group in Table 1 VII). Photooxidation and using technical further set of identical dye mediated photooxidation, disinfecting iodine complex, save 60% alcohol. fresh group with 60% alcohol storage vessel. All experiments transverse distance from the sinus node by at least lcm bovine jugular vein cut length 4cm, width 0.5 cm aortic strips. 采用STRON生物拉力仪测定牛颈静脉血管条的生物力学性能。 Measuring biomechanical properties of strips of bovine jugular vein using STRON biological tensile tester. 结果:各组的最大应力如下表2。 RESULTS: The maximum stress Table 2 below. 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 photooxidation treated group were significantly stronger than the fresh group. 去细胞处理对应力无明显影响。 No significant effect on the stress to 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。 And the blood vessel cells to stress photooxidation process was significantly lower than glutaraldehyde cross-linking group. 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 of bovine jugular vein sheet process different techniques Comparison <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: Preparation of cell binding to bovine jugular photooxidation improve the tensile strength of the tissue, but the tensile strength of the material is not as large glutaraldehyde treatment. 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 extract prepared in Example 3 Test Method for processing according to the different techniques: collecting local bovine jugular vein, using glutaraldehyde cross-linking, cross-linking and to photooxidation cells prepared photooxidation crosslinked bovine jugular vein (the same method of 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 preparation techniques, and fresh bovine jugular vein to cells treated mashed, added to tissue extract, polyacrylamide gel electrophoresis analysis of protein extracts for taking 20ixl. 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 pipe, the amount of protein extraction duct tissue and glutaraldehyde-treated group photooxidation treated group were significantly reduced, indicating that photo-oxidation functions as glutaraldehyde fixation of bovine jugular vein , a significant increase in microstructural stability, and with the increase of photo-oxidation treatment time, the amount of tissue protein extraction progressive reduction. 去细胞处理对组织稳定性无明显影响。 To cell treatment had no 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)。 Comparative Study of the immunogenicity of bovine jugular vein process for the preparation of different cross-linking techniques of Example 4: collecting local bovine jugular vein, using glutaraldehyde cross-linking, cross-linking and to photooxidation cells prepared photooxidation crosslinked 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。 Preparation of bovine jugular vein of different technologies and fresh bovine jugular vein were homogenate antigen, rabbit serum antibody titers measured immunized rabbits, and then double immunodiffusion method after mixing with complete Freund's adjuvant antigen emulsion to form a composite. 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 immunized antiserum, using immunoblotting (Western blotting) bovine jugular vein conduit Immunogenicity vitro detection crosslinked. 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 analysis: (l) SDS-PAGE: Take concentrated gel concentration was 4.0% separation gel concentration of 10% of the vertical plate electrophoresis, were added per plate of fresh bovine jugular vein, bovine PC, PH, GA crosslinking treatment jugular vein weave antigen. 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)。 Tissue antigens were associated with sample buffer before loading to i: Comparative dilution and heating treatment i (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, transferred to a membrane gel stained with Coomassie Brilliant Blue or electricity. (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) electroporation membrane: the protein band 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, after electrotransfer the gel with Coomassie Brilliant Blue staining observation protein transfer efficiency. (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) Western blot reactions: containing 1% BSA in Tween 80 / Tris-buffered saline (Tween 80, TBS) blocking NC membrane, and the NC membrane successively with 1: Reaction 12000AP goat anti-rabbit IgG,: 40 serum and 1 Finally BCIP / NBT color development reaction as a substrate, while the background bands appear lighter distilled water to terminate 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 antibody was diluted to 1: 1000, fresh bovine jugular vein tissue antiserum still significant slice display, 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 with a tissue-specific repeated with Article comprising at least six different antigens of molecular weights, indicating fresh bovine jugular vein tissue antigen of the antigenic specificity and, by photo-oxidation and cells binding to photooxidation process bovine jugular vein substantially clear band does not appear, five groups by GA crosslinked bovine jugular vein 1 groups banding, but no bands specific recurring described bovine jugular vein tissue antigens of the photo-oxidation process significantly reduce, lower antigenicity by binding to cell photooxidation bovine jugular vein (see FIG. 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)。 Method 5 anticalcification properties to photooxidation cells treated bovine jugular vein Example: collecting local bovine jugular vein, using glutaraldehyde cross-linking, cross-linking and to photooxidation cell photooxidation crosslinked 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 a flap. 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。 Select the weaning of male SD rats (provided by the Experimental Animal Center of Central South University Xiangya School of Medicine, are in line with the provisions of the national animal experiments), subcutaneous embedding, feeding 60 days, obtain specimens, spectrophotometer measured tissue calcium content by atomic absorption. 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: Results of calcium content wall and valve 253 are 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 was significant difference 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: binding to cell-mediated oxidation dye light bovine jugular produced significantly improved anti-calcification properties. 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。 Example 6 bovine jugular vein to the pulmonary artery conduit reconstruction cell binding photooxidation chamber of the right connection methods: collecting bovine jugular vein conduit, producing a light oxidation binding to cell treatment. 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。 For local application valved conduit dogs right ventricular outflow tract reconstruction, self ligation pulmonary artery pressure was measured directly before and after reconstruction, right ventricular pressure and systemic pressure, within seven days after the ultrasound, ultrasound year after blood flow and cardiac catheterization dynamics. 结果:六只犬重建后均长期存活。 Results: After six dogs were rebuilding long-term survival. 重建右室流出道后,管道内瓣膜远端血压为22/14〜37/10 mm Hg。 After the reconstruction of the right ventricular outflow tract, blood pressure distal to the valve within the conduit 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 (systolic, diastolic and mean pressure) difference was not significant (PX) .05), but were significantly higher than in the pipeline right ventricular diastolic diastolic 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 right ventricular blood pressure was 26 / 5~41 / 0 mm Hg, compared with that before the reconstruction, no significant changes in right ventricular diastolic pressure (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。 However, there was a significant increase in systolic blood pressure (p <0.05), postoperative ultrasound slender pipe valve, the valve opening and closing good, no more or very mild reflux regurgitation. 跨瓣压差小。 Transvalvular pressure difference. 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 at the killing of animals to obtain specimens of bovine jugular vein, bovine jugular vein conduit patency, the wall still has good elasticity, shape intact (Figure 4) no significant thickening of the valve leaflets, good activity (Fig. 5), the wall and the flap-like intima layer of endothelial cell growth (FIG. 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 to see, homemade bovine jugular vein conduit may be a good material outflow tract reconstruction as the right ventricle. 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 on a bovine jugular vein method photooxidation sheet in binding to cells: bovine jugular vein collected, treated cells to bind the dye mediated photo oxidation bovine jugular vein. y射线低温灭菌处理。 y-ray low-temperature sterilization process. 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。 Obtaining human umbilical vein endothelial cells, subcultured, to cultured endothelial cells in the induction of vascular endothelial growth factor. 在牛颈静脉片上种植培养。 Culture grown on bovine jugular vein sheet.

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: Growth of endothelial cells grown on a bovine jugular vein pieces and long-term survival (FIG. 9), the growth of cell density, arranged in order, 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: The cell binding to bovine jugular vein photooxidation process suitable for human endothelial cell growth.

Claims (9)

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, for the preparation of the pulmonary artery repair or reconstruction of a biological conduit with valves, comprising the following steps: 1) Get bovine jugular vein, and rinsed, disinfected; 2) dye mediated photooxidation causes the jugular vein matrix crosslinking ; 3) sterilized and stored; wherein prior to step 2 first de-treated bovine jugular vein cells obtained in the first step; decellularization agents used are non-ionic detergent, also employed in conjunction digestion enzymes and / or nuclease; digestive enzyme is trypsin; nuclease is DNase and RNase.
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, preparation method according to claim pulmonary artery repair or reconstruction of biological valved conduit for the 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, production method according to claim pulmonary vascular repair or reconstruction of biological valved conduit for the 2, characterized in that the concentration of the detergent 0. 25-1%; when the digestive enzyme is trypsin concentration 0. 05% trypsin 025~0; when the nuclease is DNase and RNase at a concentration of respectively 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 of claim preparation pulmonary artery repair or reconstruction of biological valved conduit for the 3, wherein the processing time with the detergent is 24 - 48 hours, trypsinized time 30-120 minutes, DNase and RNase mixing treatment time was 24-72 hours.
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 of claim preparation pulmonary artery repair or reconstruction of biological valved conduit for 4, characterized in that the concentration curve 100 is that the through 0.5%, a treatment time of 48 hours, the digestive enzyme is trypsin a concentration of 0.05%, the processing time was 30 minutes; and DNase and RNase at a concentration were 20u / ml and 0.2mg / ml, the processing time of 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, prepared according to the method of claim 1 to 5 for biological valved conduit repair or reconstruction of the pulmonary artery of the preceding claims, characterized in that the photooxidation reaction using methylene blue, and pH: 7 6; 0sm:. 320mosm under conditions, 150~500W suspended 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. The production method according to claim pulmonary vascular repair or reconstruction of biological valved conduit for the 6, characterized in that methylene blue solution, pH: 7.6; 0sm: 2-4 hours 320mosm soaking 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 production method according to claim pulmonary vascular repair or reconstruction of biological valved conduit for the 6, characterized in that during the photooxidation reaction, biological valved conduit at a temperature 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 a repair or reconstruction of the pulmonary artery, characterized in that it is one of the production method according to claim 8, wherein a l.
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