CN117462759A - 一种具有优异抗凝血和加快内皮化特性的组织工程血管及其制备方法 - Google Patents
一种具有优异抗凝血和加快内皮化特性的组织工程血管及其制备方法 Download PDFInfo
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Abstract
本发明属于人工血管技术领域,本发明提供了一种具有优异抗凝血和加快内皮化功能的组织工程血管及制备方法。包括如下步骤:(1)制备聚合物纤维骨架;(2)将所述聚合物纤维骨架负载功能基因载体,得到功能化的聚合物纤维骨架;(3)将所述功能化的聚合物纤维骨架植入动物皮下培育或在纤维骨架上接种细胞并培养,得到具有优异抗凝血特性和加快内皮化特性的组织工程血管。所述功能基因为携带C型钠尿肽(CNP)的慢病毒基因载体。本发明的有益效果在于,聚合物纤维骨架负载了功能基因载体,对皮下浸润细胞或培养在骨架中的细胞进行基因转染并产生CNP蛋白,CNP蛋白在组织工程血管中发挥抗凝血作用和快速内皮化效果。
Description
技术领域
本发明涉及人工血管技术领域,尤其涉及一种具有优异抗凝血和加快内皮化特性的组织工程血管及其制备方法。
背景技术
心血管疾病是人类健康的第一杀手,其发病率和致死率远高于其他疾病。血管移植是治疗心血管疾病的有效手段之一。临床上,包括乳内动脉、桡动脉和大隐静脉在内的自体血管是进行血管移植的金标准,但是大约30%的患者由于血管来源有限和尺寸不匹配等原因,没有合适的自体血管可用。
组织工程技术的发展为人体血管缺损修复和重建提供了新的解决方案,组织工程血管是一种用组织工程学方法构建的具有良好生物相容性和力学特性的血管替代物。体外组织工程是在血管支架材料上种植细胞,并通过生物反应器培养。如种植骨髓间充质干细胞、诱导多能干细胞(iPSCs)或内皮细胞,能够提高血管材料的通畅性和再生性。但是在植入动物体内后,由于体内外生长环境的变化,细胞会发生功能状态改变,甚至凋亡、脱落,因此还是会发生一定程度的血栓。
除了体外组织工程,很多学者也利用体内组织工程技术来构建人工血管,其原理是利用机体对外源物产生自发的免疫包裹反应。通常的方法是将管或棒状物植入动物皮下,待组织包裹完成后,得到体内组织工程生物管,但是该生物管力学强度不足,无法维持管状结构,不易缝合,而且植入动脉系统易出现血管膨胀和吻合口狭窄。为此,发明人前期采用在管状接收器上制备聚合物纤维骨架,将其作为模板植入动物皮下,利用宿主对外源物自发的免疫包裹反应,完成对模板材料细胞化与细胞外基质的沉积,取出已被组织包裹的模板材料,移除管状接收器,即可制备出由聚合物纤维骨架增强的体内组织工程血管。该组织工程血管在动物血管移植实验中展现了良好的通畅性和再生性。但是,该组织工程血管不具有抗凝血能力,需要给与抗凝药物来避免凝血,而且内皮细胞层的再生速度有待提高。
因此,在本发明中,发明人将抗凝血和促内皮再生的基因修饰到聚合物骨架上,利用基因局部递送和转染技术,实现聚合物纤维骨架对体外培养细胞或皮下浸润细胞的基因转染和表达,提升血管的抗凝血和促内皮化特性。
发明内容
本发明的目的在于提供一种具有优异抗凝血和促内皮化特性的组织工程血管及其制备方法。
为了实现上述发明目的,本发明提供了以下技术方案:
本发明提供了一种具有优异抗凝血和促内皮化特性的组织工程血管及制备方法,包括如下步骤:
(1)制备聚合物纤维骨架;
(2)将所述聚合物纤维骨架负载功能基因载体,得到功能化的聚合物纤维骨架;
(3)将所述功能化的聚合物纤维骨架植入动物皮下培育或在纤维骨架上接种细胞并培养,得到细胞化的具有优异抗凝血特性和加快内皮化特性的组织工程血管。
优选的,所述聚合物纤维骨架是采用熔融近场直写技术、静电纺丝技术、编织或3D打印技术制备的能够满足血管力学强度的多孔纤维骨架。
优选的,所述用于制备聚合物纤维骨架的材料为聚己内酯(PCL)、聚对二氧六环己酮(PDS)、涤纶、聚(丙交酯-乙醇酸)共聚物(PLGA)、聚乙醇酸(PGA)、聚氨基甲酸酯(PU)、聚癸二酸甘油酯(PGS)和聚乙二醇(PEO)中的一种或者多种。
优选的,所述聚合物纤维骨架表面经过涂层处理后,再进一步负载功能基因载体。
优选的,所述涂层处理的方法为,使用多聚赖氨酸溶液在所述聚合物纤维骨架表面进行聚合反应,所述多聚赖氨酸溶液的质量浓度为0.8μg/mL~500μg/mL,优选为4μg/mL~100μg/mL,以在聚合物纤维骨架表面形成呈正电性的涂层。
优选的,所述涂层处理后负载功能基因载体的方法为,使用携带抗凝和促内皮化功能基因的慢病毒基因载体与聚合物纤维骨架进行孵育,所述基因载体通过静电亲和作用结合在聚合物纤维骨架表面。
优选的,所述抗凝和促内皮化功能基因为C型钠尿肽(CNP);所述慢病毒基因载体的溶液浓度为5×104~5×108滴度/mL,优选为5×105~5×107滴度/mL。
优选的,将步骤(3)中所述功能化的聚合物纤维骨架通过植入动物皮下培养,获得具有优异抗凝血和加快内皮化特性的组织工程血管。
本发明还提供了一种按照所述的制备方法得到的具有优异抗凝血和促内皮化特性的组织工程血管。
本发明通过聚合物纤维骨架负载功能基因,提升血管的抗凝血和促内皮化特性。该高表达C型钠尿肽的组织工程血管相比于普通组织工程血管具有更加优异的促内皮化和抗凝血特性,在大鼠腹主动脉移植14天、兔子颈动脉移植28天和猪心脏搭桥30天时提高了血管通畅率和血管内皮再生速率;使用兔动静脉分流模型(AV-shunt)研究表明,高表达CNP的组织工程血管相比于普通组织工程血管形成了更少的血栓,具有更好的抗凝性能。这表明本发明提供的组织工程血管具有抗凝和快速内皮化的作用。
附图说明
图1本发明实施例和对比例制备的具有优异抗凝和促内皮化特性的组织工程血管的结构示意图,图中1指示高表达CNP的细胞,图中2指示用于负载递送CNP基因的慢病毒载体的聚合物纤维骨架。
图2实施例1中高表达CNP的组织工程血管(CNP-PB)和对比例1普通组织工程血管(PB)图像;
图3实施例1中高表达CNP的组织工程血管(CNP-PB)和对比例1普通组织工程血管(PB)的免疫荧光图像(GFP阳性显示病毒转染细胞);
图4实施例1中的PCL纤维骨架的激光共聚焦图像,其中PLL-FITC-PS是经NaOH溶液水解处理后,用带FITC基团的赖氨酸溶液浸泡处理的PCL聚合物纤维骨架;PS是只经NaOH溶液水解的PCL聚合物纤维骨架;
图5用于产生C型钠尿肽(CNP的mRNA编码序列为NM_024409.4)的慢病毒基因组示意图;
图6实施例1中富含CNP的组织工程血管(CNP-PB)和对比例1普通组织工程血管(PB)用于大鼠腹主动脉移植14天后纵切样品CD31免疫荧光染色显示的内皮覆盖情况。
图7实施例2中富含CNP的组织工程血管(CNP-PB)和对比例2普通组织工程血管(PB)用于兔颈动脉移植28天后纵切样品CD31免疫荧光染色显示的内皮覆盖情况。
图8实施例5中富含CNP的组织工程血管(CNP-PB)和对比例5普通组织工程血管(PB)用于比格犬颈动脉移植1个月后血管中段部分的内皮(vWF阳性,免疫荧光照片)再生情况。
具体实施方式
下面将结合本发明实施例的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
所述聚合物纤维骨架为本领域专业人员采用熔融纺丝、熔融近场直写技术、静电纺丝技术、编织或3D打印技术等技术制备,所述纤维骨架具有多孔结构并且能够满足血管力学强度即可,并无特殊的限制。本发明中,所述纤维骨架的纤维直径优选为0.5-100μm。
所述聚合物纤维骨架由生物医用可降解聚合物材料制备,本发明中纤维优选由聚己内酯(PCL)、涤纶、聚对二氧六环己酮(PDS)、聚(丙交酯-乙醇酸)共聚物(PLGA)、聚乙醇酸(PGA)、聚氨基甲酸酯(PU)、聚癸二酸甘油酯(PGS)与聚乙二醇(PEO)。
在本发明中,所述多聚赖氨酸在聚合物纤维骨架表面进行聚合,聚合物纤维骨架需要提供亲水性表面以利于赖氨酸在其表面聚合,对于非亲水性的聚合物骨架需要进行亲水改性处理,对于亲水的聚合物材料骨架则不用进行亲水改性处理。本发明提供的一些实施例中,优选地,使用浓度为1mol/L的NaOH溶液对疏水表面的PCL纤维骨架进行了亲水改性;本发明提供的另一些实施例中,使用了亲水性的PDS材料,没有进行亲水改性。
本发明提供的一些实施例中,没有多聚赖氨酸涂层时无法有效吸附慢病毒,进而无法实现基因递送和转染的目的;此外,多聚赖氨酸可以促进慢病毒对细胞的转染。因此,本发明对多聚赖氨酸溶液的质量浓度进行了优选。在本发明提供的另一些实施例中,对用于涂层的多聚赖氨酸溶液的质量浓度进行了优选,质量分数为0.8μg/mL~500μg/mL的多聚赖氨酸溶液用于骨架涂层后均可以实现对慢病毒的负载,优选浓度为4μg/mL~100μg/mL。
本发明提供的一些实施例中,利用病毒载体递送了CNP基因,用于在体内合成释放CNP;所述CNP慢病毒载体溶液浓度优选5×105~5×107滴度/mL。
在本发明中,将负载基因载体的聚合物纤维骨架植入到动物皮下或者接种细胞完成细胞化。优选的,将负载基因载体的聚合物纤维骨架植入到动物皮下完成细胞化,聚合物纤维骨架上负载的基因对皮下浸润细胞或纤维骨架中的种植的细胞进行基因转染,表达所需的目的基因,得到相应的组织工程血管;当采用体外细胞培养时,接种的细胞可以是成纤维细胞、内皮细胞、平滑肌细胞、干细胞、干细胞分化来的平滑肌细胞或内皮细胞中的一种或者多种。在体外流动培养期间,聚合物纤维骨架对接种在骨架表面的细胞进行转染,表达所需的目的基因,得到相应的组织工程血管。
本发明提供的组织工程血管通过使用聚合物纤维骨架负载基因载体,在血管中高表达了具有抗凝和促内皮化的CNP。CNP具有良好的抗凝血特性和加快内皮化特性,因此采用本发明所制备的组织工程血管相比于普通组织工程血管具有优异的抗凝血和加快内皮化特性。同时,本发明提供的组织工程血管中还包括了聚合物纤维骨架,可以赋予血管优良的力学特性。这种组织工程血管可用于临床血管替换、血管旁路建立或者动静脉造瘘等适应症。
为了进一步说明本发明,以下结合实施例对本发明提供的一种具有优异抗凝血和加快内皮化特性的组织工程血管及其制备方法进行详细描述
实施例1
使用PCL(Mn=80000Da),采用熔融纺丝技术在医用硅胶管上制备出纤维直径为60μm,纤维交叉角度为50°,壁厚为380μm,内径为2mm的聚合物纤维骨架。将聚合物纤维骨架用1mol/L的NaOH溶液浸泡处理8h,使其表面亲水,使用75%酒精消毒。
用20μg/mL的多聚赖氨酸溶液,浸泡处理经过亲水改性的聚合物纤维骨架过夜;将携带CNP的慢病毒基因载体稀释到终浓度为5×106滴度/mL,将聚合物纤维骨架置于3倍体积的终滴度病毒溶液中,然后将其平稳放置在4℃摇床中,70rpm孵育1h,使慢病毒载体吸附到聚合物纤维骨架表面。
将负载有CNP慢病毒基因载体的聚合物纤维骨架埋植到大鼠皮下30天,使其完成细胞化后取出。将所获得的组织工程血管在体式显微镜下进行横切拍照,观察血管的宏观形态结构(如图2中CNP-PB所示,图2中PB为对比例1的图像,对比例1见下文);然后截取3mm的血管样品置于OCT包埋剂中,进行冰冻切片,切片经PBS清洗和DAPI封片之后用荧光显微镜(488波长激发)成像,观察血管中转染的细胞(如图3中CNP-PB所示,图3中PB为对比例1的图像,对比例1见下文);从图2、3中可以得知,负载在纤维骨架上的慢病毒对浸润到骨架中的细胞进行了转染。之后进行血管其他性能的检测,并进一步用于大鼠自体腹主动脉血管移植实验(2mm内径,1.1cm长),2周后取材评估人工血管再生情况。
实施例1-5的具体参数见汇总表1。
为了展示聚合物纤维骨架表面的多聚赖氨酸涂层,本实施例还使用了带有荧光染料异硫氰酸荧光素(FITC)基团的赖氨酸溶液对亲水改性过的聚合物纤维骨架进行浸泡处理(操作步骤同上,PLL-FITC-PS),并与未亲水改性过的聚合物纤维骨架(PS)进行对比。如图4激光共聚焦图像所示,在488nm的激发波长下,纤维表面覆盖有均匀的荧光信号,表明多聚赖氨酸可以均匀地涂附在聚合物纤维表面。多聚赖氨酸涂层为进一步慢病毒在纤维表面的静电吸附提供了基础。
本实施例以及以下实施例中,使用的CNP慢病毒基因组结构如图5所示,CNP的mRNA编码序列为NM_024409.4。
实施例2
称取适量的PDS放入到3D打印机加热料筒中,设置热熔温度为220℃,以外径为2.5mm的医用不锈钢棍作为接收器,设置采用19G打印针头,设置推进气压为124kPa,打印线速度为5mm/s,接收器转速为120r/min,打印出纤维直径为100μm,厚度为200μm的PDS纤维层。打印结束后;将聚合物材料更换为PCL(Mn=40000Da),设置热熔温度为120℃,采用19G打印针头,设置推进气压为113kPa,打印线速度为5mm/s,接收器转速为120r/min,打印出纤维直径为100μm,打印厚度为200μm的PCL纤维层。
用20μg/mL的多聚赖氨酸溶液浸泡处理过夜,随后用终浓度为5×106滴度/mL的携带有CNP的慢病毒溶液孵育1h(聚合物纤维骨架置于3倍体积的终滴度病毒溶液中,然后在4℃摇床,70rpm孵育1h)。
将负载有CNP慢病毒基因载体的聚合物纤维骨架埋植到兔皮下30天,使其完成细胞化后取出,将所获得的组织工程血管进行血管性能检测,并用于兔子自体颈动脉移植实验(2.5mm内径,2.5cm长),28天后取材评估人工血管内血栓形成和再生情况。
实施例1-5的具体参数见汇总表1。
实施例3
称取适量的PCL(Mn=80000Da),融化于熔融纺丝机料筒内,设定温度为120℃,以外径为3.8mm的医用硅胶管(内衬不锈钢棒)作为接收器,控制熔融纺丝的料筒推进速度为1.2ml/h、接收器的转速为240r/min、平移速度为18.0mm/s,纺丝得到纤维直径为60μm,厚度为480μm,纤维间交叉角度为50°的PCL纤维骨架。将聚合物纤维骨架用1mol/L的NaOH溶液浸泡处理8h,使其表面亲水,使用75%酒精消毒。
使用20μg/mL的多聚赖氨酸溶液浸泡处理过夜,并进一步用终浓度为5×106滴度/mL的携带有CNP基因的慢病毒溶液孵育1h(聚合物纤维骨架置于3倍体积的终滴度病毒溶液中,然后在4℃摇床,70rpm孵育1h)。
将携带有慢病毒基因载体的PCL聚合物纤维骨架植入猪皮下30天,组织工程,从皮下取出血管,并使用该血管(内径3.8mm,长度12cm)进行猪自体心脏搭桥实验。在移植后1个月取材,评价血管内血栓形成和血管再生情况。
实施例1-5的具体参数见汇总表1。
实施例4
称取适量的PDS材料,将其溶于六氟异丙醇中得到质量分数为20%的PDS静电纺丝溶液;称取适量的PCL(Mn=80000Da),将其溶于三氯甲烷/甲醇=5:1的溶液中,得到质量分数为20%的静电纺丝溶液。将直径为2mm的PCL圆棒浸没到聚苯乙烯磺酸和吡咯溶液中,再加入氧化剂氯化铁,使聚吡咯聚合在PCL表面,使PCL棒具备导电能力。将表面有聚吡咯的PCL棒用作接收器,先使用PDS静电纺丝溶液在其表面制备纤维直径为5μm,厚度为150μm的PDS静电纺丝纤维,纺丝条件为:电压:15kV,出液速度:6mL/h,针头到接收器之间的距离为20cm。在接收完PDS纤维层后,紧接着使用PCL静电纺丝溶液在表面制备纤维直径为5μm,厚度为230μm厚的PCL纤维层,纺丝条件为:纺丝条件为:电压:11kV,出液速度:8mL/h,针头到接收器之间的距离为20cm。纺丝完成后将静电纺丝纤维骨架进行抽真空。
用20μg/mL的多聚赖氨酸溶液,浸泡处理聚合物纤维骨架过夜;将携带有CNP的慢病毒基因载体稀释到终浓度为5×107滴度/mL,然后聚合物纤维骨架置于3倍体积的终滴度病毒溶液中,之后平稳置于4℃摇床,70rpm孵育1h,使慢病毒载体吸附到聚合物纤维骨架表面。
向携带有CNP基因载体的聚合物纤维骨架中种植兔子骨髓间充质干细胞,并进一步进行体外流动培养8周,使成纤维细胞增殖并分泌细胞外基质。
将所获得的组织工程血管进行性能检测,评价血管的细胞外基质成分、力学和血液相容性,使用自体兔(对应的取成纤维细胞的兔)动静脉分流模型(AV-shunt)评价血管(内径2mm,长度2cm)的抗凝血特性。
实施例1-5的具体参数见汇总表1。
实施例5
使用PCL(Mn=80000Da),采用熔融纺丝技术,设置推进速度为3ml/h、接收器的转速为160r/min、平移速度为24mm/s,在外径为3.8mm的医用硅胶管上制备出纤维直径为100μm,纤维交叉角度为50°,厚度为480μm的聚合物纤维骨架。将聚合物纤维骨架用1mol/L的NaOH溶液浸泡处理8h,使其表面亲水,使用75%酒精消毒。
用20μg/mL的多聚赖氨酸溶液,浸泡处理经过亲水改性的聚合物纤维骨架过夜;将携带有CNP的慢病毒基因载体稀释到终浓度为5×107滴度/mL,然后聚合物纤维骨架置于3倍体积的终滴度病毒溶液中,之后平稳置于4℃摇床,70rpm孵育1h,使慢病毒载体吸附到聚合物纤维骨架表面。
将负载有CNP慢病毒基因载体的聚合物纤维骨架埋植到犬皮下30天使其完成细胞化,之后将血管从犬皮下取出进行自体颈动脉血管移植(内径3.8mm,长度4cm),并在移植后1个月取材分析血管血栓形成和血管内皮再生情况。将从皮下所获得的组织工程血管及时进行血管性能检测。
实施例1-5的具体参数见汇总表1。
表1实施例1-5组织工程血管制备参数表
同时设置以下实施例
实施例6-10中组织工程血管的制备条件除了多聚赖氨酸溶液的质量浓度不同外,其他条件均与实施例2相同,由于实施例2正好位于中间浓度参数,因此为了便于理解,实施例2也被列于表2,具体实施条件见表2:
表2实施例6-10组织工程血管制备参数表
实施例11-15中组织工程血管的制备条件除了负载CNP基因的慢病毒溶液浓度不同外,其他条件均与实施例2相同,由于实施例2正好位于中间浓度参数,因此为了便于理解,实施例2也被列于表3,具体实施条件见表3:
表3实施例11-15组织工程血管制备参数表
对比例1-5
对比例1-5中组织工程血管的制备条件除了聚合物纤维骨架不经过多聚赖氨酸溶液处理和不负载病毒外,其他条件均与实施例1-5按顺序相同,具体实施条件见表4:
表4对比例1-5组织工程血管制备参数表
以下通过相关结果的展示与分析来充分说明本发明的有益效果。
对实施例1-15所制备得到的组织工程血管的抗凝血特性和促内皮化特性进行检测,具体方法如下(结果如表5):
1.CNP含量检测:获得血管后用预冷的PBS冲洗血管样品,吸水纸吸湿称重后将组织剪碎。将剪碎的组织与按1:9重量体积的PBS加入匀浆器中,在冰上充分研磨。将匀浆液于4℃,12000rpm离心10分钟,取上清检测。使用Elisa试剂盒(Invitrogen,EH136RB)对CNP进行定量,按照说明进行标准品稀释和相关试剂配制。以每个实验组12个稀释度,每孔100μL在96孔板中上样,室温振荡孵育2.5小时,洗板,每孔加入100μL生物素偶联物,室温振荡孵育1小时,洗板,加入100μL Streptavidin-HRP溶液,室温振荡孵育45分钟,洗板,每孔加入100μL显色底物TMB,室温(25±2℃)避光显色30分钟,每孔加入50μL终止液,使用酶标仪测定450nm最大吸收波长下的OD值,依据标准曲线计算相应组织中所含CNP的量。
2.血小板粘附检测:通过血小板的黏附实验评价血管材料的抗凝性能,将血管材料纵向剪开,使其管腔内侧向上放置铺于48孔板中,每孔加入500μL人富血小板血浆(Plateletrichplasma,PRP),37℃条件下孵育1h。孵育完成后,吸弃PRP,并用PBS缓冲液漂洗3次以去除未粘附的血小板。将血管材料放置于4%多聚甲醛溶液中固定20min后进行P-选择素染色,用于评价血小板粘附的活化情况。具体步骤如下:将样品用PBS缓冲液洗3次,每次5min;加入5%封闭山羊血清在4℃条件下孵育40min;对于P-选择素的评价,在样品上滴加一抗稀释液(mouse anti P-selectin,1:50)并在4℃孵育过夜;第二天取出复温40min,用PBS缓冲液洗5次,每次5min;滴加二抗(goat anti-mouseAlexaFluor 594,1:200)室温孵育两小时,PBS缓冲液洗5次,每次5min;将样品浸入DAPI封片剂中,4℃避光反应10min;取出样品,血管内腔向上置于载玻片上,滴加DAPI至刚好覆盖样品,轻轻加上盖玻片,用胶带压紧盖玻片两端使其粘在载玻片上;48h内用激光扫描共聚焦显微镜观察管腔表面并拍照,每个视野下荧光阳性面积总和被用于激活血小板定量统计。
3.血管内皮细胞增殖实验:将从动物皮下或培养获得的组织工程血管裁成5mm长短,然后纵向剪开,使其管腔内侧向上放置铺于12孔Transwell的下层,然后将5×103的HUVECs种于上层的小室(孔径0.4μm),每日更换培养基,培养3天后使用CCK-8试剂盒检测HUVEC的增殖。具体方法为:向每个48孔中加入20μL(1:100)的CCK-8溶液,随后放置于培养箱中孵育2小时,使用酶标仪测定在450nm处的吸光度。
4.血管体内植入后抗凝性能和加快内皮化特性分析:
实施例1和对比例1,大鼠腹主动脉自体移植及其分析:使用SD大鼠,进行腹主动脉移植评估。将内径2.0mm的血管从自体背部取出,然后裁剪成长约1.1cm的血管材料,使用9-0尼龙缝合线以端端吻合的方式植入大鼠自体腹主动脉,每端间断缝合8针。2周后对植入的人工血管进行取材分析,取出的再生血管组织进行纵切处理,在体式显微镜下观察血管内腔面是否有血栓形成,取一条样品使用2.5%的戊二醛固定及梯度酒精脱水处理后,通过扫描电镜观察血管内腔面是否有内皮覆盖与凝血基质黏附。取一条样品置于组织包埋剂OCT中,使用液氮速冻后进行冰冻切片分析,使用CD31抗体和α-SMA抗体进行免疫荧光染色,血管内皮覆盖率=血管纵切内腔面CD31阳性长度/血管纵切内腔面总长度×100%。CD31免疫荧光染色结果如图6所示,从图6中可以看出实施例1实现了内皮细胞的完全覆盖,而对比例1的内皮细胞的覆盖范围不足1/2,进一步的统计数据显示实施例1和对比例1的内皮覆盖率分别为93.87%和42.93%,表明实施例1能够显著的加快内皮化。
实施例2和对比例2以及实施例6-15进行兔子颈动脉自体移植并对其进行分析:使用新西兰大耳白兔,进行颈动脉移植。将内径2.5mm的血管从自体的背部取出,然后裁剪成2.5cm,使用9-0尼龙缝合线以端端吻合的方式植入自体免颈动脉每端间断缝合8针。术后连续7天肌肉注射青霉素进行抗感染治疗(40万单位/次,2次/日)。2周后对植入的人工血管进行取材分析,取出的再生血管组织进行纵切处理,在体式显微镜下观察血管内腔面是否有血栓形成,随后使用2.5%的戊二醛固定及梯度酒精脱水处理后,通过扫描电镜观察血管内腔面是否有内皮覆盖与凝血基质黏附。取一条样品样品置于组织包埋剂OCT中,使用液氮速冻后进行冰冻切片分析,使用CD31抗体和α-SMA抗体进行免疫荧光染色,血管内皮覆盖率=血管纵切内腔面CD31阳性长度/血管纵切内腔面总长度×100%。CD31免疫荧光染色结果如图7所示,从图7中可以看出实施例2几乎实现了内皮细胞的完全覆盖,而对比例2中内皮细胞的覆盖范围大约只有1/3,进一步的统计数据显示实施例2和对比例2的内皮覆盖率分别为88.94%和33.14%,表明实施例2能够显著的加快内皮化。
实施例3和对比例3,猪心脏搭桥及其分析:使用长白猪,进行自体心脏搭桥手术。内径3.8mm的血管从自体的背部取出,然后裁剪成12cm备用,然后分离主动脉,对其进行半阻断,在阻断侧使用4-0打孔器进行打孔,将内径3.8mm,长约12cm的血管材料,使用6-0Prolene缝合线采用端侧连续缝合的方式与主动脉打孔处进行缝合随后打开心包,分离冠脉,进行冠脉侧的端侧吻合,两端吻合完毕后于冠脉缝合处近心端进行结扎,以模拟临床冠脉堵塞。检查无出血点后,关闭胸腔,缝合皮肤。术后连续7天肌肉注射青霉素进行抗感染治疗(160万单位/次,2次/日),按照文献报道饲喂阿司匹林(80g/日)和氯此格雷(75mg/日)。30天后,对植入的血管进行取材分析,从主动脉吻合口至前降支冠脉均分为4等份,并在等分位点截取长度为5mm的血管环使用福尔马林固定后进行石蜡切片分析,使用vWF抗体和α-SMA抗体分析血管内皮和平滑肌的情况,血管内皮覆盖率=来自4个样品段的横切片vWF阳性细胞覆盖总长度/血管内腔总长度×100%。
实施例4和对比例4,兔颈部构建半体内动静脉瘘模型及其分析:利用留置针、硅胶管等装置将实验动物的动脉与静脉连接起来,在体外构建一个半体内循环系统,使血液不断流经待测人工血管材料,经2h后检测材料表面血栓形成,血小板及其他血液蛋白的聚集粘附情况。
实施例5和对比例5,比格犬颈动脉血管自体移植实验及其分析:开展手术前一天,将比格犬禁食。手术当日,对比格犬肌肉注射陆眠宁(1.5mg/kg)进行麻醉诱导,气管插管后,使用异氟烷(1-4%)吸入维持麻醉。颈部区域备皮和充分消毒后,使用一次性创巾建立无菌手术区。在皮肤表面做长度为6cm的切口后,钝性分离皮下组织和肌肉层,游离长度为6cm左右的颈动脉。按照100UI/kg的剂量根据动物体重静脉推注肝素注射液,注射肝素5分钟后,使用动脉夹阻断天然血管血流。将从自体背部取出的血管裁成4cm(内径3.8mm),使用7-0Prolene缝合线将血管按照端端连续吻合方式将其与自体天然血管缝合。将血管内空气排净后,恢复颈动脉血流。用0.1%的硫酸庆大霉素生理盐水冲洗伤口后,依次缝合肌肉层和皮肤层。人工血管移植1个月后,将比格犬麻醉后颈部皮肤备皮,在血管移植部位做切口后钝性分离皮下组织和肌肉,游离并剪下人工血管及邻近的天然血管,使用含肝素的生理盐水冲洗人工血管管腔。使用体式显微镜检查血管腔内是否有血栓形成。整段人工血管均分为4等份,并在等分位点截取长度为5mm的血管环使用福尔马林固定后进行石蜡切片分析,使用vWF抗体和α-SMA抗体分析血管内皮和平滑肌的情况,血管内皮覆盖率=来自4个样品段的横切片vWF阳性细胞覆盖总长度/血管内腔总长度×100%。vWF阳性的免疫荧光照片如图8所述,从图8中可以看出实施例5实现了内皮细胞的覆盖,而对比例5完全无内皮细胞存在,进一步的统计数据显示实施例5和对比例5的内皮覆盖率分别为61.65%和28.24%,表明实施例5能够显著的加快内皮化。
表5组织工程血管的成分和性能分析(平行实验,n=5,取平均值)
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由表5可知,实施例1-5在多聚赖氨酸溶液浓度为20μg/mL,慢病毒浓度为5×106滴度/mL时,通过多聚赖氨酸涂层负载CNP基因的慢病毒载体的纤维骨架制备的组织工程血管中产生了远高于普通组织工程血管中的CNP含量(对比例1-5),高表达CNP的组织工程血管的激活血小板数量均低于普通组织工程血管,表明其具有更加优异的抗凝血性能,接种HUVEC后第三天的CCK-8实验中,组织工程高表达CNP的组织工程血管组中测得的OD450值高于普通组织工程血管组,表明高表达CNP的组织工程血管材料能够促进血管内皮细胞增殖,不同动物模型的体内血管移植和兔子AV-Shunt实验结果表明,在提升CNP含量后,血管内腔面的血栓形成更少;而且高表达CNP的组织工程血管的血管内皮细胞覆盖率也明显高于普通组织工程血管,表明其具有更好的促进血管再生的特性。
实施例6-10和实施例2表明,在病毒溶液浓度一定的情况下,未经过多聚赖氨酸溶液处理骨架的组织工程血管CNP含量(实施例6)和普通组织工程血管(对比例2)CNP含量差别不大,在多聚赖氨酸溶液的质量浓度为0.8μg/mL时,组织工程血管中增加的CNP含量十分有限,当多聚赖氨酸溶液的质量浓度大于0.8μg/mL时,随着多聚赖氨酸溶液的质量浓度增加(0.8μg/mL~100μg/mL),CNP表达量增加,当多聚赖氨酸溶液浓度超过100μg/mL时,CNP表达量不再随着多聚赖氨酸溶液浓度的增加而增加。人工血管抗血小板粘附实验数据、内皮细胞增殖实验数据的趋势与CNP含量变化基本一致,实施例6-10和实施例2的体内兔子颈动脉血管移植后血管内腔面血栓形成、血管通畅率和血管内皮覆盖率结果进一步验证了体外凝血实验和血管内皮增殖实验的结果,与CNP含量变化趋势基本一致,因此,优选多聚赖氨酸溶液浓度为4μg/mL~100μg/mL。
实施例11-15和实施例2表明,在多聚赖氨酸溶液浓度浓度一定的情况下,没有慢病毒负载的纤维骨架的组织工程血管CNP含量(实施例11)和普通组织工程血管(对比例2)中CNP含量差别不大,在5×104滴度/mL时(实施例13),CNP含量增加的极为有限,与没有慢病毒负载的纤维骨架的组织工程血管CNP含量(实施例12)基本相当,当病毒溶液为5×104-5×107滴度/mL,组织工程血管中CNP的含量显著增加,且与随着病毒浓度的增加而增加,当进一步增加病毒浓度到5×108滴度/mL时,CNP含量反而有所下降,人工血管抗血小板粘附、内皮细胞增殖实验数据的趋势与CNP含量变化基本一致。实施例11-15和实施例2的体内兔子颈动脉血管移植后血管内腔面血栓形成、血管通畅率和血管内皮覆盖率结果进一步验证了体外凝血实验和血管内皮增殖实验的结果,与CNP含量变化趋势基本一致。因此,负载CNP的慢病毒溶液浓度优选为5×105-5×107滴度/mL。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和修饰,这些改进和修饰也应视为本发明的保护范围。
Claims (9)
1.一种具有优异抗凝血和加快内皮化特性的组织工程血管的制备方法,其特征在于,包括如下步骤:
(1)制备聚合物纤维骨架;
(2)将所述聚合物纤维骨架负载功能基因载体,得到功能化的聚合物纤维骨架;
(3)将所述功能化的聚合物纤维骨架植入动物皮下培育或在纤维骨架上接种细胞并培养,得到细胞化的具有优异抗凝血特性和加快内皮化特性的组织工程血管。
2.如权利要求1所述的制备方法,其特征在于,所述聚合物纤维骨架是采用熔融近场直写技术、静电纺丝技术、编织或3D打印技术制备的多孔纤维骨架。
3.如权利要求2所述的制备方法,其特征在于,所述用于制备聚合物纤维骨架的材料为聚己内酯、涤纶、聚对二氧六环己酮、聚(丙交酯-乙醇酸)共聚物、聚乙醇酸、聚氨基甲酸酯、聚癸二酸甘油酯和聚乙二醇中的一种或者多种。
4.如权利要求1所述的制备方法,其特征在于,所述聚合物纤维骨架表面经过涂层处理后,再进一步负载功能基因载体。
5.如权利要求4所述的制备方法,其特征在于,所述涂层处理的方法为,使用多聚赖氨酸溶液在所述聚合物纤维骨架表面进行聚合反应,所述多聚赖氨酸溶液的质量浓度为0.8μg/mL~500μg/mL,在聚合物纤维骨架表面形成呈正电性的涂层。
6.如权利要求5所述的制备方法,其特征在于,所述涂层处理后负载功能基因载体的方法为,使用携带抗凝和加快内皮化功能基因的慢病毒基因载体溶液与聚合物纤维骨架进行孵育,所述慢病毒基因载体通过静电亲和作用结合在聚合物纤维骨架表面。
7.如权利要求6所述的制备方法,其特征在于,所述抗凝和加快内皮化功能基因为C型钠尿肽。
8.如权利要求6的制备方法,其特征在于,所述慢病毒基因载体溶液浓度为5×104~5×108滴度/mL。
9.一种按照权利要求1~8任一项所述的制备方法得到的具有优异抗凝血和加快内皮化特性的组织工程血管。
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