CN114681662A - 重组胶原蛋白在制备止血材料中的应用 - Google Patents
重组胶原蛋白在制备止血材料中的应用 Download PDFInfo
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Abstract
本发明公开了一种重组胶原蛋白在制备止血材料中的应用。本发明采用保藏编号为CGMCC No.5021的巴斯德毕赤酵母Pichia pastoris发酵产生的重组胶原蛋白，其能显著减少组织损伤出血时间和出血量，显著减轻肝脏病理损伤，下调肝脏损伤造成的谷草转氨酶、谷丙转氨酶、乳酸脱氢酶、白细胞介素6、肿瘤坏死因子α、转化生长因子‑β、直接胆红素和总胆红素含量，具有较好的抗肝出血和较好的肝损伤修复作用。
Description
技术领域
本发明属于医学材料技术领域，涉及一种重组胶原蛋白在制备止血材料中的应用。
背景技术
人体止血机制包括血管收缩、血小板和血液凝固。血管收缩、血小板聚集局部形成血栓并释放大量促凝物使血液凝固止血。轻度出血能通过压迫止血，但有些如医学手术过程中的重度创伤出血，仅靠压迫止血和人体自然修复几乎不可能。
止血粉剂是一种粉末状材料，能够吸收组织溢泌物与血液中的止血成分相结合，具有良好的止血修复性能。目前通常使用的止血粉主要为壳聚糖止血粉，虽然壳聚糖能够较快的止血，但是止血后，创伤周围可能会出现炎细胞浸润。
胶原蛋白是临床常用的止血材料，具有高拉伸强度、生物相容性、生物降解性、低刺激性、低抗原性、低细胞毒性以及促进细胞增殖分化等特点，是一种具有良好性能的生物材料，目前在医学界应用十分广泛。
目前使用的胶原蛋白止血材料来源主要包括动物来源的胶原蛋白或基因工程菌产生的重组胶原蛋白。动物来源的胶原在使用过程中面临免疫原性的问题，限制其在临床上的应用。基因工程菌产生的重组人源胶原蛋白解决了免疫原性的问题。中国专利申请CN111187347A公开了一种重组胶原蛋白、重组胶原海绵材料及其制备方法和应用，该重组胶原蛋白吸湿性高、止血效果显著、生物相容性佳，但其应用时需经物理交联和化学交联才能避免材料在体内残留过久引起的炎症风险。
发明内容
本发明的目的在于提供了一种重组胶原蛋白在制备止血材料中的应用。
本发明中，所述的重组胶原蛋白由保藏编号为CGMCC No.5021的巴斯德毕赤酵母Pichia pastoris发酵产生，已在中国专利201110327865.5充分公开。
本发明中，所述的止血材料为本领域常规的止血材料，包括但不限于止血粉、止血海绵、止血敷料等。
本发明中，所述的止血材料为创伤止血材料。
在本发明的具体实施方式中，采用的止血材料为重组胶原蛋白止血粉。
本发明的重组胶原蛋白止血粉具有比表面积大、吸水性好的特点。采用的重组胶原蛋白，其基因序列完全来源于人胶原基因序列，由毕赤酵母基因工程菌构建表达，无内毒素，亲水性佳，能快速吸附于创面，具有良好的止血作用，能显著减少组织损伤出血时间和出血量，显著减轻肝脏病理损伤，下调肝脏损伤造成的谷草转氨酶、谷丙转氨酶、乳酸脱氢酶、白细胞介素6、肿瘤坏死因子α、转化生长因子-β、直接胆红素和总胆红素含量，具有较好的抗肝出血和较好的肝损伤修复作用，且不引起炎症反应。
附图说明
图1为各实验组止血后，大鼠肝脏粘连情况图。
图2为各实验组病理切片HE染色结果图。
图3为各实验组对大鼠肝脏损伤后的病理学改变评分统计图，mean±SD，n＝8。
图4为各实验组的大鼠肝脏肝功能指标ALT、AST和炎症因子IL-6、TNF-α和TGF-β及DBIL、TBIL含量的影响结果图，means±SD，n＝8，**P<0.01，与空白组比较；**P<0.01，与模型组比较。
具体实施方式
下面结合具体实施例和附图对本发明作进一步详述。
实施例1
一、重组胶原蛋白对大鼠肝脏出血模型的止血实验
(1)取SD大鼠48只，雄性，随机分为4组，每组12只，分别为空白组、受试药(重组胶原蛋白)组、模型组和阳性对照组。
(2)大鼠禁食12h后，以3％水合氯醛腹腔注射麻醉(1mL/100g)，腹部备皮，5％碘酒消毒手术区域。
(3)用手术刀在大鼠腹部中正作一2.5cm纵行切口，逐层分离，充分暴露肝脏右叶，在肝右叶下缘切取一1cm×0.2cm×0.3cm肝组织。
(4)提前备好无菌纱布，并使用电子天平测量纱布初始重量。
(5)切除肝脏组织后，模型组立即使用无菌纱布吸取流出的血液，打开计时器计时。
(6)阳性对照组在创面迅速均匀涂抹壳聚糖止血粉(约0.1g每只)后，使用无菌纱布吸取流出的血液，并打开计时器计时。
(7)受试药组在创面迅速敷用重组胶原蛋白固体(约0.1g每只)后，使用无菌纱布吸取流出的血液，并打开计时器计时。
(8)各组待出血完全停止时，终止计时器，此时的时间即为各组的出血时间。
(9)测量各组无菌纱布的最终重量，减去各自的初始重量，即为各组的出血量。
(10)术后将肝脏回纳至腹中，用缝线逐层关闭腹腔，5％碘酒消毒创面。
(11)每只大鼠单笼饲养，术后3d，每日腹腔注射每只10万单位青霉素钠，观察大鼠有无感染征兆。
(12)一周后大鼠均存活，未出现明显感染情况。将所有大鼠再次麻醉，腹主动脉取血，试剂盒测量血清中肝功能和炎症因子水平。
二、实验结果及分析
1、病理切片分析
(1)切取肝脏创面愈合组织，经10％甲醛固定、乙醇脱水、石蜡包埋等过程后，使用切片机将肝脏组织切成5μm厚度切片进行保存。
(2)待染色时将切片脱蜡水化，使用HE染色，经乙醇脱水、二甲苯透明、中性树胶进行封片后，在显微镜下观察病理切片。
3、炎症因子水平分析
(1)腹主动脉取血，3000rpm离心10min，收集血清，贮存于-80℃低温冰箱，用于生化法检测ALT(谷丙转氨酶)、AST(谷草转氨酶)含量及ELISA法检测IL-6(白介素-6)、TNF-α(肿瘤坏死因子-α)、TGF-β(转化生长因子-β)、LDH(乳酸脱氢酶)、DBIL(直接胆红素)及TBIL(总胆红素)含量。
(2)数据均数±标准差(SD)表示。采用SPSS25.0进行统计学分析。采用单因素ANOVA法分析各组间大鼠肝脏出血时间、出血重量数据及生化和ELISA试剂盒数据。P<0.05时的比较差异被认为是显著的。
由图1可知，肝脏切除一周后，模型组肝脏与周边组织出现了明显的粘连，而重组人胶原蛋白和壳聚糖止血粉治疗后肝脏粘连现象显著减少，说明重组胶原蛋白和壳聚糖均具有较好的肝脏保护作用。
表1受试药重组胶原蛋白对大鼠肝脏出血时间和出血重量的影响(mean±SD，n＝8)
注：*P<0.05,**P<0.01，与模型组比较.
由表1可知，与模型组相比，重组胶原蛋白止血粉和壳聚糖止血粉均能显著减少大鼠肝脏出血时间(p<0.05,p<0.01)和出血重量(p<0.01)，说明重组胶原蛋白和壳聚糖均具有较好的抗凝血和肝脏保护作用。
图2为各实验组病理切片HE染色结果图，黑色箭头指示坏死灶，红色箭头指示炎症细胞浸润。由图2的HE染色及图3的病理评分结果可知，与空白组相比，模型组大鼠肝脏肝细胞排列紊乱，细胞轻度水肿，可见较多散在分布的小坏死灶。汇管区及肝小叶内见较多炎症细胞浸润伴局灶淋巴细胞聚集，并见较多肉芽组织增生，同时出现了大量的炎细胞浸润，阳性药壳聚糖止血粉组偶见小静脉旁炎细胞浸润，而受试药重组胶原蛋白止血粉组未见明显异常。病理评分结果显示模型组与空白对照组有显著性差异(p<0.01)，重组胶原蛋白止血粉组与模型组也有显著性差异(p<0.05)，然而阳性药壳聚糖止血粉的病理评分与模型组相比无显著性差异，说明重组胶原蛋白能有效改善大鼠肝脏损伤后的病理学变化，且效果优于阳性药壳聚糖止血粉。
由图4可见，与空白组相比，模型组大鼠血清中肝功能指标ALT、AST、DBIL及TBIL含量显著上升，同时模型组大鼠血清中炎症因子IL-6、TNF-α和TGF-β显著上升，LDH含量显著上升；阳性药壳聚糖止血粉及受试药重组胶原蛋白止血粉均能下调上述指标，并且重组胶原蛋白止血粉效果略优于壳聚糖止血粉，但无显著性差异。
本发明采用重组胶原蛋白与目前市面上常用的壳聚糖止血粉进行比较，创面出血时间以及出血量直观证明了重组胶原蛋白的止血效果。此外，通过观察大鼠术后肝脏的组织学变化，检测肝脏肝功能指标谷草转氨酶(AST)和谷丙转氨酶(ALT)(两者的异常升高是诊断肝炎的标准)、乳酸脱氢酶(LDH，肝损伤后常显著或中度增加)、白细胞介素6(IL-6)、肿瘤坏死因子α(TNF-α)、转化生长因子-β(TGF-β)(炎症因子，两者的异常升高是诊断肝炎的标准)、直接胆红素(DBIL)和总胆红素(TBIL)(两者是最常见的肝生化指标之一)含量等指标，从定性与定量两方面证实了重组胶原蛋白对机体的影响与壳聚糖止血粉相似，能够缓解肝出血及肝损伤，但不同于壳聚糖，重组胶原蛋白不会造成明显的不良反应。
综上所述，本发明的重组胶原蛋白能够显著减少大鼠肝损伤的出血时间和出血量，显著减轻肝脏病理损伤，下调肝脏损伤造成的AST、ALT、LDH、IL-6、TNF-α、TGF-β、DBIL和TBIL含量，显示出较好的抗肝出血及肝损伤修复作用，并且相比于壳聚糖止血粉，重组胶原蛋白能有效改善大鼠肝脏损伤后的病理学变化，效果优于壳聚糖。
Claims (4)
1.重组胶原蛋白在制备止血材料中的应用，其特征在于，所述的重组胶原蛋白由保藏编号为CGMCC No.5021的巴斯德毕赤酵母Pichia pastoris发酵产生。
2.根据权利要求1所述的应用，其特征在于，所述的止血材料为止血粉、止血海绵或止血敷料。
3.根据权利要求1所述的应用，其特征在于，所述的止血材料为创伤止血材料。
4.根据权利要求1所述的应用，其特征在于，所述的止血材料为重组胶原蛋白止血粉。
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