大鼠骨髓干细胞种植于豚鼠脱细胞动脉构建组织工程血管的免疫原性研究

发布时间：2018-02-16 05:53

  本文关键词： 骨髓干细胞 组织工程血管 免疫原性 IgG 补体C3　出处：《新乡医学院》2012年硕士论文　论文类型：学位论文

【摘要】：背景：临床治疗中,良好的血管移植材料一直是影响血管移植手术治疗效果的重要因素,亦是相关研究的热点内容。组织工程血管(tissue-engineering blood vessels)的构建方法是选择合适的种子细胞如骨髓干细胞(bone marrow stem cell, BMSC)种植于管型支架上,从而构建成具有细胞生长的人工血管,应用于血管移植,具有较大发展潜力。但组织工程血管因选择的血管来源或种子细胞不同,其免疫原性亦有较大差别,影响手术治疗效果。 目的：将大鼠骨髓干细胞种植于脱细胞处理后的豚鼠血管上,构建简单的组织工程血管,包埋入大鼠皮下。术后定期抽取大鼠血液并取出大鼠皮下移植物,检测抗豚鼠血管IgG、补体C3血清浓度和移植物表面沉积情况,研究大鼠骨髓干细胞种植于豚鼠脱细胞动脉支架方法构建组织工程血管的免疫原性。 方法：使用密度梯度离心法结合贴壁筛选法获取大鼠骨髓干细胞,进行体外培养。鉴定为骨髓干细胞后,种植于酶-去污剂与辐照结合方法进行脱细胞处理的豚鼠血管上,构建简单的组织工程血管。选取健康纯系雄性SD大鼠200只,体重约200-250g,随机分为4组,皮下包埋不同移植物：骨髓干细胞种植组(A组,种植大鼠骨髓干细胞的脱细胞豚鼠动脉,n=50)、单纯脱细胞组(B组,单纯脱细胞的豚鼠动脉,n=50)、新鲜异种血管组(C组,新鲜豚鼠动脉,n=50)、假手术组(D组,不包埋任何标本,n=50)。术后第3d、7d、15d、30d、60d每组处死10只大鼠,获取大鼠血清并取出移植物。HE染色观察豚鼠血管脱细胞处理效果及移植物表面炎症反应强度,ELISA方法检测各组大鼠血清中抗豚鼠血管IgG和补体C3浓度,免疫组化方法检测移植物表面抗豚鼠血管IgG和补体C3沉积情况,研究其免疫原性。 结果： 1、HE染色：HE染色显示脱细胞处理的豚鼠血管为白色半透明,质地柔软,未见细胞及细胞碎片成分,纤维网结构基本完整。新鲜豚鼠血管中可见许多蓝色深染的细胞核及正常豚鼠血管结构,未见明显空白区域。 每组移植物中均可见明显炎症细胞浸润,A组及B组的炎症浸润程度明显低于C组。C组中的正常豚鼠细胞逐渐较少,至术后15d完全消失。 2、ELISA法检测血清中抗豚鼠血管IgG：A组、B组、C组大鼠血清中抗豚鼠血管IgG浓度升高。术后3d各组之间无统计学差异(A组102.59±8.87,B组100.04±8.48,C组103.49±12.8,D组101.09±13.79)μg/ml(P0.05)。术后7d、15d、30d A组(116.63±9.72,123.79±9.12,110.76±12.44)μg/ml、B组(112.77±9.63,122.54±17.15,116.23±16.49)μg/ml高于D组(98.41±16.49,102.54±10.61,97.73±9.71)μg/ml(P0.05),术后60dA组(108.47±12.70)μg/ml、B组(105.44±11.59)μg/ml与D组(100.55±16.10)μg/ml无统计学差异(P0.05)。C组于术后7d、15d、30d、60d(121.14±6.75,138.03±17.'70,141.09±18.93,133.28±16.13)uμg/ml高于D组(P0.05),于术后15d、30d、60d高于A组、B组(P0.05)。术后各时间点A组与B组无统计学差异(P0.05)。 3、ELISA法检测血清中补体C3：A组、B组、C组大鼠血清中补体C3浓度减低。术后3d各组无统计学差异(A组158.57±21.98,B组154.47±18.27,C组156.88±16.67, D组157.89±13.57)(P0.05)。术后7d、15d、30d、60d C组(130.38±16.85,106.58±11.62,105.25±10.71,113.84±11.05)μ g/ml低于A组(152.61±21.84,130.94±16.55,127.71±15.10,134.79±16.20)μ g/ml、B组(153.10±15.81,135.24±24.56,125.18±11.88,137.25±21.17)μg/ml、D组(154.45±19.69,161.74±23.78,153.33±24.41,156.90±22.20)u g/ml(P0.05)。术后15d、30d、60dA组、B组低于D组(P0.05)。术后各时间点A组与B组无统计学差异(P0.05)。 4、免疫组化法检测移植物表面抗豚鼠血管IgG沉积：各组移植物表面有明显的抗豚鼠血管IgG沉积。术后3d各组平均光密度(average optical, AO)无统计学差异(A组0.1506±0.0394,B组0.1632±0.0340,C组0.1668±0.0510)(P0.05)。术后7d、15d、30d、60d C组平均光密度(0.2157±0.0452,0.2563±0.0442,0.2752±0.0579,0.2545±0.0466)高于A组(0.1633±0.0388,0.1787±0.0532,0.1692±0.0406,0.1633±0.0282)、B组(0.1555±0.0348,0.1740±0.0364,0.1698±0.0330,0.1621±0.0349)(P0.05)。术后各时间点A组较B组均无统计学差异(P0.05)。 5、免疫组化法检测移植物表面补体C3沉积：结果显示移植物表面有明显的大鼠补体C3沉积。术后3d、7d、15d各组平均光密度无统计学差异(A组0.1304±0.0313,0.1552±0.0425,0.1612±0.0358；B组0.1316±0.0254,0.1535±0.0396,0.1603±0.0591；C组0.1671±0.0422,0.1766±0.0493,0.2401±0.0706)(P0.05)。术后30d、60d C组平均光密度(0.2674±0.0788,0.2044±0.0494)高于A组(0.1597±0.0439,0.1382±0.0299)、B组(0.1517±0.0404,0.1407±0.0288)(P0.05)。术后各时间点A组较B组无统计学差异∽0.05)。 结论： 1、实验组大鼠在进行皮下包埋手术之后,血清中抗豚鼠血管IgG浓度及补钵C3浓度均有明显变化,且在移植物表面有明显的抗豚鼠血管IgG和补体C3沉积。 2、骨髓干细胞种植组及单纯脱细胞组标本的免疫排斥反应强度明显低于新鲜异种血管组。 3、同种骨髓干细胞种植于异种脱细胞血管支架方法构建的组织工程血管,具有较低的免疫原性,可作为较好的血管移植材料构建方法。
[Abstract]:Background: clinical treatment, vascular graft material good is always an important factor affecting the treatment effect of vascular graft surgery, also is a hot topic of research. Vascular tissue engineering (tissue-engineering blood vessels) construction method is to choose suitable seed cells such as bone marrow stem cells (bone marrow stem cell, BMSC) grown in tube type the bracket, which were constructed with artificial vascular cell growth, applied to vascular transplantation, with great potential for development. But because of the choice of vascular tissue engineering blood vessels or seed cells from different sources, its immunogenicity also has a larger difference between the effect of surgical treatment.
Objective: the guinea pig vascular rat bone marrow stem cells implanted on the acellular treatment on vascular tissue engineering research is simple, embedded in the rat subcutaneous. Postoperative blood regularly from rats and remove the rat skin down plants, the detection of anti guinea pig vascular IgG, complement C3 and serum concentrations of plant surface deposition shift. Study of rat bone marrow stem cells immunogenicity in guinea pigs and planting method of cell scaffold artery construction of vascular tissue engineering.
Methods: using density gradient centrifugation method and adherence screening method for rat bone marrow stem cells were cultured in vitro. Identification of bone marrow stem cells, grown in enzyme detergent and irradiation combined with removal of guinea pig vascular cell treatment, construction of vascular tissue engineering. Simple healthy inbred male SD rats 200, weighing about 200-250g, were randomly divided into 4 groups, subcutaneous embedding of different grafts: bone marrow stem cell implant group (A group, cultivation of rat bone marrow stem cells acellular guinea pig artery, n=50), acellular group (B group, acellular guinea pig artery, n=50), fresh porcine blood group (group C, fresh guinea pig artery, n=50), sham operation group (group D, without embedding any specimens, n=50). After the 3D, 7d, 15d, 30d, 60d group of 10 rats were killed to obtain the rat serum and grafts were observed by.HE staining in vascular acellular pig the treatment effect and graft table The surface inflammatory reaction intensity, anti guinea pig vascular IgG and complement C3 ELISA methods to detect the serum concentration of rats, immunohistochemistry was used to detect vascular graft surface anti guinea pig IgG and complement C3 deposition and Study on its immunogenicity.
Result锛，

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