本文關(guān)鍵詞： 骨髓干細(xì)胞 組織工程血管 免疫原性 IgG 補(bǔ)體C3　出處：《新鄉(xiāng)醫(yī)學(xué)院》2012年碩士論文　論文類型：學(xué)位論文

【摘要】：背景：臨床治療中,良好的血管移植材料一直是影響血管移植手術(shù)治療效果的重要因素,亦是相關(guān)研究的熱點(diǎn)內(nèi)容。組織工程血管(tissue-engineering blood vessels)的構(gòu)建方法是選擇合適的種子細(xì)胞如骨髓干細(xì)胞(bone marrow stem cell, BMSC)種植于管型支架上,從而構(gòu)建成具有細(xì)胞生長(zhǎng)的人工血管,應(yīng)用于血管移植,具有較大發(fā)展?jié)摿�。但組織工程血管因選擇的血管來(lái)源或種子細(xì)胞不同,其免疫原性亦有較大差別,影響手術(shù)治療效果。 目的：將大鼠骨髓干細(xì)胞種植于脫細(xì)胞處理后的豚鼠血管上,構(gòu)建簡(jiǎn)單的組織工程血管,包埋入大鼠皮下。術(shù)后定期抽取大鼠血液并取出大鼠皮下移植物,檢測(cè)抗豚鼠血管IgG、補(bǔ)體C3血清濃度和移植物表面沉積情況,研究大鼠骨髓干細(xì)胞種植于豚鼠脫細(xì)胞動(dòng)脈支架方法構(gòu)建組織工程血管的免疫原性。 方法：使用密度梯度離心法結(jié)合貼壁篩選法獲取大鼠骨髓干細(xì)胞,進(jìn)行體外培養(yǎng)。鑒定為骨髓干細(xì)胞后,種植于酶-去污劑與輻照結(jié)合方法進(jìn)行脫細(xì)胞處理的豚鼠血管上,構(gòu)建簡(jiǎn)單的組織工程血管。選取健康純系雄性SD大鼠200只,體重約200-250g,隨機(jī)分為4組,皮下包埋不同移植物：骨髓干細(xì)胞種植組(A組,種植大鼠骨髓干細(xì)胞的脫細(xì)胞豚鼠動(dòng)脈,n=50)、單純脫細(xì)胞組(B組,單純脫細(xì)胞的豚鼠動(dòng)脈,n=50)、新鮮異種血管組(C組,新鮮豚鼠動(dòng)脈,n=50)、假手術(shù)組(D組,不包埋任何標(biāo)本,n=50)。術(shù)后第3d、7d、15d、30d、60d每組處死10只大鼠,獲取大鼠血清并取出移植物。HE染色觀察豚鼠血管脫細(xì)胞處理效果及移植物表面炎癥反應(yīng)強(qiáng)度,ELISA方法檢測(cè)各組大鼠血清中抗豚鼠血管IgG和補(bǔ)體C3濃度,免疫組化方法檢測(cè)移植物表面抗豚鼠血管IgG和補(bǔ)體C3沉積情況,研究其免疫原性。 結(jié)果： 1、HE染色：HE染色顯示脫細(xì)胞處理的豚鼠血管為白色半透明,質(zhì)地柔軟,未見(jiàn)細(xì)胞及細(xì)胞碎片成分,纖維網(wǎng)結(jié)構(gòu)基本完整。新鮮豚鼠血管中可見(jiàn)許多藍(lán)色深染的細(xì)胞核及正常豚鼠血管結(jié)構(gòu),未見(jiàn)明顯空白區(qū)域。 每組移植物中均可見(jiàn)明顯炎癥細(xì)胞浸潤(rùn),A組及B組的炎癥浸潤(rùn)程度明顯低于C組。C組中的正常豚鼠細(xì)胞逐漸較少,至術(shù)后15d完全消失。 2、ELISA法檢測(cè)血清中抗豚鼠血管IgG：A組、B組、C組大鼠血清中抗豚鼠血管IgG濃度升高。術(shù)后3d各組之間無(wú)統(tǒng)計(jì)學(xué)差異(A組102.59±8.87,B組100.04±8.48,C組103.49±12.8,D組101.09±13.79)μg/ml(P0.05)。術(shù)后7d、15d、30d A組(116.63±9.72,123.79±9.12,110.76±12.44)μg/ml、B組(112.77±9.63,122.54±17.15,116.23±16.49)μg/ml高于D組(98.41±16.49,102.54±10.61,97.73±9.71)μg/ml(P0.05),術(shù)后60dA組(108.47±12.70)μg/ml、B組(105.44±11.59)μg/ml與D組(100.55±16.10)μg/ml無(wú)統(tǒng)計(jì)學(xué)差異(P0.05)。C組于術(shù)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組(P0.05),于術(shù)后15d、30d、60d高于A組、B組(P0.05)。術(shù)后各時(shí)間點(diǎn)A組與B組無(wú)統(tǒng)計(jì)學(xué)差異(P0.05)。 3、ELISA法檢測(cè)血清中補(bǔ)體C3：A組、B組、C組大鼠血清中補(bǔ)體C3濃度減低。術(shù)后3d各組無(wú)統(tǒng)計(jì)學(xué)差異(A組158.57±21.98,B組154.47±18.27,C組156.88±16.67, D組157.89±13.57)(P0.05)。術(shù)后7d、15d、30d、60d C組(130.38±16.85,106.58±11.62,105.25±10.71,113.84±11.05)μ g/ml低于A組(152.61±21.84,130.94±16.55,127.71±15.10,134.79±16.20)μ g/ml、B組(153.10±15.81,135.24±24.56,125.18±11.88,137.25±21.17)μg/ml、D組(154.45±19.69,161.74±23.78,153.33±24.41,156.90±22.20)u g/ml(P0.05)。術(shù)后15d、30d、60dA組、B組低于D組(P0.05)。術(shù)后各時(shí)間點(diǎn)A組與B組無(wú)統(tǒng)計(jì)學(xué)差異(P0.05)。 4、免疫組化法檢測(cè)移植物表面抗豚鼠血管IgG沉積：各組移植物表面有明顯的抗豚鼠血管IgG沉積。術(shù)后3d各組平均光密度(average optical, AO)無(wú)統(tǒng)計(jì)學(xué)差異(A組0.1506±0.0394,B組0.1632±0.0340,C組0.1668±0.0510)(P0.05)。術(shù)后7d、15d、30d、60d C組平均光密度(0.2157±0.0452,0.2563±0.0442,0.2752±0.0579,0.2545±0.0466)高于A組(0.1633±0.0388,0.1787±0.0532,0.1692±0.0406,0.1633±0.0282)、B組(0.1555±0.0348,0.1740±0.0364,0.1698±0.0330,0.1621±0.0349)(P0.05)。術(shù)后各時(shí)間點(diǎn)A組較B組均無(wú)統(tǒng)計(jì)學(xué)差異(P0.05)。 5、免疫組化法檢測(cè)移植物表面補(bǔ)體C3沉積：結(jié)果顯示移植物表面有明顯的大鼠補(bǔ)體C3沉積。術(shù)后3d、7d、15d各組平均光密度無(wú)統(tǒng)計(jì)學(xué)差異(A組0.1304±0.0313,0.1552±0.0425,0.1612±0.0358；B組0.1316±0.0254,0.1535±0.0396,0.1603±0.0591；C組0.1671±0.0422,0.1766±0.0493,0.2401±0.0706)(P0.05)。術(shù)后30d、60d C組平均光密度(0.2674±0.0788,0.2044±0.0494)高于A組(0.1597±0.0439,0.1382±0.0299)、B組(0.1517±0.0404,0.1407±0.0288)(P0.05)。術(shù)后各時(shí)間點(diǎn)A組較B組無(wú)統(tǒng)計(jì)學(xué)差異∽0.05)。 結(jié)論： 1、實(shí)驗(yàn)組大鼠在進(jìn)行皮下包埋手術(shù)之后,血清中抗豚鼠血管IgG濃度及補(bǔ)缽C3濃度均有明顯變化,且在移植物表面有明顯的抗豚鼠血管IgG和補(bǔ)體C3沉積。 2、骨髓干細(xì)胞種植組及單純脫細(xì)胞組標(biāo)本的免疫排斥反應(yīng)強(qiáng)度明顯低于新鮮異種血管組。 3、同種骨髓干細(xì)胞種植于異種脫細(xì)胞血管支架方法構(gòu)建的組織工程血管,具有較低的免疫原性,可作為較好的血管移植材料構(gòu)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錛，

本文編號(hào)：1514810