改良去细胞方法对猪源性逆转录病毒的影响  

Effects of modified acellularization process on porcine endogenous retrovirus in porcine aorta valves

作　　者：顾春虎[1] 刘维永[1] 易定华[1] 王云雅[1] 张近宝[1] 卫向阳[1] 欧阳辉[1] 刘星光[1] 谭红梅[1] 

机构地区：[1]第四军医大学西京医院心脏外科,西安710032

出　　处：《中华医学杂志》2005年第26期1827-1830,共4页National Medical Journal of China

基　　金：国家自然科学基金资助项目(30271292)

摘　　要：目的观察改良的去细胞方法对猪主动脉瓣膜的猪源性逆转录病毒的影响。方法用0.1%胰酶消化猪主动脉瓣后,再用TritonX100DNA酶/RNA酶洗脱消化,结合溶液渗透压改变等去除细胞;取新鲜猪瓣、猪外周血标本和去细胞猪瓣各20只,降主动脉内移植去细胞猪瓣后的犬外周血10例,以PK15细胞系为对照,采用PCR和RTPCR法检测猪源性逆转录病毒。结果经去细胞处理后猪瓣内的细胞成分完全去除,纤维支架和力学性能保持良好;新鲜猪瓣及猪外周血标本均检测到219bp的扩增条带,与Medline公布的猪源性逆转录病毒有90%～95%同源性;去细胞猪瓣及移植了去细胞猪瓣犬的外周血,均未测到该病毒。结论改良的去细胞方法可以去除猪心瓣膜的猪源性逆转录病毒,能够有效预防该病毒物种间的交叉感染。Objective to study the effect of modified acellularization process on porcine endogenous retrovirus (PERV) in porcine aorta valves (PAVs).Methods Twenty aortic valves of pig were put into 0.1% trypsin solution, hypotonic and hypertonic TritonX-100, DNAse solution, RNAse solution, and Hanks solution in succession so as to remove the cells. The specimens of PAV were to undergo gross observation and microscopy before and after the acellularization procedure. Fracture test was made. Primers specific for the conservative gag gene of PERV were designed PCR and RT-PCR were used to detect the expression of gag. In addition, 20 samples of native PAV were collected. Peripheral mononuclear cells (PBMCs). Were isolated from 20 samples of porcine peripheral blood. Ten dogs underwent acellularized PAV replacement; 3 months later, samples of the dogs' peripheral blood were collected. Porcine kidney cells of the line PK15 were used as positive controls. Results Microscopy showed that all the cells were removed from the acellularized PAVs. Histological analysis showed that the major structural components were maintained. There was no significant difference in fracture strength between the native and acellularized PAVs (P>0.05). PCR and RT-PCR showed a PERV 219 bp DNA fragment, 90%~95% homologous with the published PERV gene, in the genomic DNA of all native PAVs, pig PBMCs, and PK15 cells, but not in the acellularized PAVs and dog PBMCs. Conclusion PERV exists in all native PAVs. The modified acellularization process succeeds in removing all the cell component and PERV in the PAVs, thus preventing cross-species transmission of PERV.

关 键 词：逆转录病毒 改良 Medline 去细胞猪 PCR法检测 主动脉瓣膜 猪主动脉瓣 胰酶消化 RNA酶 DNA酶 主动脉内 细胞成分 力学性能 纤维支架 猪心瓣膜 交叉感染 有效预防 血标本 外周血 去除 渗透压 细胞系 RT- 同源性 新鲜 移植 

分 类 号：Q939.4[生物学—微生物学]