摘要
背景:猪异种移植过程中可能发生猪内源性反转录病毒跨种间传播的潜在危险,由此所带来的异种移植生物安全性问题逐渐受到关注。目的:观察去细胞过程对猪膀胱内源性反转录病毒的影响,以评价猪源性膀胱无细胞基质生物工程材料在异种移植中的生物安全性。设计、时间及地点:基因工程与组织工程动物体内实验,于2006-07/2007-03在中国医学科学院北京协和医院中心实验室完成。材料:正常健康雄性杂种犬8只,随机分为对照组2只,修补组6只,用于膀胱修补替代实验。取材0.5h内的猪新鲜膀胱标本采集自屠宰厂。方法:采用低渗-去污剂洗涤-核酸酶消化法对新鲜猪膀胱进行脱细胞处理,制备膀胱无细胞基质移植物。对照组犬切除50%膀胱后原位直接缝合,不使用任何材料修补;修补组犬切除50%膀胱后,分别用面积约为原膀胱30%,40%,50%的膀胱无细胞基质移植物进行替代修补。分别于移植前及移植后1个月抽取犬静脉血提取RNA和DNA,以猪Beta-actin序列片段扩增产物作为内参照,针对内源性反转录病毒gag区序列,选用特异性引物,采用PCR和RT-PCR法检测上述提取的DNA和RNA是否存在内源性反转录病毒序列片段,并对扩增产物进行测序用于同源性分析。主要观察指标:苏木精-伊红染色观察猪膀胱去细胞效果及DNA含量。PCR及RT-PCR扩增及测序结果。结果:经去细胞处理后,猪膀胱内的细胞成分完全去除,三维纤维支架保持完整,DNA含量低于1%。新鲜猪膀胱PCR和RT-PCR均检测到362bp扩增条带,与Medline公布的内源性反转录病毒有94%的同源性;所制备的生物工程材料膀胱无细胞基质移植物、及其移植前后的犬外周血样本均未检测到内源性反转录病毒序列的表达。结论:低渗-去污剂洗涤-核酸酶消化法制备的膀胱无细胞基质移植物可以去除猪膀胱内源性反转录病毒,能够有效预防内源性反转录病毒在
BACKGROUND： There are potential risks for interspecies spreading of porcine endogenous retrovirus during xenotransplantation. Therefore, the safety of xenotransplantation becomes a focus. OBJECTIVE： To observe the biologic safety of porcine endogenous retrovirus in xenotransplantation with porcine bladder acellular matrix. DESIGN, TIME AND SETTING： Gene engineering and tissue engineering in vivo animal trials were performed at Central Laboratory of Peking Union Medical College Hospital, Chinese Academy of Medical Sciences between July 2006 and March 2007. MATERIALS： Eight healthy male hybrid dogs were randomly divided into control group （n=2） and experimental group （n=6） for bladder acellular matrix grafting. Fresh porcine bladder samples within 0.5 hour were harvested from butchery. METHODS： A hypotension and detergent and nucleic acid enzymatic digestion extraction process was used to remove all cellular components from the porcine bladder to prepare bladder acellular matrix graft. The dogs in control group underwent semi-cystectomy and in situ suturation. The experimental animals underwent 30%, 40% and 50% bladder acellular matrix grafting after semi-cystectomy. Blood was drawn before and 1 month after transplantation to extract RNA and DNA. With porcine Beta-actin sequence fragment amplified product as internal control, porcine endogenous retrovirus gag sequences were detected in 6 fresh porcine bladder and 6 bladder acellular matrix grafts and 5 dog peripheral blood with postgraft bladder acellular matrix graft by polymerase chain reaction （PCR） and reverse transcription polymerase chain reaction （RT-PCR） using special specific primers. MAIN OUTCOME MEASURES： Acellular effect and DNA contents were observed using HE staining. PCR and RT-PCR amplification and sequencing results were observed. RESULTS： All cell components were removed from the bladder acellular matrix. Histological analysis showed that the major structure components were maintained. The contents of the DNA in bladd
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2008年第40期7845-7849,共5页
Journal of Clinical Rehabilitative Tissue Engineering Research
