RNA干扰抑制猪内源性逆转录病毒表达及其多克隆抗体制备的研究
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摘要
第一部分兔抗猪内源性逆转录病毒P15E多克隆抗体的制备
研究背景和目的
猪器官、组织和细胞作为临床移植的供体源具有广泛和良好的应用前景,可缓解临床器官移植的器官相对短缺；也可为临床治疗1型胰岛素依赖型糖尿病,帕金森病、亨廷顿病和癫痛病等顽固性神经疾患提供一种治疗方法。猪肝细胞也可作为猪肝细胞型生物人工肝的生物成分治疗急性爆发性肝炎肝功能衰竭和终末期肝功能衰竭的过渡性措施。自Dr.Clivepatienee(BioTransplantxnc首席科学家)1997年报道猪内源性逆转求病毒(porcine endogenous retrovirus, PERV)感染体外培养人源细胞以来,猪异种移植或猪肝细胞型生物人工肝治疗过程中可能发生PERV种间传播的潜在危险引起众多学者的广泛关注。
Patience等首先证实至少有三种PERV亚型在体外培养能够感染人源细胞,Takeuchi和Martin等也证实PERV i可以感染体外培养的人源细胞株。
1998年,Denner J等报道逆转录病毒的一个结构蛋白即跨膜蛋白P15E是一个活性成分,在体内和体外可以抵制免疫反应；P15E的检测可以作为检测PERV表达及其是否具有感染性的一个诊断指标。国内及国际抗体市场上并未见应用于检测PERV P15E的特异性抗体,本研究在国外报道的PERV P15E抗原位点的基础上,设计并制作了兔抗猪PERV P15E多克隆抗体,该抗体用于检测PERV P15E在蛋白方面的表达水平具有很高的灵敏度及特异笥。该抗体的研发,对检测临床器官移植和猪肝细胞型生物人工肝中PERV的表达和传播具有重要意义。
方法
根据文献报告获得了PERV P15E抗原表位E1和E2蛋白序列,再根据其蛋白序列反推获得了P15E E1和E2的核酸序列。P15E核酸序列最小长序317bp,为增强其抗原性以及表位的完整性,我们将P15E前方100bp核酸序列一起扩增,通过PCR扩增方法扩增得到了P15E编码区序列,然后将其连接到PGEX-4T-1载体上,转化到大肠杆菌BL21(DE3)中,再通过自身诱导法诱导表达重组质粒的融合蛋白通过割胶回收纯化融合蛋白。免疫新西兰大白兔制备多克隆抗体,Western blot不ELISA检测抗体活性和滴度。
结果
兔抗猪PERV P15E多克隆抗体可以特异性检测P15E抗原蛋白以及猪肾细胞系PK15中P15E蛋白的表达,而人肾细胞系HEK293中则检测不到该蛋白表达,证实本抗体特异性强。ELISA检测兔抗猪PERV P15E多克隆抗体滴度效价达到了1：100.000。
结论
本研究所获得的兔抗猪PERV P15E多克隆抗体不仅效价高,而且特异性强,可以作为检测临床器官移植和猪肝细胞型生物人工肝中PERV表达和传播的重要工具。
第二部分逆转录病毒介导的RNA干扰抑制西藏小型猪成纤维细胞中PERV表达的研究
研究背景和目的
异种移植在治疗人类疾病方面提供了极为丰富的器官、组织和细胞来源。因为猪来源方便,易于繁育,与人体解剖和生理有很高的相似性,可选择合适大小的器官和丰富且功能良好肝细胞,成为了异种移植和生物人工肝的首选。然而,对于异种移植和生物人工肝而言,作为异种来源的猪器官、组织和细胞,其内存在猪内源性逆转录病毒(porcine endogenous retrovirus, PERV), PERV是一种γ逆转录病毒,它整合于猪的染色体中:根据猪种的不同,其PERV拷贝数和mRNA的表达量也有很大差异。目前用于抵制PERV表达的猪成纤维细胞均来源于具有高PERVmRNA表达背景的德国大白猪,经RNA干扰技术进行基因沉默后,虽然其表达量下降,但是由于RNA干扰技术存在“脱靶效应”,所以这种具有PERV mRNA高表达背景的核供体细胞在一定程度上仍然具有较高的潜在感染风险。
为了获得一个极低量PERV表达的猪成纤维细胞,为下一步建立转基因猪提供安全的核供体细胞。本研究选用具有PERV mRNA低表达的中国西藏小型猪成纤维细胞,同时利用RNA干扰技术,获得了稳定表达shRNA且具有PERV极低量表达的猪成纤维细胞,为下一步建立转基因猪提供了更为安全的核供体细胞。
方法
针对高度保守的PERV-gag和PERV-pol区域,设计siRNA并构建于pSUPERretro-GFP/Neo表达载体后转染PERV-HEK293细胞,并利用实时荧光定量RT-PCR、Western blot和细胞免疫化学检测PERV表变化,筛选获得最有效的shRNA片段通过逆转录病毒表达载体导入西藏小型猪成纤维细胞中,并进行抗性筛选;最后在利用实时荧光定量RT-PCR、Western blot(?)细胞免疫化学检测阳性克隆西藏小型猪成纤维细胞中PERV表达量。数据处理：所得实验数据用均数±标准差(x±SD)表示,取检验性显著水准a=0.05,用SPSS13.0统计软件进行统计学分析,对实时荧光定量RT-PCR结果进行单因素方差分析、样本方差齐性时选用LSD法进行组间多重比较,方差不齐时则选用Tamhane's T2法进行比较以及独立样本t检验进行两样本均数的比较。
结果
建立了表达PERV的PERV-HEK293细胞模型,该细胞模型不仅在mRNA水平表达PERV,而且在基因组也整合有PERV基因shRNA转染PERV-HEK293细胞后,利用SPSS13.0软件对其实时荧光定量RT-PCR结果进行单因素方差分析显示：shRNA转染影响PERV-HEK293细胞中PERV表达(F=583.823,P<0.001),组间多重比较(Tamhane'sT2法)显示：shRNA-pol3转染PERV-HEK293细胞后PERVmRNA相对表达量为(16.733±0.961)%与阴性对照组(91.467±6.312)%,相比具有显著差异性(P<0.001)；PERV-HEK293细胞中PERV蛋白表达也显著下降；shRNA-pol3经逆转录病毒载体导入西藏小型猪成纤维细胞后,表现出较高的干扰效率,两样本独立t检验分析显示：干扰后的西藏小型猪成纤维细胞与未干扰的西藏小型猪成纤维细胞相比具有显著差异性(t=27.032,P<0.001),其PERV P15E蛋白表达也显著下降。
结论
稳定表达shRNA的西藏小型猪成纤维细胞具有了较前更低的PERV表达量,为我们下一步培育稳定表达shRNA的转基因猪提供了更为安全核供体细胞。
第三部分多重RNA干扰技术抑制中国实验用小型猪成纤维细胞中PERV表达的研究
研究背景和目的
猪细胞和组织中存在有猪内源性逆转录病毒(porcine endogenous retrovirus, PERV)。体外培养实验和感染性实验已经证实PERV可以感染人源细胞。Van der Laan等将猪胰岛细胞移植于非肥胖性糖尿病SCID鼠腹腔中,并检测鼠组织PERV mRNA的表达,结果发现多种组织发生PERV感染,这是移植猪组织后种间交叉感染、体内PERV主动复制的首次报道。
为了最大程度地抑制PERV表达和传播的可能性,人们采用了不同策略,如前面第二部分所提及利用RNAF扰技术去抑制PERV mRNA和蛋白表达,是目前抑制PERV表达的首选方法；目前学者多通过病毒载体将siRNA导入猪成纤维细胞中进行干扰,达到siRNA在细胞中稳定表达,如Dieckhoff等利用慢病毒载体将siRNA导入猪成纤维细胞中对PERV进行干扰：我们在前期也利用逆转录病毒载体将siRNA导入细胞中进行PERV干扰,然而,我们在干扰PERV表达的同时又将携带siRNA的病毒载体导入了细胞,从而引起了另一种安全隐患。慢病毒载体被认为是目前转染效率最高的载体,广泛应用于基因治疗、细胞工程等领域。但由于慢病毒载体整合位点的不确定性,可能会导致插入突变甚至激活原癌基因。Howe SJ等报道了在接受慢病毒介导基因治疗的10名SCID-X1患者,因携慢病毒载体将调控序列和正常基因整合在LMO-2基因上游35kb处,导致LMO-2过表达造成急性T淋巴细胞性白血病。尽管研究者已通过多种方法来提高慢病毒载体的安全性,如改变LTR序列、采用灭活病毒、分离结构蛋白基因与包装基因等,但是仍不能完全排除致瘤的可能性。Nyberg, S.L等曾报道过生物人工肝治疗过程中,治疗用细胞进入患者体内。细胞的变形运动、治疗过程中滤过膜破损都可能导致永生化细胞进入患者体内,永生化细胞裂解后释放出基因片段也可能进入患者血浆中。可见在进行细胞基因修饰的过程中,病毒载体的利用带来了极大的安全隐患。
为了达到不使用病毒载体又可以稳定高效的抑制PERV之达的目的,我们利用了Perv-silencer表达载体,该载体具有4个不同哺乳动物启动子(hU6、hH1、mU6和h7SK),4个启动子分别操纵的shRNA转录结构单元(启动子-shRNA(?)义链—loop环-shRNA反义链—终止信号),不同启动子转录各自相应的shRNA结构单元,有效避免意外重组,而且每条shRNA能够有效转录。实现了一个载体编码4条shRNA,可以起到了对PERV-gag不PERV-pol区域的多重干扰。此外,在猪成纤维细胞的选择上,我们在前期研究的基础上选取具有PERV mRNA(?)表达且不含PERV-C的中国实验用小型猪成纤维细胞；针对PERV-gag和-pol的高度保守区域,设计并筛选到了4个干扰效率最高siRNAs片段,将其构建于同一表达载体后,利用电穿孔技术将其导入中国实验用小型猪成纤维细胞中,通过抗性筛选,进而获得可以稳定抑制PERV表达的猪成纤维细胞,为下一步建立转基因猪提供了更为安全的核供体细胞。
方法
在本研究中,我们针对PERV高度保守的gag和pol区域,设计了8个small interfering RNA (siRNA)片段,将8个siRNA片段直接转染PK15细胞,进行PERV干扰,利用实时荧光定量RT-PCR、Western blot筛选出了能够抑制PERV表达最有效的4个siRNA片段,将这4个siRNA片段构建于同一载体perv-Silencer中,通过电穿孔技术导入PK15细胞进行多重干扰的初步验证,利用实时荧光定量RT-PCR和细胞免疫荧光验证PERV表达变化；经前面筛选和验证后,将重组表达载体通过电穿孔技术稳定转染到了中国实验用小型猪成纤维细胞中进行多重siRNA干扰,利用实时荧光定量RT-PCR、Western blot和细胞免疫荧光检测其PERV表达量。将获得的经基因修饰后的中国实验用小型猪成纤维细胞培养上清与HEK293细胞共培养,并通过RT-PCR检测共培养后的HEK293细胞中PERV表达。数据处理：所得实验数据用均数±标准差(x±SD)表示,取检验性显著水准a=0.05,用SPSS13.0统计软件进行统计学分析,对实时荧光定量RT-PCR结果进行单因素方差分析、样本方差齐性时选用LSD法进行组间多重比较,方差不齐时则选用Tamhane's T2法进行比较。
结果
利用SPSS13.0软件对其实时荧光定量RT-PCR结果进行单因素方差分析显示:siRNA直接转染影响PK15细胞中PERV表达(F=690.784,P<0.001),组间多重比较(Tamhane's T2法)显示：siRNA-pol2, siRNA-po13, siRNA-gagl和siRNA-gag4转染PK15细胞后PERVmRNA相对表达量分别为：(17.967±0.779)%,(17.917±1.094)%,(16.067±1.041)%,(18.100±0.832)%,与阴性对照组(98.317±1.074)%,相比具有显著差异性(P<0.001)；其相对应的PERV P15E蛋白水平也显著下降；重组Perv-silencer表达载体转染PK15细胞后,对PK15细胞中PERV的表达具有很好的干扰效果,实时荧光定量RT-PCR结果进行单因素方差分析显示：重组Perv-silencer表达载体转染影响PK15细胞中PERV表达(F=25615.576,P<0.001),组间多重比较(LSD法)显示：重组Perv-silencer表达载体转染PK15细胞后PERV-gag和PERV-pol mRNA相对表达量分别为：(10.317±0.591)%和(9.733±0.568)%,与阴性对照组(98.317±1.074)%,相比具有显著差异性(P<0.001)；免疫荧光检测其P15E蛋白表达也显著下降；重组Perv-silencer表达载体稳定转染中国实验用小型猪成纤维细胞后,实时荧光定量RT-PCR结果进行单因素方差分析显示：重组Perv-silencer表达载体稳定转染影响中国实验用小型猪成纤维细胞中PERV表达(F=5131.238,P<0.001),组间多重比较(Tamhane'sT2法)显示：重组Perv-silencer表达载体转染中国实验用小型猪成纤维细胞后PERV-gag和PERV-pol mRNA相对表达量分别为：(7.100±1.037)%和(7.650±1.118)%,与阴性对照组(96.112±2.623)%,相比具有显著差异性(P<0.001)；Western blot和免疫荧光检测其P15E蛋白显著下降；基因修饰后的中国实验用小型猪成纤维细胞培养上清液与HEK293细胞共培养结果显示HEK293细胞未感染PERV。
结论
本实难获得的稳定表达siRNA的中国实验用小型猪成纤维细胞具有极低量的PERV表达,且没有PERV-A/C感染的风险；由于本实验中未使用病毒载体,所以也避免了病毒载体进入细胞中所引起的安全隐患,可以作为下一步培育更为安全的转基因猪的核供体细胞。
Part I Preparation of rabbit anti-porcine endogenous retrovirus P15E polyclonal antibody
Background and Objection
The organs, tissues, and cells of swine have broad prospects been used as a donor source in clinical transplant. This has helped overcome the shortage of organs available for clinical transplant and provide a clinical treatment of some resistant nerve disorders just like the type-I insulin-dependent diabetes mellitus. Parkinson's disease, Huntington's disease, epilepsy and so on. The hepatocytes can also be used as a biotic component in the swine hepatocyte-based biartificial liver to treat the acute fulminant hepatitis liver function failure and a transitional measure to end-stage liver failure. Since1997, Dr. Clivepatienee (Bio Transplantxnc. Chief scientist) had reported that the porcine endogenous retrovirus (PERV) had infected the human cells cultured in vitro, it has attracted widespread attention from scholars and practitioners about the potential risk of PERV infection during swine xenotransplantation or hepatocyte-based biartificial liver.
It was Patience who first confirmed that there are at least three subtypes of PERV that can infect the human cells in vitro. Takeuchi. Martin and some other scholars also prove that PERV can infect human cell lines in vitro.
Since1998, Denner J had reported that one of the viral structural proteins, the transmembrane envelope protein (p15E), is the active component, and that purified p15E is able to inhibit a whole range of in yitro and in vivo immune responses. Besides, in the detection of PERV protein expression levels, no specific antibodies, that can detect the PERV P15E, can be seen in both domestic and international antibody market. Based on the PERV P15E antigen site reported abroad, this study designed and produced the polyclonal antibody of rabbit anti-pig PERV P15E, which has high sensibility and specificity in the detection of PERV protein expression levels. The research and development of this antibody will have prominent importance on detecting the expression and transmission of PERV in clinical organ transplantation and swine hepatocytc-based biartificial liver.
Methods
We got the epitopes of E1and E2protein sequences through literatures, based on which we traced back to the nucleic acid sequence of E1and E2, of P15E. The minimum length of P15E nucleic acid sequence is317bp. In order to enhance the antigencity of the nucleic acid sequence and the integrity of the epitope. we amplified100p nucleic acid of the anterior of P15E. and got the sequence of P15E coding region by PCR. After that, we connected it into the PEGX-4T-1vector, transforming it to the colon bacillus BL21(DE3).Through the self-induction method, the BL21(DE3) can express the fusion protein in recombinant plasmid, and then purified the protein through rubber tapping and recycling. Finally, we immunized the New Zealand rabbits with the fusion protein to prepare polyclonal antibodies and detected the activity and titer with Western blot and ELISΔ.
Results
The rabbit-anti-pig polyclonal antibody can specifically detect the expression of P15E antigenic protein and P15E protein in PK15cells. However, the expression of P15E cannot be detected in HEK.293cells. which can prove the high specificity of the rabbit-anti-pig polyclonal antibody. Detection using ELISA revealed that the antibody titer drop degree is higher than1:100,000.
Conclusion
The rabbit-anti-pig polyclonal antibody obtained in this study, possess high titer as well as specificity. It can become essential implement in detecting the expression and transmission of PERV in clinical organ transplant or swine hepatocyte-based biartificial liver.
Part II Retrovirus-mediated RNA interference inhibition of PERV expression in Tibetan miniature pig fibroblasts
Background and Objection
Xenotransplants provide rich alternative sources of organs, tissues and cells that can be potentially used for the treatment of human diseases. Pig have become the first choice for xenotransplantation and nonhuman cell source provider for bioartificial liver because they are readily available and of high similarity in anatomy and physiology to human beings. However, the porcine endogenous retrovirus (PERV) was expressed in all of pig's organs. tissues and cells. PERV is a gamma retrovirus that is integrated into porcine chromosomes. and large variations of its expression have been observed among the different pig breeds. The pig fibroblasts currently used are derived from the pig breeds which have a high background expression of PERV. Although PERV expression levels are significantly reduced after gene silencing via RNAi. these nuclear donor cells, which have a high expression of PERV, still present a highly potential risk for infection with human beings due to the "off-target effects" of RNAi technology.
In order to obtain a pig fibroblasts with an extremely low PERV expression, preparing a safe nuclear donor cells for the next step to create transgenic pigs. We selected Chinese Tibetan miniature pig fibroblasts which have a low PERV mRNA expression. Meanwhile. applying the RNAi technology to obtain the pig fibroblasts that stably expressed the shRNA and the PERV at very low level. These modified pig fibroblasts may serve as safer nuclear donor cells for the generation of transgenic pigs.
Methods
We designed the small interfering RNAs (siRNAs) that target the highly conserved PERV-gag and PERV-pol domains. Then we constructed them into the expression vector pSUPERretro-GFP/Neo and transfected the PERV-HEK293cells. Quantitative real-time RT-PCR, Western blot and Immunocytochemistry are used to detect the change of PERV expression. We selected the most effective shRNA fragment and inserted it into the Tibetan miniature pig fibroblasts by retroviral expression vector for the resistance screening. At last, we applied the Quantitative real-time RT-PCR, Western blot and Immunocytochemistry to detect the PERV expression in positive clones Tibetan miniature pig fibroblasts. Date processing: Results were expressed as mean values士standard deviation(x±SD).A valtie of a=0.05was considered the standard of inspectability significant.and SPSS13.0software was used for statistics analysis.The results of Quantitative real-time RT-PCR were analysed by one-way ANOVA.LSD method was used For multiple comparisons between groups for homogeneity of variance; otherwise Tamhane's T2method was used.The independent samples T test was for two samples mean comparison.
Results
We constructed the PERV-HEK293cell model expressing PERV gene,and this model can not only express PERV on the mRNA level,but also integrated in the genome.After transfecting the PERV-HEK293cells with shRNA. we utilized the SPSS13.0t0analyze the result of Quantitative real-time RT-PCR.One-way ANOVA showed that the transfection of shRNA can affect the PERV expression in PERV-HEK293cells (F=583.823,P<0.001). Groups multiple comparison(Tamhane's, T2method)indicated that after the transfection of shRNA-pol3to PERV-HEK293cells,the relative PERV mRNA expression was (16.733±0.961)%.compared with negative control(91.467±6.312)%.indicating significant variation(P<0.001).The PERV protein expression in PERV-HEK293cells also decreased significantly.After inserted the shRNA-pol3into the Tibetan miniature pig fibroblasts by retrovirus vector,the results indicated high intcrference emciency of shRNA.Independent samples T test showed that there was a significant variation between the modified Tibetan miniature pig fibroblasts and wild fibroblasts (t=27.032,P<0.001).The PERV P15E protein expression in modified Tibetan miniature pig fibroblasts also decreased significantly.
Conclusion
The Tibetan miniature pig fibroblasts. stably expressed shRNA. was expressed lower PERV than before. They can serve as nuclear donor cells for the generation of safer transgenic pigs that stably express shRNA.
Part III Knockdown of porcine endogenous retrovirus expression by multiple RNA interference in Chinese Experimental Miniature Pig fibroblasts
Background and Objection
Swine tissues and cells contain porcine endogenous retrovirus (PERV). Multiple experiments, such as in vitro culture and infection-based examination. have confirmed that PERV can infect human cells. Van der Laan et al. transplanted swine islet cells into the enterocoelia of non-obese diabetic SCID mice and examined the level of PERV mRNA expression. They found that different types of tissues were infected with PERV, and theirs is considered the first report on cross-species infection and active internal replication of PERV following swine xenotransplantation.
To minimize the possibility of the transmission of PERV scientists have used different strategies to inhibit or eliminate PERV expression. The best choice for inhibit the PERV mRNA and protein expression is to use RNA interference technology, as it mentioned in Part II. Many scientists using the virus vector to insert the siRNA into the pig fibroblas, making it expressed steadily in cells. For example, Dieckhoff etc. used the lentiviral vector to insert siRNA into the pig fibroblasts to inhibit the PERV expression. However, the viral vector was inserted into the cell the time we try to inhibit the PERV expression, which will bring about another kind of potential risk. The lentiviral vector is thought to be the most effective vector in transfection. widely used in genetic therapy. cell engineering and so on. However, as the uncertainty of the virus vector integration sites. it may result that the inserted gene's mutation or even activation of the oncogene. Howe SJ etc. reported that the10SCID-Ⅸ patients got the acute lymphoblastic leukemia. They were accepting the genetic therapy mediated by virus, and the letiviral vector caused the integration of the normal gene and the regulatory sequences in the upstream of LMO-2,35kb. Although many methods have been used to improve the safety of the letiviral vector by scholars, for example, altering the LTR sequences, inactivating the virus, separating the structural protein gene, packaging gene and so on. We can still not exclude the potential risk of tumorigenicity. Neyberg. S.L. etc. once reported that through the therapy of biartiticial liver, the cells for treatment can go into our bodies. The deformation movement of cells, filtration membrane damaged in the course of treatment can cause the immortal cells entering the human body. On the other hand, the gene segments released by the cells after splitting can go into the plasma of the patients. As we can see. viral vector bring about great potential risk in the modified gene cells.
In order to inhibit the PERV expression stably and efficiently without the viral vector, we used the Perv-silencer expressing vector which has four promoters from different mammal species (hU6, hHl, mU6and h7SK). The different promoters were responsible for transcription of the corresponding shRNA structure unit, prevention of accidental recombinant, and effective transcription of every strand of shRNA. Thus, a single vector encoding four strands of shRNA and multiple interfering PERV-gag and PERV-pol domains was obtained. Besides, in the selection of pig fibroblasts, we chose the Chinese experimental miniature pig fibroblasts on the basis of our previous studies. The Chinese experimental miniature pig fibroblasts are with low PERV mRNA express and absence of the PERV-C provirus. We selected four siRNAs that target the PERV-gag and PERV-pol conserved domains:these siRNAs have the highest interference efficiency. The four siRNAs were integrated into a single vector and inserted into the selected fibroblasts by electroporation.The fibroblasts that were found to stably suppress PERV expression by resistance screening were used as a more secure nuclear donor cells for cultivate the transgenic pigs.
Methods
In this study, we designed8small interfering RNAs (siRNAs) that target the PERV-gag and PERV-pol conserved domains. We used them to transfect the PK15cells directly. Meanwhile, we used the Quantitative real-time RT-PCR and Western blot selecting four siRNAs which display the highest efficiency in inhibiting the expression of PERV. Then we constructed them into one single vector which was inserted into PK15cells via electroporation to validate the multiple RNA interference preliminarily. Quantitative real-time RT-PCR and immunofluorescence experiment were used to testify the variation of PERV expression. After previous screening and testing, we inserted the recombinant vector into the Chinese experimental miniature pig fibroblasts for multiple RNA interference. Quantitative real-time RT-PCR, Western blot and immunofluorescence were used to detect the PERV expression. We co-cultivated the HEK293cells with the supernate of the modified Chinese experimental miniature pig fibroblasts and detected the PERV expression of HEK293after co-cultivation by RT-PCR. Data processing:Results were expressed as mean values±standard deviation (x±SD), A value of a=0.05was considered the standard of inspectability significant. We used the SPSS13.0software for statistics analysis. The results of Quantitative real-time RT-PCR were analysed by one-way ANOVA. LSD method was used for multiple comparisons between groups for homogeneity of variance:otherwise Tamhane's T2method was used.
Results
We utilized the SPSS13.0to analyze the result of Quantitative real-time RT-PCR. One-way ANOVA showed that directly transfection of siRNA can affect the PERV expression in PK15cells (F-690.784, P<0.001). Groups multiple comparison(Tamhane's, T2method) indicated that after transfection of siRNA-pol2, siRNA-po13, siRNA-gagl and siRNA-gag4to PK15cells, the relative PERV mRNA expression were (17.967±0.779)%,(17.917±1.094)%,(16.367±1.041)%,(18.100±0.832)%, compared with negative control (98.317±1.074)%, indicating significant variation (P<0.001). The PERV protein expression in PK15cells also decreased significantly. The PERV expression in PK15cells had been disturbed greatly after being transfected with the recombinant Perv-silencer expressing vector. Using the one-way ANOVA to analyze the results of Quantitative real-time RT-PCR demonstrated that transfecting PK15cells with recombinant Perv-silencer expressing vector affected the PERV expression (F=25615.576,P<0.001), Groups multiple comparison (LSD method) showed that after transfection with recombinant Perv-silencer expression vector to PK15cells, the relative PERV-gag and PERV-pol mRNA expression were (10.317±0.591)%and (9.733±0.568)%, compared with negative control (98.317±1.074)%. indicating significant variation (P<0.001).Immunofluorescence detection of its P15E protein expression was also significantly decreased; Finally, we stably transfected Chinese experimental miniature pig fibroblasts with recombinant Perv-silencer expression vector, using the one-way ANOVA to analyze the results of Quantitative real-time RT-PCR.The results showed that recombinant Perv-silencer expression vector can affect PERV expression in Chinese experimental miniature pig fibroblasts (F=5131.238, P<0.001), Groups multiple comparison (Tamhane's. T2method) displayed that After Chinese experimental miniature pig fibroblasts were transfected with recombinant Perv-silencer expression vectors. the relative PERV-gag和PERV-pol mRNA expression were (7.100±1.037)%and (7.650±1.118)%. compared with negative control (96.112±2.623)%. indicating significant variation (P<0.001):Western blot and immunofluorescence detection of the P15E protein decreased significantly. No HEK293cells were infected with PERV when co-cultured with the supernate of the gene modified pig fibroblasts.
Conclusion
The Chinese experimental miniature pig fibroblasts, obtained in this study, presented lower level of PERV expression compared with original cells and avoided the risk of PERV-A/C infection. Because no viral vectors were used in this experiment, so we avoided the potential risk of the viral vector going into the cells. Hence, they can serve as nucleus donor cells for transgenic pigs in subsequent studies.
研究背景和目的
猪器官、组织和细胞作为临床移植的供体源具有广泛和良好的应用前景,可缓解临床器官移植的器官相对短缺；也可为临床治疗1型胰岛素依赖型糖尿病,帕金森病、亨廷顿病和癫痛病等顽固性神经疾患提供一种治疗方法。猪肝细胞也可作为猪肝细胞型生物人工肝的生物成分治疗急性爆发性肝炎肝功能衰竭和终末期肝功能衰竭的过渡性措施。自Dr.Clivepatienee(BioTransplantxnc首席科学家)1997年报道猪内源性逆转求病毒(porcine endogenous retrovirus, PERV)感染体外培养人源细胞以来,猪异种移植或猪肝细胞型生物人工肝治疗过程中可能发生PERV种间传播的潜在危险引起众多学者的广泛关注。
Patience等首先证实至少有三种PERV亚型在体外培养能够感染人源细胞,Takeuchi和Martin等也证实PERV i可以感染体外培养的人源细胞株。
1998年,Denner J等报道逆转录病毒的一个结构蛋白即跨膜蛋白P15E是一个活性成分,在体内和体外可以抵制免疫反应；P15E的检测可以作为检测PERV表达及其是否具有感染性的一个诊断指标。国内及国际抗体市场上并未见应用于检测PERV P15E的特异性抗体,本研究在国外报道的PERV P15E抗原位点的基础上,设计并制作了兔抗猪PERV P15E多克隆抗体,该抗体用于检测PERV P15E在蛋白方面的表达水平具有很高的灵敏度及特异笥。该抗体的研发,对检测临床器官移植和猪肝细胞型生物人工肝中PERV的表达和传播具有重要意义。
方法
根据文献报告获得了PERV P15E抗原表位E1和E2蛋白序列,再根据其蛋白序列反推获得了P15E E1和E2的核酸序列。P15E核酸序列最小长序317bp,为增强其抗原性以及表位的完整性,我们将P15E前方100bp核酸序列一起扩增,通过PCR扩增方法扩增得到了P15E编码区序列,然后将其连接到PGEX-4T-1载体上,转化到大肠杆菌BL21(DE3)中,再通过自身诱导法诱导表达重组质粒的融合蛋白通过割胶回收纯化融合蛋白。免疫新西兰大白兔制备多克隆抗体,Western blot不ELISA检测抗体活性和滴度。
结果
兔抗猪PERV P15E多克隆抗体可以特异性检测P15E抗原蛋白以及猪肾细胞系PK15中P15E蛋白的表达,而人肾细胞系HEK293中则检测不到该蛋白表达,证实本抗体特异性强。ELISA检测兔抗猪PERV P15E多克隆抗体滴度效价达到了1：100.000。
结论
本研究所获得的兔抗猪PERV P15E多克隆抗体不仅效价高,而且特异性强,可以作为检测临床器官移植和猪肝细胞型生物人工肝中PERV表达和传播的重要工具。
第二部分逆转录病毒介导的RNA干扰抑制西藏小型猪成纤维细胞中PERV表达的研究
研究背景和目的
异种移植在治疗人类疾病方面提供了极为丰富的器官、组织和细胞来源。因为猪来源方便,易于繁育,与人体解剖和生理有很高的相似性,可选择合适大小的器官和丰富且功能良好肝细胞,成为了异种移植和生物人工肝的首选。然而,对于异种移植和生物人工肝而言,作为异种来源的猪器官、组织和细胞,其内存在猪内源性逆转录病毒(porcine endogenous retrovirus, PERV), PERV是一种γ逆转录病毒,它整合于猪的染色体中:根据猪种的不同,其PERV拷贝数和mRNA的表达量也有很大差异。目前用于抵制PERV表达的猪成纤维细胞均来源于具有高PERVmRNA表达背景的德国大白猪,经RNA干扰技术进行基因沉默后,虽然其表达量下降,但是由于RNA干扰技术存在“脱靶效应”,所以这种具有PERV mRNA高表达背景的核供体细胞在一定程度上仍然具有较高的潜在感染风险。
为了获得一个极低量PERV表达的猪成纤维细胞,为下一步建立转基因猪提供安全的核供体细胞。本研究选用具有PERV mRNA低表达的中国西藏小型猪成纤维细胞,同时利用RNA干扰技术,获得了稳定表达shRNA且具有PERV极低量表达的猪成纤维细胞,为下一步建立转基因猪提供了更为安全的核供体细胞。
方法
针对高度保守的PERV-gag和PERV-pol区域,设计siRNA并构建于pSUPERretro-GFP/Neo表达载体后转染PERV-HEK293细胞,并利用实时荧光定量RT-PCR、Western blot和细胞免疫化学检测PERV表变化,筛选获得最有效的shRNA片段通过逆转录病毒表达载体导入西藏小型猪成纤维细胞中,并进行抗性筛选;最后在利用实时荧光定量RT-PCR、Western blot(?)细胞免疫化学检测阳性克隆西藏小型猪成纤维细胞中PERV表达量。数据处理：所得实验数据用均数±标准差(x±SD)表示,取检验性显著水准a=0.05,用SPSS13.0统计软件进行统计学分析,对实时荧光定量RT-PCR结果进行单因素方差分析、样本方差齐性时选用LSD法进行组间多重比较,方差不齐时则选用Tamhane's T2法进行比较以及独立样本t检验进行两样本均数的比较。
结果
建立了表达PERV的PERV-HEK293细胞模型,该细胞模型不仅在mRNA水平表达PERV,而且在基因组也整合有PERV基因shRNA转染PERV-HEK293细胞后,利用SPSS13.0软件对其实时荧光定量RT-PCR结果进行单因素方差分析显示：shRNA转染影响PERV-HEK293细胞中PERV表达(F=583.823,P<0.001),组间多重比较(Tamhane'sT2法)显示：shRNA-pol3转染PERV-HEK293细胞后PERVmRNA相对表达量为(16.733±0.961)%与阴性对照组(91.467±6.312)%,相比具有显著差异性(P<0.001)；PERV-HEK293细胞中PERV蛋白表达也显著下降；shRNA-pol3经逆转录病毒载体导入西藏小型猪成纤维细胞后,表现出较高的干扰效率,两样本独立t检验分析显示：干扰后的西藏小型猪成纤维细胞与未干扰的西藏小型猪成纤维细胞相比具有显著差异性(t=27.032,P<0.001),其PERV P15E蛋白表达也显著下降。
结论
稳定表达shRNA的西藏小型猪成纤维细胞具有了较前更低的PERV表达量,为我们下一步培育稳定表达shRNA的转基因猪提供了更为安全核供体细胞。
第三部分多重RNA干扰技术抑制中国实验用小型猪成纤维细胞中PERV表达的研究
研究背景和目的
猪细胞和组织中存在有猪内源性逆转录病毒(porcine endogenous retrovirus, PERV)。体外培养实验和感染性实验已经证实PERV可以感染人源细胞。Van der Laan等将猪胰岛细胞移植于非肥胖性糖尿病SCID鼠腹腔中,并检测鼠组织PERV mRNA的表达,结果发现多种组织发生PERV感染,这是移植猪组织后种间交叉感染、体内PERV主动复制的首次报道。
为了最大程度地抑制PERV表达和传播的可能性,人们采用了不同策略,如前面第二部分所提及利用RNAF扰技术去抑制PERV mRNA和蛋白表达,是目前抑制PERV表达的首选方法；目前学者多通过病毒载体将siRNA导入猪成纤维细胞中进行干扰,达到siRNA在细胞中稳定表达,如Dieckhoff等利用慢病毒载体将siRNA导入猪成纤维细胞中对PERV进行干扰：我们在前期也利用逆转录病毒载体将siRNA导入细胞中进行PERV干扰,然而,我们在干扰PERV表达的同时又将携带siRNA的病毒载体导入了细胞,从而引起了另一种安全隐患。慢病毒载体被认为是目前转染效率最高的载体,广泛应用于基因治疗、细胞工程等领域。但由于慢病毒载体整合位点的不确定性,可能会导致插入突变甚至激活原癌基因。Howe SJ等报道了在接受慢病毒介导基因治疗的10名SCID-X1患者,因携慢病毒载体将调控序列和正常基因整合在LMO-2基因上游35kb处,导致LMO-2过表达造成急性T淋巴细胞性白血病。尽管研究者已通过多种方法来提高慢病毒载体的安全性,如改变LTR序列、采用灭活病毒、分离结构蛋白基因与包装基因等,但是仍不能完全排除致瘤的可能性。Nyberg, S.L等曾报道过生物人工肝治疗过程中,治疗用细胞进入患者体内。细胞的变形运动、治疗过程中滤过膜破损都可能导致永生化细胞进入患者体内,永生化细胞裂解后释放出基因片段也可能进入患者血浆中。可见在进行细胞基因修饰的过程中,病毒载体的利用带来了极大的安全隐患。
为了达到不使用病毒载体又可以稳定高效的抑制PERV之达的目的,我们利用了Perv-silencer表达载体,该载体具有4个不同哺乳动物启动子(hU6、hH1、mU6和h7SK),4个启动子分别操纵的shRNA转录结构单元(启动子-shRNA(?)义链—loop环-shRNA反义链—终止信号),不同启动子转录各自相应的shRNA结构单元,有效避免意外重组,而且每条shRNA能够有效转录。实现了一个载体编码4条shRNA,可以起到了对PERV-gag不PERV-pol区域的多重干扰。此外,在猪成纤维细胞的选择上,我们在前期研究的基础上选取具有PERV mRNA(?)表达且不含PERV-C的中国实验用小型猪成纤维细胞；针对PERV-gag和-pol的高度保守区域,设计并筛选到了4个干扰效率最高siRNAs片段,将其构建于同一表达载体后,利用电穿孔技术将其导入中国实验用小型猪成纤维细胞中,通过抗性筛选,进而获得可以稳定抑制PERV表达的猪成纤维细胞,为下一步建立转基因猪提供了更为安全的核供体细胞。
方法
在本研究中,我们针对PERV高度保守的gag和pol区域,设计了8个small interfering RNA (siRNA)片段,将8个siRNA片段直接转染PK15细胞,进行PERV干扰,利用实时荧光定量RT-PCR、Western blot筛选出了能够抑制PERV表达最有效的4个siRNA片段,将这4个siRNA片段构建于同一载体perv-Silencer中,通过电穿孔技术导入PK15细胞进行多重干扰的初步验证,利用实时荧光定量RT-PCR和细胞免疫荧光验证PERV表达变化；经前面筛选和验证后,将重组表达载体通过电穿孔技术稳定转染到了中国实验用小型猪成纤维细胞中进行多重siRNA干扰,利用实时荧光定量RT-PCR、Western blot和细胞免疫荧光检测其PERV表达量。将获得的经基因修饰后的中国实验用小型猪成纤维细胞培养上清与HEK293细胞共培养,并通过RT-PCR检测共培养后的HEK293细胞中PERV表达。数据处理：所得实验数据用均数±标准差(x±SD)表示,取检验性显著水准a=0.05,用SPSS13.0统计软件进行统计学分析,对实时荧光定量RT-PCR结果进行单因素方差分析、样本方差齐性时选用LSD法进行组间多重比较,方差不齐时则选用Tamhane's T2法进行比较。
结果
利用SPSS13.0软件对其实时荧光定量RT-PCR结果进行单因素方差分析显示:siRNA直接转染影响PK15细胞中PERV表达(F=690.784,P<0.001),组间多重比较(Tamhane's T2法)显示：siRNA-pol2, siRNA-po13, siRNA-gagl和siRNA-gag4转染PK15细胞后PERVmRNA相对表达量分别为：(17.967±0.779)%,(17.917±1.094)%,(16.067±1.041)%,(18.100±0.832)%,与阴性对照组(98.317±1.074)%,相比具有显著差异性(P<0.001)；其相对应的PERV P15E蛋白水平也显著下降；重组Perv-silencer表达载体转染PK15细胞后,对PK15细胞中PERV的表达具有很好的干扰效果,实时荧光定量RT-PCR结果进行单因素方差分析显示：重组Perv-silencer表达载体转染影响PK15细胞中PERV表达(F=25615.576,P<0.001),组间多重比较(LSD法)显示：重组Perv-silencer表达载体转染PK15细胞后PERV-gag和PERV-pol mRNA相对表达量分别为：(10.317±0.591)%和(9.733±0.568)%,与阴性对照组(98.317±1.074)%,相比具有显著差异性(P<0.001)；免疫荧光检测其P15E蛋白表达也显著下降；重组Perv-silencer表达载体稳定转染中国实验用小型猪成纤维细胞后,实时荧光定量RT-PCR结果进行单因素方差分析显示：重组Perv-silencer表达载体稳定转染影响中国实验用小型猪成纤维细胞中PERV表达(F=5131.238,P<0.001),组间多重比较(Tamhane'sT2法)显示：重组Perv-silencer表达载体转染中国实验用小型猪成纤维细胞后PERV-gag和PERV-pol mRNA相对表达量分别为：(7.100±1.037)%和(7.650±1.118)%,与阴性对照组(96.112±2.623)%,相比具有显著差异性(P<0.001)；Western blot和免疫荧光检测其P15E蛋白显著下降；基因修饰后的中国实验用小型猪成纤维细胞培养上清液与HEK293细胞共培养结果显示HEK293细胞未感染PERV。
结论
本实难获得的稳定表达siRNA的中国实验用小型猪成纤维细胞具有极低量的PERV表达,且没有PERV-A/C感染的风险；由于本实验中未使用病毒载体,所以也避免了病毒载体进入细胞中所引起的安全隐患,可以作为下一步培育更为安全的转基因猪的核供体细胞。
Part I Preparation of rabbit anti-porcine endogenous retrovirus P15E polyclonal antibody
Background and Objection
The organs, tissues, and cells of swine have broad prospects been used as a donor source in clinical transplant. This has helped overcome the shortage of organs available for clinical transplant and provide a clinical treatment of some resistant nerve disorders just like the type-I insulin-dependent diabetes mellitus. Parkinson's disease, Huntington's disease, epilepsy and so on. The hepatocytes can also be used as a biotic component in the swine hepatocyte-based biartificial liver to treat the acute fulminant hepatitis liver function failure and a transitional measure to end-stage liver failure. Since1997, Dr. Clivepatienee (Bio Transplantxnc. Chief scientist) had reported that the porcine endogenous retrovirus (PERV) had infected the human cells cultured in vitro, it has attracted widespread attention from scholars and practitioners about the potential risk of PERV infection during swine xenotransplantation or hepatocyte-based biartificial liver.
It was Patience who first confirmed that there are at least three subtypes of PERV that can infect the human cells in vitro. Takeuchi. Martin and some other scholars also prove that PERV can infect human cell lines in vitro.
Since1998, Denner J had reported that one of the viral structural proteins, the transmembrane envelope protein (p15E), is the active component, and that purified p15E is able to inhibit a whole range of in yitro and in vivo immune responses. Besides, in the detection of PERV protein expression levels, no specific antibodies, that can detect the PERV P15E, can be seen in both domestic and international antibody market. Based on the PERV P15E antigen site reported abroad, this study designed and produced the polyclonal antibody of rabbit anti-pig PERV P15E, which has high sensibility and specificity in the detection of PERV protein expression levels. The research and development of this antibody will have prominent importance on detecting the expression and transmission of PERV in clinical organ transplantation and swine hepatocytc-based biartificial liver.
Methods
We got the epitopes of E1and E2protein sequences through literatures, based on which we traced back to the nucleic acid sequence of E1and E2, of P15E. The minimum length of P15E nucleic acid sequence is317bp. In order to enhance the antigencity of the nucleic acid sequence and the integrity of the epitope. we amplified100p nucleic acid of the anterior of P15E. and got the sequence of P15E coding region by PCR. After that, we connected it into the PEGX-4T-1vector, transforming it to the colon bacillus BL21(DE3).Through the self-induction method, the BL21(DE3) can express the fusion protein in recombinant plasmid, and then purified the protein through rubber tapping and recycling. Finally, we immunized the New Zealand rabbits with the fusion protein to prepare polyclonal antibodies and detected the activity and titer with Western blot and ELISΔ.
Results
The rabbit-anti-pig polyclonal antibody can specifically detect the expression of P15E antigenic protein and P15E protein in PK15cells. However, the expression of P15E cannot be detected in HEK.293cells. which can prove the high specificity of the rabbit-anti-pig polyclonal antibody. Detection using ELISA revealed that the antibody titer drop degree is higher than1:100,000.
Conclusion
The rabbit-anti-pig polyclonal antibody obtained in this study, possess high titer as well as specificity. It can become essential implement in detecting the expression and transmission of PERV in clinical organ transplant or swine hepatocyte-based biartificial liver.
Part II Retrovirus-mediated RNA interference inhibition of PERV expression in Tibetan miniature pig fibroblasts
Background and Objection
Xenotransplants provide rich alternative sources of organs, tissues and cells that can be potentially used for the treatment of human diseases. Pig have become the first choice for xenotransplantation and nonhuman cell source provider for bioartificial liver because they are readily available and of high similarity in anatomy and physiology to human beings. However, the porcine endogenous retrovirus (PERV) was expressed in all of pig's organs. tissues and cells. PERV is a gamma retrovirus that is integrated into porcine chromosomes. and large variations of its expression have been observed among the different pig breeds. The pig fibroblasts currently used are derived from the pig breeds which have a high background expression of PERV. Although PERV expression levels are significantly reduced after gene silencing via RNAi. these nuclear donor cells, which have a high expression of PERV, still present a highly potential risk for infection with human beings due to the "off-target effects" of RNAi technology.
In order to obtain a pig fibroblasts with an extremely low PERV expression, preparing a safe nuclear donor cells for the next step to create transgenic pigs. We selected Chinese Tibetan miniature pig fibroblasts which have a low PERV mRNA expression. Meanwhile. applying the RNAi technology to obtain the pig fibroblasts that stably expressed the shRNA and the PERV at very low level. These modified pig fibroblasts may serve as safer nuclear donor cells for the generation of transgenic pigs.
Methods
We designed the small interfering RNAs (siRNAs) that target the highly conserved PERV-gag and PERV-pol domains. Then we constructed them into the expression vector pSUPERretro-GFP/Neo and transfected the PERV-HEK293cells. Quantitative real-time RT-PCR, Western blot and Immunocytochemistry are used to detect the change of PERV expression. We selected the most effective shRNA fragment and inserted it into the Tibetan miniature pig fibroblasts by retroviral expression vector for the resistance screening. At last, we applied the Quantitative real-time RT-PCR, Western blot and Immunocytochemistry to detect the PERV expression in positive clones Tibetan miniature pig fibroblasts. Date processing: Results were expressed as mean values士standard deviation(x±SD).A valtie of a=0.05was considered the standard of inspectability significant.and SPSS13.0software was used for statistics analysis.The results of Quantitative real-time RT-PCR were analysed by one-way ANOVA.LSD method was used For multiple comparisons between groups for homogeneity of variance; otherwise Tamhane's T2method was used.The independent samples T test was for two samples mean comparison.
Results
We constructed the PERV-HEK293cell model expressing PERV gene,and this model can not only express PERV on the mRNA level,but also integrated in the genome.After transfecting the PERV-HEK293cells with shRNA. we utilized the SPSS13.0t0analyze the result of Quantitative real-time RT-PCR.One-way ANOVA showed that the transfection of shRNA can affect the PERV expression in PERV-HEK293cells (F=583.823,P<0.001). Groups multiple comparison(Tamhane's, T2method)indicated that after the transfection of shRNA-pol3to PERV-HEK293cells,the relative PERV mRNA expression was (16.733±0.961)%.compared with negative control(91.467±6.312)%.indicating significant variation(P<0.001).The PERV protein expression in PERV-HEK293cells also decreased significantly.After inserted the shRNA-pol3into the Tibetan miniature pig fibroblasts by retrovirus vector,the results indicated high intcrference emciency of shRNA.Independent samples T test showed that there was a significant variation between the modified Tibetan miniature pig fibroblasts and wild fibroblasts (t=27.032,P<0.001).The PERV P15E protein expression in modified Tibetan miniature pig fibroblasts also decreased significantly.
Conclusion
The Tibetan miniature pig fibroblasts. stably expressed shRNA. was expressed lower PERV than before. They can serve as nuclear donor cells for the generation of safer transgenic pigs that stably express shRNA.
Part III Knockdown of porcine endogenous retrovirus expression by multiple RNA interference in Chinese Experimental Miniature Pig fibroblasts
Background and Objection
Swine tissues and cells contain porcine endogenous retrovirus (PERV). Multiple experiments, such as in vitro culture and infection-based examination. have confirmed that PERV can infect human cells. Van der Laan et al. transplanted swine islet cells into the enterocoelia of non-obese diabetic SCID mice and examined the level of PERV mRNA expression. They found that different types of tissues were infected with PERV, and theirs is considered the first report on cross-species infection and active internal replication of PERV following swine xenotransplantation.
To minimize the possibility of the transmission of PERV scientists have used different strategies to inhibit or eliminate PERV expression. The best choice for inhibit the PERV mRNA and protein expression is to use RNA interference technology, as it mentioned in Part II. Many scientists using the virus vector to insert the siRNA into the pig fibroblas, making it expressed steadily in cells. For example, Dieckhoff etc. used the lentiviral vector to insert siRNA into the pig fibroblasts to inhibit the PERV expression. However, the viral vector was inserted into the cell the time we try to inhibit the PERV expression, which will bring about another kind of potential risk. The lentiviral vector is thought to be the most effective vector in transfection. widely used in genetic therapy. cell engineering and so on. However, as the uncertainty of the virus vector integration sites. it may result that the inserted gene's mutation or even activation of the oncogene. Howe SJ etc. reported that the10SCID-Ⅸ patients got the acute lymphoblastic leukemia. They were accepting the genetic therapy mediated by virus, and the letiviral vector caused the integration of the normal gene and the regulatory sequences in the upstream of LMO-2,35kb. Although many methods have been used to improve the safety of the letiviral vector by scholars, for example, altering the LTR sequences, inactivating the virus, separating the structural protein gene, packaging gene and so on. We can still not exclude the potential risk of tumorigenicity. Neyberg. S.L. etc. once reported that through the therapy of biartiticial liver, the cells for treatment can go into our bodies. The deformation movement of cells, filtration membrane damaged in the course of treatment can cause the immortal cells entering the human body. On the other hand, the gene segments released by the cells after splitting can go into the plasma of the patients. As we can see. viral vector bring about great potential risk in the modified gene cells.
In order to inhibit the PERV expression stably and efficiently without the viral vector, we used the Perv-silencer expressing vector which has four promoters from different mammal species (hU6, hHl, mU6and h7SK). The different promoters were responsible for transcription of the corresponding shRNA structure unit, prevention of accidental recombinant, and effective transcription of every strand of shRNA. Thus, a single vector encoding four strands of shRNA and multiple interfering PERV-gag and PERV-pol domains was obtained. Besides, in the selection of pig fibroblasts, we chose the Chinese experimental miniature pig fibroblasts on the basis of our previous studies. The Chinese experimental miniature pig fibroblasts are with low PERV mRNA express and absence of the PERV-C provirus. We selected four siRNAs that target the PERV-gag and PERV-pol conserved domains:these siRNAs have the highest interference efficiency. The four siRNAs were integrated into a single vector and inserted into the selected fibroblasts by electroporation.The fibroblasts that were found to stably suppress PERV expression by resistance screening were used as a more secure nuclear donor cells for cultivate the transgenic pigs.
Methods
In this study, we designed8small interfering RNAs (siRNAs) that target the PERV-gag and PERV-pol conserved domains. We used them to transfect the PK15cells directly. Meanwhile, we used the Quantitative real-time RT-PCR and Western blot selecting four siRNAs which display the highest efficiency in inhibiting the expression of PERV. Then we constructed them into one single vector which was inserted into PK15cells via electroporation to validate the multiple RNA interference preliminarily. Quantitative real-time RT-PCR and immunofluorescence experiment were used to testify the variation of PERV expression. After previous screening and testing, we inserted the recombinant vector into the Chinese experimental miniature pig fibroblasts for multiple RNA interference. Quantitative real-time RT-PCR, Western blot and immunofluorescence were used to detect the PERV expression. We co-cultivated the HEK293cells with the supernate of the modified Chinese experimental miniature pig fibroblasts and detected the PERV expression of HEK293after co-cultivation by RT-PCR. Data processing:Results were expressed as mean values±standard deviation (x±SD), A value of a=0.05was considered the standard of inspectability significant. We used the SPSS13.0software for statistics analysis. The results of Quantitative real-time RT-PCR were analysed by one-way ANOVA. LSD method was used for multiple comparisons between groups for homogeneity of variance:otherwise Tamhane's T2method was used.
Results
We utilized the SPSS13.0to analyze the result of Quantitative real-time RT-PCR. One-way ANOVA showed that directly transfection of siRNA can affect the PERV expression in PK15cells (F-690.784, P<0.001). Groups multiple comparison(Tamhane's, T2method) indicated that after transfection of siRNA-pol2, siRNA-po13, siRNA-gagl and siRNA-gag4to PK15cells, the relative PERV mRNA expression were (17.967±0.779)%,(17.917±1.094)%,(16.367±1.041)%,(18.100±0.832)%, compared with negative control (98.317±1.074)%, indicating significant variation (P<0.001). The PERV protein expression in PK15cells also decreased significantly. The PERV expression in PK15cells had been disturbed greatly after being transfected with the recombinant Perv-silencer expressing vector. Using the one-way ANOVA to analyze the results of Quantitative real-time RT-PCR demonstrated that transfecting PK15cells with recombinant Perv-silencer expressing vector affected the PERV expression (F=25615.576,P<0.001), Groups multiple comparison (LSD method) showed that after transfection with recombinant Perv-silencer expression vector to PK15cells, the relative PERV-gag and PERV-pol mRNA expression were (10.317±0.591)%and (9.733±0.568)%, compared with negative control (98.317±1.074)%. indicating significant variation (P<0.001).Immunofluorescence detection of its P15E protein expression was also significantly decreased; Finally, we stably transfected Chinese experimental miniature pig fibroblasts with recombinant Perv-silencer expression vector, using the one-way ANOVA to analyze the results of Quantitative real-time RT-PCR.The results showed that recombinant Perv-silencer expression vector can affect PERV expression in Chinese experimental miniature pig fibroblasts (F=5131.238, P<0.001), Groups multiple comparison (Tamhane's. T2method) displayed that After Chinese experimental miniature pig fibroblasts were transfected with recombinant Perv-silencer expression vectors. the relative PERV-gag和PERV-pol mRNA expression were (7.100±1.037)%and (7.650±1.118)%. compared with negative control (96.112±2.623)%. indicating significant variation (P<0.001):Western blot and immunofluorescence detection of the P15E protein decreased significantly. No HEK293cells were infected with PERV when co-cultured with the supernate of the gene modified pig fibroblasts.
Conclusion
The Chinese experimental miniature pig fibroblasts, obtained in this study, presented lower level of PERV expression compared with original cells and avoided the risk of PERV-A/C infection. Because no viral vectors were used in this experiment, so we avoided the potential risk of the viral vector going into the cells. Hence, they can serve as nucleus donor cells for transgenic pigs in subsequent studies.
引文
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[1]Bonavita A G, Quaresma K. Cotta-de-Almeida V. et al.Hepatocyte xenotransplantation for treating liver disease. Xenotransplantation.2010,17(3):181-7
[2]Perico N, Benigni A.Remuzzi G.Xenotransplantation in the 21st century. Blood Purif,2002. 20(1):45-54
[3]Tai H C, Ezzelarab M, Hara H, et al.Progress in xenotransplantation following the introduction of gene-knockout technology. Transpl Int,2007,20(2):107-17
[4]Scobie L,Takeuchi Y.Porcine endogenous retrovirus and other viruses in xenotransplantation. Curr Opin Organ Transplant.2009.14(2):175-9
[5]Specke V. Denner J. [Porcine endogenous retroviruses (PERVs) and xenotransplantation. A risk for the recipient and for society?]. Dtsch Med Wochenschr.2003.128(23):1301-6
[6]Akiyoshi D E, Denaro M, Zhu H, et al. Identification of a full-length cDNA for an endogenous retrovirus of miniature swine. J Virol,1998,72(5):4503-7
[7]Dieckhoff B, Kessler B. Jobst D, et al. Distribution and expression of porcine endogenous retroviruses in multi-transgenic pigs generated for xenotransplantation. Xenotransplantation, 2009,16(2):64-73
[8]Mang R. Maas J. Chen X. et al.Identification of a novel type C porcine endogenous retrovirus evidence that copy number of endogenous retroviruses increases during host inbreeding..1 Gen Virol.2001,82(pt8):1829-34
[9]Huang X J. Chen P. Zeng R. et al. [Quantitative study on copy numbers of porcine endogenous retroviruses in genome of Banna miniature swine inbred line]. Beijing Da Xue Xue Bao.2006. 38(4):370-4
[10]Wu J. Ma Y. Lv M, et al. Large-scale survey of porcine endogenous retrovirus in Chinese miniature pigs. Comp Immunol Microbiol Infect Dis.2008.31(4):367-71
[11]Fujimura T, Miyagawa S, Takahagi Y, et al. Prevalence of porcine endogenous retroviruses in domestic, minature, and genetically modified pigs in Japan. Transplant Proc,2008, 40(2):594-5
[12]Ma Y. Yang Y. Lv M, et al. Real-time quantitative polymerase chain reaction with SYBR green i detection for estimating copy numbers of porcine endogenous retrovirus from Chinese miniature pigs. Transplant Proc.2010.42(5):1949-52
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[1]Deschamps J Y, Roux F A, Gouin E, et al. Reluctance of French patients with type 1 diabetes to undergo pig pancreatic islet xenotransplantation. Xenotransplantation.2005,12 (3):175-180.
[2]Dekel B, Burakova T, Arditti F D, et al. Human and porcine early kidney precursors as a new source for transplantation. Nat Med,2003,9(1):53-60.
[3]Kuddus R, Patzer J F, Lopez R, et al. Clinical and laboratory evaluation of the safety of a bioartificial liver assist device for potential transmission of porcine endogenous retrovirus. Transplantation.2002.73 (3):420-429.
[4]Moza A K. Mertsching H. Herden T. et al. Heart valves from pigs and the porcine endogenous retrovirus:Experimental and clinical data to assess the probability of porcine endogenous retrovirus infection in human subjects. J Thorac Cardiovasc Surg.2001.121(4):697-701.
[5]Schumacher J M. Ellias S A. Palmer E P, et al. Transplantation of embryonic porcine mesencephalic tissue in patients with PD. Neurology,2000.54(5):1042-1050.
[6]Tucker A W, McNeilly F, Meehan B, et al. Methods for the exclusion of circoviruses and gammaherpesviruses from pigs. Xenotransplantation,2003.10(4):343-348.
[7]Tonjes RR. Czauderna F. Fischer N. et al. molecularly cloned porcine endogenous retroviruses replicate on human cells. Transplantation Proceedings.2000.32(5):1158-1161.
[8]Li Z, Ping Y. Shengfu L. et al. Phylogenetic relationship of porcine endogenous retrovirus (PERV) in Chinese pigs with some type C retroviruses. Virus Res.2004.105(2):167-173.
[9]Lee J H. Graham Webb C. Richard Allen D M, et al. Characterizing and Mapping Porcine Endogenous Retroviruses in Westran Pigs. J Virol.2002.76(11):5548-5556.
[10]Akiyoshi, DE, Denaro M, Zhu H, et al.. Identification of a full-length cDNA for an endogenous retrovirus of miniature swine. J Virol,1998,72(5):4503-7.
[11]张立,步宏,李胜富,等.中国两种地方猪种内源性逆转录病毒的亚型分析[J].四川大学学报(医学版),2003,34(2)：197-199
[12]Denner. J. Recombinant porcine endogenous retroviruses (PERV-A/C):a new risk for xenotransplantation?Arch Virol.2008.153(8):1421-6.
[13]Takeuchi Y.Patience C.Magre S.et al.Host range and interference studies of three classes of pig endogenous retrovirus.J Virol.1998.72 (12):9986-9991.
[14]Fumihiko Fujita, Izumi Yamashita- Futsuki. Susumu Eguchi. et al.lnaclivation of porcine endogenous retrovirus by human serum as a function of complement activated through the classical pathway. Hepatol Res.2003.26 (2):106-113.
[15]Takefman D M, Spear G T, Saifuddin M. et al. Human CD59 incorporation into porcine endogenous retrovirus particles:implications for the use of tansgenic pigs for xenotransplantation. J Virol.2002.76(4):1999-2002.
[16]Irgang M. Sauer I M, Karlas A. et al. Porcine endogenous retroviruses:no infection in patients treated with a bioreactor based on porcine liver cells.J Clin Virol.2003.28 (2): 141-154.
[17]McKane B W. Ramachandran S. Yang J. et al. Xenoreactive antiGalalpha(1.3) Gal antibodies prevent porcine endogenous retrovirus infection of human in vivo. Hum Immunol. 2003.64 (7):708.
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[19]Irgang M. Karlas A. Laue C. et al. Porcine endogenous retroviruses PERV-A and PERV-B infect neither mouse cells in vitro nor SCID mice in vivo. Intervirology.2005.48(2-3): 167-73.
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[26]Tacke SJ. Kurth R, Denner J. Porcine endogenous retroviruses inhibit human immune cell function:risk for xenotransplantation? Virology.2000.268(1):87-93.
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[31]Miyagawa S. Nakatsu S, Nakagawa T, et al. Prevention of PERV infections in pig to human xenotransplantation by the RNA interference silences gene. J Biochem.2005,137(4): 503-508.
[32]Niemann H. Kues WA. Transgenic farm animals-an update. Reprod Fertil Dev,2007,19(6): 762-770.
[33]Denner J. Is porcine endogenous retrovirus (PERV) transmission still relevant? Transplant Proc,2008,40(2):587-9.
[34]Li Z. Ping Y. Shengfu L, et al.The lack of inhibition of porcine endogenous retrovirus by small interference RNA designed from the long terminal regions. Transplant Proc.2006.38(7): 2258-60.
[35]Karlas A. Kurth R. Denner J. Inhibition of porcine endogenous retroviruses by RNA interference:increasing the safety of xenotransplantation. Virology,2004.325(1):18-23
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[38]Ramsoondar J. Vaught T. Ball S. et al.Production of transgenic pigs that express porcine endogenous retrovirus small interfering RNAs. Xenotransplantation,2009.16(3):164-80
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[1]Bonavita A G, Quaresma K. Cotta-de-Almeida V. et al.Hepatocyte xenotransplantation for treating liver disease. Xenotransplantation.2010,17(3):181-7
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[3]Tai H C, Ezzelarab M, Hara H, et al.Progress in xenotransplantation following the introduction of gene-knockout technology. Transpl Int,2007,20(2):107-17
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[6]Akiyoshi D E, Denaro M, Zhu H, et al. Identification of a full-length cDNA for an endogenous retrovirus of miniature swine. J Virol,1998,72(5):4503-7
[7]Dieckhoff B, Kessler B. Jobst D, et al. Distribution and expression of porcine endogenous retroviruses in multi-transgenic pigs generated for xenotransplantation. Xenotransplantation, 2009,16(2):64-73
[8]Mang R. Maas J. Chen X. et al.Identification of a novel type C porcine endogenous retrovirus evidence that copy number of endogenous retroviruses increases during host inbreeding..1 Gen Virol.2001,82(pt8):1829-34
[9]Huang X J. Chen P. Zeng R. et al. [Quantitative study on copy numbers of porcine endogenous retroviruses in genome of Banna miniature swine inbred line]. Beijing Da Xue Xue Bao.2006. 38(4):370-4
[10]Wu J. Ma Y. Lv M, et al. Large-scale survey of porcine endogenous retrovirus in Chinese miniature pigs. Comp Immunol Microbiol Infect Dis.2008.31(4):367-71
[11]Fujimura T, Miyagawa S, Takahagi Y, et al. Prevalence of porcine endogenous retroviruses in domestic, minature, and genetically modified pigs in Japan. Transplant Proc,2008, 40(2):594-5
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[1]Deschamps J Y, Roux F A, Gouin E, et al. Reluctance of French patients with type 1 diabetes to undergo pig pancreatic islet xenotransplantation. Xenotransplantation.2005,12 (3):175-180.
[2]Dekel B, Burakova T, Arditti F D, et al. Human and porcine early kidney precursors as a new source for transplantation. Nat Med,2003,9(1):53-60.
[3]Kuddus R, Patzer J F, Lopez R, et al. Clinical and laboratory evaluation of the safety of a bioartificial liver assist device for potential transmission of porcine endogenous retrovirus. Transplantation.2002.73 (3):420-429.
[4]Moza A K. Mertsching H. Herden T. et al. Heart valves from pigs and the porcine endogenous retrovirus:Experimental and clinical data to assess the probability of porcine endogenous retrovirus infection in human subjects. J Thorac Cardiovasc Surg.2001.121(4):697-701.
[5]Schumacher J M. Ellias S A. Palmer E P, et al. Transplantation of embryonic porcine mesencephalic tissue in patients with PD. Neurology,2000.54(5):1042-1050.
[6]Tucker A W, McNeilly F, Meehan B, et al. Methods for the exclusion of circoviruses and gammaherpesviruses from pigs. Xenotransplantation,2003.10(4):343-348.
[7]Tonjes RR. Czauderna F. Fischer N. et al. molecularly cloned porcine endogenous retroviruses replicate on human cells. Transplantation Proceedings.2000.32(5):1158-1161.
[8]Li Z, Ping Y. Shengfu L. et al. Phylogenetic relationship of porcine endogenous retrovirus (PERV) in Chinese pigs with some type C retroviruses. Virus Res.2004.105(2):167-173.
[9]Lee J H. Graham Webb C. Richard Allen D M, et al. Characterizing and Mapping Porcine Endogenous Retroviruses in Westran Pigs. J Virol.2002.76(11):5548-5556.
[10]Akiyoshi, DE, Denaro M, Zhu H, et al.. Identification of a full-length cDNA for an endogenous retrovirus of miniature swine. J Virol,1998,72(5):4503-7.
[11]张立,步宏,李胜富,等.中国两种地方猪种内源性逆转录病毒的亚型分析[J].四川大学学报(医学版),2003,34(2)：197-199
[12]Denner. J. Recombinant porcine endogenous retroviruses (PERV-A/C):a new risk for xenotransplantation?Arch Virol.2008.153(8):1421-6.
[13]Takeuchi Y.Patience C.Magre S.et al.Host range and interference studies of three classes of pig endogenous retrovirus.J Virol.1998.72 (12):9986-9991.
[14]Fumihiko Fujita, Izumi Yamashita- Futsuki. Susumu Eguchi. et al.lnaclivation of porcine endogenous retrovirus by human serum as a function of complement activated through the classical pathway. Hepatol Res.2003.26 (2):106-113.
[15]Takefman D M, Spear G T, Saifuddin M. et al. Human CD59 incorporation into porcine endogenous retrovirus particles:implications for the use of tansgenic pigs for xenotransplantation. J Virol.2002.76(4):1999-2002.
[16]Irgang M. Sauer I M, Karlas A. et al. Porcine endogenous retroviruses:no infection in patients treated with a bioreactor based on porcine liver cells.J Clin Virol.2003.28 (2): 141-154.
[17]McKane B W. Ramachandran S. Yang J. et al. Xenoreactive antiGalalpha(1.3) Gal antibodies prevent porcine endogenous retrovirus infection of human in vivo. Hum Immunol. 2003.64 (7):708.
[18]Binette TM, Seeberger KL, Lyon J G, et al. Porcine endogenous retrovural nucleic acid in peripheral tissues is associated with migration of porcine cells post islet transplant. Am J Transplant,2004,4 (7):1051-1060.
[19]Irgang M. Karlas A. Laue C. et al. Porcine endogenous retroviruses PERV-A and PERV-B infect neither mouse cells in vitro nor SCID mice in vivo. Intervirology.2005.48(2-3): 167-73.
[20]Specke V. Plesker R. Wood J. et al.No in vivo infection of triple immunosuppressed non-human primates after inoculation with high titers of porcine endogenous retroviruses. Xenotransplantation.2009.16(1):34-44.
[21]Nybery SL. Hibbs JR. Hardin JA. et al. Transfer of porcine endogenous retrovirus across hollow fiber membranes. Transplantation.1999.67 (9):1251.
[22]Falasca E. Baccarani U. Pipan C. et al. Is PERV transfer across hollow fiber membranes relevant to bioartifical liver model? Transplantation.2000.69 (8):1755.
[23]Pitkin Z. Mullon C.Evidence of absence of porcine endogenous retrovirus (PERV) infection in patients treated with a bioarlificial liver support system. Artificial Organs.1999.23 (9):829-833.
[24]Shah C A, Boni J. Bisset L R. et al. Ultra-sensitive and specific detection of porcine endogenous retrovirus (PERV) using a sequencecapture real-time PCR approach. J Virol Methods.2003.109 (2):209-216.
[25]Seifarth W, Spiess B, Zeilfelder U et al. Assessment of retroviral activity using a universal retrovirus chip. J Virol Methods,2003,112 (1-2):79-91.
[26]Tacke SJ. Kurth R, Denner J. Porcine endogenous retroviruses inhibit human immune cell function:risk for xenotransplantation? Virology.2000.268(1):87-93.
[27]Oldmixon BA. Wood JC. Ericsson TA. et al. Porcine endogenous retrovirus transmission characteristics of an inbred herd of miniature swine. J Virol.2002.76(6):3045-3048.
[28]Tacke SJ. Specke V. Denner J. Differences in release and determination of subtype of porcine endogenous retroviruses produced by stimulated normal pig blood cells. Intervirology.2003. 46(1):17-24.
[29]Fiebig U, Stephan O. Kurth R. et al. Neutralizing antibodies against conserved domains of p15E of porcine endogenous retroviruses:basis for a vaccine for xenotransplantation? Virology.2003.307(2):406-413.
[30]Karlas A. Kurth R. Denner J. Inhibition of porcine endogenous retroviruses by RNA interference:increasing the safety of xenotransplantation. Virology.2004.325(1):18-23.
[31]Miyagawa S. Nakatsu S, Nakagawa T, et al. Prevention of PERV infections in pig to human xenotransplantation by the RNA interference silences gene. J Biochem.2005,137(4): 503-508.
[32]Niemann H. Kues WA. Transgenic farm animals-an update. Reprod Fertil Dev,2007,19(6): 762-770.
[33]Denner J. Is porcine endogenous retrovirus (PERV) transmission still relevant? Transplant Proc,2008,40(2):587-9.
[34]Li Z. Ping Y. Shengfu L, et al.The lack of inhibition of porcine endogenous retrovirus by small interference RNA designed from the long terminal regions. Transplant Proc.2006.38(7): 2258-60.
[35]Karlas A. Kurth R. Denner J. Inhibition of porcine endogenous retroviruses by RNA interference:increasing the safety of xenotransplantation. Virology,2004.325(1):18-23
[36]Dieckhoff B. Karlas A, Hofmann A, et al.Inhibition of porcine endogenous retroviruses (PERVs) in primary porcine cells by RNA interference using lentiviral vectors. Arch Virol. 2007,152(3):629-34.
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