【作者】 杨胜辉；

【导师】 曾庆仁；

【作者基本信息】 中南大学 ， 病原生物学， 2010， 博士

【摘要】 迄今为止,应用于血吸虫病防治实践的技术仍存在许多不足,尤在控制日本血吸虫病流行与传播方面尚无理想方法。30年前有学者提出,建立稳定生长和连续传代培养的血吸虫细胞系,可为寻求新的防治技术提供基础和条件,但通过数十年的细胞培养技术探索,一直未能实现建立可连续传代生长的血吸虫细胞系目标。本研究受哺乳动物体细胞经转导外源永生化基因后成功建立永生化细胞系的启示,开展了采用逆转录病毒载体对日本血吸虫童虫细胞进行外源基因转导的生物学理论探索,制备了载有永生化基因(hTERT)的双嗜性逆转录病毒和泛嗜性逆转录病毒,并观察了这2种逆转录病毒载体转导外源基因到日本血吸虫童虫细胞或虫体后所发生的整合、转录和表达以及对Sj细胞增殖的影响。本研究目的在于为血吸虫细胞永生化研究提供理想的转导外源基因的载体,验证外源hTERT基因能否在血吸虫体内整合、表达及其表达部位,同时探索hTERT基因表达诱导细胞增殖的可行性。[目的]探讨用双嗜性逆转录病毒载体将外源基因导入日本血吸虫(Sj)细胞的生物学理论与实验依据。[方法]从GenBank中收集褐家鼠双嗜性逆转录病毒受体(rRam-1)的氨基酸序列,应用Blastp工具对其进行序列相似性搜索,并用Cluster W2工具对相似性较高的氨基酸序列进行同源性分析；采用RPS-blast与InterproScan在线工具对rRam-1受体及其同源的氨基酸序列进行保守区域分析；进一步应用多个在线分析工具对与rRam-1同源的Sj蛋白进行蛋白二级结构、疏水性、跨膜性、信号肽、亚细胞定位以及翻译后修饰点等进行分析与预测；在生物信息学预测的基础上采用含短片段外源基因的双嗜性逆转录病毒载体感染Sj-12d童虫细胞培养物,并应用PCR与RT-PCR方法检测外源基因在Sj细胞中的整合与表达。[结果]rRam-1氨基酸残基序列相似性搜索及同源性分析显示,其氨基酸序列与多种脊椎动物的钠离子依赖性的磷酸盐运载体家族的氨基酸序列有很高的同源性,一致性均在59%以上,其中,与中国仓鼠Ram-1受体(cRam-1)和人类Ram-1 (h Ram-1)受体的氨基酸序列一致性均为93%；此外,与多种无脊椎动物磷酸盐运载体家族的氨基酸序列也有较高的同源性,氨基酸一致性在42%以上；Sj中存在2种与rRam-1受体有较高同源性的蛋白SJCHGC09605和SjCHGC05362,它们与rRam-1受体之间的氨基酸序列一致性分别为54%和61%,相似性分别为74%和72%。2种血吸虫蛋白与人类、褐家鼠及中国仓鼠的Ram-1受体蛋白处于平行的进化分枝上。保守性分析显示,2种Sj蛋白与人类、褐家鼠及中国仓鼠的Ram-1受体存在相同的PH04 Superfamily保守结构域,均为磷酸盐运载体超家族成员；二级结构显示,SJCHGC09605和SJCHGC05362蛋白中,α螺旋分别占68.97%和39.22%,跨膜区预测显示分别有7个和5个可能的区域,与疏水性预测结果完全一致；亚细胞定位及翻译后修饰位点分析显示,2种蛋白均不含信号肽序列和亚细胞定位信号,也不含糖基化、磷酸化和脂酰化等翻译后修饰位点。利用携带外源E77.43基因的双嗜性逆转录病毒感染Sj童虫细胞后,经PCR与RT-PCR检测到目的基因存在与表达,扩增的目的片段大小为330 bp,与理论值相符。[结论]双嗜性逆转录病毒rRam-1受体与日本血吸虫细胞膜上起离子转运通道或受体蛋白作用的SjCHGC09605和SjCHGC05362两种跨膜蛋白成分存在较高同源性；用载有E77.43基因的双嗜性逆转录病毒感染Sj童虫细胞获得成功,推测SjCHGC09605和SjCHGC05362两种与rRam-1受体同源的蛋白可能是Sj感染过程中起作用的分子。该结果为下一步用双嗜性逆转录病毒载体转导永生化基因至Sj细胞提供了生物学理论与实验依据。[目的]建立含永生化基因(hTERT)的稳定产逆转录病毒细胞株,观察hTERT基因转导Sj童虫细胞后的整合和表达情况以及对细胞增殖作用的影响。[方法]将从美国引进的pBABE-puro-hTERT质粒经核酸内切酶酶切、PCR扩增和测序鉴定确认；倍比稀释测定PA317细胞和NIH3T3细胞对嘌呤霉素的最高耐受浓度；用脂质体将质粒转染至PA317细胞内,经嘌呤霉素筛选获得抗性克隆并扩大培养,并通过PCR、测序、免疫荧光、Western-blot及透射电镜对抗性细胞株进行鉴定,并以NIH3T3细胞测定收集的逆转录病毒液滴度。常规制备Sj-12d童虫细胞,并在体外培养中用BrudU-ELISA法检测细胞增殖情况,PCR法检测兔线粒体特异性基因确定无宿主来源细胞污染,倍比稀释法测定Sj细胞对嘌呤霉素的最高耐受浓度；用浓缩的双嗜性逆转录病毒感染Sj-12d童虫细胞并以嘌呤霉素连续筛选培养获得抗性Sj细胞克隆,扩大培养后用PCR、RT-PCR、Western-blot检测外源hTERT基因和puror基因在细胞内的整合与表达；用3H-TdR掺入法检测嘌呤霉素抗性Sj-12d细胞的增殖能力,利用细胞计数法绘制其生长曲线,并应用TRAP-ELISA法测定其端粒酶活性。[结果]pBABE-puro-hTERT质粒经酶切、PCR和测序鉴定为目的质粒；PA317细胞和NIH3T3细胞对嘌呤霉素的最高耐受浓度为6μg/ml和3μg/ml；嘌呤霉素抗性PA317克隆扩大培养物经PCR、测序、免疫荧光以及Western-blot检测到外源hTERT基因和puror基因的整合、转录及蛋白质表达；透射电镜检测到抗性PA317细胞的培养上清及胞浆内有逆转录病毒颗粒的存在,经浓缩后测得其滴度为2×105cfu/ml。Sj-12d童虫细胞培养3d后即可见部分细胞分裂相,10-14d后可见较多分裂相,BrdU-ELISA也显示培养14d后有明显的DNA合成与增殖,其对嘌呤霉素最高耐受浓度为0.5μg/ml;双嗜性逆转录病毒感染Sj-12d细胞后经嘌呤霉素连续筛选21d可见抗性克隆形成,对扩大培养后的抗性细胞做PCR、RT-PCR和Western blot,检测到外源hTERT基因和puror基因在抗性Sj-12d细胞内的整合、转录及蛋白表达,但整合的拷贝数少,转录水平低下；3H-TdR掺入法检测显示抗性Sj-12d细胞与常规培养的Sj-12d细胞均有一定增殖能力,但二者间差异无显著性(P>0.05)；TRAP-ELISA实验未能从抗性Sj-12d细胞内检测到端粒酶活性；在培养4周内的抗性Sj-12d细胞生长相对较快,此后生长逐渐减慢,死亡细胞和退变细胞的数目逐渐增多,最后全部死亡。[结论]用pBABE-puro-hTERT逆转录病毒质粒转染PA317细胞后,成功建立含hTERT基因稳定产双嗜性逆转录病毒颗粒的细胞株；该病毒感染Sj-12d童虫细胞后可检测到外源hTERT基因和puror基因的整合、转录及蛋白表达,但整合拷贝数少,转录水平低下,未能激活Sj-12d细胞的端粒酶活性和改善细胞的增殖能力。[目的]为提高逆转录病毒对血吸虫的感染能力,探讨应用pVSV-G质粒和pBABE-puro-hTERT质粒共转染包装细胞制备泛嗜性逆转录病毒的可行性,并观察该病毒感染Sj童虫后外源基因在虫体内的整合、转录、表达及具体的表达部位情况。[方法]将pVSV-G质粒和pBABE-puro-hTERT质粒共转染GP2-293包装细胞,转染后48h收集细胞培养上清,用浓缩的上清液与Polybrene混合液感染NIH3T3细胞系,经嘌呤霉素连续筛选12d后获得抗性克隆,计数抗性克隆数目并计算病毒滴度；挑取抗性NIH3T3克隆扩大培养,以PCR检测外源hTERT基因和puror基因在细胞内的整合,采用免疫细胞化学染色法检测hTERT基因在细胞内的表达情况；将泛嗜性逆转录病毒加入到体外培养的Sj-12d童虫,感染24h后更换培养基,将虫体连续培养6d；采用PCR和Southern杂交检测外源hTERT基因在虫体内的整合,同时应用RT-PCR、Western blot及免疫组织化学染色检测外源hTERT基因在虫体内的转录、表达及定位情况。[结果]经计数后,泛嗜性逆转录病毒颗粒滴度为3.2×108,抗性NIH3T3细胞经PCR扩增出外源hTERT基因和puror基因特异性145bp和204bp目的条带,免疫细胞化学染色法检测到hTERT基因在细胞内发生了蛋白质表达,表达部位以细胞核内为主；泛嗜性逆转录病毒感染后的虫体经PCR和RT-PCR扩增出了外源hTERT基因和puror基因特异性145bp和204bp目的产物,其中,hTERT基因的转录水平较高,而puror基因的转录水平则较低,Southern杂交也显示出虫体内有外源hTERT基因的多个拷贝整合；Western blot实验显示病毒感染后的虫体内有外源hTERT基因的表达,其表达部位经免疫组织化学染色确定为在虫体的口吸盘、腹吸盘和后部体壁皮层下的表达量最多。[结论]用pVSV-G质粒和pBABE-puro-hTERT质粒共转染包装细胞后成功制备携带外源hTERT基因泛嗜性逆转录病毒,并证明该病毒感染Sj童虫活虫体后可在吸盘和后部体壁皮层下产生外源hTERT基因的多拷贝整合、转录与蛋白质表达。更多还原

【Abstract】 To date, the technologies used for schistosomiasis control still have many deficiencies. Especially, there is no ideal measure in controlling the spread of schistosomiasis japonica. Thirty years ago, some scholars have suggested that the establishment of a stable and continuous passage cell line from schistosome may provide foundation and conditions for finding new schistosomiasis prevention and control technologies. Through decades of exploration for cell culture techniques on Schistosoma japonicum (Sj), however, a continuous passage schistosome cell line has not been achieved. In this study, by an inspiration from transduction of exogenous gene with immortalization function into mammalian somatic cells and successful establishment of immortalized cell lines, we carried out exploration of a biological theory on retroviral vector used to introduce exogenous gene into schistosomula cells, prepared amphotropic retrovirus and pantropic retrovirus containing human telomerase reverse transcriptase gene (hTERT) and observed the integration, transcription, expression and cell proliferation following transduction of exogenous genes into cells or worms from Sj with the two retroviral vectors. Our purpose is to provide an ideal vector for transduction of exogenous gene in the study of schistosome cell immortalization, verify the integration and expression of hTERT gene within schistosome worm bodies and explore the feasibility of inducing cell proliferation. [Objective] To explore biologically theoretical and experimental foundation for introduction of exogenous gene into Sj cells with amphotropic retrovirus vector. [Methods] Amino acid sequence of the receptor for amphotropic murine retroviruses of Rattus norvegicus (rRam-1) was collected from GenBank and its sequence similarity search was performed with Blastp program at NCBI (the National Center for Biotechnology Information) website. Afterwards, the homology analysis were carried out for higher similarity sequences with Cluster W2 program. Conserved regions of rRam-1 receptor and its homologous amino acid sequences were analyzed with RPS-blast and InterproScan tools. Subsequently, protein secondary structures, hydrophobicity, transmembrane regions, signal peptides, subcellular localization signal and post-translation modification sites of Sj proteins homologous to rRam-1 were analyzed and forecasted with several online analysis tools. Based on bioinformatics prediction, Sj-12d cell cultures were infected with amphotropic retrovirus vector containing short segment of exogenous gene and integration and expression of the foreign gene within Sj cells were detected with PCR and RT-PCR analysis. [Results] Similarity search and homology analysis of rRam-1 showed that its amino sequence was highly homologous with amino sequences of Sodium-dependent phosphate transporter from vertebrates and the identities were all more than over 59%. Among them, the identities of amino sequences to the receptor for amphotropic murine retroviruses of Cricetulus griseus (cRam-1) and the receptor for amphotropic murine retroviruses of Homo sapiens (hRam-1) were 93%, respectively. In addition, relatively high homologies with phosphate transporter families from numerous invertebrate species were also found, and the identities were all above 42%. Two proteins of Sj, SJCHGC09605 and SjCHGC05362, were highly homologous to rRam-1 with identities of 54% and 61% and positives of 74% and 72%, respectively. The two Sj proteins were at evolution branches parallel to hRam-1, rRam-1 and cRam-1. Conservation analysis indicated that there were same conserved domain PH04 Superfamily among two Sj proteins, hRam-1, rRam-1 and cRam-1 and all proteins were members of phosphate transporter superfamily. Secondary structure analysis showed that a helixes in SJCHGC09605 and SJCHGC05362 were 68.97% and 39.22%, respectively, and transmembrane prediction showed 7 and 5 possible regions, respectively, which was exactly consistent with hydrophobicity prediction. Subcellular localization and post-translation modification site analysis indicated that the two proteins did not contain signal peptide sequence and subcellular localization signal, and that, lycosylation, phosphorylation and lipid acylation were not found. After infection of cell cultures from Sj juvenile worms with amphotropic retrovirus containing exogenous gene E77.43, the presence and expression of target gene in cells were detected by PCR and RT-PCR analysis and the amplicon of predicted size of 330 bp was generated, which is corresponding with theoretical size. [Conclusion] There were high homology between rRam-1 and either transmembrane protein SjCHGC09605 or SjCHGC05362 on Sj cellular membrane acting as ion transport channel or receptor protein. Furthermore, cell cultures from Sj juvenile worms could be successfully infected with amphotropic retrovirus containing exogenous gene E77.43, indicating the two proteins homologous to rRam-1 may play important roles during infection of Sj cells by retroviruses. These findings provided a biologically theoretical and experimental foundation for transduction of immortal gene into Sj cells with amphotropic retrovirus vector. [Objectives] To establish a cell strain which stably produce amphotropic retro virus containing immortal hTERTgene and observe the integration and expression of hTERT gene in cell cultures from Sj juvenile worm following transduction and its influence on cell multiplication. [Methods] Plasmid pBABE-puro-hTERT was identified by enzyme digestion, PCR and sequencing. The highest puromycin-resistant concentration of PA317 and NIH3T3 cells was determined by gradient dilution method. After transfection of plasmid into PA317 cell with liposome, puromycin-resistant clones were obtained following screening with puromycin and one of clones was randomly selected and cultured for continous passage. PCR, sequencing, immunofluorescence, Western-blot and transmission electron microscopy were performed to identify the puromycin-resistant cell strain and the titer of retrovirus was determined with NIH3T3 cell. Sj-12d schistosomule cells were prepared as described before and cell proliferation was measured with BrudU-ELISA method. Absence of contamination from host cells was confirmed by detection of special rabbit mitochondrium gene with PCR. The highest puromycin-resistant concentration of Sj cells was determined by gradient dilution. After infection of Sj-12d cells with concentrated amphotropic retrovirus and successive screening in puromycin-containing medium, puromycin-resistant Sj cell clones were obtained and selected for continuous cultivation. The integration and expression of exogenous genes hTERT and puror within these cells were observed by PCR, RT-PCR and Western-blot. Proliferation of puromycin-resistant Sj-12d cells was measured by 3H-TdR incorporation, and growth curve plotted by cell count and telomerase activity monitored with TRAP-ELISA method. [Results] The authenticity of pBABE-puro-hTERT plasmid was identified by enzyme digestion, PCR and sequencing. The highest puromycin-resistant concentrations of PA317 and NIH3T3 cells were 6μg/ml and 3μg/ml, respectively. The integration, transcription and expression of exogenous genes hTERT and puror could be confirmed in puromycin-resistant PA317 cell strain by PCR, sequencing, immunofluorescence and Western-blot. Retrovirus particles could be found in culture supernatant and cytoplasm of puromycin-resistant PA317 cells by transmission electron microscopy and the titer of concentrated retrovirus particles was 2×105cfu/ml following infection of NIH3T3 cell line with these virions. After cultivation for 3 days, cell division phase appeared in Sj-12d schistosomule cells and more cell division phases appeared following 10-14 days. Furthermore, DNA synthesis and proliferation were verified by BrdU-ELISA analysis in Sj schistosomule cells cultured for 14 days and the highest puromycin-resistant concentration of Sj cells was 0.5μg/ml. After successive screening for 21 days with puromycin-containing medium, puromycin-resistant clones could be found in Sj-12d cells infected with amphotropic retrovirus and one of clones was randomly selected for successive cultivation. The integration, transcription and protein expression of exogenous genes hTERT and puror within puromycin-resistant Sj-12d cells could be detected by PCR, RT-PCR and Western blot, respectively. However, the copies and transcription level of foreign genes were low within schistosomule cells.3H-TdR incorporation experiment showed that both puromycin-resistant Sj-12d cells and normal cultured Sj-12d cells exhibited some extent proliferation ability but no significant difference was found between them (P>0.05). Telomerase activity could not be detected in puromycin-resistant Sj-12d cells using TRAP-ELISA method. Within 4 weeks, cultivated cells grew fast and then slowly. The number of dead cells and degenerative cells gradually increased and, finally, the cells all died. [Conclusion] After Transfection of PA317 cell with retroviral plasmid pBABE-puro-hTERT, a cell strain, PA317/hTERT, stably producing amphotropic retoviral particles containing hTERT gene was constructed successfully. Integration, transcription and expression of exogenous genes hTERT and puror could be detected in Sj-12d cells infected with amphotropic retrovirus, but the telomerase activity within them could not be activated and the cell proliferation ability could not also be improved.[Objectives] To enhance the ability of retrovirus to infect schistosomes, explore the feasibility of preparation of pantropic retrovirus by cotransfection into packaging cell line with plasmids pVSV-G and pBABE-puro-hTERT and observe integration, transcription, expression and protein locations of exogenous genes in worm bodies post infection of Sj juvenile worms with pantropic retrovirus. [Methods] GP2-293 package cells were cotransfected with plasmids pVSV-G and pBABE-puro-hTERT, and the supernatants of cell cultures were collected 48h post transfection and then NIH3T3 cell line was infected with the concentrated supernatants mixed with polybrene. Puromycin-resistant clones were obtained through successive screening for 12d with puromycin-containing media and the numbers of clones were counted for virus titers. Thereafter, puromycin-resistant clones were randomly selected for continuous culture, integration of exogenous genes hTERT and puror in cells was detected with PCR and expression of hTERT gene was determined with immunocytochemistry stain. Pantropic retrovirus were added to in vitro cultured Sj-12d schistosomules. Medium was replaced 24h after infection and worms were successively cultured for 6d. Integration of exogenous hTERT gene in worm bodies was confirmed by PCR and Southern hybridization analysis, and, simultaneously, RT-PCR, Western blot and immunohistochemical staining were also adopted for study of transcription, expression and protein location of exogenous hTERT gene in worm bodies. [Results] After counting, the titer of pantropic retrovirus was 3.2x 108. Two fragments of predicted sizes of 145 bp and 204 bp of exogenous genes hTERT and puror were amplified from puromycin-resistant NIH3T3 cells with PCR. Protein expression of hTERT gene in cells could be detected with immunocytochemical staining and the expression location was mainly in nucleus. Specific amplicons of exogenous genes hTERT and puror in size of 145 bp and 204 bp were generated from virus-treated schistosomule genomic DNAs using PCR and RT-PCR. Southern hybridization showed that multi-copies of exogenous hTERT gene were integrated into worm chromosomes. Western blot displayed that exogenous hTERT gene was expressed in worm bodies post infection with virus and most expressions were found in subtegumental regions of oral sucker, ventral sucker and the posterior of Sj larval worms by immunohistochemical staining. [Conclussion] Pantropic retrovirus was successfully prepared following cotransfection into packaging cell line with plasmids pVSV-G and pBABE-puro-hTERT and multicopy integration, transcription and protein expression of exogenous hTERT gene within subtegumental regions of suckers and the posterior of Sj larval worms was confirmed post infection of live Sj juvenile worms with this virus.更多还原