【作者】 韩冰；

【导师】 丁义涛；

【作者基本信息】 南京大学 ， 外科学， 2011， 博士

【摘要】 第一部分：乳糖酰基壳聚糖纳米纤维支架上猪肝细胞和MSCs共培养体系的PERVs分泌及体外感染性分析目的：明确乳糖酰基壳聚糖纳米纤维支架上猪肝细胞和MSCs共培养体系的PERV分泌情况及体外感染能力。方法：将新鲜分离的猪肝细胞和传代至第3-5代MSCs细胞单独或2:1共培养接种于有或无乳糖酰基壳聚糖纳米纤维支架的6孔培养板,定为单纯肝细胞组(Hep)、肝细胞及MSCs共培养组(Co)、乳糖酰基壳聚糖纳米纤维支架+肝细胞组(Nano),乳糖酰基壳聚糖纳米纤维支架+肝细胞及MSCs共培养组(Nano-Co),连续培养7天。每天收集培养液检测逆转录酶(RT)活性,并通过RT-PCR和实时定量PCR测定PERV RNA水平。通过western blot检测培养液中PERV颗粒有无。细胞培养液体外孵育HEK293细胞以测定其感染性。结果：Hep组和Nano组PERV分泌10小时和第2天最高,后呈逐渐下降趋势,前者PERV分泌持续5天,而后者延长分泌至第6天。Co组和Nano-Co组PERV分泌在第2天后随时间逐渐增多,第6天达到PERV分泌高峰。经比较,Nano-Co组从第2天后PERV分泌量明显高于单纯肝细胞,且从第2天后每天PERV分泌量显著高于10H。体外感染实验发现Co组和Nano-Co在后期有微嵌合细胞外,并无HEK293细胞感染PERV。结论：猪肝细胞和MSCs共培养体系在两种培养条件下均能分泌PERV,而在乳糖酰基壳聚糖纳米纤维支架上培养2天后PERV分泌量随时间逐渐增多,且后期体外感染实验出现微嵌合状态,但并无表现出明显体外感染性。由此提示猪肝细胞和MSCs共培养体系置于乳糖酰基壳聚糖纳米纤维支架上培养2天内使用较为合理。第二部分：不同孔径血浆成分交换柱对新型BAL系统PERV透过情况的影响目的：检测采用不同孔径的血浆交换柱的PERV透过情况；为新型BAL系统血浆交换柱孔径的选择提供数据参考。方法：将猪肝细胞和MSCs以2:1混合共培养于基于乳糖酰基壳聚糖纳米膜支架的多层平板型生物反应器中,并构建新型BAL系统。依据血浆成分交换柱膜孔径将实验分为10nm组、20nm组、30nm组和35nm组。细胞灌入后4小时开始培养液循环,循环48小时。检测三循环培养液内逆转录酶(RT)活性、PERV RNA以及western blot测定PERV颗粒。同时用培养液体外孵育HEK293细胞以测定其感染性。结果：除30nm和35nm组在循环48小时后出现PERV RNA透过血浆成分交换柱和血浆分离柱外,在各孔径组各循环时间点均无PERV RNA、PERV蛋白和RT活性检出,提示无PERV透过各种孔径的血浆成分交换柱。体外感染实验除个别出现微嵌合状态外并无HEK293细胞感染PERV。结论：在拟定治疗期6小时内,并未见感染性PERV颗粒透过血浆成分交换柱,但在膜孔径>20nm的血浆成分交换柱中48小时时有PERV RNA的透过。所以,从病毒安全性方面考虑,采用膜孔径≤20nm的血浆成分交换柱是安全的。第三部分：新型BAL治疗应用的病毒安全性研究目的：评估新型BAL治疗应用的病毒安全性。方法：采用氨基半乳糖诱导建立急性肝功能衰竭犬动物模型。以基于乳糖酰基壳聚糖纳米纤维支架的多层平板型反应器为装置基础,以猪肝细胞-MSCs共培养体系为种子细胞构建新型BAL,并于建模第2天治疗实验犬,治疗时间3小时,抽取治疗前、治疗中和治疗后BAL系统内血浆及犬全血,并于治疗后1年处死取新、肝、脾、肺、肾组织。检测血浆、PBMCs以及各组织中PERV RNA、DNA、逆转录酶(RT)活性,组织样本加行western blot和免疫组化测定PERV衣壳蛋白gag p30。此外,各时间点血浆体外孵育HEK293细胞以测定其感染性。结果：PERV RNA、DNA、RT活性出现于循环3血浆中,提示循环3中存在细胞释放的PERVs.其余血浆样本均未检出PERV RNA、DNA、RT活性以及抗PERV抗体,而且体外感染中均无HEK293感染。此外,PBMCs和组织标本中各项PERV检验也均为阴性,提示治疗期间并无PERV透过血浆成分交换柱而感染实验犬。结论：新型BAL治疗急性肝功能衰竭实验犬模型后并无动物感染PERV,说明新型BAL在治疗应用中具有可靠地在体病毒安全性,为将来该系统的临床试验提供了安全性证据。更多还原

【Abstract】 Part 1:Production and infectivity of PERVs by the co-culture system of porcine hepatocytes and MSCs with galactosylated chitosan nanofiber scaffoldsAIM:To detect the production and infectivity of PERVs released by the co-culture system of porcine hepatocytes and MSCs with galactosylated chitosan nanofiber scaffolds.METHODS:The primary porcine hepatocytes or the mixed suspension of hepatocytes and MSCs during passages 3 to 5 (2:1) was cultured for 7 days in the 6 well cell culture plates with or without the galactosylated chitosan nanofiber scaffolds (defined as Hepatocytes (Hep) group, nanofiber scaffold (Nano) group, co-culture (Co) group and nanofiber scaffold & co-culture (Nano-Co) group, respectively). The culture media were collected daily to detect the reverse transcriptase (RT) activity with RT activity assay kits, the PERV RNA by RT-PCR and real-time PCR with the PERV specific primers, and the PERV protein gag p30 with Western blotting. In vitro infectivity of the supernatant was tested by incubating HEK293 cells.RESULTS:Two peaks of PERV expression were found at 10H and day 2 and followed by a regular decline in Hep group and Nano group. Presence of the virus lasted five days in the former group and six days in the later group. But in Co group and Nano-Co group, the PERV release gradually increased after 2 days to the peaks in Day 6. By Comparision, the virus levels in the Nano-Co group after two days were obviously higher than that in the Hep group or than that in the Nano-Co group at 10H.. No HEK293 cell was infected in vitro by the supernatant, although some microchimerism was observed in Co group and Nano-Co group.CONCLUSION:PERV release was confirmed in the co-culture system of porcine hepatocytes and MSCs, and when the cells were cultured with galactosylated chitosan nanofiber scaffolds the viruses increased gradually over time with a significant higher production after 2 days than simple primary hepatocytes or its level at 10H in Nano-Co group. However, no obvious in vitro infectivity was found although microchimerism appeared in the last few days. The results suggest it reasonable that the co-culture system of porcine hepatocytes and MSCs with galactosylated chitosan nanofiber scaffolds be used within two days after inoculation.Part 2:The influence of the plasma component separators with different membrane pore sizes on the transfer of PERVs in the novel BALAIM:To detect the transfer of PERVs across the plasma component separators with different membrane pore sizes, and to find a plasma component separator with suitable pore size for the novel BAL.METHODS:The mixed suspension of hepatocytes and MSCs during passages 3 to 5 (2:1) was perfused into the novel multi-layer radial-flow bioreactor based on galactosylated chitosan nanofiber scaffolds to establish a new BAL system composed of three circuits. The BAL systems were divided into four groups according to the membrane pore size of the plasma component separators, that is, l0nm group,20nm group,30nm group and 35nm group. The media flow was initiated 4 hours after the cells were seeded and lasted 48 hours. The culture media in each circuit were collected at regular intervals to detect the reverse transcriptase (RT) activity with RT activity assay kits, the PERV RNA by RT-PCR and real-time PCR with the PERV specific primers, and the PERV protein gag p30 with Western blotting. In vitro infectivity of the supernatant was tested by incubating HEK293 cells.RESULTS:Except the transmission of PERV RNA through the plasma component separators and plasmafilters at 48 hours in 30nm group and 35nm group, no PERV RNA, PERV capsid protein, and RT activity was detected in the supernatant in the external circuit of all groups at any time point, suggesting no PERV particles across the plasma component separators with different membrane pore sizes. In addition, no HEK293 cell was infected in vitro by the supernatant, although some microchimerism was observed with the media from the Circuit 3.CONCLUSION:No transfer of infectious PERVs across the plasma component separators with different membrane pore sizes was found during the proposed duration of the treatment, but PERV RNA was able to pass through the plasma component separators with membrane pore sizes>20nm. Therefore, it may be safer in the virology to apply the plasma component separators with mambrane pore sizes≤20nm.Part 3:Study on the virological safety of the novel BAL in therapeutic applicationAIM:To evaluate the virological safety of the novel BAL in therapeutic application.METHODS:Five dogs of galactosamine-induced ALF were treated for 3 hours with the new BAL based on the new multi-layer radial-flow bioreactor containing galactosylated chitosan nanofiber scaffolds and co-system of porcine hepatocytes and MSCs in the second day after models established. The plasma in each circuit and the whole blood of the canines were collected before, during and after the treatment, and the dogs were sacrificed to retrieve the tissue of hearts, livers, spleen, lung and kidneys. These fuild samples were used to detect the PERV RNA, DNA, and RT activity, and the immunohistochemisty and western blot were performed to find the PERVs capsid protein gag p30 in the tissue. Furthermore, the HEK293 cells were incubated by the plasma to determine their In vitro infectivity.RESULTS:PERV RNA, DNA and RT activity found in the plasma of Circuit 3 revealed PERV particles released by the co-cultured cells existing in Circuit 3. No PERV RNA, DNA, RT activity and anti-PERV antibody was detected in he other plasma including that from the other circuits and the whole blood of the dogs, and no HEK293 cells was infected by all the plasma samples in vitro. In addition, the results of all PERV-related analysis in the PBMCs and the tissue were negative, suggesting no transfer of PERVs across the plasma component separators and no experimental dogs infected.CONCLUSION:No evidence of transmission of PERVs into the ALF canines with the treatment of the new BAL was observed, which proved the reliable virological safety of the new BAL.更多还原