新型生物人工肝病毒安全性的实验探讨

发布时间：2018-11-15 16:11

【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。
[Abstract]:The first part: the secretion and in vitro infectivity of porcine liver cells and MSCs in the co-culture system of the porcine liver cell and the MSCs on the lactose-based chitosan nanofiber scaffold Objective: To clarify the secretion and in vitro sense of PERV in the co-culture of porcine liver cells and MSCs on the chitosan nanofiber scaffold of lactose-based chitosan. The method comprises the following steps of: co-culturing the freshly isolated pig liver cells and the 3-5 generation MSCs in a single or a 2: 1 co-culture to a 6-hole culture plate with or without a lactose-based chitosan nano-fiber scaffold, and is characterized in that the pure hepatocyte group (Hep), the liver cell and the MSCs Culture group (Co), lactose-based chitosan nanofiber scaffold + hepatocyte group (Nano), lactose-based chitosan nanofiber scaffold + liver cell and MSCs co-culture group (Nano-Co) and continuously culturing for 7 days. The activity of the reverse transcriptase (RT) is detected by the culture solution every day, and the PE is determined by RT-PCR and real-time quantitative PCR. The level of RV RNA was detected by western blot. The presence or absence of ERV granules. HEK293 cells were incubated outside the cell culture liquid. The results showed that PEV in the Hep group and the Nano group were secreted for 10 hours and the second day, and then gradually decreased, the former PEV was secreted for 5 days. The latter was extended to day 6. The secretion of PERV in the Co group and the Nano-Co group increased with time after day 2, and 6 The peak of PEV secretion was reached on the day of day. After comparison, the amount of PERV secreted by the Nano-Co group was significantly higher than that of the pure hepatocytes after the second day, and the PER was per day after the second day. The amount of V secretion was significantly higher than that of 10H. In vitro infection experiments, the Co group and the Nano-Co were found to have microchimera cells in the later stage, and there was no HEK. Conclusion: The co-culture system of porcine liver cell and MSCs can secrete PERV under two conditions of culture, and the amount of PERV secretion increases with time after 2 days on the lactose-based chitosan nano-fiber scaffold. In the later in-vitro infection experiments, the microchimerism was observed. The results showed that the co-culture system of the pig liver cells and the MSCs was put into the lactose-based chitosan nano-fiber. The two-day use of the dimensional scaffold is more reasonable. The second part: the exchange of plasma components of different pore sizes The effect of the column on the transmission of the PEV of the new BAL system is to detect the PEV transmission of the plasma exchange column with different pore sizes; to The selection of the plasma exchange column aperture of the novel BAL system provides a data reference. Methods: The porcine liver cells and MSCs were co-cultured in 2: 1 co-culture on the basis of lactose-based chitosan. in a multi-layer flat plate type bioreactor of a rice membrane support, a novel BAL system is constructed, The experiment was divided into 10nm group, 20nm group, 30nm group and 3 5 nm group. The culture medium was circulated for 48 hours after the cell was injected for 4 hours. The activity of reverse transcriptase (RT) in the three-cycle culture solution was detected, P ERV RNA and Western blot for the determination of PERV The HEK293 cells were incubated at the same time to determine the infectivity of the HEK293 cells in addition to the culture liquid. Results: PEV RNA was not present at each cycle time point in each aperture group except for the plasma component exchange column and the plasma separation column in the group of 30nm and 35nm after 48 hours of circulation, and the PERV was not present at each cycle time point in each aperture group. The protein and RT activity were detected, suggesting that no PERV was passed through the plasma component exchange column of various apertures. In vitro The infection experiment showed no HEK293 cell-infected PERV in addition to the individual microchimerism. Conclusion: During the proposed treatment period, no infectious PERV particles are seen to pass through the plasma component exchange column but at 48 hours in the plasma component exchange column of the membrane pore size of 20nm, there was a permeation of the PERV RNA. in order to take into account the safety of the virus, the membrane pore diameter 2020n is used, The plasma component exchange column of m is safe. The third part Sub-point: The purpose of the study on the safety of the new type of BAL in the treatment of viral safety: an evaluation The invention relates to a method for estimating the virus safety of a novel BAL treatment application. The method comprises the following steps of: using amino galactose to induce the establishment of an animal model of an acute liver failure dog, taking a multi-layer flat plate type reactor based on a lactose-based chitosan nanofiber scaffold as a device basis, and taking the porcine liver cell-M as a base, The SCs co-culture system is a new type of BAL for seed cells, and the experimental dogs are treated for 3 hours on the second day of modeling. The whole blood of plasma and dog in the BAL system before and after treatment and after treatment was taken and the new, liver, spleen, lung, and kidney tissues were sacrificed after 1 year of treatment. The plasma, PBMCs and PEV RNA, DNA, and reversal in the tissues were detected. Enzyme (RT) activity, tissue sample plus western blot and immunity The PERV capsid protein gag p30 was measured in group. In addition, the HEK293 cells were incubated in vitro for each time point plasma to determine its infectivity. Results: The activity of PERV RNA, DNA and RT in circulating 3 plasma showed the presence of PERVs in circulating 3. PEV RNA, DNA, RT activity and anti-PEV antibody were not detected in plasma samples, and there were no HEK293 infection in the in vitro infection. In addition, P The PRV test in the BMCs and the tissue specimens was also negative, suggesting that no PERV was infected with the experimental dog during the treatment period through the plasma component exchange column. Conclusion: The new type of BAL is used to treat acute liver failure experimental canine model and
【学位授予单位】：南京大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R373

【相似文献】