【摘要】：第一部分：乳糖�；鶜ぞ厶羌{米纖維支架上豬肝細胞和MSCs共培養(yǎng)體系的PERVs分泌及體外感染性分析 目的：明確乳糖酰基殼聚糖納米纖維支架上豬肝細胞和MSCs共培養(yǎng)體系的PERV分泌情況及體外感染能力。 方法：將新鮮分離的豬肝細胞和傳代至第3-5代MSCs細胞單獨或2:1共培養(yǎng)接種于有或無乳糖�；鶜ぞ厶羌{米纖維支架的6孔培養(yǎng)板,定為單純肝細胞組(Hep)、肝細胞及MSCs共培養(yǎng)組(Co)、乳糖�；鶜ぞ厶羌{米纖維支架+肝細胞組(Nano),乳糖�；鶜ぞ厶羌{米纖維支架+肝細胞及MSCs共培養(yǎng)組(Nano-Co),連續(xù)培養(yǎng)7天。每天收集培養(yǎng)液檢測逆轉錄酶(RT)活性,并通過RT-PCR和實時定量PCR測定PERV RNA水平。通過western blot檢測培養(yǎng)液中PERV顆粒有無。細胞培養(yǎng)液體外孵育HEK293細胞以測定其感染性。 結果：Hep組和Nano組PERV分泌10小時和第2天最高,后呈逐漸下降趨勢,前者PERV分泌持續(xù)5天,而后者延長分泌至第6天。Co組和Nano-Co組PERV分泌在第2天后隨時間逐漸增多,第6天達到PERV分泌高峰。經(jīng)比較,Nano-Co組從第2天后PERV分泌量明顯高于單純肝細胞,且從第2天后每天PERV分泌量顯著高于10H。體外感染實驗發(fā)現(xiàn)Co組和Nano-Co在后期有微嵌合細胞外,并無HEK293細胞感染PERV。 結論：豬肝細胞和MSCs共培養(yǎng)體系在兩種培養(yǎng)條件下均能分泌PERV,而在乳糖�；鶜ぞ厶羌{米纖維支架上培養(yǎng)2天后PERV分泌量隨時間逐漸增多,且后期體外感染實驗出現(xiàn)微嵌合狀態(tài),但并無表現(xiàn)出明顯體外感染性。由此提示豬肝細胞和MSCs共培養(yǎng)體系置于乳糖�；鶜ぞ厶羌{米纖維支架上培養(yǎng)2天內(nèi)使用較為合理。 第二部分：不同孔徑血漿成分交換柱對新型BAL系統(tǒng)PERV透過情況的影響 目的：檢測采用不同孔徑的血漿交換柱的PERV透過情況；為新型BAL系統(tǒng)血漿交換柱孔徑的選擇提供數(shù)據(jù)參考。 方法：將豬肝細胞和MSCs以2:1混合共培養(yǎng)于基于乳糖�；鶜ぞ厶羌{米膜支架的多層平板型生物反應器中,并構建新型BAL系統(tǒng)。依據(jù)血漿成分交換柱膜孔徑將實驗分為10nm組、20nm組、30nm組和35nm組。細胞灌入后4小時開始培養(yǎng)液循環(huán),循環(huán)48小時。檢測三循環(huán)培養(yǎng)液內(nèi)逆轉錄酶(RT)活性、PERV RNA以及western blot測定PERV顆粒。同時用培養(yǎng)液體外孵育HEK293細胞以測定其感染性。 結果：除30nm和35nm組在循環(huán)48小時后出現(xiàn)PERV RNA透過血漿成分交換柱和血漿分離柱外,在各孔徑組各循環(huán)時間點均無PERV RNA、PERV蛋白和RT活性檢出,提示無PERV透過各種孔徑的血漿成分交換柱。體外感染實驗除個別出現(xiàn)微嵌合狀態(tài)外并無HEK293細胞感染PERV。 結論：在擬定治療期6小時內(nèi),并未見感染性PERV顆粒透過血漿成分交換柱,但在膜孔徑20nm的血漿成分交換柱中48小時時有PERV RNA的透過。所以,從病毒安全性方面考慮,采用膜孔徑≤20nm的血漿成分交換柱是安全的。 第三部分：新型BAL治療應用的病毒安全性研究 目的：評估新型BAL治療應用的病毒安全性。 方法：采用氨基半乳糖誘導建立急性肝功能衰竭犬動物模型。以基于乳糖�；鶜ぞ厶羌{米纖維支架的多層平板型反應器為裝置基礎,以豬肝細胞-MSCs共培養(yǎng)體系為種子細胞構建新型BAL,并于建模第2天治療實驗犬,治療時間3小時,抽取治療前、治療中和治療后BAL系統(tǒng)內(nèi)血漿及犬全血,并于治療后1年處死取新、肝、脾、肺、腎組織。檢測血漿、PBMCs以及各組織中PERV RNA、DNA、逆轉錄酶(RT)活性,組織樣本加行western blot和免疫組化測定PERV衣殼蛋白gag p30。此外,各時間點血漿體外孵育HEK293細胞以測定其感染性。 結果：PERV RNA、DNA、RT活性出現(xiàn)于循環(huán)3血漿中,提示循環(huán)3中存在細胞釋放的PERVs.其余血漿樣本均未檢出PERV RNA、DNA、RT活性以及抗PERV抗體,而且體外感染中均無HEK293感染。此外,PBMCs和組織標本中各項PERV檢驗也均為陰性,提示治療期間并無PERV透過血漿成分交換柱而感染實驗犬。 結論：新型BAL治療急性肝功能衰竭實驗犬模型后并無動物感染PERV,說明新型BAL在治療應用中具有可靠地在體病毒安全性,為將來該系統(tǒng)的臨床試驗提供了安全性證據(jù)。
[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
【學位授予單位】：南京大學
【學位級別】：博士
【學位授予年份】：2011
【分類號】：R373

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