新生大鼠肝细胞脾内移植联合rALR治疗重症肝炎的研究
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摘要
目的:文献报道用胶原酶非灌注消化离体大鼠肝脏，其所得肝细胞数量少,活性较差且有大量的杂细胞污染，给进一步的研究造成了难以克服的影响。本研究针对以上不足加以改进，以提高肝细胞的纯度及质量，为后续研究奠定基础；并同时探索肝细胞的最佳冻存、复苏条件，评价冻存细胞作为供者细胞的可行性，为今后临床应用时出现供者细胞短缺提供解决办法。
方法: 使用胶原酶体外非灌注法短时间分次震荡消化新生大鼠肝组织,获取上清消化液，非连续性梯度离心纯化新生大鼠肝细胞。比较采用rALR条件培养液和无rALR条件培养液培养后,肝细胞的生存时间，并采用流式细胞仪对原代新生大鼠肝细胞和成年大鼠肝细胞通过溴化丙啶染色进行DNA直方图细胞周期分析。用不同浓度的二甲亚枫冻存新生大鼠肝细胞并在不同时间点复苏，比较新生大鼠肝细胞的复苏存活状态。
结果: 用该法分离的新生大鼠肝细胞经台盼蓝拒染试验证实存活率在85%以上，非连续性梯度离心肝细胞，使其纯度高达98%。添加rALR后的条件培养液可使新生大鼠肝细胞的培养存活时间达到12天,较未使用rALR的条件培养液存活时间延长了5天。白蛋白的合成能力可保持较高水平达9天左右。DNA直方图显示经rALR处理后的新生大鼠肝细胞和对照相比：G2-M期细胞比例为13.49%vs11.56%。原代成年大鼠肝细胞经rALR处理后和对照相比：G2-M期细胞比例为
4.57%vs4.12%。肝细胞的最佳冻存条件为培养基中加入二甲亚枫至终浓度为10%，冻存时间以不超过3周为宜。
结论: 用我们改进的非灌注法分离纯化的肝细胞性能良好，可作为肝细胞移植的供体; 对肝细胞分离后冻存条件的观察，为肝细胞移植的临床应用提供了一个参考指标。
关键词: 新生大鼠;肝细胞;分离纯化;冻存分析
Objective: Lots of literatures had reported the methods that digested the liver from mammals through non-perfusion for hepatocyte preparation, but hepatocyte viability and yield obtained by above methods were not satisfactory，moreover, contaminated with lots of other cells so that further research was hampered. So, we tried to improve method of isolation and purification in order to establish the foundation for hepatocyte transplantation and quested the best way of hepatocyte cryopreservation and revivfication and assessed the viability of hepatocyte cryopreserved in order to resolve the shortage of donor cell in future clinic.
Methods: Performing enzymatic digestion of newborn rat liver tissues by three sequential steps in vitro and gaining supernatant, we applied discontinous gradient centrifugation method to purify hepatocytes, and the living times of hepatocytes were compared when cultured with rALR or not.We analyzed the histogram of DNA of newborn rat hepatocyte and primary adult rat hepatocyte for cell cycle study by flow cytometer. We used different concentrations DMSO as cryopreservation solutions for hepatocyte, revived hepatocytes at various intervals in our experiment, and assessed the viability of hepatocyte cryopreserved by trypan blue exclusion.
Results: The hepatocyte viability using our non-perfusion method
was 85% approximately by trypan blue exclusion，and the hepatocyte purification reached above 98% to avoid the contamination with mesothelial cell. Through the comparison of adding rALR to culture or not,we found out the living time of hepatocyte cultured with rALR was 5 days longer than without rALR. The histogram of DNA showed that the percent for G2-M phase of newborn rat hepatocyte cultured with rALR was higher (13.49% VS 11.56%) compared to that without rALR.At the same time, the results for primary adult rat hepatocyte was 4.57%vs4.12% . The function of albumin synthesis could have kept at the high levels for 9 days. The best way for cryopreservation was adding 10% DMSO in the medium for 3 weeks.
Conclusions: The newborn rat's hepatocyte isolated and purified by our method had a good function, which could be used as donors for hepatocyte transplantation. It may be useful for the near future clinic applications that the investigation of hepatocyte's cryopreservation and revivfication.
KEY WORDS: newborn rat;hepatocytes;isolation and purificatrion;cryopreservation
方法: 使用胶原酶体外非灌注法短时间分次震荡消化新生大鼠肝组织,获取上清消化液，非连续性梯度离心纯化新生大鼠肝细胞。比较采用rALR条件培养液和无rALR条件培养液培养后,肝细胞的生存时间，并采用流式细胞仪对原代新生大鼠肝细胞和成年大鼠肝细胞通过溴化丙啶染色进行DNA直方图细胞周期分析。用不同浓度的二甲亚枫冻存新生大鼠肝细胞并在不同时间点复苏，比较新生大鼠肝细胞的复苏存活状态。
结果: 用该法分离的新生大鼠肝细胞经台盼蓝拒染试验证实存活率在85%以上，非连续性梯度离心肝细胞，使其纯度高达98%。添加rALR后的条件培养液可使新生大鼠肝细胞的培养存活时间达到12天,较未使用rALR的条件培养液存活时间延长了5天。白蛋白的合成能力可保持较高水平达9天左右。DNA直方图显示经rALR处理后的新生大鼠肝细胞和对照相比：G2-M期细胞比例为13.49%vs11.56%。原代成年大鼠肝细胞经rALR处理后和对照相比：G2-M期细胞比例为
4.57%vs4.12%。肝细胞的最佳冻存条件为培养基中加入二甲亚枫至终浓度为10%，冻存时间以不超过3周为宜。
结论: 用我们改进的非灌注法分离纯化的肝细胞性能良好，可作为肝细胞移植的供体; 对肝细胞分离后冻存条件的观察，为肝细胞移植的临床应用提供了一个参考指标。
关键词: 新生大鼠;肝细胞;分离纯化;冻存分析
Objective: Lots of literatures had reported the methods that digested the liver from mammals through non-perfusion for hepatocyte preparation, but hepatocyte viability and yield obtained by above methods were not satisfactory，moreover, contaminated with lots of other cells so that further research was hampered. So, we tried to improve method of isolation and purification in order to establish the foundation for hepatocyte transplantation and quested the best way of hepatocyte cryopreservation and revivfication and assessed the viability of hepatocyte cryopreserved in order to resolve the shortage of donor cell in future clinic.
Methods: Performing enzymatic digestion of newborn rat liver tissues by three sequential steps in vitro and gaining supernatant, we applied discontinous gradient centrifugation method to purify hepatocytes, and the living times of hepatocytes were compared when cultured with rALR or not.We analyzed the histogram of DNA of newborn rat hepatocyte and primary adult rat hepatocyte for cell cycle study by flow cytometer. We used different concentrations DMSO as cryopreservation solutions for hepatocyte, revived hepatocytes at various intervals in our experiment, and assessed the viability of hepatocyte cryopreserved by trypan blue exclusion.
Results: The hepatocyte viability using our non-perfusion method
was 85% approximately by trypan blue exclusion，and the hepatocyte purification reached above 98% to avoid the contamination with mesothelial cell. Through the comparison of adding rALR to culture or not,we found out the living time of hepatocyte cultured with rALR was 5 days longer than without rALR. The histogram of DNA showed that the percent for G2-M phase of newborn rat hepatocyte cultured with rALR was higher (13.49% VS 11.56%) compared to that without rALR.At the same time, the results for primary adult rat hepatocyte was 4.57%vs4.12% . The function of albumin synthesis could have kept at the high levels for 9 days. The best way for cryopreservation was adding 10% DMSO in the medium for 3 weeks.
Conclusions: The newborn rat's hepatocyte isolated and purified by our method had a good function, which could be used as donors for hepatocyte transplantation. It may be useful for the near future clinic applications that the investigation of hepatocyte's cryopreservation and revivfication.
KEY WORDS: newborn rat;hepatocytes;isolation and purificatrion;cryopreservation
引文
1. 顾长海，王宇明。急性肝衰竭。成都；四川科学技术出版社，1997；11-21
2．Nikola LV, Lorenzo P, Alessandro A, et al. Changes of liver-resident NK cells during liver regeneration in rats. J Immunology 1995; 154:6324-6338
3．Blazka ME, Elwell MR, Holladay SD, et al. Histopathology of acetaminophen-induced liver changes: ole of interleukin 1 alpha and tumor necrosis factor alpha. Toxicol Pathol 1996; 24:181-189
4．Tamura F, Masuhara A, Sakaida I, et al. FK506 promates liver regeneration by suppressing natural killer cell activity. J Gastroenterol Hepatol 1998; 13:703-708
5．Boulton RA, Alison MR, Golding M, et al. Augmentation of the early phase of liver regeneration after 70% partial hepatectomy in rats following selective kuffer cell depletion. J Hepatol 1998; 29:271-280
6．Takeshita K, Ishibashi H, Suzuki M, et al. Hepatocellular transplantation for metabolic support in experimental acute ischemic liver failure in rats. Cell Transplant 1993; 2:319-324
7．Demetriou AA, Reisner A, Sanchez J, et al. Transplantation of microcarrier-attached hepatocytes into 90% partially hepatectomized rats. Hepatology 1988; 8:1006-1009
8．Mito M, Susano M, Sawa M, et al. Hepatocyte transplantation for hepatic failure. Transplant Rev 1993; 7:35-43
9．Michio Hagiya, Antonio Francavilla, Lorenzo Polimeno, et al. Cloning and sequence analysis of the rat augmenter of liver regeneration(ALR) gene: Expression of biologically active recombinant ALR and demonstration of tissue distribution. Proc. Natl. Acad. Sci. USA 1994; 91:8142-8146
10．刘 杞，王志毅，罗 娅等。人肝再生增强因子阅读框的cDNA克隆和序列分析。中华肝脏病杂志。1999; 7(3):156-158
11．刘 杞，石小枫，罗 娅等。重组人肝再生增强因子原核表达载体的构建及在大肠杆菌中的表达。中华肝脏病杂志 2000；8（1）：9-11
12．Francavilla A, Vujanovic NL, Polimeno L, et al. The in vivo effect of hepatotrophic factors augmenter of liver regeneration, hepatocyte growth factor, and insulin-like growth factor-II on liver natural killer cell functions. Hepatology 1997; 25:411-415
13．Tanigawa K, Sakaida I, Masuhara M, et al. Augmenter of liver regeneration (ALR) may promote liver regeneration by reducing natural killer (NK) cell activity in human liver diseases. J Gastroenterol 2000;
35:112-119
14．Adams GA, Maestri M, Squiers EC, et al. Augmenter of liver regeneration enhances the success rats of fetal pancreas transplantation in rodents. Transplantation 1998; 65:32-36
2．Nikola LV, Lorenzo P, Alessandro A, et al. Changes of liver-resident NK cells during liver regeneration in rats. J Immunology 1995; 154:6324-6338
3．Blazka ME, Elwell MR, Holladay SD, et al. Histopathology of acetaminophen-induced liver changes: ole of interleukin 1 alpha and tumor necrosis factor alpha. Toxicol Pathol 1996; 24:181-189
4．Tamura F, Masuhara A, Sakaida I, et al. FK506 promates liver regeneration by suppressing natural killer cell activity. J Gastroenterol Hepatol 1998; 13:703-708
5．Boulton RA, Alison MR, Golding M, et al. Augmentation of the early phase of liver regeneration after 70% partial hepatectomy in rats following selective kuffer cell depletion. J Hepatol 1998; 29:271-280
6．Takeshita K, Ishibashi H, Suzuki M, et al. Hepatocellular transplantation for metabolic support in experimental acute ischemic liver failure in rats. Cell Transplant 1993; 2:319-324
7．Demetriou AA, Reisner A, Sanchez J, et al. Transplantation of microcarrier-attached hepatocytes into 90% partially hepatectomized rats. Hepatology 1988; 8:1006-1009
8．Mito M, Susano M, Sawa M, et al. Hepatocyte transplantation for hepatic failure. Transplant Rev 1993; 7:35-43
9．Michio Hagiya, Antonio Francavilla, Lorenzo Polimeno, et al. Cloning and sequence analysis of the rat augmenter of liver regeneration(ALR) gene: Expression of biologically active recombinant ALR and demonstration of tissue distribution. Proc. Natl. Acad. Sci. USA 1994; 91:8142-8146
10．刘 杞，王志毅，罗 娅等。人肝再生增强因子阅读框的cDNA克隆和序列分析。中华肝脏病杂志。1999; 7(3):156-158
11．刘 杞，石小枫，罗 娅等。重组人肝再生增强因子原核表达载体的构建及在大肠杆菌中的表达。中华肝脏病杂志 2000；8（1）：9-11
12．Francavilla A, Vujanovic NL, Polimeno L, et al. The in vivo effect of hepatotrophic factors augmenter of liver regeneration, hepatocyte growth factor, and insulin-like growth factor-II on liver natural killer cell functions. Hepatology 1997; 25:411-415
13．Tanigawa K, Sakaida I, Masuhara M, et al. Augmenter of liver regeneration (ALR) may promote liver regeneration by reducing natural killer (NK) cell activity in human liver diseases. J Gastroenterol 2000;
35:112-119
14．Adams GA, Maestri M, Squiers EC, et al. Augmenter of liver regeneration enhances the success rats of fetal pancreas transplantation in rodents. Transplantation 1998; 65:32-36