新生大鼠肝细胞脾内移植联合rALR治疗重症肝炎的研究

【作者】 袁刚；

【导师】 刘杞；

【作者基本信息】 重庆医科大学 ， 内科学， 2003， 硕士

【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<WP=7>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;冻存分析更多还原

【Abstract】 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<WP=11>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更多还原