三维拟生态网片用于猪肝细胞高密度培养的可行性研究

发布时间：2018-12-20 14:29

【摘要】：目的通过观察猪肝细胞的功能和形态学特征，探讨一种三维拟生态培养网片用于猪肝细胞高密度培养的可行性。方法采用两步法分离乳猪肝细胞，，使用由美国Cordlife公司生产的以一种三维拟生态培养网片为基础的细胞培养装置进行乳猪肝细胞培养，共进行肝细胞培养试验4次，于培养开始后0、2、4、6、8小时观察培养液中各项生化指标、白蛋白、安定和尿素氮的浓度以及培养装置进出口氧分压差的变化，分析O-2、2-4、4-6、6-8小时段安定清除率和尿素合成率的变化，观察肝细胞在培养网片中的形态学特征。结果以培养开始后不同时间分组，分为0、2、4、6、8小时组，各组之间培养液中谷丙转氨酶、谷草转氨酶、乳酸脱氢酶、总胆红素浓度以及培养装置进出口氧分压差的均数无明显差异(P＞0.05)，尿素氮和安定浓度的均数存在明显差异(P＜0.05)；以不同时间段分组，分为0-2、2-4、4-6、6-8小时组，各组之间尿素氮合成率和安定清除率存在明显差异(P＜0.05)，随着时间的延长而有所下降；倒置相差显微镜下可观察到肝细胞典型的形态学特征。肝细胞在培养网片中的培养密度达到了10~7/ml。结论生长在培养网片中的肝细胞保持了稳定的生化活性、良好的物质转化代谢能力和生物合成能力、典型的细胞生理代谢特点和生长形态学特征，该培养网片能为肝细胞提供更接近体内的培养环境，实现了肝细胞的高密度和高活性培养。今后可进一步探讨以此培养网片为基础构建一种新的肝细胞培养系统，以及其应用于生物人工肝的研究。
[Abstract]:Objective to observe the function and morphological characteristics of porcine hepatocytes and to explore the feasibility of using a three-dimensional ecologically cultured mesh for high density culture of porcine hepatocytes. Methods the porcine hepatocytes were isolated by two steps method. The hepatocytes were cultured with a three dimensional ecological culture mesh device produced by Cordlife Company of USA. The hepatocyte culture experiments were carried out for 4 times. The changes of biochemical indexes, concentration of albumin, diazepam and urea nitrogen, and the difference of oxygen partial pressure between the inlet and outlet of the culture unit were observed at 0 ~ 2 ~ 4 ~ 4 ~ 6 ~ (-1) h after culture. The changes of diazepam clearance rate and urea synthesis rate in 6-8 hours of O-2O2-4O4-6-6 were analyzed, and the morphological characteristics of hepatocytes in cultured mesh were observed. Results according to the different time groups after culture, they were divided into two groups, one group was treated with alanine aminotransferase (alt), the other was aspartate aminotransferase (AST) and lactate dehydrogenase (lactate dehydrogenase). There was no significant difference in the concentration of total bilirubin and the difference of oxygen partial pressure (P > 0.05), but there was significant difference between the concentration of urea nitrogen and diazepam (P < 0.05). There were significant differences in urea nitrogen synthesis rate and diazepam clearance rate between groups (P < 0.05). Typical morphological features of hepatocytes can be observed under inverted phase contrast microscope. The density of hepatocytes in the cultured mesh was 10 7 / ml. Conclusion the hepatocytes growing in the cultured meshes maintain stable biochemical activity, good ability of substance transformation, metabolism and biosynthesis, typical characteristics of cell physiological metabolism and growth morphology. This culture mesh can provide a more close culture environment for hepatocytes and achieve the high density and high activity of hepatocytes. In the future, a new hepatocyte culture system based on the culture mesh can be further explored, and its application to bioartificial liver can be studied.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R329

【相似文献】