三维拟生态培养网片培养猪肝细胞的功能和形态观察

目的通过观察猪肝细胞的功能和形态,探讨一种三维拟生态培养网片用于猪肝细胞培养的可行性。方法两步法分离乳猪肝细胞,接种于培养网片进行培养,观察培养基中各项生化指标、白蛋白以及培养装置进出口氧分压差的变化,分析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特征。结果以培养时间分组,分为0、2、4、6、8h组,各组培养基中谷丙转氨酶(AST)、谷草转氨酶(ALT)、乳酸脱氢酶(LDH)、总胆红素浓度(TBIL)以及培养装置进出口氧分压差的均数无明显差异(P>0.05);以不同时间段分组,分为0-2、2-4、4-6、6-8h组,尿素氮合成率和安定清除率均保持较高水平,组间存在明显差异(P<0.05),随着时间的延长而有所下降;培养结束时可观察到网片中肝细胞生长的典型形态特征。肝细胞在培养网片中的培养密度达到了108/ml以上。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。

A biomimetic 3-Dimentinal culture matrix for high-density culture of swine hepatocytes

Objective To study the feasibility of a biomimetic 3-Dimentinal culture matrix for culture of porcine hepatocyte by observing the function and morphological characteristic of the cultured cell.Methods Porcine hepatocyte were isolated by two-step perfusion and cultured in a unique cell culture setting based on biomimetic 3-Dimentinal culture matrix four times.Observing the changes of AST,ALT,LDH,TBIL,albumin in setting and oxygen pressure difference between the exit and entry of setting,analyzing the changes of diazepam clearance and urea（BUN） synthesizing rate,observing morphological characteristics of hepatocytes in culture matrix.Results During the total period of culture,there were no significant difference in concentration average of AST,ALT,TBIL,LDH and oxygen pressure difference average among different groups according to different time（P 0.05）;there were significant difference in diazepam clearance and urea synthesizing rate among different groups according to different period（P 0.05）.The typical cellular morphological characteristics of hepatocytes could be observed in culture matrix.The density of cultured hepatocytes reached 108/ml.Conclusions The hepatocytes in culture matrix keep steady biochemical activity,good substance metabolic transformation and biosynthesis ability,typical cell physiological and morphological characteristics,this culture matrix achieved high density and activity culture of hepatocytes,which maybe more eligible for use in the study of bioartificial liver.