目的 建立大鼠原代肝細(xì)胞提取鑒定體系,比較對乙酰氨基酚(APAP)對大鼠原代肝細(xì)胞和永生化細(xì)胞BRL-3A的毒性作用。方法 采用膠原酶原位兩步灌流法提取大鼠原代肝細(xì)胞,通過過碘酸雪夫染色(PAS)和肝細(xì)胞雙核結(jié)構(gòu)進(jìn)行鑒定;CCK 8法測定APAP對大鼠原代肝細(xì)胞及BRL-3A細(xì)胞毒性作用的IC₅₀;光學(xué)顯微鏡透射電鏡觀察APAP對2種細(xì)胞的損傷情況;全自動生化分析儀測定細(xì)胞上清AST、ALT、LDH、ALP、ALB、BUN、TP、GLU 8項(xiàng)生化指標(biāo)的變化。結(jié)果 PAS糖原染色鑒定獲取的大鼠原代肝細(xì)胞,雙核結(jié)構(gòu),細(xì)胞存活率浮動在80%~95%;最佳接種密度為60000/cm²,在第3~5天為對數(shù)生長期;APAP作用于大鼠原代肝細(xì)胞24 h的IC₅₀為18.03 mmol/L,95%置信區(qū)間為(17.28~18.81)mmol/L,作用于BRL-3A的IC₅₀為20.05 mmol/L,95%置信區(qū)間為(18.99~21.17)mmol/L;透射電鏡結(jié)果顯示,在30 mmol/L APAP作用下,2種細(xì)胞細(xì)胞器腫脹,核膜破裂,細(xì)胞膜邊界模糊不清;與對照組比較,大鼠原代肝細(xì)胞分泌的天冬氨酸氨基轉(zhuǎn)移酶(AST)、尿素氮(BUN)、葡萄糖(GLU)、堿性磷酸酶(ALP)、乳酸脫氫酶(LDH)隨著APAP濃度增加產(chǎn)生顯著變化,而BRL-3A細(xì)胞幾乎所有的酶學(xué)指標(biāo)變化均差異不顯著。結(jié)論 與永生化細(xì)胞BRL-3A比較,大鼠原代肝細(xì)胞更能體現(xiàn)藥物的肝臟毒性作用,但其體外培養(yǎng)存活時間較短;BRL-3A細(xì)胞缺少肝臟重要酶類,增加細(xì)胞內(nèi)肝臟酶類是提升其作為肝臟毒性篩選模型的更好手段之一。

Objective To establish a rat primary hepatocytes isolation and identification system, and carry out studies in rat primary hepatocytes and BRL-3A cells for liver toxicity characteristic of early drug evaluation. Methods Rat primary hepatocytes were isolated by two-step in situ collagenase perfusion method, and then were identified by PAS staining and its dual-nuclei structure; The IC₅₀ values, which was hepatocelluar toxicity of APAP in rat primary hepatocytes, was evaluated by CCK8 assay; Damage to the two kinds of cells from the drug was observed using inverted phase contrast microscope, hematoxylin eosin (HE) staining, and transmission electron microscopy (SEM); Automatic biochemical analyzer was used to detect the contents of ALT, AST, ALP, LDH, TP, ALB, GLU, and BUN changes in cell supernatants after administration. Results PAS staining shows positive involvement of two nuclei in some cells, cell viability was ranged between 80% and 95%. Rat primary hepatocytes grew best with a cell density of 60000/cm², 3-5 d was ogarithmic growth phase; Primary rat hepatocytes BRL-3A were exposed to concentration of APAP showed that IC₅₀ values were 18.03 mmol/L, 95%CI=(17.28, 8.81) mmol/L and 20.05 mmol/L, 95%CI=(18.99, 21.17) mmol/L. In the high- dose groups, transmission electron microscopy revealed that cells were organelles swelling, nuclear membrane rupture and the nucleus were almost invisible. Meanwhile, compared with control group, with drug concentration increased, the values of aspartate aminotransferase (AST), urea nitrogen (BUN), glucose (GLU), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH) were significantly changed, while other targets were no significantly changed in BRL-3A cells. Conclusion Compared with immortalized BRL-3A cells, primary hepatocytes can be more reflective model with the liver toxicity. However, primary hepatocytes cannot live for a long time and lack of extensive metabolic enzymes. Thus, increasing the kinds of liver enzymes of immortalized cells is a better means to boost BRL-3A cells as liver toxicity screening model.

重大新藥創(chuàng)制科技重大專項(xiàng)(2013ZX09302303,2012ZX09301-001-008);北京市科委基金項(xiàng)目(Z131100006513010)