Modulation of pro-apoptotic (Bax) and anti-apoptotic (Bcl-2) gene expression in isolated porcine hepatocytes perfused within a radial-flow bioreactor after low-temperature storing
Modulation of pro-apoptotic (Bax) and anti-apoptotic (Bcl-2) gene expression in isolated porcine hepatocytes perfused within a radial-flow bioreactor after low-temperature storing
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DOI：
10.1177/039139880302600208
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发表时间：
2003-02-01
影响因子：
1.7
通讯作者：
Gambari, R
中科院分区：
文献类型：
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作者：
Mischiati, C;Puviani, AC;Gambari, R
由于可用的人类肝脏稀缺，目前正在评估猪肝细胞作为体外生物人工肝（BAL）的异种细胞来源。低温储存分离的猪肝细胞可以支持BAL使用的装载细胞的生物反应器的储备，并且可以提供随时可以在患者床边使用的生物反应器。对于该技术的发展，确保低温储存和温热再灌注后的细胞活力和分化功能至关重要。我们比较了在径向流生物反应器（RFB）中灌注的分离的猪肝细胞的细胞活力、功能活性和凋亡，这些猪肝细胞在4 ℃下储存，然后在37 ℃下再灌注。RFB加载8 × 10（9），大于或等于90%的活肝细胞，37 ℃下3 h。然后用4 ℃的威斯康星州大学溶液（UW）冲洗RFB，随后储存24小时或48小时。然后在37 ℃下用再循环培养基加血清再灌注RFB 8小时。在反复注射利多卡因后，通过检测流出液培养基中的单乙基甘氨酰二甲基苯胺（MEGX），研究了冷藏前后细胞色素P450（CYP 1A 2）的活性。再灌注实验后，收获肝细胞用于总RNA分离。逆转录-聚合酶链反应（RT-PCR），以扩增Bcl-2和Bax基因的特异性mRNA，通过使用适当的引物; β-肌动蛋白引物作为对照。通过北方印迹分析提取总RNA，并使用RT-PCR扩增用于Bcl-2、Bax和β-肌动蛋白RNA信使检测。新鲜分离的肝细胞灌注到RFB中表现出MEGX的逐步增加，而Bax表达的损失被Bcl-2表达的增加所抵消，与起始肝细胞相比。在4 ℃储存和温热再灌注后，MEGX的产生在储存24 h和48 h的生物反应器中保持不变，Bcl-2的急剧增加和Bax mRNA的减少。我们的研究表明，冷冻肝细胞生物反应器是一种合适的策略，以保持分离的肝细胞的活力和功能，长达48小时的时间长度是兼容的长距离交付的即用型生物反应器。
Due to the scarcity of available human livers, porcine hepatocytes are currently being evaluated as a xenogeneic cell source for extracorporeal bioartificial liver (BAL). Hypothermic storage of isolated porcine hepatocytes could support stocking of cell-loaded bioreactors for BAL use and may provide bioreactors ready to be used at the patient's bedside. For the development of this technology, it is of utmost importance to ensure cell viability and differentiated functions after low-temperature storage and following warm reperfusion. We compared cell viability, functional activity and apoptosis in isolated porcine hepatocytes which were perfused within a radial-flow bioreactor (RFB), stored at 4degreesC and then reperfused at 37degreesC. RFBs were loaded with 8 x 10(9), greater than or equal to 90% viable hepatocytes at 37degreesC for 3 h. RFBs were then flushed with 4 0 C University of Wisconsin solution (UW) and subsequently stored for 24 h or 48 h. RFBs were then reperfused for 8 h with recirculating medium plus serum at 37degreesC. Cytochrome P450 (CYP) activity was studied before and after cold storage by means of monoethylglycinexylide (MEGX) detection in the effluent medium, after repeated lidocaine injections. After reperfusion experiments, hepatocytes were harvested for total RNA isolation. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used in order to amplify specific mRNAs for Bcl-2 and Bax genes, by using appropriate primers; beta-actin primers were used as control. Total RNA was extracted by northern blotting analysis and for Bcl-2, Bax and beta-actin RNA messenger detection, RT-PCR amplification was used. Freshly isolated hepatocytes perfused into the RFB showed a progressive increase of MEGX while a loss in Bax expression was paralleled by an increase in Bcl-2 expression, in comparison to starting hepatocytes. After 4degrees C storage and warm reperfusion, MEGX production was preserved in 24 h- and 48 h-stored bioreactors as well as a sharp increase of Bcl-2 and a decrease of Bax mRNAs. Our study suggests that refrigeration of hepatocyte-bioreactors is a suitable strategy to maintain both viability and function of isolated hepatocytes,,for up to 48 h a time-length that is compatible with long-distance delivery of ready-to-use bioreactors.