适用于海藻酸钠-壳聚糖微囊化肝细胞RNA提取的破囊方法

目的验证不同破囊液裂解载肝细胞海藻酸钠一壳聚糖（AC）微囊获取高纯度RNA的可行性。方法破囊液A基本成分为0．1mol／L二水合柠檬酸三钠；破囊液B的基本成分为0．2mol／L碳酸氢钠和0．06mol／L二水合柠檬酸三钠。将载可逆性永生化人肝细胞（Hepli4）的AC微囊分别用破囊液A（A组，n=5）和破囊液B（B组，n=5）处理。破囊后，Trizol法提取总RNA，RT-PCR法检测肝脏特异性功能基因。结果A组细胞中可见微囊碎片残留．而B组细胞中基本观察不到微囊碎片。A组和B组RNA样品的A—A。差异无统计学意义（P〉0．05），A260/A230差异有统计学意义（P〈0．05）。B组RNA获得率为（8．69±0．59）μg／10。细胞，A组为（2．39＋0．32）μg／10。，两组比较差异有统计学意义（t=21．09，P〈0．05）。以B组的RNA为模板，可检测到谷氨酰胺合成酶（GS）、尿苷二磷酸葡萄糖醛酸转移酶（UGTIAl）、白蛋白（ALB）等肝脏特异性功能基因。结论以碳酸氢钠-二水合柠檬酸三钠破囊液处理载肝细胞AC微囊，能避免微囊成分污染，获得高纯度RNA。该破囊液可应用于AC微囊化肝细胞的基因表达研究。

Microcapsule-broken method suitable for RNA isolation from alginate-chitosan encapsulated hepatocytes

Objective To test the feasibility of different microcapsule-broken solutions （MBS） for high quality RNA isolation from Alginate-chitosan （AC） encapsulated hepatocytes. Methods MBS A was basically composed of 0.1mol/L Na3C6I-I507 ＂2H20; MBS B was basically composed of 0.2 mol/L NaHCO3 and 0.06 mol/L Na3C6HsOT＂ 2H20. AC microcapsules loaded with reversibly immortalized human hepatocytes （Hepli4） were treated with MBS A （group A, n=5） or MBS B （group B, n=5）, respectively. Total cellular RNA was isolated using Trizol reagent. Expression of liver-specific genes in Hepli4 cells was analyzed by RT-PCR. Results Fragments of the mi- crocapsules could be seen in the cells of group A. However, no fragments of the microcapsules were found in the cells of group B.Between two groups, A~o/Azs0 ratios detected from the RNA samples showed no statistical differ- ences （P〉0.05）, A~o/A~0 ratios in group A were significantly lower （P〈0.05）o The quantities of RNA in group B were （8.69＋-0.59） p.g/iO6 cells, were significantly higher than that in group A （2.39＋-0.32） p,g/106 cells（t=21.09, P〈0.05 ）. By using RNA of group B as a template, liver-specific genes, such as glutamine synthetase （GS）, uridine diphosphate glucuronyltransferase （UGTI A1 ）, albumin （ALB）, could be detected. Conclusions By treating hepa- tocyte-loaded AC with MBS composed of NaHCO3 and Na3C6HsOT＂2H20, it could thoroughly remove, ingredients from microcapsules, and obtain a highly purified RNA. This MBS might be useful in analyzing gene expression of AC encapsulated hepatocytes.