CAJ | 학술논문

目的验证不同破囊液裂解载肝细胞海藻酸钠-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0.1 mol/L二水合柠檬酸三钠;破囊液B的基本成分为0.2 mol/L碳酸氢钠和0.06 mol/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A260/A280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/106细胞,A组为(2.39±0.32) μg/106,两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(UGT1A1)、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.
목적 험증불동파낭액렬해재간세포해조산납-각취당(AC)미낭획취고순도RNA적가행성.방법 파낭액A기본성분위0.1 mol/L이수합저몽산삼납;파낭액B적기본성분위0.2 mol/L탄산경납화0.06 mol/L이수합저몽산삼납.장재가역성영생화인간세포(Hepli4)적AC미낭분별용파낭액A(A조,n=5)화파낭액B(B조,n=5)처리.파낭후,Trizol법제취총RNA,RT-PCR법검측간장특이성공능기인.결과 A조세포중가견미낭쇄편잔류,이B조세포중기본관찰불도미낭쇄편.A조화B조RNA양품적A260/A280차이무통계학의의(P＞0.05),A260/A230차이유통계학의의(P＜0.05).B조RNA획득솔위(8.69±0.59) μg/106세포,A조위(2.39±0.32) μg/106,량조비교차이유통계학의의(t=21.09,P＜0.05) 이B조적RNA위모판,가검측도곡안선알합성매(GS)、뇨감이린산포도당철산전이매(UGT1A1)、백단백(ALB)등간장특이성공능기인.결론 이탄산경납-이수합저몽산삼납파낭액처리재간세포AC미낭,능피면미낭성분오염,획득고순도RNA.해파낭액가응용우AC미낭화간세포적기인표체연구.
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 Na3C6H5O7·2H2O; MBS B was basically composed of 0.2 mol/L NaHCO3 and 0.06 mol/L Na3C6H5O7· 2H2O.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 microcapsules 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,A260/A280 ratios detected from the RNA samples showed no statistical differences (P＞0.05),A260/A230 ratios in group A were significantly lower (P＜0.05) . The quantities of RNA in group B were (8.69±0.59) μg/106 cells,were significantly higher than that in group A (2.39±0.32) μg/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 (UGT1A1),albumin (ALB),could be detected.Conclusions By treating hepatocyte-loaded AC with MBS composed of NaHCO3 and Na3C6H5O7·2H2O,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.