鼠重组IL-12腺病毒经脂质体包埋转染Kupffer细胞的实验研究

发布时间：2019-02-13 06:31

【摘要】： 目的:(1)体外分离培养并鉴定SD大鼠肝枯否细胞,得到较高纯度及细胞活力的KCs,为进一步实验提供充足的KCs。(2)构建鼠重组IL-12腺病毒,得到能高度及稳定表达IL-12的腺病毒载体,为进一步基因治疗提供有效基因转运载体。(3)利用KCs的吞噬作用,检测经脂质体包埋的鼠重组IL-12腺病毒在KCs中IL-12的表达,为靶向治疗肝癌肝内微转移灶的治疗提供实验依据。 方法:(1)采用Ⅳ型胶原酶离体灌注消化、重复离心等方法将大鼠肝脏非实质细胞(nonparenchymal cells,NPC)和实质细胞(肝细胞)(parenchymal cells)分离开来,并将NPC进行贴壁培养,进一步分离出其中的KCs,相差显微镜下观察KCs的贴壁及生长情况,台盼蓝染色判断细胞产率和存活率。KCs的鉴定:运用latex beads颗粒进行KCs吞噬功能实验;荧光显微镜观察首次换液后的KCs,置328nm波长下观察细胞自发荧光的情况。(2)利用细菌内重组系统AdEasyTM System,及腺病毒载体pCA13-mILl2质粒,构建鼠重组IL-12腺病毒(Adv-mIL12)。PCR法、酶切鉴定证实重组是否正确。50%组织培养感染剂量(TCID50 )法测定病毒滴度。(3)细胞培养板中培养KCs,共28孔,细胞浓度为1X106/ml。
[Abstract]:Objective: (1) to isolate and identify Kupffer cells of SD rat liver in vitro, and obtain KCs, with high purity and cell viability to provide sufficient KCs. (2) to construct recombinant IL-12 adenovirus. An adenovirus vector with high and stable expression of IL-12 was obtained to provide an effective gene transport vector for further gene therapy. (3) the phagocytosis of KCs was utilized. The expression of IL-12 in liposome embedded rat recombinant IL-12 adenovirus in KCs was detected to provide experimental evidence for the targeted treatment of intrahepatic micrometastasis of hepatocellular carcinoma (HCC). Methods: (1) Rat liver non-parenchymal cells (nonparenchymal cells,NPC) and parenchymal cells () (parenchymal cells) were isolated by type 鈪，

本文编号：2421279