【摘要】： 目的:(1)體外分離培養(yǎng)并鑒定SD大鼠肝枯否細(xì)胞,得到較高純度及細(xì)胞活力的KCs,為進(jìn)一步實(shí)驗(yàn)提供充足的KCs。(2)構(gòu)建鼠重組IL-12腺病毒,得到能高度及穩(wěn)定表達(dá)IL-12的腺病毒載體,為進(jìn)一步基因治療提供有效基因轉(zhuǎn)運(yùn)載體。(3)利用KCs的吞噬作用,檢測(cè)經(jīng)脂質(zhì)體包埋的鼠重組IL-12腺病毒在KCs中IL-12的表達(dá),為靶向治療肝癌肝內(nèi)微轉(zhuǎn)移灶的治療提供實(shí)驗(yàn)依據(jù)。 方法:(1)采用Ⅳ型膠原酶離體灌注消化、重復(fù)離心等方法將大鼠肝臟非實(shí)質(zhì)細(xì)胞(nonparenchymal cells,NPC)和實(shí)質(zhì)細(xì)胞(肝細(xì)胞)(parenchymal cells)分離開(kāi)來(lái),并將NPC進(jìn)行貼壁培養(yǎng),進(jìn)一步分離出其中的KCs,相差顯微鏡下觀察KCs的貼壁及生長(zhǎng)情況,臺(tái)盼藍(lán)染色判斷細(xì)胞產(chǎn)率和存活率。KCs的鑒定:運(yùn)用latex beads顆粒進(jìn)行KCs吞噬功能實(shí)驗(yàn);熒光顯微鏡觀察首次換液后的KCs,置328nm波長(zhǎng)下觀察細(xì)胞自發(fā)熒光的情況。(2)利用細(xì)菌內(nèi)重組系統(tǒng)AdEasyTM System,及腺病毒載體pCA13-mILl2質(zhì)粒,構(gòu)建鼠重組IL-12腺病毒(Adv-mIL12)。PCR法、酶切鑒定證實(shí)重組是否正確。50%組織培養(yǎng)感染劑量(TCID50 )法測(cè)定病毒滴度。(3)細(xì)胞培養(yǎng)板中培養(yǎng)KCs,共28孔,細(xì)胞濃度為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，

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