肝臟細(xì)胞分類組成和肝臟巨噬細(xì)胞分離富集方案

1.肝臟細(xì)胞分類

肝臟是人體最大腺體，包含兩大類細(xì)胞，分別是肝實(shí)質(zhì)細(xì)胞（PC）和肝非實(shí)質(zhì)細(xì)胞（NPC）。

肝臟實(shí)質(zhì)細(xì)胞包括了肝細(xì)胞和膽管細(xì)胞。肝細(xì)胞約占所有肝細(xì)胞的70%，并負(fù)責(zé)大部分肝代謝功能。

肝非實(shí)質(zhì)細(xì)胞約占總肝細(xì)胞的30%。肝非實(shí)質(zhì)細(xì)胞包括巨噬細(xì)胞（Kupffer cells, KC）比例約35%、肝竇內(nèi)皮細(xì)胞（Liver endothelial cells, LEC）比例約45%和肝成纖維細(xì)胞（Liver fibroblasts）。也就是說(shuō)巨噬細(xì)胞和肝竇內(nèi)皮細(xì)胞一起占據(jù)了80%。

2.肝臟內(nèi)不同細(xì)胞分離富集

2.1肝組織灌洗：一方面可以去除肝細(xì)胞的紅細(xì)胞，并通過膠原蛋白酶灌注提高肝組織的利用率，另一方面可以收集肝臟血液中的白細(xì)胞等其他免疫細(xì)胞。肝臟灌洗替代的方式可以將肝臟組織剪成2-4 毫米的小塊，加入適量的酶并在最佳溫度（通常為 37°C）下孵育適當(dāng)?shù)臅r(shí)間，間歇混合。最終將組織消化后通過篩網(wǎng)過濾細(xì)胞懸浮液。

2.2根據(jù)肝臟中各細(xì)胞的物理特性對(duì)肝內(nèi)細(xì)胞進(jìn)行分離和提純。

例如由于粒徑的差異，肝細(xì)胞的粒徑普遍在100um左右，而KC、LEC、成纖維細(xì)胞的粒徑普遍小于100um，因此可以40um或70um的細(xì)胞篩網(wǎng)篩去除不需要的肝細(xì)胞，也可以使用50g離心5min將肝細(xì)胞（會(huì)沉淀）和肝臟其他細(xì)胞（繼續(xù)懸浮）分離開來(lái)。由于各細(xì)胞的密度差異，死細(xì)胞或細(xì)胞殘?jiān)捎诿芏刃。趯游鲆褐幸苿?dòng)慢，因此使用不同密度梯度的層析液可以有效地分離死細(xì)胞和肝臟內(nèi)各成分的活細(xì)胞。

2.3根據(jù)肝臟中各細(xì)胞的黏附速度進(jìn)行分離

肝臟中KC與其他非實(shí)質(zhì)細(xì)胞相比黏附速度很快，因此將肝臟非實(shí)質(zhì)細(xì)胞提取放置在細(xì)胞培養(yǎng)箱中約30min，KC就會(huì)貼壁，而其他類型的細(xì)胞不會(huì)貼壁，此時(shí)吸走細(xì)胞培養(yǎng)基，留在培養(yǎng)瓶底的大多數(shù)為KC。反復(fù)操作可利于收集更多KC。

2.4根據(jù)肝臟細(xì)胞中各細(xì)胞的黏附能力進(jìn)行分離

肝臟中成纖維細(xì)胞的黏附能力明顯強(qiáng)于肝臟中其他細(xì)胞。因此如果細(xì)胞懸液中僅有成纖維細(xì)胞和目標(biāo)細(xì)胞，例如LEC或KC等。可以使用胰酶短時(shí)間消化后立即收集細(xì)胞，反復(fù)多次操作利于提高目標(biāo)細(xì)胞的比例。

2.5根據(jù)肝臟細(xì)胞中各細(xì)胞的生長(zhǎng)速度輔助提純

肝臟中成纖維細(xì)胞的生長(zhǎng)能力很強(qiáng)，如果樣本分離不純使得目標(biāo)細(xì)胞中摻雜了成纖維細(xì)胞，可以使用含1%FBS培養(yǎng)基限制成纖維細(xì)胞的生長(zhǎng)，當(dāng)細(xì)胞密度大于50%后，再通過胰酶快速消化的方法純化目標(biāo)細(xì)胞。

3.肝臟內(nèi)不同細(xì)胞分離的器械、試劑或耗材

3.1 器械：準(zhǔn)備鑷子、刀片、留置針、50ml注射器/恒流泵（強(qiáng)烈建議用恒流泵）、外科線、外科剪、轉(zhuǎn)運(yùn)盒、冰塊。

3.2 試劑：DMEM培養(yǎng)基、RPMI培養(yǎng)基、Hanks液、PBS、血清、肝細(xì)胞培養(yǎng)基（或購(gòu)買地塞米松、胰島素、白蛋白、谷氨酰胺等）、EDTA/肝素、雙抗/三抗、膠原蛋白酶Ⅳ型、層析液Percoll、乙醇。

3.3 耗材：培養(yǎng)皿、培養(yǎng)瓶、細(xì)胞篩網(wǎng)、移液管、移液槍、50ml和15ml離心管。

4.肝臟灌注，密度梯度離心方法Intrahepatic macrophages isolation

Primary mouse macrophages were isolated from the mouse liver. Briefly, mouse livers were first perfused in situ with Ca2+ and Mg2+-free Hank’s balanced salt solution (HBSS) containing 0.5 mM EGTA through the portal vein and then again with HBSS containing 0.05% collagenase IV. The digested livers were torn open and then gently separated with forceps until they were in suspension. The cell suspensions were filtered with a 70-μm cell filter, and the hepatocytes were removed by differential centrifugation (50×g, 5 min; 72×g, 5 min). To obtain nonparenchymal cells, the supernatant was collected and centrifuged further (300×g, 5 min; 650×g, 7 min). The pellets from both centrifugation steps were pooled and resuspended in DMEM containing 2% fetal bovine serum (FBS). The cell suspension was transferred to a 25%/50% Percoll gradient and centrifuged at 1200×g for 30 min without braking. The macrophage fraction was enriched in the interface between the 25% and 50% Percoll layers. Macrophages were purified by using a mouse F4/80 positive selection EasySep kit (EasySep™ Mouse F4/80 Positive Selection Kit; 100-0659) in accordance with the manufacturer’s instructions.