ISOLATION AND LIPID BIOCHEMICAL-CHARACTERIZATION OF STRATUM-CORNEUM MEMBRANE COMPLEXES - IMPLICATIONS FOR THE CUTANEOUS PERMEABILITY BARRIER
ISOLATION AND LIPID BIOCHEMICAL-CHARACTERIZATION OF STRATUM-CORNEUM MEMBRANE COMPLEXES - IMPLICATIONS FOR THE CUTANEOUS PERMEABILITY BARRIER
复制标题
DOI：
10.1111/1523-1747.ep12505953
复制
发表时间：
1982-01-01
影响因子：
6.5
通讯作者：
ELIAS, PM
中科院分区：
文献类型：
--
作者：
GRAYSON, S;ELIAS, PM
尽管大量的组织化学和超微结构证据表明哺乳动物角质层是一个由脂肪耗竭的角质细胞包埋在中性富脂基质中的异质组织，但关于这一层中脂质的分布仍然存在争议。对分离的小鼠角质层膜复合体进行了细胞分级和脂质生化研究。用葡萄球菌表皮溶解毒素处理新生小鼠，用胰酶去除残留的颗粒细胞，获得完整、均一的角质层。将生成的薄片混合在一起，在-196度下粉碎。C，并在斯坦斯特德细胞破碎机中以20-30,000磅/平方英寸的速度均质。用0.005%枯草杆菌蛋白酶VIII型，在pH 8.8的Tris-HCl缓冲液中，在25度下处理60分钟。C从质膜下剥离角蛋白细丝。当超微结构检查时，所产生的相对膜复合体的均一群体显示被捕获的细胞间脂双层。整个角质层片层含有10%的脂肪/总脂肪+蛋白质重量；纯化的膜复合体含有apprx。50%的脂质，是整个细胞膜部分中脂质浓度的5倍。从现有的定量数据来看，角质层细胞质域含有3%的脂肪，而膜复合体占角质层总脂肪的80%以上。分离的膜复合体的冷冻断裂复制品显示与整个角质层相同的细胞间膜双层，并通过脂类溶剂提取去除。从膜复合体中提取的脂类的TLC显示出与整张提取液中类似的脂类的光谱和重量分布。角质层细胞膜区优先于整个组织富含脂质，这证实了早期的形态学研究，即在角化过程中，脂类在膜域中逐渐分离。这种不同的结构/化学组织对哺乳动物皮肤屏障功能、角质层凝聚力和经皮转运途径具有深远的影响。
Despite substantial histochemical and ultrastructural evidence suggesting that mammalian stratum corneum is a heterogenous tissue composed of lipid-depleted corneocytes embedded in a neutral lipid-enriched matrix, controversy remains about the distribution of lipids in this layer. Cell fractionation and lipid biochemistry of isolated stratum corneum membrane complexes from mice was performed. Intact homogeneous sheets of stratum corneum were obtained from neonatal mice by treatment with the staphylococcal epidermolytic toxin and removal of residual granular cells with trypsin. The resultant sheets were pooled, pulverized at -196.degree. C and homogenized at 20-30,000 psi in a Stansted cell disrupter. Treatment with 0.005% Subtilisin (protease-type VIII) in Tris-HCl buffer, pH 8.8, for 60 min at 25.degree. C stripped keratin filaments from beneath the plasma membrane. The resulting homogeneous population of apposed membrane complexes revealed trapped intercellular lipid bilayers when it was examined ultrastructurally. Whole stratum corneum sheets contained 10% lipid/total lipid + protein weight; purified membrane complexes contained .apprx. 50% lipid, a 5-fold concentration of lipid in membrane fractions over whole cells. From available quantitative data stratum corneum cytoplasmic domains contained < 3% lipid, while membrane complexes accounted for over 80% of the total stratum corneum lipid. Freeze-fracture replicas of isolated membrane complexes revealed intercellular membrane bilayers that were identical to those encountered in whole stratum corneum, and removed by lipid solvent extraction. TLC of lipids extracted from membrane complexes revealed a similar spectrum and weight distribution of lipids as encountered in extracts from whole sheets. Stratum corneum cell membrane regions are preferentially enriched in lipids over whole tissue, confirming earlier morphological studies that lipids are progressively segregated in membrane domains during cornification. This heterogeneous structural/chemical organization has profound implications for mammalian skin barrier function, stratum corneum cohesion, and for avenues of percutaneous transport.