CAJ | 학술논문

目的建立人Ⅱ型肺泡上皮细胞(ATⅡ)分离、纯化、原代培养及鉴定的方法,探索体外培养的表型维持情况.方法取手术切除的边缘肺组织,用胰酶、弹性蛋白酶联合法消化分离人ATⅡ细胞粗悬液,经过滤、差速贴壁、密度梯度分离、磁珠分选纯化.用肺表面活性蛋白C前体(pro-SP-C)免疫荧光、溶酶体绿色荧光染料(GreendND-26)荧光探针染色以及透射电镜鉴定人ATⅡ细胞;用pro-SP-C免疫荧光法和GreenDND-26荧光探针染色法评估细胞纯度;锥虫蓝染色法评估细胞活性;并运用逆转录-聚合酶链反应(RT-PCR)检测体外培养过程中肺表面活性蛋白(SP-A、SP-B、SP-C、SP-D)的表达情况.结果人ATⅡ细胞产量为(5～10)×105/g;锥虫蓝染色细胞活性为(93±2)％;pro-SP-C免疫荧光、GreendND-26荧光探针鉴定、评估细胞纯度一致,为98％左右,透射电镜下观察ATⅡ细胞特有的板层小体结构清晰可见.SP-A、SP-C表达持续时间在24d左右(SP-A 16d:0.52±0.03,20d:0.35±0.02,24d:0.26±0.01,28d:0.10±0.08;SP-C 16d:0.68±0.16,20d:0.31±0.04,24d:0.18±0.06,28d:0.14±0.09),SP-B、SP-D表达持续时间可在28d左右(SP-B 16d:1.05±0.17,20d:0.76±0.35,24d:0.55±0.15,28d:0.36±0.19;SP-D 16d:0.52±0.19,20d:0.33±0.12,24d:0.31±0.04,28d:0.23±0.02).结论成功建立了一种分离、纯化及鉴定人ATⅡ细胞的方法,此方法能较持久维持ATⅡ细胞表型.
목적 건립인Ⅱ형폐포상피세포(ATⅡ)분리、순화、원대배양급감정적방법,탐색체외배양적표형유지정황.방법 취수술절제적변연폐조직,용이매、탄성단백매연합법소화분리인ATⅡ세포조현액,경과려、차속첩벽、밀도제도분리、자주분선순화.용폐표면활성단백C전체(pro-SP-C)면역형광、용매체록색형광염료(GreendND-26)형광탐침염색이급투사전경감정인ATⅡ세포;용pro-SP-C면역형광법화GreenDND-26형광탐침염색법평고세포순도;추충람염색법평고세포활성;병운용역전록-취합매련반응(RT-PCR)검측체외배양과정중폐표면활성단백(SP-A、SP-B、SP-C、SP-D)적표체정황.결과 인ATⅡ세포산량위(5～10)×105/g;추충람염색세포활성위(93±2)％;pro-SP-C면역형광、GreendND-26형광탐침감정、평고세포순도일치,위98％좌우,투사전경하관찰ATⅡ세포특유적판층소체결구청석가견.SP-A、SP-C표체지속시간재24d좌우(SP-A 16d:0.52±0.03,20d:0.35±0.02,24d:0.26±0.01,28d:0.10±0.08;SP-C 16d:0.68±0.16,20d:0.31±0.04,24d:0.18±0.06,28d:0.14±0.09),SP-B、SP-D표체지속시간가재28d좌우(SP-B 16d:1.05±0.17,20d:0.76±0.35,24d:0.55±0.15,28d:0.36±0.19;SP-D 16d:0.52±0.19,20d:0.33±0.12,24d:0.31±0.04,28d:0.23±0.02).결론 성공건립료일충분리、순화급감정인ATⅡ세포적방법,차방법능교지구유지ATⅡ세포표형.
Objective To establish a method of isolate,purify,primary culture and identify human alveolar type Ⅱ cells (AT Ⅱ ) in vitro,as well as its possible maintaining phenotype characteristics.Methods The marginal lung tissue was collected.AT Ⅱ cells were isolated with trypsin and elastase,purified by a series of steps,such as,cell sieve filtration,differential adhesion,gradient separation and anti-CD14 beads separation.AT Ⅱ cells were identified with immunofluorescence of human pro-surfactant-associated protein C (pro-SP-C),Green DND-26 probe and electron microscope.The purity of AT Ⅱ cells was measured by immunofluorescence of human pro-SP-C and Green DND-26 probe.The viability of AT Ⅱ cells was measured by trypan blue staining.The phenotypes (SP-A,SP-B,SP-C,SP-D ) were monitored with reverse transcription-polymerase chain reaction ( RT-PCR ) at different time points.Results The output of AT Ⅱ cells from lung tissue was (5-10) × 105/g,and the cell viability was (93 ± 2 )％ with trypan blue staining,the cell purity was about 98％ with pro-SP-C immunofluorescence and Green DND-26 fluorescent probe,the lamellar bodies were clearly observed with transmission electron microscope.In the aspect of phenotypes maintaining,the time of surfactant expression was about 24 days [ SP-A:0.52 ± 0.03 (day 16),0.35 0.02 ( day 20),0.26 ± 0.01 (day 24),0.10 ± 0.08 (day 28 );SP-C:0.68 ± 0.16 (day16),0.31 ± 0.04 (day 20),0.18 ± 0.06 (day 24),0.14 ±0.09 (day 28)],and the longest one was more than 28 days [SP-B:1.05 ±0.17 (day 16),0.76 ±0.35 (day 20),0.55 ± 0.15 (day 24),0.36 ± 0.19 (day 28);SP-D:0.52 ± 0.19 (day 16),0.33 ± 0.12 (day 20),0.31 ±0.04 (day 24),0.23 ± 0.02 (day 28)].Conclusion We successfully established a procedure to separate,purify,identify of AT Ⅱ cells,which retain primary phenotypic characteristics over long period.