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目的观察肺泡Ⅱ型上皮细胞(AECⅡ)体外培养的生长状况及转分化过程。方法通过差速贴壁法对原代培养的AECⅡ进行分离、纯化,并将AECⅡ随机分为第0天、第2天、第4天、第6天、第8天8个时间点。利用倒置显微镜观察细胞生长情况,记录细胞数目,绘制生长曲线;锥虫蓝染色观察细胞活力;流式细胞仪观察细胞生长周期和细胞凋亡情况;电子显微镜观察细胞超微结构;采用免疫荧光技术,共聚焦显微镜观察肺表面活性蛋白(SP-C)、水通道蛋白5(AQP5)的阳性表达。结果倒置显微镜下第0天、第2天、第4天、第6天及第8天细胞数分别为(4.02±0.99)×108L-1、(10.41±0.24)×108L-1、(27.90±1.91)×108L-1、(27.12±0.85)×108L-1及(26.29±1.59)×108L-1,第2天与第0天、第4天与第2天细胞数比较差异均有统计学意义(Pa<0.05)。第2天、第4天、第6天及第8天的细胞活力分别为(97.00±0.71)%、(97.20±0.84)%、(95.00±0.71)%及(92.80±1.30)%,第6天与第4天、第8天与第6天比较差异有统计学意义(Pa<0.05)。细胞周期:空气组细胞G1期、S期细胞所占比率分别在第6天、第4天最高,但各时间点与前一时间点比较差异均无统计学意义(Pa>0.05)。细胞凋亡:空气组Annexin-V+/PI-亚群在第4天所占比例最高,与第2天及第6天比较差异均有统计学意义(Pa<0.05);Annexin-V+/PI+亚群细胞所占比例在第8天时最高,但与第6天比较差异无统计学意义(P>0.05),而第6天与第4天比较差异有统计学意义(P<0.05)。细胞超微结构:第6天时可见介于AECⅡ和AECⅠ之间的中间状态细胞,此类细胞在细胞表面有微绒毛,但胞质内无板层小体或胞质内有板层小体但细胞表面无微绒毛。免疫荧光:第6天可见部分细胞胞质内有呈绿色荧光的SP-C阳性表达,荧光强度较第2天、第4天减弱,伴有胞膜点状分布的呈红色荧光的AQP5阳性表达,细胞表达强度不等,表达AQP5相对较弱的细胞表达SP-C的强度相对增强。结论原代培养的AECⅡ在培养早期增殖能力最强,细胞增殖分化能力在培养晚期降低。体外培养的AECⅡ有向AECⅠ转分化的能力。
Objective To observe the growth and transdifferentiation of alveolar type Ⅱ epithelial cells (AECⅡ) cultured in vitro. Methods The primary cultured AECⅡwas isolated and purified by differential adherence method, and AECⅡwas randomly divided into 8 time points on day 0, day 2, day 4, day 6 and day 8. The cell growth was observed by inverted microscope, the cell number was recorded and the growth curve was drawn. The cell viability was observed by trypan blue staining. The cell growth cycle and apoptosis were observed by flow cytometry. The ultrastructure of cells was observed by electron microscopy. , The positive expression of pulmonary surfactant protein (SP-C) and aquaporin 5 (AQP5) were observed by confocal microscopy. Results The numbers of cells on day 0, day 2, day 4, day 6 and day 8 in the inverted microscope were (4.02 ± 0.99) × 108L-1, (10.41 ± 0.24) × 108L-1, (27.90 ± 1.91) × 108L-1, (27.12 ± 0.85) × 108L-1 and (26.29 ± 1.59) × 108L-1 respectively. There were statistically significant differences in the number of cells between day 2 and day 0, day 4 and day 2 Significance (Pa <0.05). The cell viability at day 2, day 4, day 6 and day 8 were (97.00 ± 0.71)%, (97.20 ± 0.84)%, (95.00 ± 0.71)% and (92.80 ± 1.30)%, respectively Day and day 4, day 8 and day 6 were significantly different (Pa <0.05). Cell cycle: The percentage of cells in G1 phase and S phase in air group was the highest on the 6th day and the 4th day, respectively, but there was no significant difference between each time point and the previous time point (P> 0.05). Apoptosis: The percentage of Annexin-V + / PI-subpopulation in air group was the highest on day 4, with statistical significance (P <0.05) on day 2 and day 6; The proportion of the group of cells was the highest on the 8th day, but no significant difference compared with the 6th day (P> 0.05). The difference between the 6th day and the 4th day was statistically significant (P <0.05). Cell ultrastructure: At day 6, intermediate cells between AECII and AECI were seen. These cells had microvilli on the cell surface, but there were no lamellar bodies in the cytoplasm or lamellar bodies in the cytoplasm No microvilli on the cell surface. Immunofluorescence: On the 6th day, some of the cytoplasm of the cells showed positive green fluorescent SP-C expression. The fluorescence intensity was weaker than that on the 2nd day and the 4th day. AQP5 positive expression with dot fluorescence , The intensity of cell expression ranged from that of cells expressing relatively weak AQP5 to SP-C. Conclusion The primary cultured AECⅡhad the strongest proliferative ability in early stage of culture, and the ability of proliferation and differentiation of cells decreased in the late stage of culture. In vitro cultured AECII has the ability to transdifferentiate to AECI.