不完全射频消融后人缺氧诱导因子-1α调控Ⅱ型肺泡上皮细胞增殖能力的研究

Regulation of type Ⅱ alveolar epithelial cell proliferation in vitro by hypoxia- inducible factor- 1α after incomplete radiofrequency ablation

目的观察不完全射频消融后缺氧诱导因子-1α（HIF-1α）对Ⅱ型肺泡上皮细胞（ATⅡ）增殖能力和生物学功能的影响。方法取小鼠ATⅡ细胞分为4组，进行体外培养，培养温度分别为37、50、60、70℃，模拟体内射频消融后消融灶周围不同区域肺组织的温度变化。采用噻唑蓝（MTT）法和划痕实验观察4组ATⅡ的增殖和迁移，应用基因转染技术将HIF-1α转入ATⅡ，检测HIF-1α表达对ATⅡ调控增殖作用和生物学功能的影响。通过实时定量聚合酶链反应（Real—timePCR）检测HIF-1α mRNA在ATⅡ中的表达水平。采用HIF-1α特异性抑制剂YC-1作用于ATⅡ，抑制ATⅡ中的HIF-1αt表达，采用Westernblot和Real—timePCR测定转染后ATⅡ中行溴化脱氧尿嘧啶核苷（BrdUrd）、碱性磷酸酶（AKP）和活性标志物表面活性蛋白C（SPC）的表达变化，观察YC-1对ATⅡ增殖和生长的影响。结果（1）经37、50、60、70℃分别加热10min和30min时，ATⅡ增殖能力的意志率分别为0、（18．79±2．56）％、（43．27±4．94）％、（58．72±6．19）％和0、（44．58±3．81）％、（65．49±7．03）％、（75．13±6．45）％。不同组间比较差异有统计学意义（P〈0．05），同一组间不同作用时间比较差异也有统计学意义（P〈0．05）。（2）划痕实验：经高温处理的ATⅡ迁移能力低于正常细胞（P〈0．05）。（3）Real—timePCR：随着处理ATⅡ的温度越高，ATⅡ中HIF-1α表达越强，而BrdUrd、AKP和SPC等反映ATⅡ增殖再生能力指标的激活被抑制，差异有统计学意义（P〈0．05）。（4）通过转染实验将HIF-1α真核表达载体成功导入ATⅡ，HIF-1α转染的ATⅡ逐渐出现凋亡，48h后ATⅡ数量明显减少。将A组分别与B、C组比较，组问细胞数量的差异有统计学意义（P〈0．05）。（5）将HIF-1α特异性抑制剂YC-1作用于3组ATⅡ细胞，Real—timePCR法检测A组的Brdurd、AKP和SPCmRNA表达水平明显升高，A组为2．87±0．47、2．46±0．51和2．92±0．55，B组为1．71±0．32、1．63±0．29和1．85±0．38，C组为1．62±0．30、1．56±0．25和1．80±0．31。显示YC-1抑制了HIF-1α的表达，ATII的增殖能力显著提高，差异具有统计学意义（P〈0．05）。结论培养温度的升高可以抑制ATⅡ的增殖能力，ATⅡ转染HIF-1α后增殖能力减弱，抑制HIF-1α表达后ATⅡ的增殖能力有所恢复。

Objective To study effect of hypoxia - inducible factor - 1α （ HIF - 1α） on type Ⅱ alveolar epithelial cells （ AT Ⅱ ） proliferation in vitro after incomplete radiofrequency ablation. Methods The mice AT Ⅱ cells were divided into 4 groups, and euhured in vitro at 37, 50, 60, or 70 ℃, mimieing in vivo temperature changes after radiofrequeney ablation ablation in lung tissues around the focus of differ- ent regions. By the MTF method and scratch test, AT Ⅱ proliferation and migration were observed in the 4 groups. HIF-1α was transfected into AT Ⅱ cells, and the regulatory effects of the expression of HIF-1α on AT Ⅱ proliferation and biological functions. The expression level of HIF - 1α mRNA in AT Ⅱ was de- tected by real - time quantitative polymerase chain reaction （ Real - time PCR） technology. AT Ⅱ cells were treated with the HIF - 1α specific inhibitor YC - 1 to inhibit the expression of HIF - 1α. Western blotting and Real -time PCR were used to detect the expression change of alkaline phosphatase （AKP）, surfactant protein C （SPC） and Bromodeoxyuridine （BrdUrd） after transfection. The effects of YC - 1 on the proliferation and growth of AT Ⅱ were observed. Results （ 1 ） With 37, 50, 60, 70 ℃ respectively heated 10 min and 30 min, the proliferation rate of AT Ⅱ were 0, （18.79 ± 2.56）%, （43.27 ± 4.94）%, （58.72 ±6. 19）% and 0, （4.458 ±3.81）%,（65.49±7.03）% and （75. 13 ±6.45）%. The higher the temperature, the proliferation activity of AT Ⅱ subtype worse. There were significant differ- cnces between different groups （P 〈 0. 05 ）, and there was significant difference between different time in the same group （ P 〈 0. 05 ）. （2） The scratch test ： After high temperature treatment, the migration ability of AT Ⅱ was lower than that of normal cells （ P 〈 O. 05 ）. （ 3 ） Real - time PCR ： With the increase of tem- perature, the HIF - 1α expression was increased in ATⅡ , while BrdUrd, AKP and SPC were inhibited in AT Ⅱ （P 〈 0. 05 ）. （4） After the HIF - 1α eukaryotie expression vector was successfully transfected into AT Ⅱ , HIF - 1α - transfected AT 11 exhibited apoptosis, and the number of AT Ⅱ was significantly re- duced at 48 h. The difference of number of AT U in A group and B, C group were statisticaug significant （P〈0. 05）. （5） The HIF-1α specific inhibitor YC-1 role in 3 groups of ATⅡ cells. Real- time PCR method tor detection of group A with BrdUrd, AKP and SPC mRNA expression level was significantly im- proved： group A was 2. 87 ±0. 47, 246 ±0. 51 and 2. 92 ±0. 55, group B was 1.71±0. 32, 1.63±0. 29 and 1.85 ±0.38, group C was 1.62±0.30, 1.56 ±0.25 and 1.80 ＋0. 31. YC-1 inhibited the expression of AT and the proliferation of HIF-1α Ⅱ was signifieantly improved, and the difference was statistically sig- nificant （ P 〈 0. 05 ）. Conclusion The increase of culture temperature can inhibit the AT Ⅱ proliferation. Transfection of HIF -1α into AT Ⅱ can inhibiti the proliferation of AT Ⅱ. The inhibition of HIF - 1α can rcstrere the proliferation of AT Ⅱ to some extent.