FasL基因表达对缺氧状态下直肠癌细胞增殖凋亡的影响

目的探讨直肠癌侵袭转移过程中FasL基因表达对细胞增殖凋亡的影响。方法采用北京军区总医院普通外科实验室构建的4组不同侵袭力的人直肠癌HR-8348细胞亚型HR-8348B、HR-8348L、HR-8348F和HR-8348As,并用化学缺氧法构建上述4组细胞的缺氧12h模型,以Western印迹法验证各组细胞内FasL蛋白的表达水平和缺氧状态下细胞内FasL蛋白的表达,流式细胞仪测定细胞周期分布并计算细胞增殖指数,四甲基偶氮唑盐（MTT）法测定细胞增殖能力改变并计算细胞抑制率,末端标记法（TUNEL）测定细胞凋亡状况。结果Western印迹显示FasL蛋白于40000处显色。常氧条件下HR-8348F细胞内FasL的表达水平显著高于HR-8348B、HR-8348L和HR-8348As（F=361.149。P〈0.01）;缺氧12h时各组细胞均生长良好,形态较常氧状态皱缩;HR-834F,细胞内FasL的表达水平仍显著高于其他3组（F=278.766,P〈0.01）,但其自身常氧与缺氧状态下FasL的水平差异无统计学意义（t=1.762,P〉0.05）。缺氧12h后各组细胞增殖均受到抑制,HR-8348F细胞的增殖指数（60.43±3.72）显著高于HR-8348B（40.01±3.30）、HR-8348L（41.30±4.06）和HR-8348As（35.87±4.39）（F=39.477,P〈0.01）,增殖抑制率（17.30±1.98）和凋亡指数（13.10±1.04）显著低于HR-8348B（33.70±4.33和21.60±1.31）、HR-8348L（34.20±3.92和20.10±1.15）、HR-8348As（38.00±4.55和23.90±1.23）细胞（F分别为28.811和76.462,P〈0.01）。结论缺氧环境中,直肠癌细胞FasL表达增强可导致细胞增殖加速、凋亡减少和侵袭能力增强。促进细胞对微环境缺氧的耐受。     

缺氧诱导因子-1α对低氧状态下前列腺癌PC-3细胞株增殖及侵袭的影响

目的 研究缺氧诱导因子-1α(HIF-1α)对低氧状态下前列腺癌PC-3细胞株增殖及侵袭的影响。 方法 利用转染试剂Fermentas将人HIF-1α重组表达质粒pcDNA3.1-HIF-1α转染PC-3细胞株后,低氧环境培养,采用G418筛选,建立稳定表达HIF-1α基因的细胞株,分别命名为pcD-NA3.1-HIF-1 α-PC-3、pcDNA3.1-PC-3及PC-3组。采用RT-PCR和蛋白质印迹法检测3组细胞HIF-1α mRNA和蛋白的表达情况;噻唑盐法测定细胞生长;transwell小室检测侵袭能力。 结果 与pcDNA3.1-PC-3组和PC-3组相比,pcDNA3.1-HIF-1 α-PC-3组细胞内HIF-1α mRNA条带增强不明显,pcDNA3.1-HIF-1α-PC-3组细胞内HIF-1α蛋白的条带明显增强,HIF-1α过表达的PC-3细胞增殖速度明显增快,侵袭细胞数明显增多。 结论 HIF-1α过表达对PC-3细胞株的增殖及侵袭具有促进作用。     

FasL基因表达对缺氧状态下直肠癌细胞增殖凋亡的影响

Objective To elucidate the effect of FasL gene expression on the proliferation and apoptosis of hypoxic rectal carcinoma cells. Methods The normoxic expression level of FasL in HR-8348 subtype cells (HR-8348B, HR-8348L, HR-8348F and HR-8348As) with different invasive power were verified by Western blot. Hypoxia models for HR-8348B, HR-8348L, HR-8348F and HR-8348As were constructed with chemical modeling, then the FasL levels in all groups at 12 h after hypoxia were quantitated by Western blot. Distribution of different cell life cycles was determined with flow cytometry. Cell reproductive activities were detected with MTT method, and cell apoptesis was assessed with TUNEL. Results FasL protein was pigmentized at the position of 40 000 by Western blot, and the expression level of FasL was significantly higher in HR-8348F cells than those in HR-8348B, HR-8348L and HR-8348As cells(F=361.149, P<0.01) in normoxia. At 12 h after hypoxia, the FasL level was also significantly higher in HR-8348F cells than those in other groups (F=278.766, P<0.01), but was not markedly different as compared to themselves in normoxia (t=1.762, P>0.05). The proliferation index was significantly higher in HR-8348F (60.43±3.72) than those in HR-8348B (40.01±3.30), HR-8348L (41.30±4.06) and HR-8348As cells (35.87±4.39), respectively (F=39.477, P<0.01). However, both inhibition rate of proliferation and apoptotic index were remarkably lower in HR-8348F (17.30±1.98 and 13.10±1.04) than those in HR-834B (33.70±4.33 and 21.60±1.31), HR-8348L (34.20±3.92 and 20.10±1.15), and HR-8348As (38.00±4.55 and 23.90±1.23), respectively (F=28.811 and 76.462, respectively, P<0.01). Conclusion The expression enhancement of intracellular FasL in rectal carcinoma in hypoxia can lead to accelerated proliferation and reduced apeptosis of cells, which will promote tumor cells to adapt microenvironmental hypoxia.     

FasL基因表达对缺氧状态下直肠癌细胞增殖凋亡的影响

Objective To elucidate the effect of FasL gene expression on the proliferation and apoptosis of hypoxic rectal carcinoma cells. Methods The normoxic expression level of FasL in HR-8348 subtype cells (HR-8348B, HR-8348L, HR-8348F and HR-8348As) with different invasive power were verified by Western blot. Hypoxia models for HR-8348B, HR-8348L, HR-8348F and HR-8348As were constructed with chemical modeling, then the FasL levels in all groups at 12 h after hypoxia were quantitated by Western blot. Distribution of different cell life cycles was determined with flow cytometry. Cell reproductive activities were detected with MTT method, and cell apoptesis was assessed with TUNEL. Results FasL protein was pigmentized at the position of 40 000 by Western blot, and the expression level of FasL was significantly higher in HR-8348F cells than those in HR-8348B, HR-8348L and HR-8348As cells(F=361.149, P<0.01) in normoxia. At 12 h after hypoxia, the FasL level was also significantly higher in HR-8348F cells than those in other groups (F=278.766, P<0.01), but was not markedly different as compared to themselves in normoxia (t=1.762, P>0.05). The proliferation index was significantly higher in HR-8348F (60.43±3.72) than those in HR-8348B (40.01±3.30), HR-8348L (41.30±4.06) and HR-8348As cells (35.87±4.39), respectively (F=39.477, P<0.01). However, both inhibition rate of proliferation and apoptotic index were remarkably lower in HR-8348F (17.30±1.98 and 13.10±1.04) than those in HR-834B (33.70±4.33 and 21.60±1.31), HR-8348L (34.20±3.92 and 20.10±1.15), and HR-8348As (38.00±4.55 and 23.90±1.23), respectively (F=28.811 and 76.462, respectively, P<0.01). Conclusion The expression enhancement of intracellular FasL in rectal carcinoma in hypoxia can lead to accelerated proliferation and reduced apeptosis of cells, which will promote tumor cells to adapt microenvironmental hypoxia.     

FasL基因表达对缺氧状态下直肠癌细胞增殖凋亡的影响

Objective To elucidate the effect of FasL gene expression on the proliferation and apoptosis of hypoxic rectal carcinoma cells. Methods The normoxic expression level of FasL in HR-8348 subtype cells (HR-8348B, HR-8348L, HR-8348F and HR-8348As) with different invasive power were verified by Western blot. Hypoxia models for HR-8348B, HR-8348L, HR-8348F and HR-8348As were constructed with chemical modeling, then the FasL levels in all groups at 12 h after hypoxia were quantitated by Western blot. Distribution of different cell life cycles was determined with flow cytometry. Cell reproductive activities were detected with MTT method, and cell apoptesis was assessed with TUNEL. Results FasL protein was pigmentized at the position of 40 000 by Western blot, and the expression level of FasL was significantly higher in HR-8348F cells than those in HR-8348B, HR-8348L and HR-8348As cells(F=361.149, P<0.01) in normoxia. At 12 h after hypoxia, the FasL level was also significantly higher in HR-8348F cells than those in other groups (F=278.766, P<0.01), but was not markedly different as compared to themselves in normoxia (t=1.762, P>0.05). The proliferation index was significantly higher in HR-8348F (60.43±3.72) than those in HR-8348B (40.01±3.30), HR-8348L (41.30±4.06) and HR-8348As cells (35.87±4.39), respectively (F=39.477, P<0.01). However, both inhibition rate of proliferation and apoptotic index were remarkably lower in HR-8348F (17.30±1.98 and 13.10±1.04) than those in HR-834B (33.70±4.33 and 21.60±1.31), HR-8348L (34.20±3.92 and 20.10±1.15), and HR-8348As (38.00±4.55 and 23.90±1.23), respectively (F=28.811 and 76.462, respectively, P<0.01). Conclusion The expression enhancement of intracellular FasL in rectal carcinoma in hypoxia can lead to accelerated proliferation and reduced apeptosis of cells, which will promote tumor cells to adapt microenvironmental hypoxia.     

FasL基因表达对缺氧状态下直肠癌细胞增殖凋亡的影响

Objective To elucidate the effect of FasL gene expression on the proliferation and apoptosis of hypoxic rectal carcinoma cells. Methods The normoxic expression level of FasL in HR-8348 subtype cells (HR-8348B, HR-8348L, HR-8348F and HR-8348As) with different invasive power were verified by Western blot. Hypoxia models for HR-8348B, HR-8348L, HR-8348F and HR-8348As were constructed with chemical modeling, then the FasL levels in all groups at 12 h after hypoxia were quantitated by Western blot. Distribution of different cell life cycles was determined with flow cytometry. Cell reproductive activities were detected with MTT method, and cell apoptesis was assessed with TUNEL. Results FasL protein was pigmentized at the position of 40 000 by Western blot, and the expression level of FasL was significantly higher in HR-8348F cells than those in HR-8348B, HR-8348L and HR-8348As cells(F=361.149, P<0.01) in normoxia. At 12 h after hypoxia, the FasL level was also significantly higher in HR-8348F cells than those in other groups (F=278.766, P<0.01), but was not markedly different as compared to themselves in normoxia (t=1.762, P>0.05). The proliferation index was significantly higher in HR-8348F (60.43±3.72) than those in HR-8348B (40.01±3.30), HR-8348L (41.30±4.06) and HR-8348As cells (35.87±4.39), respectively (F=39.477, P<0.01). However, both inhibition rate of proliferation and apoptotic index were remarkably lower in HR-8348F (17.30±1.98 and 13.10±1.04) than those in HR-834B (33.70±4.33 and 21.60±1.31), HR-8348L (34.20±3.92 and 20.10±1.15), and HR-8348As (38.00±4.55 and 23.90±1.23), respectively (F=28.811 and 76.462, respectively, P<0.01). Conclusion The expression enhancement of intracellular FasL in rectal carcinoma in hypoxia can lead to accelerated proliferation and reduced apeptosis of cells, which will promote tumor cells to adapt microenvironmental hypoxia.     

FasL基因表达对缺氧状态下直肠癌细胞增殖凋亡的影响

Objective To elucidate the effect of FasL gene expression on the proliferation and apoptosis of hypoxic rectal carcinoma cells. Methods The normoxic expression level of FasL in HR-8348 subtype cells (HR-8348B, HR-8348L, HR-8348F and HR-8348As) with different invasive power were verified by Western blot. Hypoxia models for HR-8348B, HR-8348L, HR-8348F and HR-8348As were constructed with chemical modeling, then the FasL levels in all groups at 12 h after hypoxia were quantitated by Western blot. Distribution of different cell life cycles was determined with flow cytometry. Cell reproductive activities were detected with MTT method, and cell apoptesis was assessed with TUNEL. Results FasL protein was pigmentized at the position of 40 000 by Western blot, and the expression level of FasL was significantly higher in HR-8348F cells than those in HR-8348B, HR-8348L and HR-8348As cells(F=361.149, P<0.01) in normoxia. At 12 h after hypoxia, the FasL level was also significantly higher in HR-8348F cells than those in other groups (F=278.766, P<0.01), but was not markedly different as compared to themselves in normoxia (t=1.762, P>0.05). The proliferation index was significantly higher in HR-8348F (60.43±3.72) than those in HR-8348B (40.01±3.30), HR-8348L (41.30±4.06) and HR-8348As cells (35.87±4.39), respectively (F=39.477, P<0.01). However, both inhibition rate of proliferation and apoptotic index were remarkably lower in HR-8348F (17.30±1.98 and 13.10±1.04) than those in HR-834B (33.70±4.33 and 21.60±1.31), HR-8348L (34.20±3.92 and 20.10±1.15), and HR-8348As (38.00±4.55 and 23.90±1.23), respectively (F=28.811 and 76.462, respectively, P<0.01). Conclusion The expression enhancement of intracellular FasL in rectal carcinoma in hypoxia can lead to accelerated proliferation and reduced apeptosis of cells, which will promote tumor cells to adapt microenvironmental hypoxia.     

FasL基因表达对缺氧状态下直肠癌细胞增殖凋亡的影响

Objective To elucidate the effect of FasL gene expression on the proliferation and apoptosis of hypoxic rectal carcinoma cells. Methods The normoxic expression level of FasL in HR-8348 subtype cells (HR-8348B, HR-8348L, HR-8348F and HR-8348As) with different invasive power were verified by Western blot. Hypoxia models for HR-8348B, HR-8348L, HR-8348F and HR-8348As were constructed with chemical modeling, then the FasL levels in all groups at 12 h after hypoxia were quantitated by Western blot. Distribution of different cell life cycles was determined with flow cytometry. Cell reproductive activities were detected with MTT method, and cell apoptesis was assessed with TUNEL. Results FasL protein was pigmentized at the position of 40 000 by Western blot, and the expression level of FasL was significantly higher in HR-8348F cells than those in HR-8348B, HR-8348L and HR-8348As cells(F=361.149, P<0.01) in normoxia. At 12 h after hypoxia, the FasL level was also significantly higher in HR-8348F cells than those in other groups (F=278.766, P<0.01), but was not markedly different as compared to themselves in normoxia (t=1.762, P>0.05). The proliferation index was significantly higher in HR-8348F (60.43±3.72) than those in HR-8348B (40.01±3.30), HR-8348L (41.30±4.06) and HR-8348As cells (35.87±4.39), respectively (F=39.477, P<0.01). However, both inhibition rate of proliferation and apoptotic index were remarkably lower in HR-8348F (17.30±1.98 and 13.10±1.04) than those in HR-834B (33.70±4.33 and 21.60±1.31), HR-8348L (34.20±3.92 and 20.10±1.15), and HR-8348As (38.00±4.55 and 23.90±1.23), respectively (F=28.811 and 76.462, respectively, P<0.01). Conclusion The expression enhancement of intracellular FasL in rectal carcinoma in hypoxia can lead to accelerated proliferation and reduced apeptosis of cells, which will promote tumor cells to adapt microenvironmental hypoxia.     

FasL基因表达对缺氧状态下直肠癌细胞增殖凋亡的影响

Objective To elucidate the effect of FasL gene expression on the proliferation and apoptosis of hypoxic rectal carcinoma cells. Methods The normoxic expression level of FasL in HR-8348 subtype cells (HR-8348B, HR-8348L, HR-8348F and HR-8348As) with different invasive power were verified by Western blot. Hypoxia models for HR-8348B, HR-8348L, HR-8348F and HR-8348As were constructed with chemical modeling, then the FasL levels in all groups at 12 h after hypoxia were quantitated by Western blot. Distribution of different cell life cycles was determined with flow cytometry. Cell reproductive activities were detected with MTT method, and cell apoptesis was assessed with TUNEL. Results FasL protein was pigmentized at the position of 40 000 by Western blot, and the expression level of FasL was significantly higher in HR-8348F cells than those in HR-8348B, HR-8348L and HR-8348As cells(F=361.149, P<0.01) in normoxia. At 12 h after hypoxia, the FasL level was also significantly higher in HR-8348F cells than those in other groups (F=278.766, P<0.01), but was not markedly different as compared to themselves in normoxia (t=1.762, P>0.05). The proliferation index was significantly higher in HR-8348F (60.43±3.72) than those in HR-8348B (40.01±3.30), HR-8348L (41.30±4.06) and HR-8348As cells (35.87±4.39), respectively (F=39.477, P<0.01). However, both inhibition rate of proliferation and apoptotic index were remarkably lower in HR-8348F (17.30±1.98 and 13.10±1.04) than those in HR-834B (33.70±4.33 and 21.60±1.31), HR-8348L (34.20±3.92 and 20.10±1.15), and HR-8348As (38.00±4.55 and 23.90±1.23), respectively (F=28.811 and 76.462, respectively, P<0.01). Conclusion The expression enhancement of intracellular FasL in rectal carcinoma in hypoxia can lead to accelerated proliferation and reduced apeptosis of cells, which will promote tumor cells to adapt microenvironmental hypoxia.     

FasL基因表达对缺氧状态下直肠癌细胞增殖凋亡的影响

Objective To elucidate the effect of FasL gene expression on the proliferation and apoptosis of hypoxic rectal carcinoma cells. Methods The normoxic expression level of FasL in HR-8348 subtype cells (HR-8348B, HR-8348L, HR-8348F and HR-8348As) with different invasive power were verified by Western blot. Hypoxia models for HR-8348B, HR-8348L, HR-8348F and HR-8348As were constructed with chemical modeling, then the FasL levels in all groups at 12 h after hypoxia were quantitated by Western blot. Distribution of different cell life cycles was determined with flow cytometry. Cell reproductive activities were detected with MTT method, and cell apoptesis was assessed with TUNEL. Results FasL protein was pigmentized at the position of 40 000 by Western blot, and the expression level of FasL was significantly higher in HR-8348F cells than those in HR-8348B, HR-8348L and HR-8348As cells(F=361.149, P<0.01) in normoxia. At 12 h after hypoxia, the FasL level was also significantly higher in HR-8348F cells than those in other groups (F=278.766, P<0.01), but was not markedly different as compared to themselves in normoxia (t=1.762, P>0.05). The proliferation index was significantly higher in HR-8348F (60.43±3.72) than those in HR-8348B (40.01±3.30), HR-8348L (41.30±4.06) and HR-8348As cells (35.87±4.39), respectively (F=39.477, P<0.01). However, both inhibition rate of proliferation and apoptotic index were remarkably lower in HR-8348F (17.30±1.98 and 13.10±1.04) than those in HR-834B (33.70±4.33 and 21.60±1.31), HR-8348L (34.20±3.92 and 20.10±1.15), and HR-8348As (38.00±4.55 and 23.90±1.23), respectively (F=28.811 and 76.462, respectively, P<0.01). Conclusion The expression enhancement of intracellular FasL in rectal carcinoma in hypoxia can lead to accelerated proliferation and reduced apeptosis of cells, which will promote tumor cells to adapt microenvironmental hypoxia.     

FasL基因表达对结直肠癌细胞肝转移影响的研究

目的　探讨FasL基因表达对结直肠癌细胞生物学行为的影响及在肝转移中的作用。方法　采用RT PCR方法检测大肠癌原发灶、癌旁肠黏膜、肝转移灶中FasL基因表达。用细胞转染方法 ,将FasLcDNA转染人直肠癌细胞HR 8348,采用四唑蓝法观测FasL表达对癌细胞生长抑制率及对5 FU、卡铂杀伤作用的影响。　结果　结直肠癌原发灶 (5 8例 )、癌旁肠黏膜 (5 8例 )、肝转移灶 (2 8例 )中FasL基因表达阳性率分别为 2 4 % (14 /5 8)、14 % (8/5 8)、10 0 % (2 8/2 8)。肝转移灶中FasL表达阳性率高于癌原发灶 (χ2 =4 3 4 9,P <0 0 1)和癌旁肠黏膜组织 (χ2 =5 7 6 6 ,P <0 0 1)。肝转移组原发灶FasL表达阳性率高于无肝转移组 (χ2 =3 96 ,P <0 0 5 )。转染HR 8348细胞FasL表达为阳性。用 5 FU、卡铂杀伤FasL转染细胞和未转染细胞 ,2组癌细胞生长抑制率有显著性差异 (t=9 0 2、t=11 93,P <0 0 1)。在相同化疗药物浓度下 ,FasL阳性HR 8348细胞存活率高于对照组癌细胞。　结论　FasL阳性癌细胞对化疗药物有较强的耐受性。FasL基因表达能使癌细胞逃避免疫监视和杀伤并对化疗药物产生抗性 ,促进结直肠癌发生肝转移。     

FasL cDNA转染对直肠癌细胞顺铂耐药性的影响

目的:探讨FasLcDNA转染和表达对直肠癌细胞耐药性的影响。方法:用RT-PCR方法克隆人FasL全长cDNA,构建pcDNA3.1-FasL真核表达载体,用脂质体法转染HR-8348人直肠癌细胞,采用MTT法检测顺铂对转染和未转染直肠癌细胞的生长抑制率。结果:DNA测序证实克隆FasLcDNA898bp与GeneBank序列完全一致。构建真核表达载体转染HR-8348细胞后,FasLmRNA表达明显增强。在不同浓度顺铂(1、5、10、20、40mg/L)的作用下,FasL转染组直肠癌细胞抑制率分别为11.0%、25.4%、31.2%、37.8%、42.4%:对照组癌细胞抑制率分别为26.1%、34.4%、37.6%、42.9%、53.2%,其差异有显著性意义(t=4.43,P<0.05)。结论:FasL转染HR-8348细胞可增强癌细胞的耐药性,减弱顺铂对HR-8348细胞的杀伤作用。     

反义缺氧诱导因子-1α对胰腺癌细胞BxPC-3化疗敏感性的影响

目的： 观察反义缺氧诱导因子-1α(HIF-1α)对胰腺癌细胞BxPC-3化疗敏感性的影响。方法：实验分组：（1）缺氧条件下(0.5% O2)体外培养4h，未转染反义HIF-1α质粒的BxPC-3细胞设为缺氧对照组；（2）常氧条件下体外培养，未转染反义HIF-1α质粒的BxPC-3 细胞设为常氧对照组；（3）缺氧条件下(0.5% O2)体外培养4h，稳定转染反义HIF-1α质粒的BxPC-3细胞设为实验组。采用逆转录聚合酶链反应 (RT-PCR)和免疫印迹(Western Blot)检测各组的HIF-1α和survivin表达情况。 流式细胞术和MTT比色法检测不同剂量的化疗药物(5-氟尿嘧啶、阿霉素、吉西他宾)对各组的凋亡率和生长抑制率的影响。 结果：实验组HIF-1α和survivin的表达明显降低 (P<0.05)，与对照组相比，实验组的凋亡率、抑制率与剂量成正比，高剂量引起高抑制(P<0.05)。 结论：反义HIF-1α可能通过阻断survivin的表达而增强胰腺癌对化疗的敏感性。据此可望通过阻断HIF-1α的表达为胰腺癌基因治疗提供一种新途径。     

二甲基乙二酰基甘氨酸对缺氧复氧诱导的肾小管上皮细胞损伤的保护作用及其机制

目的 通过脯氨酸羟化酶抑制剂二甲基乙二酰基甘氨酸（DMOG）稳定缺氧诱导因子1α（HIF-1α）表达,探讨其对缺氧复氧诱导的肾小管上皮细胞（HKC）损伤的保护作用及其机制。 方法 制作无糖缺氧复氧细胞损伤模型,用不同浓度的DMOG预处理,锥虫蓝染色和乳酸脱氢酶（LDH）活性方法检测细胞活力及损伤;Annexin V和PI染色流式细胞仪技术检测细胞凋亡;实时荧光定量PCR方法检测红细胞生成素（EPO）、热休克蛋白70（HSP70）和血红素氧合酶1（HO-1） mRNA的表达;Western印迹法检测HIF-1α、活性caspase-3和Bcl-2蛋白表达。 结果 正常情况下HKC细胞内几乎无HIF-1α蛋白表达,DMOG刺激6 h后HIF-1α蛋白及其靶基因EPO、HSP70和HO-1 mRNA表达均显著上调（均P ＜ 0.01）,且呈浓度依赖性。500 μmol/L或1 mmol/L DMOG预处理可明显改善缺氧复氧诱导的细胞损伤,表现为细胞存活率升高（95.6%±1.8%、96.1%±1.0%比 83.3%±3.1%）;培养上清液中LDH 活性下降;细胞凋亡减少（8.6%±2.7%、6.1%±2.3%比19.2%±4.0%）（均P ＜ 0.05）。另外,细胞内活性caspase-3蛋白表达显著下调,而Bcl-2蛋白表达则显著上调（均P ＜ 0.05）。 结论 DMOG预处理可稳定肾小管上皮细胞内HIF-1α表达,对缺氧复氧诱导的肾小管上皮细胞损伤具有一定保护作用。其机制可能与促进EPO、HSP70和HO-1表达,抑制caspase-3活化,上调Bcl-2表达有关。     

缺氧应激对HepG2细胞中甲硫氨酸腺苷转移酶2A基因表达的影响

目的 观察体外缺氧条件下肝癌细胞株HepG2中缺氧诱导因子-1α(HIF-1α)和甲硫氨酸腺苷转移酶2A(MAT2A)的表达,探讨HIF-1α在低氧条件下对MAT2A基因表达的调控作用.方法 构建人MAT2A启动子真核表达载体质粒,CoCl2化学模拟肿瘤缺氧环境,检测缺氧条件下MAT2A启动子活性,HIF-1α和MAT2A在HepG细胞中的共定位、mRNA和蛋白水平的表达.以及小干扰RNA(siRNA)沉默HIF-1α后对MAT2A基因表达的影响.结果　缺氧24 h能使HepG2细胞活性增加到高峰(41.26±2.34),同时MAT2A基因的表达也较常氧时显著升高(P＜0.01),siRNA转染HepG2细胞后能显著下调HIF-1α蛋白表达(抑制率90%),并导致MAT2A基因的表达也受到明显抑制.结论　缺氧促使HepG2细胞中HIF-1α在蛋白水平表达升高,缺氧时HIF-1α能上调MAT2A基因的表达水平.

Abstract：

Objective To observe the expression of hypoixa inducible factor-1α (HIF-α) and methionine adenosyltransferase-2A (MAT2A) gene in HepG2 cells under hypoxia in vitro, and explore the regulation of MAT2A gene expression by HIF-1α. Methods Human MAT2A promoter eukaryotic expression vector was constructed. CoCl2 was used as a chemical hypoxia-inducible reagent to mimic tumor bypoxic microenvironment. MAT2A promoter activity, and mRNA and protein expression of HIF-α were detected in the HepG2 cells transfected with RNA interference (RNAi) originated by small interfering RNA (siRNA). The change of MAT2A gene expression was observed after HIF-1α gene silencing. Results Under hypoxia for 24 h, HepG2 cell activity was increased to (41.26 ± 2. 34 ), and the mRNA and protein expression levels of MAT2A were up-regulated (P ＜0. 01 ). After siRNA targeting, the HIF-1α was downregulated efficiently in HepG2 cells (inhibition ratio: 90% ), and MAT2A gene was down-regulated as well. Conclusion Hypoxia can increase protein level of HIF-1α in HepG2 cells, and HIF-1α up-regulates the gene expression of MAT2A.     

短发夹状RNA抑制缺氧诱导因子1α在前列腺癌细胞中的表达

目的:构建产生缺氧诱导因子1α(HIF-1α)特异性短发夹状RNA(shRNA)的载体并检测其抑制作用,探讨该技术在前列腺癌治疗中的应用前景。方法:设计、合成针对HIF-1α的特异性shRNA模板序列,将其插入含有U6启动子的psilencerTM2.1-U6载体中,得到shRNA表达载体psilencer-HIF,在脂质体介导下转染前列腺癌细胞株PC-3M,应用绿色荧光蛋白质粒pEGFP共转染评价转染效率,RT-PCR及Western印迹检测其对HIF-1α表达的影响。结果:重组质粒psilencer-HIF经测序分析证实与设计的完全一致,共转染24 h后质粒转染效率为(89.26±4.72)%,其产生的shRNA在PC-3M细胞中能诱导RNA干扰(RNAi),转染后48 h HIF-1αmRNA和蛋白水平分别下降82.09%和81.61%(P<0.01);而阴性对照组HIF-1α表达水平无明显改变(P>0.05)。结论:成功构建了HIF-1α基因shRNA表达质粒,其可有效抑制前列腺癌细胞中的HIF-1α的表达,为前列腺癌的基因治疗提供了新思路。     

双启动子引导自杀基因靶向杀伤5-FU耐药肿瘤细胞的研究

目的：探讨胸苷酸合成酶（TS）基因启动子和p16基因启动子引导胸苷激酶（TK）自杀基因靶向杀伤5－FU耐药肿瘤细胞的作用。方法：构建TS、p16双启动子重组表达载体,将TK基因插入TS、p16启动子之间,转染耐药人直肠癌细胞系HR－8348、外周血单个核细胞。通过克隆形成实验、细胞存活率测定和裸鼠移植瘤治疗实验,观察双启动了引导TK基因特异杀伤肿瘤细胞的作用。结果：将TS、p16双启动子重组质粒载体转入耐药HR－8348细胞,检测TS、TK基因表达阳性,TK基因与TS表达一致。转染组和对照组肿瘤细胞集落形成分别为：9／300、92／300。转染组瘤细胞集落形成率明显降低（t=33.885,P＜0．01）,癌细胞生长抑制率显著提高。对裸鼠移植瘤的生长抑制率为74．5％。在转染的外周血单个核细胞,p16表达阳性,TS、TK表达阴性。转染双启动子重组质粒对正常外周血单个核细胞无损伤作用。结论：TS和p16双启动子可引导TK基因靶向性杀伤5－FU耐药肿瘤细胞,保护肌体正常细胞,提高自杀基因治疗的安全性。     

瘢痕疙瘩成纤维细胞培养中缺氧与缺氧诱导因子-1α表达的相关性

目的研究缺氧程度与瘢痕疙瘩中缺氧诱导因子-1α（hypoxia inducible factor,HIF－1α）基因表达的量化关系和关联性,进一步探讨缺氧通过HIF-1a途径促进异常瘢痕形成的机制。方法设立不同O2浓度组,在常氧及不同程度缺氧条件下培养瘢痕疙瘩成纤维细胞,应用Western印迹技术检测比较各组间HIF－1α基因蛋白的表达,进行数据处理和统计学分析。结果瘢痕疙瘩成纤维细胞在常氧（20％）、不同缺氧浓度（10％、5％、1％）下．HIF－1α蛋白表达相对含量（HIF－1α／β肌动蛋白）分别为0．007±0．006、0．133±0．006、0．537±0．015和0．903土0．021,表明缺氧系统中瘢痕疙瘩成纤维细胞HIF—1α蛋白表达明显上调。结论缺氧条件促进了瘢痕疙瘩中HIF-1α蛋白的表达,而且表达的量与缺氧程度呈正相关。缺氧通过HIF－1α基因途径与异常瘢痕的形成密切相关。     

缺氧对肠上皮细胞缺氧诱导因子1α活化影响的实验研究

目的 了解缺氧对肠上皮细胞缺氧诱导因子1α(HIF-1α)活化的影响. 方法 将肠上皮细胞分为常氧处理(正常对照)、缺氧(设缺氧1、2、6、12、24 h)及缺氧+寡霉素处理(分别用浓度为5、10、20、40μg/mL寡霉素处理1 h,再缺氧6 h).采用蛋白质印迹法检测HIF-1α蛋白表达,免疫荧光法观察HIF-1α向细胞核转位的情况. 结果 与正常对照(0.08±0.07)相比较,缺氧1 h肠上皮细胞HIF-1α蛋白表达(0.52±0.30)即显著升高(P＜0.05),6 h达峰值(2.37±1.08,P＜0.05),同时HIF-1α向细胞核转位也明显增加.寡霉素呈剂量依赖性地抑制缺氧引起的肠上皮细胞HIF-1α蛋白表达增加,用5、10、20及40μg/,mL寡霉素处理的缺氧肠上皮细胞HIF-1α蛋白表达量分别为1.62±0.96、1.48±0.56、1.08±0.36及0.58±0.11,均较单纯缺氧6 h(2.67±1.38)显著降低(P＜0.05),HIF-1α向细胞核转位也被抑制. 结论 呼吸链抑制剂寡霉素可抑制缺氧肠上皮细胞HIF-1α活化,线粒体呼吸链可能在其发生机制中具有重要作用.