目的 探讨重楼皂苷Ⅰ（PPI）对慢性髓系白血病细胞（K562）细胞的抑制作用及可能的作用机制。方法 采用CCK-8法筛选药物最适浓度,将培养时间为24 h的药物最适浓度作为后续实验的干预浓度。分组如下：（1）空白组;（2）PPI组;（3）抑制剂组;（4）PPI+抑制剂组。采用CCK-8法检测细胞增殖率;AO/EB染色观察细胞形态;流式细胞术检测凋亡率;ROS检测试剂盒检测活性氧（ROS）含量、还原型谷胱甘肽含量检测试剂盒检测谷胱甘肽（GSH）含量、细胞亚铁比色法测试盒检测细胞亚铁（Fe²⁺）含量;qRT-PCR法和蛋白免疫印迹法检测各组肿瘤蛋白53（p53）、钠氯离子依赖性氨基酸转运蛋白11（SLC7A11）、谷胱甘肽过氧化物酶4（GPX4）mRNA及蛋白表达量。结果 PPI抑制K562细胞的生长,且呈一定的剂量及时间依赖性（与同时间段的对照组0μmol/L比较,均P<0.01）。与空白组相比,PPI抑制K562细胞的增殖,提高了凋亡率,而铁死亡抑制剂（Ferrostian-1）的使用逆转了PPI对K562凋亡的促进作用（P<0.01）。与空白组相比,PPI组ROS、Fe²⁺含量升高,GSH含量下降,而铁死亡抑制剂的使用可下调ROS、Fe²⁺,上调GSH的含量（P<0.01）。PPI组较空白组p53 mRNA和蛋白表达水平升高,而SLC7A11、GPX4 mRNA和蛋白表达水平下降（P<0.05）;PPI+抑制剂组细胞较重楼皂苷组p53 mRNA和蛋白表达水平下降,而SLC7A11、GPX4 mRNA和蛋白表达水平升高（P<0.05）。结论 PPI能够有效抑制K562细胞增殖,促进K562细胞铁死亡,其分子机制可能与p53信号通路的调控有关。

Objective To investigate the inhibitory effect of polyphyllin I（PPI）on chronic myeloid leukemia cells（K562）and its possible mechanism．Methods K562 cell line was cultured in suitable environment,and the optimal concentration of the drug was screened by CCK-8 method．The optimal concentration of the drug cultured for 24 hours was used as the intervention concentration of the follow-up experiment．Cells were divided into the following groups：（1）blank group,（2）saponins group,（3）inhibitor group and（4）saponins + inhibitor group．The cell proliferation rate was detected by CCK-8 method．The cell morphology was observed by AO/EB staining．The apoptosis rate was detected by flow cytometry．The contents of reactive oxygen species（ROS）,glutathione（GSH）and ferrous（Fe²⁺）in different groups were detected,and the expression of mRNA and protein in different groups were detected by qRT- PCR and Western blot respectively．Results PPI significantly inhibited the growth of K562 cells in a dose-and time-dependent manner．Compared with the blank group,PPI significantly inhibited the proliferation of K562 cells and increased the apoptosis rate of K562 cells,while the use of ferroptosis inhibitor（Ferrostian-1）reversed the promoting effect of PPI on apoptosis of K562 cells．Compared with the blank group,the contents of reactive oxygen species（ROS）and ferrous iron（Fe²⁺）increased and the content of glutathione（GSH）decreased in the saponins group．The use of Ferrostian-1 could down-regulate the contents of ROS and Fe²⁺ and increase the content of GSH in the cells treated with the drug．Compared with the blank group,the expression of p53 mRNA and protein in the saponins group increased,while the expression of SLC7A11,GPX4 mRNA and protein decreased．The expression of p53 mRNA and protein in the saponins + inhibitor group was lower than that in the saponins group,while the expression levels of SLC7A11,GPX4 mRNA and protein increased．Conclusions PPI can effectively inhibit the proliferation of K562 cells and promote ferroptosis in K562 cells．The molecular mechanism can be related to the regulation of p53 signal pathway．

目的 探讨链霉菌抗生物素蛋白-过氧化物酶（SP）免疫组化染色检测胰岛素样生长因子-1（IGF-1）及组织P53蛋白的表达在结直肠腺癌中意义。方法 选取乌鲁木齐市中医医院2020年1月—2023年12月收治的169例结直肠腺癌患者为恶性组,另选取我院同期收治的169例良性结直肠肿瘤患者为良性组。取手术或活检病理组织应用SP免疫组化染色检测2组肿瘤组织IGF-1及P53蛋白表达水平,对比良性组与恶性组间差异,分析不同临床分期、淋巴结转移情况及组织分化程度患者的IGF-1水平、IGF-1mRNA及P53蛋白阳性率。结果 SP免疫组化染色检测IGF-1阳性率、P53蛋白阳性率,恶性组（72.78%、47.93%）均高于良性组（14.79%、6.51%）,对比差异有统计学意义（χ²=115.440、χ²=73.180,P<0.05）;Ⅳ期患者IGF-1阳性率（96.77%）及P53蛋白阳性率（77.42%）高于Ⅲ期（85.11%、63.83%）、Ⅱ期（62.69%、31.34%）及Ⅰ期患者（45.83%、25.00%）,对比差异有统计学意义（χ²=24.860,χ²=28.000,P<0.05）,各亚组两两比较差异无统计学意义（IGF-1阳性与阴性,Ⅰ期 vs Ⅲ期,χ²=12.110,P<0.001;Ⅰ期 vs Ⅳ期,χ²=16.060,P<0.001;Ⅱ期 vs Ⅲ期,χ²=6.880,P=0.009;P53蛋白阳性与阴性Ⅰ期 vs Ⅲ期,χ²=9.580,P<0.002;Ⅰ期 vs Ⅳ期,χ²=14.9990,P<0.001;Ⅱ期 vs Ⅲ期,χ²=11.790,P=0.001;）;有淋巴结转移患者IGF-1阳性率（85.71%）及P53蛋白阳性率（71.43%）高于无淋巴结转移患者（14.79%、40.94%）,对比差异有统计学意义（χ²=4.720、11.740,P<0.05）;低分化患者IGF-1阳性率（93.48%）及P53蛋白阳性率（71.74%）高于中分化患者（81.18%、54.12%）、高分化患者（52.63%、31.58%）,对比差异有统计学意义（χ²=21.250、13.510,P<0.05）。结论 SP免疫组化染色检测提示结直肠腺癌患者IGF-1、P53蛋白阳性率高于良性肿瘤患者,且临床分期高、淋巴结转移与肿瘤组织低分化者的IGF-1、P53蛋白阳性率高,因此IGF-1、P53有望成为结直肠腺癌诊治的重要检测指标。

Objective Exploring the significance of immunohistochemical staining with streptavidin-perosidase（SP）to detect the expression of insulin-like growth factor-1（IGF-1）and tissue P53 in colorectal adenocarcinoma．Methods A total of 169 patients with colorectal adenocarcinoma admitted to a hospital from January 2020 to December 2023 were selected and divided into the malignant group．Other 169 patients with benign colorectal tumors admitted to a hospital during the same period were selected and divided into the benign group．SP immunohistochemistry staining was used to detect and compare the expression levels of IGF-1 and P53 proteins in two groups of tumor tissues obtained in surgery or biopsy．And the IGF-1 levels,IGF-1 mRNA,and P53 positivity rates in patients with different clinical stages,lymph node metastasis,and tissue differentiation levels were compared．Results There was a significant difference in the positive rates of IGF-1mRNA and P53 detected by SP immunohistochemistry staining,and malignant group（72.78%,47.93%）were higher than the benign group（14.79%,6.51%）,which were statistically significant（χ²=115.440,73.180,P<0.05）．The positive rates of IGF-1 and P53 were 96.77% and 77.42% in stage IV,which were higher than those in Stage III（85.11%,63.83%）,II（62.69%,31.34%）and I（45.83%,25.00%）,the differences were statistically significant（χ²=24.860,28.000,P<0.05）．The expression levels of IGF-1 and P53 in colorectal adenocarcinoma patients with different lymph node metastases showed significant differences,the positive rates of IGF-1（85.71%）and P53（71.43%）in patients with lymph node metastasis were higher than those without lymph node metastasis（14.79%,40.94%）,and the differences were statistically significant（χ²=4.720,11.740,P<0.05）．There were significant differences in the expression levels of IGF-1 and P53 among patients with colorectal adenocarcinoma of different degrees of differentiation,the positive rates of IGF-1（93.48%）and P53（71.74%）were significantly higher than those of moderately differentiated（81.18%,54.12%）and well differentiated（52.63%,31.58%）,and the differences were statistically significant（χ²=21.250,13.510,P<0.05）．Conclusion sThrough SP immunohistochemical staining,it was found that the positivity rates of IGF-1 and P53 in colorectal adenocarcinoma patients were higher than those in benign tumor patients,and those with high the clinical stage,lymph node metastasis,and low differentiation of tumor tissue,had higher the positivity rates of IGF-1 and P53．Therefore,IGF-1 and P53 are expected to become important monitoring indicators for the diagnosis and treatment of colorectal adenocarcinoma．

目的 观察低氧对p53RFP基因表达及启动子活性的影响,探索低氧反应元件(HRE)是否参与了低氧对p53RFP基因启动子活性的调控。方法 将HEK293细胞置于低氧条件下培养,实时定量PCR及Western blot 检测p53RFP mRNA及蛋白表达水平;通过定点突变技术构建HRE突变型p53RFP启动子双荧光素酶报告基因载体并转染HEK293细胞,分别置于常氧和低氧条件下培养,双荧光素酶报告基因检测系统检测启动子活性。结果 低氧处理不同时间点p53RFP mRNA 表达水平均显著增加,差异有统计学意义(F=96.493,P<0.001);低氧组p53RFP 及HIF-1α蛋白表达量均呈时间依赖性递增。成功构建了HRE 突变型p53RFP 启动子双荧光素酶报告基因载体,双荧光素酶报告基因检测显示,与常氧组相比,低氧条件下野生型p53RFP基因启动子活性增加(t=-19.504,P<0.001),HRE单突变或双突变后启动子活性均较野生型降低(F=160.891,P<0.001)。结论 低氧可诱导p53RFP基因表达上调;成功构建了HRE 突变型p53RFP 双荧光素酶报告基因载体,HRE 位点可能在低氧调控p53RFP基因启动子活性中发挥关键作用。

Objective To investigate the effect of hypoxia on p53RFP gene expression and p53RFP promoter activity, and explore whether hypoxia responsive element (HRE) is involved in the regulation of p53RFP gene promoter activity by hypoxia. Methods HEK293 cells were cultured under normoxia and hypoxia conditions, p53RFP expressions at mRNA and protein levels were detected by real-time PCR and Western blot respectively. The HRE mutant p53RFP promoter dual luciferase reporter gene vector was constructed by site-directed mutagenesis technology and transfected into HEK293 cells under normoxia and hypoxia conditions. The promoter activities were detected by dual luciferase reporter system. Results Compared with the normoxia group, the expression of p53RFP mRNA in the hypoxia group increased significantly at different time points, and the difference was statistically significant(F=96.493,P<0.001); the expression of p53RFP and HIF-1α protein in the hypoxia group increased in a time-dependent manner. The luciferase reporter vectors containing p53RFP with mutant HRE were successfully constructed. The dual luciferase reporter gene assay showed that the activity of the wild-type p53RFP gene promoter was significantly increased under hypoxic conditions compared to normoxic condition(t=-19.504,P<0.001),and the promoter activity of p53RFP with HRE single mutation or double mutation were both significantly lower than that of wild type under hypoxic condition (F=160.891,P<0.001). Conclusions p53RFP gene expression was induced by hypoxia; the p53RFP promoter with mutant HRE dual luciferase reporter gene vectors were successfully constructed, and the HRE locus may play a key role in the hypoxia regulation of p53RFP gene promoter activity.

目的 观察比较术中及术前化疗干预对进展期胃恶性肿瘤手术患者p53、ki-67表达及预后的影响,为临床化疗时间的选择提供理论依据。方法 自2014年8月—2015年5月,我院共收入胃恶性肿瘤患者40例,将40例患者随机分为两组,每组各20例,保证两组患者在性别、年龄、胃癌分期等方面可比,无统计学差异(P＞0.05),标记为Ⅰ组和Ⅱ组。Ⅰ组20例患者于术前进行化疗干预,Ⅱ组在术中给予化疗干预。观察比较两组患者p53、ki-67表达状况及预后。结果 Ⅰ组及Ⅱ组治疗后p53及ki-67均比治疗前升高,差异有统计学意义(P<0.05)。但是治疗后,Ⅰ组和Ⅱ组的p53表达状况组间差异不明显,无统计学意义(P>0.05)。治疗前后,AI差异有统计学意义(P<0.05)。Ⅰ组效果明显好于Ⅱ组,两者差异有统计学意义(P <0.05)。术后六个月、一年随访时发现两组复发率、死亡率差别不大,无统计学意义(P>0.05),术后两年随访发现Ⅱ组复发率、死亡率明显低于Ⅰ组,差异有统计学意义(P<0.05)。结论 术中化疗的疗效优于术前化疗,患者预后较术前化疗好。

Objective To observe the effect of intraoperative and preoperative chemotherapy on the expression of p53, Ki-67 and prognosis in patients with advanced gastric cancer. Methods 40 cases of advanced gastric cancer in our hospital from Aug 2014 to May 2015 were enrolled in the study, and were divide into 2 groups randomly. In group I, 20 patients received chemotherapy intervention befoerer operation, and the other group received chemotherapy intervention during operation. The expressions and prognosis of p53 and Ki-67 were observed and compared between the two groups. Results Group Ⅰ and group Ⅱ after treatment, p53 and Ki-67 were higher than that before treatment, with statistical significance(P<0.05). However, there was no significant difference in the expression of p53 between group Ⅰ and group Ⅱ after treatment, and there was no significant difference(P>0.05). Before and after treatment, the difference of AI was significant, with statistical significance (P<0.05). The effect of group Ⅰ was obviously better than that of group Ⅱ, the difference was statistically significant(P<0.05). Six monthse after the operation and one year follow-up found two groups of recurrence rate and mortality rate had no significant difference(P>0.05). After two years follow-up found the group Ⅱ recurrence rate, mortality was lower than in group Ⅰ (P<0.05). Conclusion The effect of intraoperative chemotherapy is better than that of preoperative chemotherapy, and the prognosis is better than that of preoperative chemotherapy.

       目的  探讨重楼皂苷Ⅰ（PPI）对慢性髓系白血病细胞（K562）细胞的抑制作用及可能的作用机制。方法  采用CCK-8法筛选药物最适浓度，将培养时间为24 h的药物最适浓度作为后续实验的干预浓度。分组如下：（1）空白组；（2）PPI组；（3）抑制剂组；（4）PPI+抑制剂组。采用CCK-8法检测细胞增殖率；AO/EB染色观察细胞形态；流式细胞术检测凋亡率；ROS检测试剂盒检测活性氧（ROS）含量、还原型谷胱甘肽含量检测试剂盒检测谷胱甘肽（GSH）含量、细胞亚铁比色法测试盒检测细胞亚铁（Fe²⁺）含量；qRT-PCR法和蛋白免疫印迹法检测各组肿瘤蛋白53（p53）、钠氯离子依赖性氨基酸转运蛋白11（SLC7A11）、谷胱甘肽过氧化物酶4（GPX4）mRNA及蛋白表达量。结果  PPI抑制K562细胞的生长，且呈一定的剂量及时间依赖性（与同时间段的对照组0μmol/L比较，均P＜0.01）。与空白组相比，PPI抑制K562细胞的增殖，提高了凋亡率，而铁死亡抑制剂（Ferrostian-1）的使用逆转了PPI对K562凋亡的促进作用（P＜0.01）。与空白组相比，PPI组ROS、Fe²⁺含量升高，GSH含量下降，而铁死亡抑制剂的使用可下调ROS、Fe²⁺，上调GSH的含量（P＜0.01）。PPI组较空白组p53 mRNA和蛋白表达水平升高，而SLC7A11、GPX4 mRNA和蛋白表达水平下降（P＜0.05）；PPI+抑制剂组细胞较重楼皂苷组p53 mRNA和蛋白表达水平下降，而SLC7A11、GPX4 mRNA和蛋白表达水平升高（P＜0.05）。结论  PPI能够有效抑制K562细胞增殖，促进K562细胞铁死亡，其分子机制可能与p53信号通路的调控有关。

       Objective  To investigate the inhibitory effect of polyphyllin I（PPI）on chronic myeloid leukemia cells（K562）and its possible mechanism．Methods  K562 cell line was cultured in suitable environment，and the optimal concentration of the drug was screened by CCK-8 method．The optimal concentration of the drug cultured for 24 hours was used as the intervention concentration of the follow-up experiment．Cells were divided into the following groups：（1）blank group，（2）saponins group，（3）inhibitor group and（4）saponins + inhibitor group．The cell proliferation rate was detected by CCK-8 method．The cell morphology was observed by AO/EB staining．The apoptosis rate was detected by flow cytometry．The contents of  reactive oxygen species（ROS），glutathione（GSH）and ferrous（Fe²⁺）in different groups were detected，and the expression of mRNA and protein in different groups were detected by qRT- PCR and Western blot respectively．Results  PPI significantly inhibited the growth of K562 cells in a dose-and time-dependent manner．Compared with the blank group，PPI significantly inhibited the proliferation of K562 cells and increased the apoptosis rate of K562 cells，while the use of ferroptosis inhibitor（Ferrostian-1）reversed the promoting effect of PPI on apoptosis of K562 cells．Compared with the blank group，the contents of reactive oxygen species（ROS）and ferrous iron（Fe²⁺）increased and the content of glutathione（GSH）decreased in the saponins group．The use of Ferrostian-1 could down-regulate the contents of ROS and Fe²⁺ and increase the content of GSH in the cells treated with the drug．Compared with the blank group，the expression of p53 mRNA and protein in the saponins group increased，while the expression of SLC7A11，GPX4 mRNA and protein decreased．The expression of p53 mRNA and protein in the saponins + inhibitor group was lower than that in the saponins group，while the expression levels of SLC7A11，GPX4 mRNA and protein increased．Conclusions  PPI can effectively inhibit the proliferation of K562 cells and promote ferroptosis in K562 cells．The molecular mechanism can be related to the regulation of p53 signal pathway．