SERPINH1 promoted the proliferation and metastasis of colorectal cancer by activating PI3K/Akt/mTOR signaling pathway

BACKGROUND Serpin peptidase inhibitor clade H member 1(SERPINH1)was initially recognized as an oncogene implicated in various human malignancies.Nevertheless,the clinical relevance and functional implications of SERPINH1 in colorectal cancer(CRC)remain largely elusive.AIM To investigate the effects of SERPINH1 on CRC cells and its specific mechanism.METHODS Quantitative real-time polymerase chain reaction,western blotting analysis,The Cancer Genome Atlas data mining and immunohistochemistry were employed to examine SERPINH1 expression in CRC cell lines and tissues.A series of in-vitro assays were performed to demonstrate the function of SERPINH1 and its possible mechanisms in CRC.RESULTS SERPINH1 demonstrated elevated expression levels in both CRC cells and tissues,manifested at both mRNA and protein tiers.Elevated SERPINH1 levels correlated closely with advanced T stage,lymph node involvement,and distant metastasis,exhibiting a significant association with poorer overall survival among CRC patients.Subsequent investigations unveiled that SERPINH1 overexpression notably bolstered CRC cell proliferation,invasion,and migration in vitro,while conversely,SERPINH1 knockdown elicited the opposite effects.Gene set enrichment analysis underscored a correlation between SERPINH1 upregulation and genes associated with cell cycle regulation.Our findings underscored the capacity of heightened SERPINH1 levels to expedite G1/S phase cell cycle progression via phosphatidylinositol 3-kinase/AKT/mechanistic target of rapamycin pathway activation,thereby facilitating CRC cell invasion and migration.CONCLUSION These findings imply a crucial involvement of SERPINH1 in the advancement and escalation of CRC,potentially positioning it as a novel candidate for prognostic assessment and therapeutic intervention in CRC management.

MiR-451 inhibits proliferation of esophageal carcinoma cellline EC9706 by targeting CDKN2D and MAP3K1

AIM To investigate the underlying molecularmechanisms of miR-451 to inhibit proliferation ofesophageal carcinoma cell line EC9706.METHODS： Assays for cell growth, apoptosis andinvasion were used to evaluate the effects of miR-451expression on EC cells. Luciferase reporter and Westernblot assays were used to test whether cyclin-dependentkinase inhibitor 2D （CDKN2D） and MAP3K1 act as majortargets of miR-451.RESULTS： The results showed that CDKN2D andMAP3K1 are direct targets of miR-451. CDKN2D andMAP3K1 overexpression reversed the effect of miR-451.MiR-451 inhibited the proliferation of EC9706 bytargeting CDKN2D and MAP3K1.CONCLUSION： These findings suggest that miR-451might be a novel prognostic biomarker and a potentialtarget for the treatment of esophageal squamous cellcarcinoma in the future.

Inhibitory Effect of Histone Deacetylase Inhibitor on the Proliferation of Leukemia Cells and Its Anti-Tumor Pharmacology

The aim of this study was to explore the inhibitory effect of histone deacetylase inhibitor (HDACI) on the proliferation of leukemia cells. The two kinds of leukemia cells (human promyelocytic leukemia cell (HL-60) and human acute myelogenous leukemia cell (KG-1)) were selected for in vitro research. Besides, Chidamide, a kind of benzamide HDACI, was applied to induce and culture the HL-60 and KG-1 cells, and the anti-tumor cell proliferation activity of Chidamide on HL-60 and KG-1 was detected by the methyl thiazolyl tetrazolium (MTT) assay, which was 5.6 and 6.1 in turn. The cell scratch experiment verified that Chidamide had the metastasis inhibitory effect on HL-60 and KG-1 cells. Flow cytometry was employed to measure the percentage of apoptotic cells, and it was found that the percentage of apoptotic cells was 55.6% ± 1% and 48.6% ± 1% in sequence after HL-60 and KG-1 cells were treated with Chidamide for 36 hours. The number of auto-phagosomes was determined by transmission electron microscopy showing that the number of auto-phagosomes in HL-60 and KG-1 cells was 12 ± 1 and 10 ± 1, respectively after the induction process of Chidamide. The phosphorylated histone H2AX protein (γ-H2AX) recognition antibody immunofluorescence method was adopted to determine the deoxyribonucleic acid (DNA) damage, and the positive rates of HL-60 and KG-1 cells reached 28.41% and 26.35%, respectively after Chidamide treatment. Therefore, Chidamide, as a kind of HDACI, could effectively inhibit the proliferation of leukemia cells, so that the results of this experiment had a good guiding meaning for the clinical diagnosis and treatment of leukemia.

EFFECTS OF BLEOMYCIN A5 COMBINED WITH CALMODU-LIN INHIBITOR ON THE PROLIFERATION OF S-180 CELLS IN VITRO

The effects of bleomycin A5 (BLM A5) alone and combined with calmodulin inhibitor N-(4-aminobutyl)-5-chloro-2-naphthalene sulfonamide (W-13) on the proliferation on S-180 cells in vitro were studied. IC50 of BLM used alone for the cells was about 2.63 μg/ml, but it was reduced to 1/3.8 and 1/9.5 of 2.63 μg/ml when plus W-13 1, 5 μg/ml respectively. The results indicated that nontoxic doses of W-13 enhanced the hinibition of cell proliferation under the condition of BLM 0.5 - 2.5 μg/ ml. In colony forming test, the survival fraction of S-180 cells treated with BLM plus W-13 was decreased to 1/87 - 240 of that of the cells treated with BLM alone. The results suggest that W-13 can enhance antitumor activity of BLM in vitro and may be used as an synergist of BLM A5 in vivo.

血清sMICA、PCNA、GASP-1、TIMP-1在非小细胞肺癌患者中的表达及相关性分析

目的探讨血清可溶性MHC-I类链相关蛋白A(sMICA)、增殖细胞核抗原(PCNA)、G蛋白偶联受体相关分选蛋白1(GASP-1)、组织金属蛋白酶抑制剂1(TIMP-1)在非小细胞肺癌(NSCLC)患者中的表达及与病理分型的相关性。方法2020年7月至2022年8月诊治的86例NSCLC患者作为研究对象,并设立为观察组,同期选取43例健康体检者设立为对照组;并根据不同病理分型将观察组分为腺癌组(n=33)和鳞癌组(n=53),对比血清sMICA、PCNA、GASP-1、TIMP-1;并采用Logistic回归模型分析sMICA、PCNA、GASP-1、TIMP-1对非小细胞肺癌的影响;采用ROC曲线模型分析sMICA、PCNA、GASP-1、TIMP-1诊断非小细胞肺癌的AUC、敏感度及特异度。结果观察组的sMICA、PCNA、GASP-1、TIMP-1均高于对照组(P<0.05)。腺癌组的sMICA、PCNA、GASP-1、TIMP-1均高于鳞癌组(P<0.05)。二元Logistic回归模型分析显示,sMICA、PCNA、GASP-1、TIMP-1高表达会对非小细胞肺癌的发生产生影响(P<0.05)。ROC曲线分析显示,sMICA、PCNA、GASP-1、TIMP-1及四项联合诊断NSCLC的AUC值分别为(0.750、0.654、0.819、0.788、0.843,P均<0.05),敏感度分别为57.00%、46.50%、67.40%、90.70%、79.10%;特异度分别为93.00%、93.00%、88.40%、58.10%、86.00%。结论sMICA、PCNA、GASP-1、TIMP-1在NSCLC患者中呈高表达趋势,其表达水平会随病理分型而升高。

STAT3抑制剂stattic对小鼠结肠癌CT26细胞增殖和凋亡的影响

目的探讨信号转导和转录激活因子3(STAT3)抑制剂盐酸萘替芬(stattic)对小鼠结肠癌CT26细胞增殖和凋亡的影响和作用机制。方法采用0μmol/L、1μmol/L、5μmol/L、10μmol/L stattic溶液处理小鼠结肠癌CT26细胞,通过CCK-8实验、细胞克隆形成实验、细胞划痕实验、Transwell侵袭实验以及流式细胞术检测细胞活力、增殖、迁移、侵袭、周期和凋亡情况;利用Western blot法检测stattic对小鼠结肠癌细胞磷酸化STAT3(p-STAT3)表达的影响;通过实时荧光定量多聚核苷酶链式反应(RT-qPCR)检测stattic作用后CT26细胞B淋巴细胞瘤-2(Bcl-2)和人跨膜受体蛋白Notch-1(Notch-1)的表达。结果与0μmol/L组相比,stattic溶液组CT26细胞的活力及增殖能力降低(P<0.001)、迁移率和侵袭率降低(P<0.001),细胞凋亡率随浓度增加而增加(P<0.0001);stattic能将CT26细胞周期阻断于G1期,进而阻止CT26细胞的增殖;Western blot结果显示stattic抑制CT26细胞p-STAT3的表达(P<0.05);RT-qPCR检测结果表明stattic下调CT26细胞Bcl-2和Notch1的表达(P<0.05)。结论stattic通过阻断STAT3信号,抑制p-STAT3蛋白的表达,下调下游抗凋亡分子Bcl-2和Notch1信号分子的表达从而抑制CT26细胞增殖促进细胞凋亡。

The cancer-testis gene,MEIOB,sensitizes triple-negative breast cancer to PARP1 inhibitors by inducing homologous recombination deficiency

Objective:The newly defined cancer-testis(CT)gene,MEIOB,was previously found to play key roles in DNA double-strand break(DSB)repair.In this study,we aimed to investigate the effects and mechanisms of MEIOB in the carcinogenesis of triple-negative breast cancers(TNBCs).Methods:The Cancer Genome Atlas database was used to quantify the expression of MEIOB.Cox regression analysis was used to evaluate the association between MEIOB expression and the prognosis of human TNBC.The effects of MEIOB on cell proliferation and migration in TNBCs were also assessed in vitro.Patient-derived xenograft(PDX)models were used to assess the sensitivity of breast cancers with active MEIOB to PARP1 inhibitors.Results:We confirmed MEIOB as a CT gene whose expression was restricted to the testes and breast tumors,especially TNBCs.Its activation was significantly associated with poor survival in breast cancer patients[overall,hazard ratio(HR)=1.90(1.16–2.06);TNBCs:HR=7.05(1.16–41.80)].In addition,we found that MEIOB was oncogenic and significantly promoted the proliferation of TNBC cells.Further analysis showed that MEIOB participated in DSB repair in TNBCs.However,in contrast to its function in meiosis,it mediated homologous recombination deficiency(HRD)through the activation of poly ADP-ribose polymerase(PARP)1 by interacting with YBX1.Furthermore,activated MEIOB was shown to confer sensitivity to PARP inhibitors,which was confirmed in PDX models.Conclusions:MEIOB played an oncogenic role in TNBC through its involvement in HRD.In addition,dysregulation of MEIOB sensitized TNBC cells to PARP inhibitors,so MEIOB may be a therapeutic target of PARP1 inhibitors in TNBC.

Bowman-Birk inhibitors from legumes as colorectal chemopreventive agents

Aberrant functioning of serine proteases in inflammatory and carcinogenic processes within the gastrointestinal tract(GIT)has prompted scientists to investigate the potential of serine protease inhibitors,both natural and synthetic,as modulators of their proteolytic activities.Protease inhibitors of the Bowman-Birk type,a major protease inhibitor family in legume seeds,which inhibit potently and specifically trypsin-and chymotrypsin-like proteases,are currently being investigated as colorectal chemopreventive agents.Physiologically relevant amounts of Bowman-Birk inhibitors(BBI)can reach the large intestine in active form due to their extraordinary resistance to extreme conditions within the GIT.Studies in animal models have proven that dietary BBI from several legume sources,including soybean,pea,lentil and chickpea,can prevent or suppress carcinogenic and inflammatory processes within the GIT.Although the therapeutic targets and the action mechanism of BBI have not yet been elucidated,the emerging evidence suggests that BBI exert their preventive properties via protease inhibition;in this sense,serine proteases should be considered as primary targets in early stages of carcinogenesis.The validation of candidate serine proteases as therapeutic targets together with the identification,within the wide array of natural BBI variants,of the most potent and specific protease inhibitors,are necessary to better understand the potential of this protein family as colorectal chemopreventive agents.

THE INCREASE IN PLASMINOGEN ACTIVATOR INHIBITOR TYPE-1 EXPRESSION BY STIMULATION OF ACTIVATORS FOR PEROXISOME PROLIFERATOR-ACTIVATED RECEPTORS IN HUMAN ENDOTHELIAL CELLS

Objective.To investigate the effect of peroxis ome proliferator-activated recept ors(PPARs )activators on plasminogen activator inhibitor ty pe-1(PAI-1)expression in human umbilical vein e ndothelial cells and the possi-ble mechanism.Methods.Human umbilical vein endothelial ce lls(HUVECs )were obtained from normal fetus,and cul-tured conventionally.Then the HUVECs were exposed to test agents(linolenic acid,linoleic acid,oleic acid,stearic acid and prostaglandin J 2 respectively)in varying concentrations with fresh media.RT -PCR and ELISA were applied to determine the expression of PPARs and PAI-1in HUVECs.Results.PPARα,PPARδand PPARγmRNA were detected by using RT-PCR in HUVECs.Treatment of HUVECs with PPARαand PPARγactivators---linolenic acid,linoleic acid,oleic acid and prostaglandin J 2 respectively,but not with stearic a cid could augment PAI-I mRNA expression and protein secretion in a concentration-dependent manner.However,the mRNA expressions of 3subclasses of PPAR with their activators in HUVECs were not changed compared w ith controls.Conclusion.HUVECs express PPARs.PPARs activators may increase PAI-1expression in ECs,but the underlying mechanism remains uncle ar.Although PPARs expression was not enhanced after stimulated by their activators in ECs,the role of functionally active PPARs in regulating PA I-1expression in ECs needs to be further investigated by using transient gen e transfection assay.

Effect of cis-9,trans-11-conjugated linoleic acid on cell cycle of gastric adenocarcinoma cell line(SGC-7901)

AIM: To determine the effect of cis-9, trans-1l-conjugated linoleic acid ( c9, tl 1-CLA) on the cell cycle of gastric cancer cells( SGC-7901 ) and its possible mechanism in inhibition cancer growth.METHODS: Using cell culture and immunocytochemicaltechniques, we examined the cell growth, DNA synthesis,expression of PCNA, cyclin A, B1, D1, pl6nink4a and p21clp/wafl ofSGC-7901 cells which were heated with various c9, tll-CLAconcentrations (25,50,100 and 200μnol@L-1)of c9, tll-CLA for 24and 48 h, with a negative control (0.1% ethane).RESULTS: The cell growth and DNA synthesis of SGC-7901cells were inhibited by c9, tll-CLA. SGC-7901 cells. Eightday after treatment with various concentrations of c9, tl1-CLA mentioned above, the inhibition rates were 5.92 % ,20.15 % ,75.61% and 82.44 % ,respectively and inhibitory effeotof c9, tll-CLA on DNA synthesis (except for 25 tmol/L,24h) showed significantly less 3H-TdR incorporation than thatin the negative controls (P ＜ 0. 05 and P ＜ 0. 01).Immunocytochemical staining demonstrated that SGC-7901cells preincubated in media supplemented with different c9,tl1-CLA concentrations at various times significantlydecreased the expressions of PCNA (the expression rateswere7.2-3.0 %,24 h and 9.1-0.9 % at 48 h, respectively),Cyclin A (l1.0-2.3 %, 24 h and 8.5-0.5 % ,48 h), B1 (4.8-1.8% at 24 h and 5.5-0.6 % at 48 h)and D1 (3.6-1.4 % at 24 hand 3.7 %-0 at 48 h) as compared with those in the negativecontrols(the expressions of PCNA, Cyclin A, B1 and D1were 6.5 % at 24h and 9.0 % at 48 h, 4.2% at 24h and5.1% at 48 h, 9.5 % at 24h and 6.0 % at 48 h,respectively)(P＜ 0.01), whereas the expressions of p16ink4a and p21clp/waf1,cyclin-dependent kinases inhibltors(CDKI), were increased.CONCLUSION: The cell growth and proliferation of SGC-7901cell is inhibited by c9, tll-CLA via blocking the cell cycle,with reduced expressions of cyclin A, B1 and D1 andenhanced expressions of CDKI( p16ink4a and p21cip/waf1).

端粒酶抑制因子X1过表达对人食管癌EC1细胞增殖及凋亡的影响

目的探究端粒酶抑制因子X1(PinX1)对人食管癌EC1细胞增殖及凋亡的影响。方法体外培养人食管癌细胞系EC1细胞株,并将其分为空白对照(NG)组(空白对照细胞)、pcDNA组(转染阴性对照质粒)、pcDNA-PinX1组(转染PinX1过表达质粒)、N-乙酰-L-半胱氨酸(NAC)组(加入信号通路抑制剂)。采用实时荧光定量PCR法检测各组PinX1 mRNA的表达水平。分别采用CCK-8法和流式细胞术检测各组细胞的增殖、凋亡情况。采用荧光探针法检测各组活性氧簇(ROS)水平。采用蛋白质印迹法检测各组腺苷酸激活蛋白激酶/信号转导与转录激活因子3(AMPK/STAT3)信号通路相关蛋白的表达水平。结果经成功转染细胞后,与NG组、pcDNA组比较,pcDNA-PinX1组细胞的增殖抑制率、凋亡率、ROS水平、p-AMPK/AMPK均升高,而p-STAT3/STAT3均降低,差异均有统计学意义(P均<0.05)。与pcDNA-PinX1组比较,NAC组细胞的增殖抑制率、凋亡率、ROS水平、p-AMPK/AMPK均降低,p-STAT3/STAT3升高,差异均有统计学意义(P均<0.05)。结论PinX1过表达可抑制人食管癌EC1细胞增殖并诱导其凋亡,可能通过促进ROS/AMPK/STAT3信号通路活化来实现。

Circadian variation in expression of G_1 phase cyclins D_1 and E and cyclin-dependent kinase inhibitors p16 and p21 in human bowel mucosa

瞄准：评估在大肠上皮细胞的细胞的增长率是否被生理节奏的节奏描绘。方法：在 2003 年 1 月和 2004 年 12 月之间，作为受不了被诊断的二十个病人主要，将切除更低的直肠的有能力的、非变形的腺癌，渗入括约肌机制，经历了腹会阴切除术，全部的中央直肠的切除和永久左肠骨的结肠开口术。在每六个小时( 00:00 ， 06:00 ， 12:00 ， 18:00 和 24:00 )从结肠开口术粘膜获得的修理福尔马林、嵌入石蜡的活体检视标本，我们象 G ( 1 )阶段的表示一样学习了 G ( 1 )阶段 cyclins ( D ( 1 )和 E )的表示房间的指示物骑车的cyclin依赖的激酶( CDK )禁止者 p16 和 p21 在用免疫的结肠的上皮细胞的前进组织化学的方法。结果：两 cyclins 的表示显示出在 00:00 和 18:00 获得他们的最低、最高的价值的类似的生理节奏的时尚，分别地(P【0.001 ) 。在 CDK 禁止者蛋白质 p16 和 p21 的表示的生理节奏的节奏也被观察，与分别地在 12:00 和 18:00 (P【0.001 ) 获得的底层。当建筑群 cyclins D (1 )-p21 和 E-p21 被检验时， cyclins 的表示不利地在整个日子被相关到 p21 表示。什么时候建筑群 cyclins D (1 )-p16 和 E-p16 被检验， p16 表示的高水平在 00:00， 06:00 和 24:00 被相关到 cyclin 表示的底层。同时，两 cyclins 的最高的表示层次在 18:00 被相关到 p16 表示的高水平。结论：结肠的上皮细胞似乎在下午期间进入房间周期的 G (1 ) 阶段(在 12:00 和 18:00 之间) 与在 18:00 获得的最高的率。从一个临床的观点，当最小化毒性时，现在的结果建议在下午力量的 G (1 ) 阶段 specific 反癌症治疗最大化他们的反肿瘤效果。

Assessment of the tumor malignancy after immunotherapy combined with EGFR-TKIs drug treatment of EGFR-mutant advanced non-small cell lung cancer

Objective:To study the effect of immunotherapy combined with epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) drug therapy on the tumor malignancy in patients with EGFR-mutant advanced non-small cell lung cancer (NSCLC).Methods:Patients with EGFR-mutant advanced NSCLC who were treated in Xi'an No.1 Hospital and Shaanxi Provincial Tumor Hospital between May 2014 and August 2015 were selected as the research subjects and randomly divided into DC-CIK group and control group, the former received immunotherapy combined with EGFR-TKI drug therapy, and the latter received EGFR-TKI drug therapy. Before and after treatment, the cancer cell viability-related marker levels in serum as well as the antitumor immune response marker molecule expression in peripheral blood mononuclear cells were determined respectively, and the proliferation-related gene expression in lung cancer lesions were determined after treatment.Results: 2 weeks and 4 weeks after treatment, serum CEA, Cyfra21-1 and SCCA levels of both groups were significantly lower than those before treatment and serum CEA, Cyfra21-1 and SCCA levels of DC-CIK group were significantly lower than those of control group, CD3, CD4 and CD8 fluorescence intensity in peripheral blood mononuclear cells of DC-CIK group were significantly higher than those before treatment, and the CD3, CD4 and CD8 fluorescence intensity in peripheral blood mononuclear cells of control group were not significantly different from those before treatment;TCF3, MEF2D and cFLIP(L) expression in lung cancer lesions of DC-CIK group were significantly lower than those of control group while FRMD8, PDCD5, caspase-3 and caspase-8 expression were significantly higher than those of control group.Conclusion:Immunotherapy combined with EGFR-TKI drug treatment of EGFR-mutant advanced non-small cell lung cancer (NSCLC) can enhance the antitumor immune response and promote cancer cell apoptosis.

丝氨酸肽酶抑制因子Kazal型1对肝癌细胞增殖的影响及其机制

目的探讨丝氨酸肽酶抑制因子Kazal型1(SPINK1)对肝癌细胞RH-35增殖的影响及其分子机制。方法利用基因表达综合(GEO)数据库分析Spink1基因在肝癌患者和大鼠肝癌模型中的表达,用重组大鼠SPINK1蛋白(rrSPINK1)处理RH-35细胞,采用MTT、流式细胞术、5-乙炔基-2’-脱氧尿苷(EdU)等方法检测SPINK1对RH-35细胞增殖的影响,Real-time PCR和Western blotting检测SPINK1信号通路、细胞增殖和凋亡相关基因的表达。结果SPINK1在肝癌和大鼠肝癌模型中显著高表达;与对照组相比,rrSPINK1处理的RH-35细胞活力,EdU阳性细胞率,S期和G_2/M期细胞比例明显增加;而RH-35细胞凋亡无显著变化;rrSPINK1上调RH-35细胞中p38 MAPK和STAT信号通路相关基因/蛋白的表达;并且在肝癌中Spink1基因的表达与Mapk13、Stat1和Stat3基因正相关。结论SPINK1促进肝癌细胞中p38 MAPK和Janus激酶/信号转导及转录激活因子(JAK/STAT)的表达,促进肝癌细胞的增殖。

STAT3抑制剂S3I-201对人肝癌BEL-7402细胞增殖、凋亡及细胞周期的影响

目的 研究STAT3小分子抑制剂S3I-201对人肝癌细胞系BEL-7402增殖、凋亡与细胞周期的影响和初步的作用机制。方法 分别采用细胞活性检测试剂盒cell couting kit-8(CCK-8)和EdU细胞增殖法检测S3I-201对人肝癌细胞BEL-7402细胞增殖的影响,应用流式细胞术分析S3I-201对BEL-7402的细胞凋亡和周期的影响,通过Western blotting检测凋亡相关蛋白Bcl-2、Bax的表达。结果 CCK-8及EdU结果显示,S3I-201能抑制BEL-7402细胞的生长和增殖(P<0.05),使BEL-7402细胞阻滞于S期,具有诱导细胞凋亡的作用,上调Bax的表达,下调Bcl-2的表达(P<0.05)。结论 STAT3小分子抑制剂S3I-201通过促进BEL-7402凋亡、上调促凋亡蛋白Bax、下调抗凋亡蛋白Bcl-2的表达,并将BEL-7402阻滞于S期而发挥抗肿瘤效应。

复方藤梨汤含药血清对人脑胶质瘤U251细胞增殖、凋亡及上皮间质转化的影响

目的探究复方藤梨汤含药血清对人脑胶质瘤U251细胞增殖、凋亡及上皮间质转化的影响。方法将24只雄性SD大鼠分为对照组和低、中、高剂量组[复方藤梨汤6、12、24g/(kg·d)],每组6只,制备含药血清处理人脑胶质瘤U251细胞。采用CCK-8法检测细胞活力,集落形成实验检测细胞克隆形成能力,流式细胞术检测细胞周期分布与细胞凋亡,定量聚合酶链反应检测原癌基因C-myc、细胞周期蛋白D1(CyclinD1)基因表达,Western blot法检测细胞凋亡蛋白[B细胞淋巴瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、胱天蛋白酶(Caspase)-3、Caspase-8]以及β-连环蛋白(β-catenin)、上皮性黏附蛋白(E-cadherin)及波形蛋白(Vimentin)表达。结果选取体积分数10%的含药血清为最终剂量。与对照组比较,低、中、高剂量组细胞活力、克隆形成数、C-myc mRNA、CyclinD1 mRNA、Vimentin表达水平降低,处于S期细胞比例、Bax/Bcl-2比值、Caspase-3表达水平升高(P<0.05);低、高剂量组G1期细胞比例降低(P<0.05);中、高剂量组G2期细胞比例、β-catenin表达水平降低,Caspase-8和E-cadherin表达水平升高(P<0.05);高剂量组细胞凋亡率升高(P<0.05)。结论复方藤梨汤含药血清可促使人脑胶质瘤U251细胞阻滞于S期,抑制细胞增殖,诱导细胞凋亡,通过调节β-catenin、E-cadherin和Vimentin表达抑制上皮间质转化。

ANO1抑制剂在AngⅡ诱导的VSMC增殖中的作用研究

目的研究钙激活氯通道蛋白1(ANO1)抑制剂在血管紧张素Ⅱ(AngⅡ)诱导的血管平滑肌细胞(VSMC)增殖中的作用。方法研究时间为2019年7月至2022年6月。体外培养大鼠胸主动脉平滑肌细胞A7r5细胞株,AngⅡ刺激A7r5细胞建立细胞增殖模型,分别采用10 nmol/L、100 nmol/L、500 nmol/L不同浓度ANO1抑制剂(A01)干预,同时设置对照组(以等量生理盐水干预),采用Hoechst 33342荧光染色法观察ANO1抑制剂干预后AngⅡ诱导的VSMC形态学变化,蛋白免疫印迹法检测ANO1抑制剂干预后AngⅡ诱导的VSMC细胞凋亡相关蛋白表达水平。多组间比较采用单因素方差分析,组间两两比较采用LSD-t检验。结果Hoechst 33342荧光染色结果显示,随着ANO1抑制剂浓度增加,呈亮蓝色的细胞愈来愈多(染色质凝聚、出现凋亡小体),即细胞凋亡数量越多。低剂量组、中剂量组、高剂量组凋亡率分别为(17.60±1.36)%、(36.30±3.50)%、(61.25±6.33)%,均高于对照组凋亡率(4.32±0.51)%,组间差异均有统计学意义(均P<0.05),即AngⅡ诱导的VSMC增殖后凋亡与ANO1抑制剂浓度之间存在剂量依赖性。蛋白免疫印迹法检测ANO1抑制剂干预后AngⅡ诱导的VSMC凋亡相关蛋白结果显示,不同组间EPK、AKT、CDK4、Cyclin D1蛋白相对表达量主效应差异有统计学意义(P<0.05);与对照组比较,低剂量组、中剂量组、高剂量组EPK、AKT、CDK4、Cyclin D1蛋白相对表达量均降低,且呈剂量依赖性,各组间差异均有统计学意义(均P<0.05)。结论ANO1抑制剂可显著抑制AngⅡ诱导的VSMC增殖作用,其机制可能与抑制EPK/AKT信号通路及细胞周期进程有关。

奥希替尼联合放疗对EGFR基因突变晚期NSCLC患者血清PCNA、TSGF水平和预后的影响

目的:探讨奥希替尼联合放疗对表皮生长因子受体(EGFR)基因突变晚期非小细胞肺癌(NSCLC)患者血清细胞增殖抗原(PCNA)、肿瘤特异性生长因子(TSGF)和预后的影响。方法:根据治疗方案不同将100例EGFR基因突变晚期NSCLC患者分为联合组与对照组,每组各50例;联合组予以奥希替尼联合放疗治疗,对照组予以单药奥希替尼治疗。比较两组近期疗效、远期生存期、治疗前和治疗3个月后细胞角蛋白19片段(CYFRA21-1)、糖类抗原125(CA125)和PCNA、TSGF表达及不良反应发生情况。结果:联合组的客观缓解率高于对照组,但差异无统计学意义(P>0.05)。治疗3个月后,联合组血清CYFRA21-1、CA125和PCNA、TSGF水平均低于对照组(P<0.05)。患者随访6~36个月,联合组中位无进展生存期、总生存期分别为14.9个月、25.2个月,优于对照组的10.1个月、18.8个月,差异均有统计学意义(P<0.05)。两组不良反应发生率比较,差异无统计学意义(P>0.05)。结论:奥希替尼联合放疗治疗EGFR基因突变晚期NSCLC可有效降低血清PCNA、TSGF水平,改善患者生存预后,且不增加不良反应。

选择性MAO-B抑制剂TM-5对胶质瘤U87细胞自噬和凋亡的影响

目的探讨选择性单胺氧化酶B(MAO-B)抑制剂TM-5对胶质瘤U87细胞体外自噬及凋亡的影响。方法正常培养胶质瘤细胞株U87,随机分为6组:对照组不做处理,5、10、25、50、100μmoL/L TM-5组分别予以5、10、25、50、100μmoL/L TM-5处理细胞,每组设3个复孔。采用噻唑蓝(MTT)法检测细胞活力;流式细胞术检测细胞凋亡;实时荧光定量聚合酶链反应(qRT-PCR)检测促凋亡蛋白Bcl-2相关蛋白X(Bax)、抑凋亡蛋白B淋巴细胞瘤-2(Bcl-2)的基因表达;蛋白质免疫印迹(WB)检测细胞凋亡相关蛋白Bax和切割后半胱氨酸蛋白酶3(cleaved caspase-3)以及自噬相关标志蛋白Beclin-1、自噬相关蛋白1轻链-Ⅱ(LC3-Ⅱ)和P62表达。结果与对照组比较,10、25、50、100μmoL/L TM-5组U87细胞存活率降低(P<0.05),选取5、10、25μmoL/L TM-5组(细胞存活率>50%)进行后续实验。与对照组比较,10、25μmoL/L TM-5组相对细胞凋亡率、细胞中LC3-Ⅱ和Bax蛋白相对表达量升高,差异有统计学意义(P<0.05);5、10、25μmoL/L TM-5组U87细胞中促凋亡基因Bax mRNA相对表达水平、Beclin-1和cleaved caspase-3蛋白相对表达量升高,抑凋亡基因Bcl-2 mRNA、P62蛋白相对表达量降低,差异有统计学意义(P<0.05)。结论TM-5可抑制胶质瘤U87细胞存活,可上调Beclin-1、LC3-Ⅱ、Bax和cleaved caspase-3,下调Bcl-2和P62表达,诱导细胞自噬和凋亡。

第3代酪氨酸激酶抑制剂二线治疗伴T790M突变晚期肺腺癌142例生存预后影响因素

目的探讨第3代酪氨酸激酶抑制剂(EGFR-TKIs)二线治疗伴T790M突变晚期肺腺癌病人生存预后影响因素。方法回顾性纳入2017年6月至2020年6月于镇江市第一人民医院接受奥希替尼二线治疗伴T790M突变晚期肺腺癌病人共142例,分析临床病理资料和随访生存资料,采用单因素和多因素法评价病人无进展生存时间独立影响因素。结果单因素分析结果显示,一代EGFR-TKI治疗后无进展生存时间[9.0月比13.0月]和人类增殖细胞核抗原Ki-67表达水平分别为<30%、30%~60%、>60%的无进展生存时间[16.0月比12.0月比9.0月]与奥希替尼二线治疗伴T790M突变晚期肺腺癌病人无进展生存时间有关(P<0.05);Cox回归模型分析结果显示,一代EGFR-TKI治疗后无进展生存时间和Ki-67表达水平是奥希替尼二线治疗伴T790M突变晚期肺腺癌病人无进展生存时间独立影响因素(P<0.05)。结论第3代EGFR-TKIs二线治疗伴T790M突变晚期肺腺癌病人生存预后与一代EGFR-TKI治疗后无进展生存时间和Ki-67表达水平关系密切;其中一代EGFR-TKI治疗后无进展生存时间>10个月和Ki-67表达水平<30%者二线治疗后无进展生存时间往往更长。