目的 研究结肠癌组织中转录因子KLF8的表达及下调KLF8的表达对结肠癌细胞的影响。方法 收集结肠癌组织和癌旁正常组织,检测KLF8的蛋白含量;培养结肠癌Lovo细胞株,转染KLF8 siRNA后检测细胞侵袭、迁移以及上皮-间质转化(EMT)。结果 结肠癌组织中KLF8的蛋白含量高于癌旁正常组织;转染KLF8 siRNA的结肠癌细胞组迁移距离低于阴性对照组,且侵袭至transwell微孔膜外侧面的细胞数少于阴性对照组;转染KLF8 siRNA的结肠癌细胞组内E-cadherin的表达升高,Vimentin、N-cadherin的蛋白含量低于阴性对照组。结论 结肠癌组织中KLF8的表达量升高,下调结肠癌细胞中KLF8的表达可抑制结肠癌细胞侵袭、迁移及上皮-间质转化过程。

Objective To study the expression of transcription factor KLF8 in colorectal cancer tissue and its effect of downregulation KLF8 on colorectal cancer cell. Methods Collecting cancer tissues and adjacent normal color tissue and detecting the protein level of KLF8. Culturing the colorectal cancer Lovo cell lines and detecting cell invasion, cell migration and epithelial-mesenchymal transition after transfecting of KLF8 siRNA. Results KLF8 was highly expressed in colorectal cancer tissues compared with adjacent normal colon tissue. After transfection of KLF8 siRNA, the migration distance of colorectal cancer cell and the cell population transferred to the lateral surface of transwell microporous membrane were lower than those of negative control siRNA. E-cadherin of KLF8 siRNA group were higher than those of negative control siRNA group. Vimentin and N-cadherin were lower than those of negative control siRNA group. Conclusion The expression of KLF8 in colorectal cancer tissue is elevated;downregulation of KLF8 expression in colorectal cancer cell lines may inhibit cell invasion, cell migration and epithelial-mesenchymal transition processes.

目的 研究NR3C1(核受体亚科3,C组,成员1)又称糖皮质激素受体(GR)表达量对前列腺癌恶性程度的影响及其与前列腺癌生化复发的相关性。方法 通过组织芯片免疫组化染色检测的方法检验NR3C1在不同恶性程度前列腺癌组织的表达情况,结合Taylor数据库分析NR3C1表达水平与前列腺癌临床病理特征关系,再采用Kaplan-Meier法分析NR3C1对前列腺癌生化复发生存率的影响,最后用Cox回归分析临床病理特征与生化复发的相关性。结果 组织芯片免疫组化结果显示NR3C1在Gleason评分低的前列腺癌组织中表达高于Gleason评分高的前列腺癌组织(P=0.028)。结合Taylor公用数据库分析,NR3C1在前列腺癌组织中的表达低于癌旁组织(P＜0.001),NR3C1在Gleason评分低的前列腺癌组织中表达高于Gleason评分高的前列腺癌组织(P=0.005),NR3C1低表达与PSA复发(P=0.028)和转移(P=0.003)相关。Kaplan-Meier结果提示:NR3C1高表达组患者术后的生化复发生存率更高(P=0.043),总体生存率没有明显区别(P=0.872)。单因素分析结果显示:NR3C1(P=0.002),病理分期(P＜0.001),Gleason评分(P＜0.001),是否转移(P=0.012)是前列腺癌生化复发的影响因素。多因素分析结果显示:高Gleason 评分(P=0.017)和转移(P<0.001)均为生化复发危险因素。结论 NR3C1影响前列腺癌的发病进程,检验NR3C1的表达情况,能预测前列腺癌患者生化复发的概率,可协助判断前列腺癌预后。

Objective We study the role of NR3C1 (nuclear receptor subfamily 3,group C,member 1) in PCa progression,and the correlation between its expression level and the biochemical recurrence of PCa. Methods Immunohistochemistry was used to detect the expression of NR3C1 in PCa tissues of different degrees of malignancy. The associations of NR3C1 expression and clinical pathological features were analyzed using the Taylor dataset. Kaplan-Meier was used to detect the relationship between NR3C1 expression and biochemical recurrence survival rate in PCa. Cox-regressive analysis was used to detect the relationship between clinical pathological features and biochemical recurrence. Results Immunohistochemistry analysis showed the expression of NR3C1 was higher in which its Gleason Score was lower(P=0.028). Base on the Taylor dataset,the expression of NR3C1 was higher in the adjacent benign tissues than that in PCa(P＜0.001). The expression of NR3C1 was higher in which its Gleason Score was lower(P=0.005). Furthermore,low NR3C1 expression was associated with PSA failure(P=0.028) and Metastasis(P=0.003). Kaplan-Meier showed the biochemical recurrence-free time of PCa patients in low NR3C1 expression groups reduced(P=0.043). The overall survival time of PCa patients was not correlated to NR3C1 expression levels(P=0.872). Single factor analysis showed the biochemical recurrence is associated with NR3C1 expression(P=0.002),pathological stage(P＜0.001),Gleason score(P＜0.001), Metastasis status(P=0.012). Multivariate analysis by Cox regression further identified the high Gleason Score(P=0.017) and Metastasis status (P<0.001)were hazards of the biochemical recurrence. Conclusion Our study showed that the expression of NR3C1 critically connected with the process of PCa,which indicated that we can predict the probability of the biochemical recurrence and determine the prognosis of prostate cancer by detecting the expression of NR3C1 in PCa patients.

目的 分析人脑胶质瘤组织中O6—甲基鸟嘌呤—DNA甲基转移酶(MGMT)、DNA拓扑异构酶Ⅱ(TopoⅡ)基因的表达情况及其对化疗敏感性的影响。方法 收集医院2012年4月—2018年6月期间进行开颅手术切除的新鲜人脑胶质瘤标本80例和同期经颅脑手术治疗的脑外伤或脑出血内减压切除的正常脑组织30例。采用免疫组化法检测两种标本中MGMT和TopoⅡ基因表达情况,并通过脑胶质瘤U251和U87细胞培养、体外药物(替莫唑胺)干预、Transwell体外侵袭实验分析其对化疗敏感性的影响。结果 胶质瘤标本、正常脑组织MGMT和TopoⅡ基因表达程度分布比较差异均有统计学意义(P＜0.05),且二者MGMT基因阳性表达率分别为63.75%、3.33%,TopoⅡ基因阳性表达率分别为55.00%、0.00%,差异有统计学意义(P＜0.05)。MGMT、TopoⅡ基因均在细胞核显示为阳性染色。体外药物干预的实验组、未进行药物干预的阴性对照组干预前U251、U87细胞穿膜细胞计数比较无统计学意义(P＞0.05),但干预后实验组U251、U87细胞穿膜细胞计数高于阴性对照组(P＜0.05),干预后实验组U251、U87细胞有更强的侵袭力。结论 MGMT和TopoⅡ基因在人脑胶质瘤标本中阳性表达率高,且可能参与促进脑胶质瘤细胞侵袭,影响肿瘤化疗敏感性。

Objective To analyze the expression of O6-methylguanine-DNA methyltransferase (MGMT) and DNA topoisomerase II (Topo II) genes in human brain gliomas and its influence on the chemosensitivity. Methods A total of 80 fresh human brain glioma specimens resected by craniotomy and 30 normal brain tissues resected by craniocerebral operation for traumatic brain injury or decompression for cerebral hemorrhage during the period from April 2012 to June 2017 were collected. The expression of MGMT and Topo II genes in the two kinds of specimens was detected by immunohistochemical method, and the influence on chemosensitivity was analyzed through brain glioma U251 and U87 cell culture, in vitro drug (temozolomide) intervention and Transwell invasion in vitro. Results There was a significant difference in the expression of MGMT and Topo II genes in glioma specimens and normal brain tissues (P<0.05). The positive expression rates of MGMT gene in the two kinds of specimens were 63.75% and 3.33% respectively while the positive expression rates of Topo II gene were 55.00% and 0.00%, respectively (P<0.05). Both of MGMT and Topo II genes were displayed in the nucleus as positive staining. There was no significant difference in transmembrane cell count of U251 and U87 cells between the experimental group given drug intervention in vitro and negative control group without drug intervention before the intervention (P>0.05). However, the transmembrane cell count of U251 and U87 cells in the experimental group after intervention was higher than that in the negative control group (P<0.05). After intervetion, U251 and U87 cells were with stronger invasiveness in the experimental group. Conclusion The positive expression rates of MGMT and Topo II genes are high in human brain glioma specimens. They may be involved in promoting glioma cell invasion, affecting tumor chemosensitivity.

目的 探究叶酸对子宫内膜癌作用的靶基因。方法 通过转录组测序筛选叶酸作用下子宫内膜癌细胞中的差异基因,生存分析寻找对子宫内膜癌具有生存意义的差异基因,qPCR及western blot检测其在叶酸作用下的表达。结果 转录组测序发现36个差异基因,生存分析发现FMN1,TRIB3,INHBE及NRBP2的表达对子宫内膜癌具有生存意义,qPCR及western blot验证叶酸作用下NRBP2在子宫内膜癌细胞中的表达下调。结论 叶酸下调子宫内膜癌中NRBP2基因的表达,NRBP2可能是叶酸对子宫内膜癌作用的靶标。

Objective To explore the target genes of folic acid on endometrial carcinoma. Methods The differential genes in endometrial cancer cells treated with folic acid were screened by transcriptome sequencing. Survival analysis was used to find the differential genes with survival significance. QPCR and western blot were used to detect their expression under the action of folic acid. Results 36 differential genes were found by transcriptional sequencing. Survival analysis showed that the expression of FMN1,TRIB3,INHBE and NRBP2 had survival significance in endometrial carcinoma. QPCR and western blot confirmed that the expression of NRBP2 in endometrial cancer cells was down-regulated by folic acid. Conclusion Folic acid down-regulates the expression of NRBP2 gene in endometrial carcinoma, and NRBP2 may be the target of the effect of folic acid on endometrial carcinoma.

目的 应用iTRAQ联合质谱技术筛选COPD大鼠肺组织差异表达蛋白。方法 20只雄性SD大鼠(200~220 g),随机分为对照组和模型组,每组10只,采用熏烟法建立COPD大鼠模型。观察大鼠肺组织病理学改变,测定肺功能,BALF白细胞数,肺组织总蛋白iTRAQ标记后质谱鉴定,用生物信息学方法分析蛋白表达变化。结果 与对照组相比,模型组大鼠支气管黏膜下肌层增厚,肺内可见大量炎性细胞浸润,肺功能降低,BALF白细胞数升高(均P<0.05)。质谱鉴定出4 916种蛋白,筛选出468个差异表达蛋白,其中285个表达上调,183个表达下调。筛选了上皮细胞粘着连接蛋白、fMLP、整合素等与COPD相关蛋白。结论 基于iTRAQ技术的蛋白质组学方法筛选出COPD大鼠差异表达蛋白,为进一步研究COPD的发生机制奠定了基础。

Objective iTRAQ and mass spectrometry were used to screen the differentially expressed proteins in the lung of COPD rats. Methods 20 male SD rats (200-220g)were randomly divided into control group and treatment group, with 10 rats in each group. COPD rat model was established by smoking. The lung function, the number of BALF leukocytes, the total protein iTRAQ in lung tissue were measured and identified by mass spectrometry. The differentially expressed proteins were identified by bioinformatic analysis. Results Compared with the control group, the submucous layer of bronchus in the model group was thickened, a large number of inflammatory cells were seen in the lung, the lung function was reduced, and the number of BALF leukocytes was increased. 4 916 proteins were identified by mass spectrometry, 468 differentially expressed proteins were screened, 285 of which were up-regulated and 183 down regulated. Among them, the important COPD related proteins were epithelial adhesion connexin, fMLP and integrins. Conclusion iTRAQ technology screened out the differentially expressed proteins of COPD rats, which laid the foundation for the further study of COPD mechanism

目的 探讨Meis1在人子宫内膜细胞中的表达及其受雌、孕激素调控的规律。方法 通过免疫细胞化学和western blot的方法检测雌、孕激素对体外培养的在子宫内膜基质细胞(ESC)及Ishikawa细胞中Meis1的表达及调控。结果 Meis1在ESC和Ishikawa细胞均有表达,且均表达于细胞核中;在ESC中,E2、P4和 E2＋P4三组中Meis1平均蛋白表达水平均高于对照组(P<0.05)。Meis1在E2、P4和 E2＋P4组之间的表达水平亦差异有统计学意义(P<0.05),表达强度E2＋P4组>P4组>E2组;在Ishikawa细胞中,E2、P4和 E2＋P4使Meis1表达增强,表达强度P4组>E2＋P4组>E2组,但与对照组比较无差异(P>0.05),E2、P4和 E2＋P4各组间亦无差异(P>0.05)。结论 转录因子Meis1在ESC和Ishikawa细胞中受到雌、孕激素的调控,可能在子宫内膜容受性分子网络的构建中发挥着重要的作用。

Objective To investegate the expression and regulation discipline of Meis1 in human ESC and Ishikawa cells in vitro by estrogen and progesterone stimuli. Methods Immunocytochemistry and western blot were used to detect the expression and regulation discipline of Meis1 in human normal endometrial cells. Results Meis1 expressed both in endometrial stromal cells (ESC) and in ishikawa cells, and both situ in nucleus. In ESC, the expression of Meis1 was up-regulated by E₂/P₄ and E₂＋P₄, and the up-regulated effect may be superposition by E₂＋P₄, the differences between the groups were statistically difference(P<0.05). In Ishikawa cells, western blot showed that the expression of Meis1 was up-regulated by E₂/P₄ and E₂＋P₄. The differences weren't statistically significant when compared with the control group or between themselves(P>0.05). Conclusion The expression of transcription factor Meis1 is regularly regulated by estrogen and progesterone, which may be a key role during the formation of endometrial receptivity molecular network.

目的 探讨前列地尔联合依帕司他对糖尿病足患者创面肉芽组织肿瘤坏死因子(tumor necrosis factor-α,TNF-α)、白介素-6 (interleukin-6,IL-6)及血管内皮生长因子(vascular endothelial growth factor,VEGF)表达的影响。方法 将90例糖尿病足患者随机分为研究组和对照组,每组45例,对照组予常规治疗,研究组在常规治疗基础上给予前列地尔+依帕司他联合治疗。监测两组患者创面愈合率,患肢足背血流动力学及腓总神经传导速度,创面肉芽组织TNF-α、IL-6、VEGF含量及基因表达变化。结果 治疗后第2、4周研究组较对照组创面愈合率升高,差异有统计学意义(P<0.05);治疗后两组患者足背动脉血流动力学及腓总神经传导速度均有改善,而研究组疗效更明显(P<0.05);治疗后研究组患者创面肉芽组织TNF-α、IL-6含量及基因表达较对照组降低,VEGF含量及基因表达则升高(P<0.05)。结论 前列地尔联合依帕司他联合治疗可改善糖尿病足患者足背动脉血流动力学,促进受损神经功能恢复;降低糖尿病足患者创面肉芽组织TNF-α、IL-6表达,减轻免疫损伤;增加VEGF基因表达,促进血管生成,加速创伤愈合。

Objective To investigate the effects of alprostadil combined with epalrestat on the expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) in granulation tissue in patients with diabetic foot patients.Methods Totally 90 cases with diabetic foot were randomly divided into study group (45 cases)and control group(45 cases). The control group received conventional treatment for 4 weeks and the study group additionally received combination of alprostadil and epalatone for 4 weeks. The curative rate of wound healing, limb dorsal hemodynamics, peroneal nerve conduction velocity and the levels of TNF-α, IL-6 and VEGF in the granulation tissue of the wound were monitored in the two groups.Results The wound healing rate of the study group was significantly higher than that of the control group at the 2nd and 4th week after treatment (P<0.05). After treatment, the hemodynamics and peroneal nerve conduction velocity were improved (P<0.05). The contents and expressions of TNF-α and IL-6 in the granulation tissue of the treatment group were significantly lower than that of the control group, and the content of VEGF and gene expression were significantly increased in the study group (P<0.05).Conclusion The combination therapy of alprostadil and epalrestat may improve the hemodynamics of dorsalis pedis artery in patients with diabetic foot and promote the recovery of damaged nerve function.Also it may reduce the expression of TNF-α and IL-6 in the granulation tissue of diabetic patients and reduce the immune injury. It promotes angiogenesis and accelerates wound healing by increasing VEGF gene expression.

目的 探讨p27^Kip1蛋白的表达与梅州地区客家人食管鳞癌的关系。方法 收集我院病理科2003年1月—2008年12月的61例经手术切除的中国梅州地区客家人食管鳞癌组织及32例食管良性肿瘤组织为研究对象并包埋成蜡块。采用免疫组织化学技术检测组织中p27^Kip1蛋白的表达,结合患者的临床病理资料和随访资料,进行回顾性分析,并作出评价。结果 P27^Kip1蛋白在食管鳞癌细胞核和/或细胞浆都有表达。胞核表达阳性率为27.87%,低于食管良性肿瘤组织胞核表达（50.0%）（P<0.05）;而胞浆表达阳性率为49.18%,高于食管良性肿瘤组织胞浆表达（12.5%）（P<0.05）。无淋巴结转移的食管鳞癌p27^Kip1蛋白胞核表达阳性率为42.41%,高于有淋巴结转移者的阳性率（10.7%）,差异有统计学意义（P<0.05）;而无淋巴结转移的食管鳞癌p27^Kip1蛋白胞浆表达阳性率为42.4%,比有淋巴结转移者（57.12%）的阳性率有降低的趋势（P>0.05）。Ⅱ期（ⅡA+ⅡB）和Ⅲ期p27Kip1蛋白胞核表达阳性率分别为42.41%和10.71%,差异有统计学意义（P<0.05）;p27^Kip1蛋白胞浆表达阳性率有随着TNM分期的增高而增高的趋势（P>0.05）。结论 P27^Kip1蛋白作为肿瘤抑制因子,其在食管鳞癌的胞核和胞浆表达阳性呈相反趋势,随食管鳞癌TNM分期越高,胞核阳性率越低,胞浆阳性率越高,其可作为食管肿瘤恶性程度及进展的预测指标。

目的 研究鳞癌抗原(SCC-Ag)的表达对宫颈癌手术治疗后临床转归的预测价值。方法 选取我院2014年4月—2015年9月实施宫颈癌手术的患者52例,分别于手术前后分析所有患者的鳞癌抗原表达,对患者进行1年的随访,以发生癌细胞转移、复发或死亡为研究终点,比较预后良好的患者与预后差的患者鳞癌抗原的表达的不同。结果 Ia、Ib1、Ib2、IIa、IIb期宫颈癌患者术后SCC-Ag水平较手术前均显著降低(P＜0.05);纳入本次研究的患者宫颈癌术后复发或转移发生率为15.38%,转归良好的患者为84.62%,预后良好的患者术后SCC-Ag水平(0.91±0.27)ng/mL较发生复发或转移的患者(1.37±0.57)ng/ml显著较较低(P＜0.05)。结论 宫颈癌患者术后血清SCC-Ag水平与肿瘤的应答之间具有关联性,术后SCC-Ag水平高的患者复发与转移发生率显著高于SCC-Ag水平低的患者,鳞癌抗原的表达对宫颈癌手术后患者的转归情况具有预测价值,临床应予以重视。

目的 利用大肠杆菌原核表达系统优化表达纯化肠道病毒71型(EV71)VP3结构蛋白,为后续单克隆抗体制备及检测试剂盒研发提供前期基础。方法 采用PCR方法扩增EV71病毒VP3基因,将其插入表达载体pET28a(+),构建pET28a-VP3重组质粒,转化大肠杆菌BL21(DE3)菌株,分别在25 ℃、37 ℃下经IPTG诱导表达,重组表达的蛋白产物经凝胶电泳初步分析,比较不同温度诱导表达的蛋白产物。结果 成功构建pET28a-VP3重组质粒,不同温度下诱导表达的蛋白产物在30.5 kDa左右位置均出现目的条带;37 ℃下诱导表达的蛋白超声破碎并离心后,目的蛋白基本位于沉淀中,而25 ℃诱导表达的蛋白产物有少量目的蛋白溶解于上清液中。结论 在25 ℃或37 ℃下均能利用大肠杆菌原核表达系统有效表达EV71病毒VP3蛋白;37 ℃诱导时蛋白可融性表达低,目的蛋白获取效率较高。

Objective To express VP3 capsid protein of enterovirus 71 by using Escherichia coli prokaryotic expression system. Methods VP3 gene was amplified by PCR before inserted into pET28a(+) plasmid. Then the plasmid pET28a-VP3 was transformed and expressed in the Escherichia coli BL21 strain at 25 ℃ or 37 ℃. Finally the protein was analyzed by SDS-PAGE gel electrophoresis. Results The pET28a-VP3 plasmid was successfully constructed, and the EV71 VP3 protein was expressed. Supernatant of the production after ultrasonication and centrifugation got a little VP3 protein. Conclusion The EV71 VP3 protein was expressed. Expression at 25 ℃ may lead to the dissolution of the recombinant protein.