目的 利用分析各种浓度环氧化酶-2(COX-2)特异度抑制剂塞来昔布对食管癌EC109细胞系的作用,进而对COX-2蛋白表达的影响及对细胞凋亡能力的作用,进一步探讨塞来昔布对食管癌细胞凋亡的作用及机制。方法 使用0 μmol/L、20 μmol/L、60 μmol/L、100 μmol/L四个浓度的塞来昔布处理EC109细胞24 h,酶联免疫吸附剂测定(ELISA)法测定COX-2蛋白表达;流式细胞仪测定EC109细胞凋亡情况。结果 与0 μmol/L塞来昔布组比较,20 μmol/L、60 μmol/L、100 μmol/L塞来昔布组EC109细胞内COX-2蛋白表达不断降低(1.581±0.116;1.226±0.089,0.846±0.076,0.521±0.082)(P<0.05);而细胞凋亡率逐步上升(1.700±0.557,13.400±1.735,18.766±1.301,28.100±1.997)(P<0.05)药物浓度依赖于梯度。结论 塞来昔布是一种COX-2抑制剂,可能以浓度梯度的形式抑制COX-2蛋白的表达,从而促进EC109细胞的凋亡。

Objective The effects of celecoxib, a specific COX-2 inhibitor at various concentrations, on EC109 cell line of esophageal cancer were analyzed, and the effect and mechanism of celecoxib on apoptosis of esophageal carcinoma cells were further studied. Methods EC109 cells were treated with celecoxib at concentrations of 0 μmol/L, 20 μmol/L, 60 μmol/L and 100 μmol/L for 24 h. The protein of COX-2 in EC109 cells was determined by enzyme-linked immunosorbent assay (ELISA). Assay of EC109 cell apoptosis were determined by flow cytometry. Results Compared with the 0μmol/L celecoxib group, the expression of COX-2 protein in EC109 cells of 20μmol/L, 60μmol/L, 100μmol/L celecoxib group gradually decreased(1.581±0.116; 1.226±0.089, 0.846± 0.076, 0.521±0.082) (P<0.05); and the apoptotic rate gradually increased (1.700±0.557; 13.400±1.735, 18.766±1.301, 28.100±1.997) (P<0.05) in a drug concentration gradient-dependent manner. Conclusion The COX-2 inhibitor celecoxib may inhibit the expression of COX-2 protein in a concentration gradient and promote the apoptosis of esophageal cancer EC109 cells.

目的 检测外周血循环肿瘤细胞(circulating tumor cell, CTC)在原发性肝癌患者中的表达情况,并探讨CTC动态变化及其相对于甲胎蛋白(Alpha fetoprotein AFP)对原发性癌患者术后复发转移的预测作用。方法 收集原发性肝癌患者134例,肝脏良性病变患者72例,检测外周血 CTC 数目,同时检测AFP的表达水平,分析 CTC 与 AFP 的相关性。然后在134名原发性肝癌患者中筛选出成功行肝癌根治术的患者,共86例,检测这86名患者术前、术后外周血CTC和AFP,分析CTC和AFP对原发性肝癌术后复发转移的评估价值。结果 原发性肝癌患者外周血CTC阳性率高于肝脏良性病变患者,差异有统计学意义(P＜0.05);原发性肝癌患者CTC水平与AFP水平、淋巴结转移、肿瘤结节多少有关,与年龄、性别、肿瘤直径、分化程度、肝硬化有无、TNM分期无关;原发性肝癌患者CTC和AFP生存分析显示,原发性肝癌根治术后早期复发转移与CTC和AFP密切相关;CTC较阳性对术后复发转移具有更好的诊断价值,二者联合对复发转移预测价值最高。结论 CTC可以做为一个比传统肿瘤标志物更好的对原发性肝癌术后复发转移进行监测的指标,与肿瘤标志物联合检测预测价值更高。

Objective To detect the expression of peripheral blood circulating tumor cells CTC in patients with primary liver cancer and to explore the dynamic changes of CTC and its predictive effect on postoperative recurrence and metastasis of primary cancer. Methods The number of CTC in peripheral blood was measured in 134 patients with primary liver cancer and 72 patients with benign liver disease, the expression of AFP was detected, and the correlation between CTC and AFP was analyzed. Then 86 patients with primary liver cancer were selected from 134 patients with primary liver cancer who underwent radical hepatectomy. The values of CTC and AFP in evaluating recurrence and metastasis of primary liver cancer before and after operation were analyzed by CTC and AFP, in peripheral blood of these 86 patients. Results The positive rates of CTC in peripheral blood of patients with primary liver cancer were higher than that of patients with benign liver disease(P< 0.05). The levels of CTC in patients with primary liver cancer were related to AFP level, lymph node metastasis and the number of tumor nodules, but not to age, sex, tumor diameter, differentiation degree, liver cirrhosis and TNM stage. The survival analysis of CTC and AFP in patients with primary liver cancer showed that the early recurrence and metastasis of primary liver cancer after radical resection were closely related to the positive rate of CTC and AFP, and the positive rate of CTC was more effective than that of AFP positive in the diagnosis of recurrence and metastasis after operation, and the combination of the two had the highest predictive value for recurrence and metastasis. Conclusion CTC may be used as a better index to monitor postoperative recurrence and metastasis of primary liver cancer than traditional tumor markers. The combined detection prediction value of tumor markers is higher.

目的 体外细胞实验检测吴茱萸碱对骨肉瘤HOS细胞株细胞周期及体外增殖能力的影响。方法 通过利用浓度为0、3、6、12 μmol/L吴茱萸碱处理骨肉瘤HOS细胞48 h后,Hoechst-33258荧光染色观察不同浓度吴茱萸碱处理后HOS细胞核的形态学变化。利用流式细胞术检测3 μmol/L的吴茱萸碱处理后骨肉瘤HOS细胞的细胞周期分布变化。结果 3、6、12 μmol/L的骨肉瘤吴茱萸碱处理细胞,细胞呈现凋亡核碎裂等典型变化,而且随药物剂量增加而更趋明显。呈剂量依赖性抑制其体外增殖能力。3 μmol/L吴茱萸碱处理骨肉瘤HOS细胞0、24、48、72 h,各组细胞周期变化如下:G0/G1期:对照组(51.12±2.13)%、24 h(19.17±1.02)%、48 h(16.94±1.67)%、72 h(11.05±1.25)%;S期:对照组(32.92±0.73)%、24 h(31.00±1.42)%、48 h(32.38±3.03)%、72 h(29.18±2.87)%;G2/M期:对照组(16.01±2.26)%、24 h(49.82±0.62)%、48 h(50.6767±2.80)%、72 h(59.56±1.97)%。结论 吴茱萸碱可诱导人骨肉瘤HOS细胞发生G2/M期阻滞,而S期变化不明显。说明吴茱萸碱可以抑制骨肉瘤细胞的增殖能力,并阻滞细胞周期于G2/M期。

Objective Using transcriptome sequencing and in vitro cell assay to detect the effect of evodiamine on cell cycle and proliferation in osteosarcoma HOS cell line. Methods HOS cells were treated with evodiamine at 0, 3, 6, and 12 μmol/L for 48 hours, Hoechst-33258 fluorescence staining was used to observe the morphological changes of HOS nuclei after treatment with different concentrations of evodiamine.The cell cycle distribution of HOS cells treated with 3 μmol/L evodiamine was detected by flow cytometry. Results 3,6,12 μmol/L osteosarcoma treated with evodiamine, the cells showed typical changes such as apoptotic nuclear fragmentation, and it became more obvious with the increase of drug dosage. Inhibition of proliferation in vitro in a dose-dependent manner.HOS cells were treated with 3 μmol/L evodiamine for 0, 24, 48, 72 h. The cell cycle changes of each group were as follows: G0/G1 phase: control group(51.12±2.13)%, 24 h(19.17±1.02)%, 48 h(16.94±1.67) %, 72 h(11.05±1.25)%;S phase: control group(32.92±0.73)%, 24 h(31.00±1.42)%, 48 h(32.38±3.03)%, 72 h(29.18±2.87)%;G2/M period: control group(16.01±2.26)%, 24 h(49.82±0.62)%, 48 h(50.6767±2.80)%, 72 h(59.56±1.97)%. Conclusion Analysis of the above results revealed that evodiamine can induce G2/M phase arrest in human osteosarcoma HOS cells, but the S phase changes are not obvious. It indicated that evodiamine would inhibit the proliferation of osteosarcoma cells and block the cell cycle in G2/M phase.

目的 研究过表达miR-29a-3p对IL-22诱导的HaCaT细胞增殖的影响。方法 将HaCaT细胞分为Cell组、IL-22组、IL-22+NC组和IL-22+ miR-29a-3p组,荧光定量PCR检测miR-29a-3p的表达水平,CCK8检测细胞的活力,流式细胞仪检测细胞凋亡及周期。结果 与0 μg/L组相比,25 μg/L组、50 μg/L组和100 μg/L组HaCaT细胞的增殖率在24 h、48 h和72 h均出现升高(F值分别为33.27、36.19、52.29,均P<0.000 1)。与0 μg/L组相比,miR-29a-3p在50 μg/L组和100 μg/L组HaCaT中的表达水平降低(F=129,P<0.000 1),分别降低83%和80%。与IL-22+NC组相比,IL-22+ miR-29a-3p组的增殖率在24 h、48 h和72 h均降低(P值分别为0.002 1、0.001 6、0.023 1),细胞总凋亡率增加(6.67±1.06 vs 30.55±1.86,P=0.000 1),G1期细胞比例增加(P=0.000 1),S期细胞比例降低(P=0.000 1)。结论 IL-22可降低HaCaT中miR-29a-3p的表达量,过表达miR-29a-3p通过促进凋亡和引起细胞G1期阻滞抑制IL-22诱发的HaCaT细胞过度增生。

Objective To investigate the effect of miR-29a-3p overexpression on IL-22-induced proliferation of HaCaT cells. Methods HaCaT cells were divided into four groups, Cell group, IL-22 group, IL-22 +NC group and IL-22+miR-29a-3p group. The expression level of miR-29a-3p was detected by fluorescence quantitative PCR. Cell viability was detected by CCK8. Apoptosis and cell cycle were detected by flow cytometry. Results Compared with the 0 g/L group, the proliferation rate of HaCaT cells in the 25 μg/L group, 50 μg/L group and 100 μg/L group was increased at 24 h, 48 h and 72 h (F value was 33.27, 36.19, 52.29,respectively, all P<0.000 1). Compared with the 0 μg/L group, miR-29a-3p expression level in HaCaT in 50 μg/L and 100 μg/L groups was decreased (F=129, P<0.000 1), with a decrease of 83% and 80%, respectively. Compared with the IL-22+NC group, proliferation rate of IL-22+miR-29a-3p group was decreased at 24 h, 48 h and 72 h (P value was 0.002 1, 0.001 6, 0.023 1, respectively), total apoptosis rate was increased (6.67±1.06 vs 30.55±1.86, P=0.000 1), cell proportion in G1 phase was increased (P=0.000 1), and the cell proportion in S phase was decreased (P=0.000 1). Conclusion Il-22 can reduce miR-29a-3p expression level in HaCaT, and miR-29a-3p overexpression can inhibit the excessive proliferation induced by IL-22 in HaCaT cells by promoting apoptosis and inducing G1 phase arrest.

目的 探讨骨髓间充质干细胞源性微泡(BMSC-MV)修复大鼠早发性卵巢功能不全的自噬机制。方法 大鼠骨髓分离培养骨髓间充质干细胞;超速离心法从骨髓间充质干细胞培养液中分离微泡;腹腔注射顺铂溶液制备早发性卵巢功能不全(POI)模型,制备后3 d尾静脉取血ELISA检测血清雌二醇(E2)及卵泡刺激素(FSH);尾静脉注射BMSC-MV移植治疗POI大鼠模型,移植后28 d尾静脉取血ELISA检测E2、FSH及抗苗勒管激素(AMH),同时取卵巢组织检测自噬相关蛋白LC3及P62。结果 模型对照组及微泡移植组在模型制备后3 d的E2 含量低于正常对照组,FSH 含量高于正常对照组(P<0.001);微泡移植组在移植后28 d的E2、AMH含量高于模型对照组(P<0.001),FSH含量低于模型对照组(P<0.001);微泡移植组的LC3较模型对照组表达升高,而P62表达降低(P<0.001)。结论 BMSC-MV介导自噬修复大鼠早发性卵巢功能不全。

Objective To investigate the autophagy mechanism of bone marrow mesenchymal stem cell-derived microvesicle (BMSC-MV) in repairing premature ovarian dysfunction in rats. Methods The whole bone marrow adherence method was used to isolate,culture and identify BMSCs of SD rats. Microvesicles were isolated from bone marrow mesenchymal stem cell by ultracentrifugation. Premature ovarian insufficiency (POI) model was prepared by intraperitoneal injection of cisplatin solution,and serum estradiol (E2) and follicular stimulating hormone (FSH) were detected by ELISA from tail vein 3 days after preparation. Rat model of POI was treated with BMSC-MV transplantation by tail vein. Blood from tail vein was collected 28 days after transplantation to detect E2,FSH and AMH by ELISA. Meanwhile,ovarian tissues were collected to detect autophagy-related proteins LC3 and P62. Results The E2 content of the model control group and the microvesicle transplantation group was lower than that of the normal control group,and the FSH content was higher than that of the normal control group (P<0.001). The content of E2 and AMH in the microvesicle transplantation group at 28 days after transplantation was higher than that in the model control group (P<0.001),and the content of FSH was lower than that in the model control group (P<0.001). Compared with the model control group,LC3 expression in the microvesicle transplantation group was increased,while P62 expression was decreased (P<0.001). Conclusion BMSC -MV mediate autophagy to repair premature ovarian insufficiency in rats.

目的 探讨制何首乌、巴戟天及二者配伍,对氧化型低密度脂蛋白(ox-LDL)诱导的人脐静脉内皮细胞(HUVEC)损伤的影响,以示临床。方法 建立ox-LDL诱导的HUVEC损伤模型,分别用制何首乌、巴戟天、二者配伍的水煮物干预,检测HUVEC的细胞增殖、相对活率、细胞凋亡率、细胞周期、NFκB mRNA的表达。结果 ①制何首乌、巴戟天均能抑制ox-LDL诱导的HUVEC凋亡,二者配伍的抑制作用强于单味中药制何首乌。②制何首乌、巴戟天均能延长ox-LDL诱导的HUVEC的细胞周期(S+G2)%,制何首乌、巴戟天的延长作用相似,二者配伍的延长作用强于单味中药制何首乌、巴戟天。③制何首乌组、巴戟天组的NFκB mRNA的表达量下降,制何首乌组的抑制作用强于巴戟天组,二者配伍的抑制作用强于单味中药制何首乌、巴戟天。结果 制何首乌、巴戟天均能抑制ox-LDL诱导的HUVEC损伤,二者配伍的作用强于单味中药制何首乌、巴戟天。

Objective To investigate the effects of Polygonum multiflorum praeparata, Morinda officinalis and their compatibility on ox-LDL-induced injury of human umbilical vein endothelial cells(HUVEC). Methods We established an ox-LDL-induced HUVEC injury model, made intervention with Polygonum multiflorumpraeparata, Morinda officinalis and their compatibility, the HUVEC cell proliferation, relative viability, apoptosis, cell cycle, NFκB mRNA were detected. Results ①Both Polygonum multiflorumpraeparata, Morinda officinalis reduced the apoptosis rate of HUVEC, and their compatibility had a stronger effect on reducing the apoptosis rate of HUVEC than single Polygonum multiflorumpraeparata. ②Both Polygonum multiflorumpraeparata, Morinda officinalis increased the HUVEC cell cycle (S+G2)%, the extension between Polygonum multiflorumpraeparata and Morinda officinalis was similar, and their compatibility increased HUVEC cell cycle (S+G2)%, it was stronger than single Polygonum multiflorumpraeparata and single Morinda officinalis. ③Both Polygonum multiflorumpraeparata and Morinda officinalis down-regulated the expression of NFκB mRNA in HUVEC, their compatibility down-regulated HUVEC NFκB mRNA expression,it was stronger than Polygonum multiflorumpraeparata, Morinda officinalis. Conclusion Polygonum multiflorumpraeparata, Morinda officinalis and their compatibility can inhibit ox-LDL-induced HUVEC injury, and their compatibility inhibition is stronger than single Polygonum multiflorumpraeparata, and Morinda officinalis.

目的 筛选结直肠癌（CRC）差异性表达的长链非编码RNA（lncRNA）,并进行临床标本验证,研究其对结肠癌细胞HCT116功能的作用。方法 利用lncRNA PCR芯片对3对CRC组织和癌旁对照组织筛选差异性表达的lncRNA,确定候选研究lncRNA GAPLINC,RT-qPCR对21例临床样本进行验证其表达的差异性;同时构建GAPLINC表达质粒及其沉默体siRNA转染HCT116细胞,研究其对细胞凋亡、迁移及侵袭能力的影响。结果 lncRNA芯片实验结果提示CRC组织中存在大量的差异性表达的lncRNA,其中GAPLINC在CRC组织表达稳定增加,21例临床样本进一步验证了其在肿瘤组织中表达增加（P<0.05）;转染GAPLINC表达质粒后,HCT116细胞凋亡被抑制,同时其迁移及侵袭能力增强,转染siRNA抑制GAPLINC的表达后,则出现相反的结果。结论 利用lncRNA芯片可对CRC差异性表达lncRNA进行批量筛选,GAPLINC在CRC组织中表达稳定增加,具有促癌作用,在CRC发生发展中可能起着重要作用。

Objective To screen the differentiational expression of lncRNA in CRC tissue,confirm it in large simple of clinical specimens,and study its effects on human colorectal cells HCT116 cell line. Methods We screened the lncRNA which expressed differently in 3 CRC tissues and their pair-non carcinour tissues by lncRNA arrays;chose the over expressed lncRNA which played the potential role of oncogene for further researching,and tested the difference in 21 clinical specimens by RT-qPCR. We cultured the HCT166 cells,and then constructed expressed plasmids pcCDNA3.1-GAPLINC and synthesized GAPLINC siRNA,transfected the plasmids and siRNA into HCT116 cells;to study the changes of HCT116 cells behavior,the transwell assays were carried on;the changes of apoptosis of HCT116 cells were tested by flow cytometry. Results There existed many lncRNA which expressed differently between CRC tissues and normal control tissues by lncRNA arrays,there were 21 lncRNA down expressed,and 3 lncRNA up expressed;among these lncRNA,GAPLINC over expressed stably,and its high level of expression was approved in 21 clinical specimens by the test of RT-qPCR. We constructed the expressed plasmids pcCDNA3.1-GAPLINC and synthesizing GAPLINC siRNA successfully;after transfecting pcCDNA3.1-GAPLINC into HCT116 cells,the over expression of GAPLINC increased the migration and invasion of the HCT166 cells （P<0.05）,decreased the proportion of cell apoptosis （P <0.05）;by contraries,knocked down the expression of GAPLINC inhibited invasion and migration of HCT116 cells （P<0.05）,and promoted the apoptosis of the HCT116 cells （P <0.05）. Conclusion It could screen the different expression of lncRNA in large quantities by lncRNA arrays,and GAPLINC expressed highly and stably in CRC tissues. GAPLINC played a role of oncogene,which promoted the proliferation and invasion of CRC cells,and inhibited the apoptosis of CRC cells,which meant playing an important role in the carcinoma and development of CRC.

目的 探讨血清ApoA-1浓度与食管鳞状上皮细胞癌的相关性及其预后价值。方法 对我院2010年1月—2012年10月收治的食管鳞状上皮细胞癌患者的血清ApoA-1浓度进行回顾性调查分析,与我院体检中心2009年1月—2009年4月219例正常人群的血清ApoA-1浓度作为对照组进行统计学处理分析。结果 210例食管鳞状上皮细胞癌患者组和219例健康对照组在性别和年龄比较均无统计学差异(P>0.05)。血清ApoA-1 浓度在ESCC组的pTNM和临床分期均有统计学差异(P<0.05)。与ApoA-1水平高的患者相比,ApoA-1水平低的患者的总体生存率更低(P=0.002)。结论 血清ApoA-1 水平是食管鳞状上皮细胞癌食管癌的一个重要的预后因素。

目的 采用生物信息学方法预测低氧预处理人胎盘绒毛膜间充质干细胞环状RNAs相对应的miRNA及其靶基因,并分析靶基因所参与的生物学过程和信号通路。方法 用Arraystar公司的商业软件为环状RNAs预测其相对应的miRNAs,分别用targetScan7.1和mirdbV5数据库预测miRNAs的靶基因,并取两个预测结果的合集,应用在线网站http://www.geneontology.org和http://www.genome.ad.jp/kegg对靶基因进行功能富集分析和信号通路富集分析。结果 功能富集分析表明,circRNAs的靶基因主要涉及到细胞发育、细胞分化和细胞发育调控。东京基因和基因组百科全书信号通路富集分析表明肿瘤中转录失控和有丝分裂原激活蛋白激酶(MAPK)信号通路最有意义,而且分析发现MAPK信号通路为核心通路。本研究表明,低氧预处理使得间充质干细胞中部分circRNAs的表达量发生差异性变化。结论 低氧预处理人胎盘绒毛膜间充质干细胞环状RNAs同低氧预处理间充质干细胞的生物学特性变化密切有关,为了解低氧预处理影响间充质干细胞特性发生变化的分子机制提供新思路。

Objective To predict the miRNA and its target genes of circular RNAs in hypoxia- preconditioned human palcenta chorionic mesenchymal stem cells using bioinformatics, and analyze the biological process and signaling pathway. Methods Arraystar's commercial software was used to predict the corresponding miRNAs of circular RNAs. The target genes of miRNAs were predicted by targetScan7.1 and mirdbV5 databases respectively, and an intersection of two prediction results was obtained. The online databases http://www. geneontology.org and http://www.genome.ad.jp/kegg performed functional enrichment analysis and signal pathway enrichment analysis of target genes. Results Functional enrichment analysis indicated that the target genes of circRNAs mainly involved cell development, cell differentiation and cell development regulation. The signal enrichment analysis of the Tokyo Gene and Genome Encyclopedia indicates that transcriptional misregulation in cancer and mitogen-activated protein kinase (MAPK) signaling pathway are most meaningful, and the MAPK signaling pathway is found to be the core pathway. This study showed that hypoxic preconditioning caused significant changes in the expression of mesenchymal stem cell circRNAs. Conclusion The changes of circular RNAs in hypoxia-preconditioned human placental chorionic mesenchymal stem cell is closely related to the biological characteristics of hypoxia-preconditioned mesenchymal stem cells. This study provides a new idea for understanding the molecular mechanism of hypoxic preconditioning affecting the changes of biological characteristics in mesenchymal stem cells.

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.