目的:探讨APS复发性流产患者血小板活化状态及其对内皮细胞活化的作用.方法:流式细胞术分别检测23例APS患者和31例正常早孕人群血小板CD62P和CD40L的表达水平.洗涤血小板悬液与人脐静脉内皮细胞(HUVECs)共培养6 h,ELISA法分析HUVECs培养上清IL-8的水平,荧光定量PCR法检测HUVECs细胞ICAM-1的mRNA水平.结果:APS组血小板CD62P和CD40L阳性表达百分率为(10.27±3.76)％和(13.26±5.21)％,较正常早孕组显著升高(P<0.05);APS组血小板悬液能显著促进HUVEC细胞ICAM-1基因表达水平和分泌IL-8水平.特异性CD40L单抗可以显著抑制HUVEC细胞表达ICAM-1水平和分泌IL-8水平.结论:APS流产患者外周血血小板活化程度增加,血小板-内皮细胞反应参与APS导致高凝、炎症和流产的病理过程.

Objective To analyze the expression of microRNA-126 (miR-126) and its relation with clinicopathological characteristics of colon carcinoma.Methods Resection specimens of 70 cases of colon carcinoma and 32 cases of adenomatous colon polyps were collected from January 2015 to September 2018 in Jilin Province People's Hospital.Expression of miR-126 in adenoma,carcinoma and adjacent tissue were tested by real-time fluorescence quantitative polymerase chain reaction.Patients with colon carcinoma were divided into miR-126 high expression group and low expression group according to the mean expression value.Relation between miR-126 expression and clinicopathological parameters of colon carcinoma was analyzed.Results Expression of miR-126 in colon adenoma(0.75 ± 0.09) was significantly lower than that in paracancerous tissue(1.00);the expression in high,medium and poor differentiated colon carcinoma [(0.47 ± 0.04),(0.36 ± 0.12),(0.29 ±0.07)] was significantly lower than that in adenoma(P ＜0.05 or P ＜0.01).The mean value of miR-126 expression was (0.37 ± 0.03) and the patients were divided into high expression group (19 cases) and low expression group (51 cases);tumor differentiation degree,TNM stage,lymphatic metastasis and distant metastasis showed significant differences between high expression group and low expression group (P ＜ 0.05 or P ＜ 0.01).Conclusion MiRNA-126 is low-expressed in colon carcinoma;it may play an important role in the occurrence and progression of colon carcinoma as an anti-oncogene.

Objective To investigate the expression and significance of AMHR2 in the endometrium during the im-plantation window period of the patients with IVF-ET.Methods The expression of AMHR2 in the endometrium of IVF-ET patients during the phase of corpus phase was detected by immunofluorescence staining.The endometrial stro-mal cells were isolated and cultured,and the cultured cells were treated by hCG for48h.The expression of AMHR2 was detected by method of PCR and Immunohistochemical analysis.Results The expression of AMHR2 were detected in endometrial implantation window,and the levels of AMHR2 gene and protein in endometrial cells were significantly in-creased after HCG treatment.Conclusion AMHR2 was detected in the endometrium during the implantation window of women,and level of AMHR2 were affected by HCG.AMHR2 might be a reference index to predict endometrial re-ceptivity.

目的 探讨顺铂(DDP)通过抑制受体相互作用蛋白(RIP)增强肺癌细胞 A549 对肿瘤坏死因子相关的凋亡诱导配体(TRAIL)敏感性的分子机制.方法 Western印迹检测顺铂、Nec-1作用A549细胞后RIP的蛋白表达.MTT法检测联合应用DDP+rhTRAIL和Nec-1+rhTRAIL作用后A549细胞的生长抑制率.Western印迹检测DDP+rhTRAIL和Nec-1+rhTRAIL对 A549 凋亡相关蛋白 caspase-8 的活化情况.流式细胞仪检测DDP+rhTRAIL和Nec-1 +rhTRAIL作用后对A549细胞凋亡的影响.结果 DDP和Nec-1能显著降低A549细胞中RIP的蛋白水平,DDP、Nec-1联合rhTRAIL可以实现caspase-8的活化,促进rhTRAIL对A549细胞的杀伤作用,凋亡率从(5.9 ±4.93)%提高到(60.5 ±4.93)%和(64.1±5.17)%(P<0.01).抑制率分别提高到(65.5±4.93)% 和(76.3±9.52)%(P<0.01).结论 抑制RIP蛋白活性能增加 A549 细胞对 rhTRAIL 诱导细胞凋亡的敏感性,有利于促进rhTRAIL蛋白在治疗NSCLC方面的应用.

目的:检测小细胞肺癌(SCLC)患者血清中白细胞介素9(IL-9)的水平和外周血中Th9的细胞比例,探讨Th9在SCLC疾病进展过程中的作用.方法:选择20例局限期SCLC患者(局限期SCLC组)、16例扩散期SCLC患者(扩散期SCLC组)和10名健康对照者(健康对照组).ELISA法检测各组研究对象血清中IL-9水平,流式细胞术检测各组研究对象外周血Th9细胞在CD4+T细胞中所占比例.结果:SCLC组患者外周血中Th9细胞比例高于健康对照组(t=3.731,P＜0.01),局限期SCLC患者外周血中Th9细胞比例低于扩散期SCLC组(t=2.825,P＜0.01).SCLC组患者血清IL-9水平高于健康对照组(t=4.001,P＜0.01),局限期SCLC组患者血清中IL-9水平低于扩散期SCLC组(t=3.089,P＜0.01);结论:Th9可能通过分泌IL-9直接促进SCLC的疾病进展,外周血中Th9细胞和IL-9可能成为评价小细胞肺癌的分子标志.

The current study aimed to investigate the distribution of memory and naïve T cell (TN) subsets in hepatitis B virus (HBV)‑infected patients at different immune stages and investigate the effect of interleukin 33 (IL‑33) on the regulation of the T‑cell subsets. The distributions of memory and naïve T cells were detected by flow cytometry. ELISA was conducted to assess the levels of IL‑4, IL‑5, IL‑10, IL‑12, interferon γ and tumor necrosis factor α. The expression levels of IL‑33 and HBV x protein (HBx) were measured by reverse transcription‑quantitative polymerase chain reaction and western blot analysis, respectively. By detecting TNs, central memory T cells (TCM) and effector memory T cells (TEM), it was identified that the proportions of TCM and TEM in CD4+ T cells were increased in patients with HBV. The trend observed for levels of CD8+ TCM and TEM was similar to that of CD4+ T cells in the immune tolerance and immune activation groups, however CD8+ TCM and TEM were significantly reduced in patients who underwent treatment. The CD8+ TEM cells appeared to be more sensitive to HBV activation and drug therapy. In addition, IL‑33 stimulation was observed to induce imbalances of CD8+ TN and CD8+ TEM, and while the imbalances were directly regulated by HBx, IL‑33 was not a key factor for the expression of HBx. CD8+ TEM cells may be a sensitive marker to assess the immune state of patients with HBV and the effect of clinical therapy. Treatment targeting IL‑33 may be a potential method to reverse HBV‑induced immune tolerance.

Misregulation of vascular endothelial growth factor A (VEGF‑A) has been implicated in numerous types of ovarian disease, such as polycystic ovarian syndrome, ovarian hyperstimulation syndrome, endometriosis and ovarian cancer. VEGF regulates blood vessel permeability and angiogenesis. In our previous study, VEGF‑regulated gene expression was profiled in the uterus of a transgenic mouse model with repressed VEGF expression, which indicated that VEGF is an important regulator in controlling gene expression in the uterus. The anti‑Müllerian hormone (AMH) is expressed by ovarian granulosa cells (GCs) and acts through its type 2 receptor, AMH receptor 2 (AMHR2). Serum AMH levels are used to predict ovarian reserves and the small antral follicles contribute markedly to the serum AMH level. AMH recruits primordial follicles and inhibits excessive follicular development by follicular stimulating hormone (FSH). However, AMH may be influenced by suppression of gonadotrophin secretion and VEGF inhibition. In the current study, human primary ovarian GCs were isolated from ovarian follicle fluid of in vitro fertilization/intracytoplasmic sperm injection cycles (IVF/ICSI). It was identified that the FSH receptor was consistently expressed in the isolated cells. VEGF‑A treatment stimulated AMHR2 overexpression at the gene and protein levels. In addition, VEGF induced AMHR2 expression on the surface of the isolated GCs from mature follicles. The VEGF treatment was also performed in an ovarian granulosa‑like cell line, KGN. AMH and AMHR2 are co‑expressed in normal GCs; however, as a result of VEGF misregulation, AMHR2 overexpression increases AMH binding, which may attenuate follicular or oocyte maturation. However, the associated function and underlying mechanism requires further investigation.

Objective To study the expression levels of CXCL12 and CXCR4 in patients with small cell lung cancer (SCLC),and to investigate the correlation between CXCL1 2/CXCR4 and the development of human SCLC.Methods The serum concentration of CXCL1 2 was detected by ELISA,and the expression of CXCL1 2/CXCR4 in tissues were detected by real-time PCR and Westernblot respectively in 30 SCLC patients in limited stage,32 SCLC patients in ex-tensive stage and 10 benign lung cancers (control).Then we analyzed the correlation between CXCL12/CXCR4 and clinical pathological characteristics of SCLC.Results The concentration of serum CXCL1 2 was higher in patients with limited stage than that in patients with extensive stage,both of them were showed a higher level than that in HC.The expressions of CXCL12/CXCR4 were also higher in SCLC patients than that in HC as well.The serum level of CXCL12 was associated with tumor size and lymphatic metastasis (P<0.01)and had no relations with sex or age of patients (P>0.05).Conclusion The expression of CXCL12/CXCR4 may participate in regulating the development of SCLC.

丙型肝炎是由丙型肝炎病毒（hepatitis C virus,HCV）感染机体后所引起的传染性疾病。大多数的丙型肝炎患者感染后转为慢性,病毒在体内持续存在,慢性丙型肝炎是引起肝硬化和肝癌的重要原因之一。CD3+CD4-CD8-T细胞（双阴性T细胞,DNT）是一种新发现的调节性T细胞亚群,研究发现其在移植排斥、自身免疫性疾病、肿瘤免疫、感染性疾病、过敏性疾病以及移植物抗宿主病等疾病中

目的 探讨中药组方对健康供者外周血造血干细胞的影响.方法 34例志愿健康供者应用中药组方,1次/d,1袋/次,服用1个月.采用血细胞计数仪检测外周血中单个核细胞数,流式细胞仪检测CD34+细胞数的变化;通过RT-PCR法观察服药前后外周血造血细胞生长因子白细胞介素(IL)-3、粒细胞集落刺激因子(GM-CSF)的mRNA半定量表达情况.结果 与服药前相比,外周血单核细胞、CD34+细胞数明显增高(P＜0.01),服药后IL-3、GM-CSF mRNA的半定量表达水平与服药前相比差异显著(P＜0.01).结论 中药组方可能通过增强造血生长因子IL-3、GM-CSF mRNA表达水平,刺激造血细胞分化成熟,有效动员外周血造血干细胞,供者无明显的不良反应.

Multiple drug resistance (MDR) is considered a major challenge in the clinical treatment of non-small cell lung cancer (NSCLC). Both nitric oxide synthase (iNOS) and Wnt signaling pathway participate in the regulation of drug resistance, but the interaction between them remains unclear. In the present study, we detected the activation of Wnt/β-catenin signaling in iNOS-induced drug-resistant lung cancer cells, and compared the effect of canonical and noncanonical Wnt pathway on the level of iNOS. Moreover, we investigated the expression of Wnt/β-catenin signaling downstream factors and its main inhibitors. The results indicated iNOS-induced drug resistance was possibly mediated by glutathione S-transferase-π (GST-π) and topoisomerase IIα (TOPO IIα), but not P-glycoprotein (P-gp), and this process was closely associated with the activation of canonical Wnt/β-catenin signaling, but less with noncanonical pathways. The mechanism of iNOS promoting Wnt/β-catenin pathway was mainly dependent on the inverse regulation of Dickkopf-1 (DKK-1) and secreted frizzled-related protein-1 (SFRP-1). Clarifying the relationship between iNOS and Wnt signaling may provide insight into a better understanding of the mechanism of drug resistance development in NSCLC.

目的：构建针对胰岛素生长因子结合蛋白-2（IGFBP-2）的小干扰RNA（siRNA）表达载体，研究载体介导的RNAi技术对人 U251脑胶质瘤细胞株中IGFBP-2表达的抑制作用。方法根据GenBank提供的IGFBP-2基因（NM-000597）核苷酸序列及siRNA的设计原则，设计合成4对带有BamHⅠ、HindⅢ酶切位点的发夹结构寡核苷酸序列，筛选得到576～594 nt、908～926 nt、938～956 nt 3个19 bp片段靶序列，同时随机组合出一条无关序列，应用限制性内切酶酶切和测序技术鉴定重组克隆。用脂质体介导转染人U251胶质瘤细胞株，采用RT-PCR和Western印迹方法检测细胞IGFBP-2 mRNA和蛋白表达。结果酶切筛选得到4个目的片段的阳性克隆，测序证实重组表达载体中插入序列与设计一致。 RT-PCR和Western印迹检测显示构建的siRNA表达载体可显著降低U251细胞中IGFBP-2 mRNA和蛋白表达水平。结论成功构建载体介导的IGFBP-2靶向 RNA干扰重组体，并能有效抑制U251细胞中IGFBP-2基因的表达。

目的 分析慢性丙型肝炎患者外周血树突细胞含量与HCV-RNA病毒载量的相关性,探讨HCV感染慢性化的免疫学机制.方法 流式细胞仪检测40例慢性丙型肝炎患者(CHC)外周血中髓样树突细胞(mDC)及浆样树突细胞(pDC)的含量,22例健康人作对照比较.实时荧光定量PCR法检测外周血HCV-RNA载量.结果 HCV-RNA1+ E003 IU/ml患者及HCV-RNA≥1+E003 IU/ml患者外周血mDC表达(0.25％±0.09％,0.14％±0.07％)均低于健康对照组(0.38％±0.10％),差异有统计学意义(t=4.340 9,9.222,P值均＜0.05);HCV-RNA1+ E003 IU/ml患者及HCV-RNA≥1+E003 IU/ml患者外周血pDC(0.23％±0.10％,0.13％±0.08％)均低于健康对照组(0.33％±0.09％),差异有统计学意义(t=6.552 4,11.04,P值均＜0.05);HCV RNA≥1+E003 IU/ml患者外周血mDC,pDC的表达量明显低于HCVRNA1+E003 IU/ml患者的表达,差异有统计学意义(t=4.349 6,2.671 8,P值均＜0.05).结论 CHC患者外周血DC含量与HCV-RNA病毒载量呈负相关,CHC患者免疫功能低下.

Objective To investigate the effect of silencing IGFBP-2 with RNA interference on the proliferation and apoptosis of the glioma U251 cells,and to provide a viable method for the gene therapy of glioma.Methods RNAi ex-pression vector for IFGBP-2 was constructed and transfected to U251 cell with lipofectamin.RT-PCR method was used to detect the variation of IGFBP-2 mRNA level.MTT method was used to measure the variation in the proliferation of U251 cells.Flow cytometer was used to monitor cell apoptosis and changes.Results The construction of RNAi expres-sion vector inhibited the expression of IGFBP-2 mRNA;Silencing IGFBP-2 gene with RNA interference could inhibit the proliferation of U251 cells and induce the apoptosis of U251 cells (P<0 .0 1 ).Conclusion RNAi technique can ef-fectively inhibit the expression of IGFBP-2 gene(P<0.01),hence inhibit the proliferation of U251 cells and induce the apoptosis of U251 cells.

Objective: To observe the expression of COX-2 and Survivin in hepatocellular carcinoma SMMC-7721 cells and in- vestigate the mechanism of NS-398 on inhibiting tumor cell growth. Methods: The hepatoma cell line SMMC-7721 was cultured and treated with different concentrations of NS-398. The expressions of COX-2 and Survivin mRNA were detected by RT-PCR method and the expressions of COX-2 and Survivin protein were detected by flow cytometry / financal capacity model( FCM) and Western blot method. Results: There was a high expression of COX-2 and Survivin gene /protein in SMMC-7721 cells. NS-398 inhibited the expression of COX-2 and Survivin expression in SMMC-7721 cells in the manner of dose and time dependent. Conclusion: NS-398 could inhibit proliferation and induce apoptosis of SMMC-7721 by inhibiting the expression of COX-2 and Survivin.

非编码RNAs（non-coding RNAs,ncRNAs）,包括以miRNA、siRNA和piRNA等为代表的短小RNA和长链非编码RNAs（long non-coding RNAs,lncRNAs）。近20年来,ncRNAs的发现和功能研究,大大改变了人们对RNA分子的认识。ncRNAs不仅承担了遗传信息中间载体的辅助性角色,而且在发育和基因表达调控中发挥着复杂而精确的功能[1]。目前,关于ncRNAs在疾病发生、发展和防治中的作用已有大量文献报导,研究主要集中于短ncRNAs在肿瘤、自身免疫病等疾病中的表达和调控作用[2]。虽然关于lncRNAs与疾病之间关联的研究还处于起步阶段,但其调控作用已经开始引起人们广泛的关注。

Objective To construct prokaryotic plasmid of hcTnI and obtain recombinant hcTnI by prokaryotic expression and purification.Methods The encoding sequence for mature peptide of human cardiac troponin I was amplified with RT-PCR and inserted into pQE30 vector to establish the prokaryotic expressing system.The recombinant expression plasmid pQE30-hcTnI was constructed and expressed in E.coli and hcTnI was purified by Ni2+-NTA affinity chromatography.Western Blot was used to determine the recombinant hcTnI.Results The sequence of amplified hcTnI gene was consistent with that reported in GenBank(M64247).pQE30-hcTnI induced by IPTG in E.coli M15was about 29 kD,and a special band was identified by Western blotting after Ni2+-NTA affinity chromatography.Conclusion Human cardiac troponin I gene was successfully cloned and expressed in E.coli.Highly purified hcTnI was gained by Ni2+-NTA affinity chromatography,which improved the research on anti-cTnI monoclonal antibody and establishment of quick clinical detection.

Objective To obtain recombinant enzyme donor (ED) and enzyme acceptor (EA) fragments of β-galactosidase in E.coli and establish new cloned enzyme donor immunoassays (CEDIA) for clinical use.Methods The encoding sequences of ED and EA fragments were amplified with pSV-β-galactosidase control vector as template and inserted into pGEM-Teasy vector,the target gene was chosen by double digestion,PCR and sequencing,then ED and EA fragments were inserted into the expression vector pET20b+.The competent cells of host strain of BL21 were transformed by the recombinant plasmid.The expression of the target protein was induced with IPTG and purified by Ni2+-NTA agarose column.The β-galactosidae with activity is formed.Results The cloned fragments of ED and EA were 100% consistent with that of pSV-β-galactosidase control vector.The expression vector pET20b-ED and pET20b-EA were constructed and expressed.The target protein was purified by Ni2+-NTA agarose column.The expressed fusion-protein ED fragment was 14 000 and EA fragment was 116 000 in SDS-PAGE as expected.Conclusion ED and EA proteins are prepared successfully and β-galactosidase with activity is formed.

Objective To construct prokaryotic expression plasmid of human B-type Natriurtic Peptide (BNP),then to obtain recombinant BNP.Methods The encoding sequence for human BNP was amplified with RT-PCR and inserted into pGEM-Teasy vector,choose the target gene by double digestion,PCR and sequencing,then to establish the prokaryotic expressing system then insert BNP fragments into the expression vector pGEX-6P-1.The competent cells of host strain of BL21 were transformed by the recombinant plasmid.Expression of the target protein was induced with IPTG and purified by Glutathione Sepharose 4B agarose column.Results The cloned fragment of BNP was 100% consistent with that in genbank (NM002521).The Expressed fusion-protein was 29 kD in SDS-PAGE as expected,was recognized by a commercial McAb.After preliminary purification through Ni 2+-NTA agarose affinity chronmatography.Conclusion The recombinant BNP is highly obtained in prokaryotic expressing system.

Objective: To investigate the expression of glycosyl-phosphatidyl inositol (GPI) anchored protein on peripheral blood cells from patients with paroxysmal nocturnal hemoglobinuria (PNH) and provide a basis for diagnosis and differential diagnosis of PNH. Methods: The percentages of CD55, CD59, CD16 and CD14 expression on the peripheral blood cells from 32 patients with PNH were determined by flow cytometry. Results: The expression levels of CD55 and CD59 were decreased significantly on the erythrocytes and granulocytes from patients with PNH compared with control group (P < 0.001), the expression levels of CD55 and CD59 on granulocytes were lower than those on erythrocytes (P < 0.05). The expression levels of CD55 and CD59 on the thrombocytes of PNH patients were lower than those in control group (P < 0.001). The expression levels of CD55 and CD59 on the lymphocytes from 27 PNH patients were lower than those on granulocytes and erythrocytes, the deficiency rate was 30%. The percentage of CD16 expression varied greatly from 34% to 83% on the granulocytes, there was significant difference compared with control group (P < 0.001). The deficiency of CD14 clones was detected on the monocytes from PNH patients. Conclusion: The diagnosis reliability of patients with PNH is increased by the assay of CD55 and CD59 on all kinds of cells of peripheral blood. The detection of CD16 and CD14 is also an optimal and helpful index to diagnose PNH.