运用网络药理学和分子对接方法探索黄连温胆汤治疗幽门螺杆菌相关性胃炎的分子机制.应用中药系统药理学数据库与分析平台对黄连温胆汤药物活性成分及药物作用靶点基因进行筛选.通过Genecards 数据库、DisGENET数据库查找幽门螺杆菌相关性胃炎疾病相关靶点,利用维恩在线绘图工具获得疾病与药物的共同靶点基因.利用Cytoscape 3.8.2 软件以及STRING数据库分别构建药物-化合物-靶点相互作用网络及蛋白质-蛋白质相互作用网络图.运用DAVID数据库进行基因本体功能和京都基因与基因组百科全书通路富集分析.选取度值排名前 3 的靶点蛋白和活性成分使用AutoDockTools 1.5.6 软件进行分子对接.共筛选得到槲皮素、川皮苷、柚皮素等 127 种黄连温胆汤药物活性成分,并获得Akt1、JUN、TNF-α、STAT3 等 101 个潜在靶点蛋白,涉及P53 信号通路、NOD样受体信号通路、TNF信号通路等 90条信号通路.分子对接结果显示关键靶点STAT3、TP53、Akt1 与活性成分槲皮素、川皮苷、柚皮素的亲和力较好,其中以Akt1 与柚皮素的结合能力最强.通过分析黄连温胆汤治疗幽门螺杆菌相关性胃炎的作用靶点和相关信号通路,能够为临床应用和后续实验研究提供理论基础.

Objective:To explore the clinical factors affecting the efficacy of PD-1 monoclonal antibody in the treatment of advanced non-small cell lung cancer (NSCLC) .Methods:The clinical data of 106 patients with advanced NSCLC treated with PD-1 monoclonal antibody in affiliated Cancer Hospital of Shandong First Medical University from 2019 to 2020 were reviewed retrospectively. We evaluated the survival and analyzed the clinical influencing factors.Results:The median progression-free survival (PFS) time of the 106 patients with advanced NSCLC was 10.0 months, and the median overall survival time was not reached. The median PFS of patients receiving immunotherapy alone ( n=29) was longer than that of patients receiving immunotherapy combined with other therapies ( n=77) (13.6months vs. 8.9 months, P=0.034) . The median PFS of patients with neutrophil/lymphocyte ratio (NLR) ≤5 ( n=87) , lactic dehydrogenase (LDH) ≤245 U/L ( n=71) , and prognostic nutritional index (PNI) >45 ( n=76) before the first PD-1 immunotherapy was significantly longer than that of patients with NLR >5 ( n=19) , LDH >245 U/L ( n=35) , and PNI≤45 ( n=30) (10.3 vs. 6.1 months, P=0.008; 11.2 vs. 6.5 months, P= 0.004, 11.2 vs. 8.5 months, P=0.002) . Cox proportional risk regression model analysis showed that LDH level ( HR=0.485) and PNI index ( HR=2.160) were independent influencing factors for the prognosis of advanced NSCLC treated with PD-1 monoclonal antibody (all P < 0.05) . Conclusions:LDH level and PNI index were independent influencing factors for the prognosis of advanced NSCLC treated with PD-1 monoclonal antibody.

放射性肺损伤是胸部肿瘤放疗的常见并发症,通常包括早期的放射性肺炎和晚期的放射性肺纤维化,严重影响患者的治疗和预后.目前常用的治疗放射性肺纤维化的方法包括糖皮质激素治疗、抗炎治疗、抗氧化治疗等.随着人们对放射性肺损伤的深入研究,分子靶向治疗受到广泛关注.分子靶向抑制剂是一类新兴的治疗放射性肺损伤的药物,主要针对各类细胞因子、信号通路、酪氨酸激酶受体等靶点.本文将对放射性肺损伤的发病机制及分子靶向治疗进行系统综述.

为了提高研究生的培养质量,系统地对研究生病原分子生物学技术进行优质课程建设和实践,包括师资队伍、课程目标、课程设计理念、课程内容、教学方法、教学手段、教学评价、教材建设、课程资源、课程管理、课程实践条件等,探索出提高研究生培养质量的优质课程模式.

为了解鸭乙型肝炎病毒(duck hepatitis B virus,DHBV)在我国鸭群中的感染状态,从我国养鸭业比较集中的山东、江苏2个省份10个鸭群采集临床有发病症状的150只病死鸭的心、肝、脾、肺、肾等5种脏器,以提取的组织DNA为模板通过PCR方法检测DHBV的感染情况,并进行了2株DHBV野毒SD-01和SD-02的全基因组序列测定及同源性和进化树的分析.结果显示,DHBV的群体阳性率为100.0％,个体阳性率为41.3％.被检测的器官中,肝脏的阳性率达100.0％,其他器官从高到低依次是心脏、脾脏、肺脏和肾脏,分别为77.4％,71.0％,67.7％,66.1％.SD-01和SD-02的全基因组全长分别为3 027 bp和3 021 bp,与其他15株GenBank已发表的全基因组序列同源性在89.6％～99.2％;全基因组核苷酸序列及S蛋白氨基酸序列进化树分析表明,SD01株与4株中国分离株位于同一进化分支上,而SD-02则与中国分离株DHBV-XY株以及9株美国、加拿大、德国、南非以及印度分离毒株处于同一分支.结果表明,我国目前樱桃谷鸭群中携带的DHBV毒株可能有不同的来源.

目的 了解宝鸡市人民医院耐甲氧西林金黄色葡萄球菌的临床分布特点及对抗菌药物的耐药性,为临床合理使用抗生素和医院感染控制提供依据. 方法 回顾性分析该院2012-2015年耐甲氧西林金黄色葡萄球菌(methicillin resistant Staphylococcus aureus,MRSA)的临床分布及耐药性情况.采用VITEK-2 Compact全自动微生物鉴定药敏仪对可疑菌落进行鉴定和药敏检测,使用WHONET 5.6软件分析数据. 结果 2012-2015年共分离出MRSA菌株177株,MRSA菌株检出率为28.0％,男性患者MRSA检出率(33.1％)明显高于女性(21.2％),差异有统计学意义(x2=10.97,P=0.00).MRSA菌株主要源于痰液(占50.2％)和分泌物(占45.7％),二者MRSA菌株检出率分别为35.7％、26.0％;不同科室MR-SA主要以儿科(占33.9％)、皮肤科(占14.7％)为主,各科室MRSA检出率差异有统计学意义(x2=11.202,P=0.011);各年度MRSA检出率差异有统计学意义(x2=14.898,P=0.002),2012年最高(37.2％),2014年最低(20.3％).MASA对青霉素G、苯唑西林完全耐药,对红霉素(89.8％)、克林霉素(74.6％)耐药率较高,尚未发现对利奈唑胺、万古霉素、替考拉宁耐药. 结论 2012-2015年宝鸡市人民医院MRSA检出率28.0％,低于全国平均水平,不同标本、不同科室、不同年度MRSA检出率不同.MRSA对青霉素G、苯唑西林、红霉素、克林霉素常出现耐药.

Objective To investigate the carrying rate of hepatitis B virus(HBV) in Tibetan population in Tibet Ngari Region,and to provide evidence for the prevention and control of hepatitis B virus infection in this region.Methods A total of 2 749 Tibetans in Ngari Prefecture were selected,serological markers of hepatitis B virus were detected and the detection results were analyzed statically.Results Our results showed that the HBsAg positive,immunity,infection and sensitivity rate of the Tibetans was 20.33％,23.43％,26.12％ and 50.45％,respectively.In detail,the HBV positive and infection rates of the male were significantly higher than those of the female (P ＜ 0.05).Moreover,the HBV positive and infection rates of the farmers and herdsmen were significantly higher than those of the urban dwellers (P ＜ 0.05).Conclusion The HBV positive rate of Tibetan populations in this region was significantly higher than the average HBV positive rate of China.The HBV infections were prevalent in Ngari prefecture.

Objective To ascertain the molecular mechanism by which Helicobacter pylori CagA upregulates CIP2A.Methods After AGS and GES-1 cells were separately cultured to exponential phase,AGS or GES-1 cells were separately transfected with either the recombinant plasmid WT-cagA or the control vector plasmid pcDNA3.1.The cells were then treated with a signal inhibitor of B-Raf or JNK2 and transfected with the plasmid recombinant WT-cagA.The expression of CIP2A was detected with Western blotting,and the cell clonogenic potential was determined using a cell cloning assay.Cell migration was detected with a cell migration assay.Results AGS and GES-1 cells transfected with the plasmid WT-cagA had a higher level of CIP2A expression than that in the control.After treatment with a signal inhibitor of B-Raf or JNK2 and subsequent transfection with the plasmid WT-cagA,the level of CIP2A expression in the cells was decreased.The cell clonogenic potential and cell migration decreased below the cell clonogenic potential and cell migration noted in cells transfected with the plasmid WT-cagA alone.Conclusion H.pylori CagA upregulates the expression of the oncogene CIP2A through B-Raf and JNK2,affecting the cell clonogenic potential and cell migration.

Objective To investigate the immune effects of herpes simplex virus type 2 (HSV-2) DNA vaccine of pc(pcDNA3.1-)-P6-gBCTL-TBK-1 in BALB/c mice.Methods The eukaryotic expression plasmid of pc-P6-gBCTL-TBK-1 was constructed and transfected into HeLa cells,and the expressed protein was verified by Western blotting.The BALB/c mice were immunized by the plasmids through intramuscular injection for three times,in week 0,2 and 4.In week 5,the levels of interferon (IFN-γ),interleukin-1 8 (IL-18),interleukin-4 (IL-4),interleukin-6 (IL-6) and specific HSV-2gD-P6 IgG antibody were measured by ELISA.In week 8,spleen cells of vaccinated mice were separated and the proliferation of spleen lymphocytes were detected by MTT.The activity of cytotoxic T lymphocyte (CTL) was detected by lactic dehydrogenase (LDH) method.Results After being inoculated by the plasmid of pc-P6-gBCTL-TBK-1,the mice were induced to generate higher level of specific antibody,including IFN-γ,IL-18 and IL-4;the CTL kill cells' activity and proliferation capability of splenic lymphocytes were improved.Conclusion The recombinant plasmid of pc-P6-gBCTL-TBK-1 has immunogenicity,which can induce the humoral and cellular immune responses,and TBK-1 may have good function of molecular chaperone.

通过构建口腔专业技能竞赛教学平台,实施"以赛促教、以赛促学、以赛促改"的新型教学模式,强化了口腔专业学生的基础知识和基本技能的培养,促进了理论与实践相结合,提高了学生的操作能力、创新能力和综合素质,同时提高了教师的教学能力.

目的 阐明乙型肝炎病毒(hepatitis B virus,HBV)相关肝细胞癌的癌组织与癌旁组织中HBV cccDNA全基因组的点突变模式及其与HBV cccDNA和总DNA含量的关系,寻找可以作为肝细胞癌(hepatocellular carcinoma,HCC)患者预后判断指标的突变位点. 方法 取23例HCC患者术后肝癌与癌旁组织各1份,采用Qiagen法提取DNA,采用滚环扩增HBV cccDNA,采用Taqman探针法定量测定HBV cccDNA和总DNA.采用PCR分段扩增HBV cccDNA全基因组,PCR产物纯化后测序,确定其基因型,并与标准野生株序列比对,确定HBV cccDNA的突变位点. 结果 肝癌与癌旁组织中HBV cccDNA突变频率较高的位点集中于X和C区,肝癌与癌旁组织突变个数之和大于10的位点有26个.G1719突变组HBV cccDNA和总DNA的含量均低于T1719野生型组(t=2.449,P＜0.05和t=2.525,P＜0.05),G1719突变组患者术后生存时间短于T1719野生型组(x2=8.56,P＜0.05),并且T1719G突变与患者BCLC分级相关,G1719突变组更倾向于较高的BCLC分级(x2=6.296,P＜0.05). 结论 T1719G突变可能成为HCC患者术后预后的早期预测因素,值得进一步研究.

目的探究IL-2在调控巨噬细胞极化分型方面的作用及机制。方法重组小鼠白介素-2（IL-2）刺激处于M0期的小鼠单核巨噬细胞RAW 264.7,同时以IL-4作为对照。Real-time PCR和Western blot检测M1型和M2型标志分子的表达;流式细胞术检测M1型和M2型巨噬细胞的百分比;Western blot检测Jak3和Stat5活化水平。结果经IL-2刺激后,处于M0的RAW 264.7细胞显著上调表达M1型标记分子,如IL-1β、IL-12、TNF-α和i NOS等;IL-2使巨噬细胞中M1型的比例由3.2%上升到24.6%,同时Jak3和Stat5分子的磷酸化水平显著提高。结论 IL-2具有促进巨噬细胞由M0向M1极化的作用,且该作用可能是通过Jak3-Stat5通路实现。

Objective To prepare Helicobacter pylori Omp18 protein and polyclonal antibodies against that protein Methods The Omp18 gene was amplified using PCR and cloned into the vector pMD-18T.It was then was cloned into the prokaryotic expression plasmid vector pTriEx(TM)-4.The vector was transformed into Escherichia coli JM109DE.Omp18 protein was produced and purified,and then rabbits were repeatedly administered the protein to produce polyclonal antibodies.Results The Omp18 gene was successfully cloned into the prokaryotic expression vector pTriEx(TM)-4 and the vector was successfully transformed into E.coli JM109DE.Omp18 protein was successfully produced,and purified Omp18 protein was administered to rabbits to yield polyclonal antibodies.Conclusion Omp18 protein was successfully expressed in prokaryotic cells,and polyclonal antibodies were successfully prepared in rabbits.This research will benefit study of the function and regulation of Omp18 and it will lay a foundation for detection of H.pylori and preparation of vaccines.

目的:评价PBL+ LBL双轨教学法在留学生医学微生物学教学中应用效果.方法:采用期末考试成绩和问卷调查综合评价PBL+ LBL双轨教学法的教学效果.结果:期末考试实验班学生成绩显著高于对照班学生成绩(P＜0.05).90％以上的学生认为PBL+ LBL双轨教学模式可激发学习兴趣,提高自身多重能力.结论:PBL+ LBL双轨教学法在留学生的教学效果明显优于传统LBL教学.

Objective The effects of Escherichia coli on the motility,ultrastructure,and apoptosis of human sperm in vitro were studied in order to identify the cause of male infertility and facilitate diagnosis and therapy for male infertility caused by pathogenic infections.Methods Semen samples were obtained from 30 healthy volunteers.Four groups of sperm suspensions were prepared: a normal sperm suspension serving as negative control(Group A),a sperm suspension mixed with suspended E.coli(Group B),a sperm suspension mixed with suspended E.coli treated with traditional Chinese medicine(Group C),and a sperm suspension mixed with suspended E.coli treated with furadantin(Group D).These samples were collected at 0,2,and 4 h.Motility parameters were analyzed using a computer-aided semen analysis(CASA) system.Changes in the ultrastructure of human sperm were observed with transmission electron microscopy.The rate of apoptosis of semen sperm was detected with flow cytometry combined with FITC-Annexin V/PI dual color fluorescence staining.Results Group B had significantly lower motility parameters,including straight line velocity(VSL),curve velocity(VCL),average path velocity(VAP),amplitude of lateral head displacement(ALH),and progressive motility,in comparison to the other groups at 4h(P0.01).There were no significant differences in the ultrastructure of sperm in the control group(A) and groups treated with medicine(C and D),while there were significant changes in the ultrastructure of sperm in Group B.In addition,the rate of sperm apoptosis increased significantly(P0.01) in Group B.Conclusion E.coli significantly inhibited sperm motility,changed the ultrastructure of sperm,and increased the rate of apoptosis of sperm in vitro.Therefore,E.coli infection may be one cause of male infertility.

This study aim to evaluate humoral and cellular immune response in Balb/c mice immunized by pc(pcDNA3.1)-P6-gBCTL-Hsp70 genetic vaccine. Firstly the recombinant plasmid pc-P6-gBCTL-Hsp70 was transformed into CHO cells and the expressed protein was characterized by indirect immunofluorescence and Western blotting. Then the recombinant plasmid was inoculated into Balb/c mice by intramuscular injection for three times, once of two weeks. One week after the last vaccination, the levels of specific IgG antibody, IFN-γ and IL-18 were measured by ELISA; one month after the last vaccination, spleen cells of vaccinated mice were separated and the proliferation of spleen lymphocytes was measured by MTT. We found that pc-P6-gBCTL-Hsp70vaccine could promote splenocytes proliferation, induce higher levels of IFN-γ, IL-18 and specific antibody,improve the activity of CTL kill cells of Balb/c mice. In conclusion, the recombinant clone pc-P6-gBCTL-Hsp70can effectively induce humoral and cellular immune responses, which can offer basic conditions for constructing new type of polygenic DNA vaccine.

To study whether the position change of IL18,HBsAg,and P6(NP6) in vector pcDNA3.1 affect the protein expressing.IL-18 was respectively connected with amino terminal and carboxyl terminal of fusion protein P6-S(NP6-S) to construct 4 plasmids: pc-IL18-P6-S,pc-IL18-NP6-S,pc-S-P6-IL18,and pc-S-NP6-IL18.The recombinant plasmids were analyzed by DNAstar,and then transformed into CHO cells.The expressed proteins were characterized by indirect immunofluorescence and Western blotting.Then we can determine which is the best connection.It was certified that IL18 should be connected with amino terminal of fusion protein by DNAstar.However we drew the opposite conclusion by indirect immunofluorescence.The result indicate that IL18 should be connected with carboxyl terminal of fusion protein,which offers the basic conditions for constructing new type of polygenic DNA vaccine.

Objective This study sought to explore the use of methylated XbaⅠ in gene transmitting vectors.Methods A pair of primers to amplify the vector backbone pΔSG was designed according to pIRES2-EGFP.pΔSG with potentially methylating XbaⅠ and deletion of IRES and EGFP segments and individual EGFP were obtained by PCR amplification.Recombinants of rpΔSG-EGFPV-Xho,rpΔSG-EGFPV-Xbawere screened with colony PCR and enzyme digestion after the two PCR products were digested and linked with T4 ligase.The two PCR products were also separately cloned into a pMD 18-T simple vector,and two recombinants of psT-ΔSG and psT-EGFP were identified.The EGFP in psT-EGFP was subcloned into the vector backbone of pΔSG cut out of psT-ΔSG to obtain the recombinant rpΔSG-sT-EGFPV-Xho.With the replacement of EGFP by DsRed2 in rpΔSG-sT-EGFPV-Xho,recombinant rpΔSG-sT-DsRed2V-Xho was obtained.The four final recombinants were transfected into CHO cells with Lipofectamine2000,and EGFP or DsRed2 was detected under a fluorescence microscope.Results The expression of EGFP or DsRed2 was obvious in cells transfected with the different recombinants.Conclusion The potentially methylating sequence of XbaⅠ in PCR products remained active during enzyme digestion and ligation in vitro while being protected by methylation in prokaryotic cells of genotype Dam+or/and Dcm+after transformation.The sequence had no effect on the stability and biological activity of recombinants in host cells.

Objective To determine if gastric epithelial cells produce IFN-γ and the mechanism of its production.Methods AGS gastric epithelial cells were infected with H.pylori and the production of IFN-γ was detected using RT-PCR and ELISA.AGS gastric epithelial cells were transfected with CagA plasmid and tested for IFN-γ using ELISA.MEK / ERK,Src,P38,and NF-kB inhibitors were then added to the cells transfected with CagA plasmid and they were tested for IFN-γ using ELISA.Results When AGS gastric epithelial cells were infected with H.pylori,the cells produced IFN-γ.When the cells were transfected with CagA plasmid,the cells also produced IFN-γ.When MeK/ErK,P38 and NF-kB inhibitors were added to the cells transfected with CagA plasmid,the production of IFN-γ was inhibited.Conclusion H.pylori AGS gastric epithelial cells to produce IFN-γ.As the main virulence factor for H.pylori,CagA induces cells to produce IFN-γ,and that production depends on signal pathways involving MeK / ErK,P38,and NF-kB.

AIM:To construct and express pcDNA3.1-IL-18-HSV P6 recombinant DNA vaccine, and to observe the immune responses of pcDNA3.1-IL-18-HSV P6 genetic vaccine.METHODS:Genes of P6 and IL-18 were cloned into the eukaryotic expression vector pcDNA3.1(-) respectively, and confirmed by enzyme digestion and sequence analysis, the recombinant plasmid pcDNA3.1-IL-18-HSVP6 was transformed into CHO cells. The expressed protein was characterized by indirect immunofluorescence and Western blotting. The recombinant plasmid pcDNA3.1-IL-18-HSVP6 was used to inoculate BALB/c mice by intramuscular injection for three times, once a week. One week after the last vaccination, the levels of specific IgG antibody, IFN-γ and IL-18 were detected by ELISA; One month after the last vaccination, spleen cells of vaccinated mice were separated and proliferation of spleen lymphocytes were detected by MTT assay.RESULTS:Recombinant pcDNA3.1-IL-18-HSV P6 plasmid was confirmed by enzyme digestion and DNA sequencing. After inoculated by pcDNA3.1-IL-18-HSV P6 vaccine, the mice could produce higher level of splenocytes proliferation and secrected higher level of IFN-γ, IL-18 and specific antibody.CONCLUSION:DNA vaccine of pcDNA3.1-IL-18-HSV P6 has been successfully constructed, and it can effectively induce humoral and cellular immune responses, which provided a basis for constructing new type of DNA vaccine.