Taking the virtual simulation experiment project of programmed death receptor 1 (PD-1) molecular antibody preparation and application as an example, the project bases on the basic knowledge of antibody preparation and introduces antibody application following the scientific research progress, which improves the situation that the previous experiment cannot meet the teaching objectives, helps students learn basic knowledge and cultivate scientific thinking. This paper expounds the construction and teaching methods of virtual simulation experiment from the aspects of necessity, design idea, design purpose, teaching method, implementation process, etc. In later use, the research team aim to cultivate technical development team to continue updating and maintenance.

目的 观察麦冬多糖(OJP)对转化生长因子-β1(TGF-β1)诱导的人胚胎肺成纤维(HEL)细胞表型转化的影响,探讨其分子机制.方法 将经6ng/mL诱导TGF-β1的HEL细胞作为模型组,加入25、50、100μg/mL OJP继续培养的HEL细胞作为OJP干预组.通过电镜观察细胞的超微结构;实时荧光定量RT-PCR检测α-SMA mRNA和COLⅠmRNA表达;Western blot检测α-平滑肌肌动蛋白(α-SMA)、Ⅰ型胶原蛋白(COLⅠ)、Smad2蛋白和p-Smad2蛋白表达.结果 模型组及25、50、100μg/mL OJP干预组细胞的存活率分别为(99.65±1.55)％,(88.39±1.68)％,(82.77±1.96)％和(79.48±1.74)％,OJP干预组存活率低于模型组且随浓度的增加呈剂量依赖趋势,差异有统计学意义(P均<0.05);100μg/mL OJP干预组细胞表面微绒毛减少、微丝变短,胞浆内线粒体数目下降,粗面内质网减少;模型组和100μg/mL OJP干预组α-SMA mRNA表达量为(1.00±0.09)和(0.69±0.05),COLⅠmRNA表达量为(1.02±0.11)和(0.86±0.09),100μg/mL OJP干预组细胞α-SMA和COLⅠmRNA表达较模型组下调(P均<0.05);与模型组比较,100μg/mL OJP干预组α-SMA、COLⅠ、Smad2和p-Smad2蛋白表达明显下降(P均<0.01)[α-SMA蛋白:(0.48±0.03)比(1.56±0.02);COLⅠ蛋白:(0.41±0.02)比(1.66±0.02);Smad2蛋白:(0.71±0.01)比(1.34±0.01);p-Smad2蛋白:(0.69±0.01)比(1.52±0.01),P均<0.05].结论 麦冬多糖可能通过干预TGF-β/Smads信号通路,在一定程度上抑制成纤维细胞向肌成纤维细胞的转化.

虚拟仿真技术具有真实性、互动性、多样性等特点.医学检验技术虚拟仿真平台是将医学检验技术与虚拟仿真高效、有机地进行结合,为专业理论教学与实践教学带来便利.本项目基于学校现有的教学资源与附属医院的仪器设备设计出一个集检验科、大型仪器、实验项目、诊断流程为一体的医学检验虚拟仿真平台,为培养医学检验技术专门人才提供信息平台.

Objective To establish a method for detecting concentrations of plasma cell-free DNA (cf-DNA) based on the droplet digital PCR (ddPCR),and to explore its diagnosis and treatment value in colorectal cancer.Methods ddPCR specific primers and probes for β-actin were designed to establish an absolute quantitative method for the detection of cf-DNA.The sensitivity,linearity and reproducibility of the method were validated by the detection of standards with different concentrations.The plasma cf-DNA levels were detected by this method in 68 patients with colorectal cancer,33 patients with colorectal benign tumor and 60 healthy controls,and the concetrations of carcinoembryonic antigen (CEA),carcinoembrvonic antigen 19-9 (CA19-9) were assayed by chemiluminescence,the correlation between plasma cf-DNA levels and clinicopatholoical parameters and serum CEA,CA19-9 was analyzed,and Receiver Operqting Characreristic (ROC) curves were established to illustrate the diagnostic performance.Results The ddPCR method established in this experiment can detect as low as 10pg/μl DNA template,which is approximate to 0.29copies/μl,and ddPCR was evaluated as linear in the range of 10pg/μl-10ng/μl (Y =-2.34 + 33.17X,R2 =0.999);Coefficient of Variance (CV) of intra-ssay was 9.85％,CV of inter-assay was 11.04％.The levels of cf-DNA detected by ddPCR among the plasma samples of patients with colorectal cancer,colorectal benign tumor and healthy controls were 6700 (3800-9925),3100 (2300-4000),2500 (1775-4000) copies/ml,respectively.There were significant differences between colorectal cancer and colorectal benign tumor patients (P =0.000),colorectal cancer patients and healthy individuals (P =0.000),and there was no significant difference between colorectal benign tumor patients and healthy individuals (P =0.167).No statistically significant difference of cf-DNA in age,gender,tumor sites,tumor differentiation,lymph node metastasis and the degree of tumor invasion was found..But there was significant difference in the stage TNM (P ＜0.01).There was no correlation between cf-DNA and CEA (r =0.210,P =0.099) and CA19-9(r =0.125,P =0.233).The ROC curve showed that when cut off value was 5300copies/ml,the area under the ROC curve (AUC),sensitivity and specificity of plasma cf-DNA in colorectal cancer patients were 0.931,73.3％ and 98.3％,respectively,which were significantly higher than those of CEA(0.762,51.7％,95.6％) and CA19-9 (0.548,45.0％,82.0％).Conclusion A sensitive,specific ddPCR for detecting plasma cf-DNA has been established,which is helpful for the diagnosis and prognosis of colorectal cancer.

目的 建立基于微滴式数字PCR(ddPCR)技术定量检测结直肠癌(CRC)患者血浆循环游离DNA(cfDNA)含量的方法,并初步探讨其临床应用.方法 设计β-actin基因ddPCR特异性引物和探针,通过条件优化建立cfDNA定量检测方法;用不同浓度标准品验证所建方法的特异性、灵敏度、线性及重复性;并用该法检测39例CRC患者与40例健康人群血浆标本,比较CRC患者与对照组、术前与术后组血浆cfDNA浓度差异.结果 所建方法无非特异性扩增,检测灵敏度为0.29 copies/μl,在模板浓度为10 pg/μl～ 10 ng/μl内线性良好(y=-2.34 +33.17x,r2=0.999),批内CV为9.85％,批间CV为11.04％;CRC患者血浆cfDNA结果(7.20 ±5.15) copies/μl明显高于对照组(3.38±1.97)copies/μl(P＜0.01);术后组血浆cfDNA浓度为(3.90±3.39) copies/μl,较术前组明显下降(P＜0.01).结论 建立的ddPCR定量检测血浆cfDNA方法特异性强、灵敏度高、重复性好,可为CRC患者的临床决策和病程监控提供一种无创手段.

Objective To establish a liquid biopsy technique of KRAS gene G12D mutation and to assess its diagnostic value. Methods KRAS G12D mutation was analyzed by ddPCR in plasma DNA from 52 colorectal cancer patients and compared that of to 80 healthy subjects. KRAS gene sequencing in cancerous tissue of colorectal cancer patient being set as a golden standard, we evaluated the accuracy of ddPCR and analyzed the correlation between G12D mutation rate, plasma concentration;and their clinical manifestations in CRC. Results ddPCR indicated that KRAS G12D mutation rate and concentration(26.92％, 81.5 copies/mL) in the plasma samples of colorectal cancer patients were significantly higher than that of healthy subjects (8.75％, 16 copies/mL). Colorectal cancer patients with highly differentiated adenocarcinoma showed a significantly higher number of mutant copies than medium and low differentiated adenocarcinoma(P＜0.05);M2 patients had a significantly higher number of mutant copies than N1 and NO patients (P＜0.05);The concordance rate of KRAS gene mutation between cancerous tissue and plasma ctDNA was 87.50％ in CRC.Conclusions ddPCR is a fast, noninvasive and accurate method for plasma testing of ctDNA, and the test results could be used to monitor the course of the disease and as clinical guidelines.

AIM:To observe the antiulcer effect of butyric acid and hydrogen , the main metabolites of Clos-tridium butyricum (C.butyricum), and to explore the underlying mechanism .METHODS: The mouse model of acute gastric mucosal lesion was prepared by gavage with ethanol .The mice were randomly divided into 4 groups:normal group , model group , butyric acid group and hydrogen group .The mice in butyric acid group and hydrogen group were given buty-rate and hydrogen prior to model establishment , respectively .Macroscopic observation of the pathological changes in gastric tissues was performed to evaluate the effect of the 2 metabolites of C.butyricum.Meanwhile, the mRNA expression levels of inflammatory factors, such as IL-12, RAN1 and MCP-1, were determined by RT-qPCR.The expression levels of apopto-sis-related proteins Bcl-2 and Bax were detected by immunohistochemical staining .RESULTS:The macroscopic observa-tion found that butyrate , not hydrogen , protected gastric mucosa .HE staining also showed that butyrate significantly attenu-ated the pathological damage of the gastric mucosa induced by ethanol .Compared with model group , the mRNA levels of inflammatory factors IL-12, RAN1 and MCP-1 in butyrate group significantly decreased (P<0.01).In butyrate group, the protein level of Bax was obviously decreased compared with model group (P<0.01), while the protein level of Bcl-2 was significantly increased ( P<0.01 ) .CONCLUSION: The gastric mucosa protective metabolite of C.butyricum may be butyric acid , not hydrogen .Butyric acid protects the gastric mucosa against ethanol-induced lesion by inhibiting the inflam- mation and reducing the expression ratio of Bax/Bcl-2.

Objective: To investigate the correlation between exogenous estrogens (EEs) and the devel-opment risk of HER2 positive breast cancer cells.Methods: We treated HER2-positive and ERα-negative cells SKBR3, ERα-positive and HER2-negative cells MCF-7 with 17β-estradiol. The six different concentra-tions of 17β-estradiol were 10-5, 10-6, 10-7, 10-8, 10-9, 10-10 mol/L. For each concentration condition, there were three time dose such as 24 h, 48 h, 72 h. The expression levels of HER2 and ERα were observed as the key positive marker. And then cell invasion and cell proliferation of breast cancer cells were analysed for MCF-7 and SKBR3 under 17β-estradiol treatment.Results: It showed that the transcriptional levels of HER2 and ERα were upregulated in MCF7 cells under all 18 different conditions. However, only the transcriptional levels of HER2 was upregulated in all 18 different conditions in HER2-positive and ERα-negative SKBR3. At protein expression levels, HER2 in SKBR3 cells and ERα in MCF-7 cells were expressed apparently after the breast cancer cells were stimulated by 17β-estradiol under all different conditions. Simultaneously,the prolif-eration of MCF-7 and SKBR3 were promoted obviously. With the higher concentration of 17β-estradiol treat-ment the proliferative activity of these two breast cancer cells were promoted much more obviously. As for the invasion of the breast cancer cells, MCF-7 was increased after 72 h, and SKBR3 was increased after 24 h and 48 h. Whereas the invasion of SKBR3 and MCF-7 were not up or down regulated evidently with the change of concentrations and time.Conclusion: In a word, the expression levels of HER2 in HER2 positive breast&nbsp;cancer cells SKBR3 are upregulated under different 17β-estradiol treatment conditions. Furthermore, the inva-sion and proliferation of HER2 positive breast cancer cells SKBR3 are promoted under exogenous estrogens 17β-estradiol treatment.

生命科学竞赛是培养大学生创新能力的有效途径,其选题切合实际,能够激发学生理论联系实际的创新热情;原始实验记录、论文、PPT制作和答辩多项内容的综合评分制度能够有效提高学生综合能力;开放高端科研实验室有助于开拓学生视野和提高实际操作能力.

目的 探讨三磷酸腺苷结合盒转运蛋白G2基因(ABCG2) rs2231142位点与浙南地区原发性痛风的相关性.方法 收集浙南地区原发性痛风样本508例和正常健康体检样本558例,用微滴式数字化PCR技术进行基因分型,分析该位点多态性与痛风的相关性.结果 痛风组尿酸、三酰甘油、胆固醇、尿素氮、肌酐以及收缩压水平显著高于正常组(P＜0.05).痛风组ABCG2基因rs2231142位点AA型和A等位基因频率显著高于正常组(P＜0.05).痛风组AA基因型/AC基因型尿酸、尿素氮和肌酐水平型显著高于痛风组CC基因(P＜0.05).结论 ABCG2基因rs2231142位点是浙南地区原发性痛风的易感基因位点,提示A等位基因提高了痛风患者的尿酸水平,携带AA基因型的个体更易患痛风.

Objective: This study aimed to establish a rapid screening method for gene mutation of beta Thalassemia with HRM technique and to discuss its clinical application value. Methods: Common gene mutation sites of patients with beta Thalassemia such as IVS-2-654(C>T) and -28(A>G) in Wenzhou were adopted. TA clone technology was used to construct plasmid DNA as template or genotype control. The method for identifying gene mutation of beta Thalassemia with HRM technique was established. A total of 117 suspected beta Thalassemia patients were enrolled. DNA of peripheral leukocytes was extracted for IVS-2-654(C>T) and -28(A>G) detections with HRM. The results were compared with the results of bi-directional sequencing. Results: HRM technique could be used to detect IVS-2-654(C>T) and -28(A>G) mutation sites of beta Thalassemia. There was no non-specific amplification fragment. Variable coefficient (CV) of melting temperature (Tm) detected by HRM among intra-batch and inter-batch of different genotypes was <0.1%. At least 1000 copies of DNA templates could be tested and mutation with only 10% of variation could even be detected. Among 117 patients, 45 were IVS-2-654(C>T) heterozygous mutant and 9 cases were -28(A>G) heterozygous mutant. Moreover, two locus of homozygous mutant gene were not observed, which were completely inconsistent with direct sequencing. Conclusion: The established HRM technology can be used to screen gene mutation of beta Thalassemia with superior specificity and sensitivity. It provides a universal technology platform for detecting other mutation site and SNP.

Objective To evaluate the effect of Flotrac /Vigileo(Edwards Lifesciences,USA)monitoring on clinical indexes of severe sepsis patients with continuous renal replacement therapy(CRRT),so as to improve the prognosis by reducing complications caused by liquid excessive load or dormant low blood volume,improving microcirculation and enhancing organic functional recovery.Methods Totally 96 severe sepsis patients treated with CRRT were randominzed into 2 groups,conventional treatment group(group A,n =48) and Flotrac /Vigileo monitoring group(group B,n =48).Patients in both groups were given conventional treatment of severe sepsis,in-cluding active treatment of the primary disease,broad -spectrum antibiotics,organ support therapy.With indwelling central venous cath-eter,the Aquarius continuous blood purification system(Edwards Lifesciences,USA)was adopted and CVVH model treatment was con-ducted for a successive 3 days.Conventional liquid treatment was conducted in group A based on MAP,ultrafiltration volume of urine, LAC and CVP,while in group B Flotrac /Vigileo was adopted for measuring CI,SVI,SVV.Fluid was added when SVV≥12% and ultra-filtration rate was decreased.Stepwise titration method was used for fluid therapy when SVV <12% and ultrafiltration was increased.Pa-tients’visceral function index,hemodynamic indexes,length of hospital stay and mortality were observed.Results MAP,LAC,PaO2 /FiO2 ,GREA,APACHE II score of group B after 72 -hour treatment improved more obviously than group A ,of which differences in PaO2 /FiO2 ,LAC,GREA were statistically significant.Patients’length of stay in ICU in group B was shorter than in group A,which showed statistically significant difference.The mortality rate in group B was 15.1%,which showed no statistically significant difference from group A(16.2%).Conclusions Flotrac /Vigileo monitoring used in severe sepsis patients with CRRT can improve visceral func-tion and reduce complication of CRRT via strict capacity control.

Objective:To investigate the polymorphism of apolipoproteinB(ApoB)C7673T in mormal polulation in Wenzhou Hans.Methods:Genetic poly-morphisus of ApoB C7673T in a tolal of 304 normal Wenzhou Hans were studied with polymerase chain reaction and sequence determ ination technique,cur-rent results were compared with the data on other ethnic groups.Results:The alleles genetic frequencies C and T were 96.71% and 3.29% respectively.While the genotype frequencies of ApoB C7673T polymorphisms were found to be CC(93.42%),CT(6.58%),TT(0%)respectively.Conclusion:Frequen-cies of T allele in Wenzhou Hans is similar to that of the other Hans,but significantly lower than those of the Europeans and Americans.No correlation is identified between the male and female groups.

Objective To analysis the pulmonary tissue proteomic a-mong the normal , pulmonary hypertensive induced by monocrotaline and Puerarin rats , How the Puerarin inhibits the form of pulmonary hyperten-tion and pneumoangiogram rebuilding cleared.Methods SD ras random-ly divided into 5 groups, the normal group, the solvent group, the model group in two times, monocrotaline and puerarin group ( experimental group ) , 10 in each group .The solvent group was intraperitoneal injected of ethanol mixed with 0.9% sodium chloride solution ( 50 mg · kg -1 weight ) every morning and bred three weeks .The model group was intra-peritoneal injected monocrotaline (50 mg· kg -1 weight)once and respec-tively bred 2 or 3 weeks.The experimental group, after the injection of monocrotaline solution once , was intraperitoneal injected puerarin liquid (40 mg· kg -1 weight ) every morning in three weeks .Set up the pulmo-nary hypertensive rat model sucessfully , two dimensional gel electropho-resis of the total proteins from these five group , gel enzymolysis.Matrix-assisted laser desorption ionization -time of flight mass spectrometry ( MALDI-TOF-MS) and retrieval databank in NCBInr and discuss the results.Results The pulmonary hypertention , pneumoangiogram rebuil-ding , the right ventricle hypertrophy was inhibited by puerarin.The MALDI-TOF-TOF-MS discovered that the elongation factor 2 express&nbsp;very little in normal group and express a great quantity in 3-week-monocrotaline group , but in puerarin group ex-press down quickly.Conclusion The puerarin could inhibit the form of pulmonary hypertention by interfering in elon-gation factor 2 , it worth to study more about it.

目的 分离筛选出一株对甲胺磷敏感的双歧杆菌菌株.方法 双歧杆菌菌株传代筛选,取一系列浓度(4.0、2.0、1.0、0.5和0.25 μg/mL)的甲胺磷农药标准溶液30 μL加入含TPY液体培养基的检测管溶液内,双歧杆菌菌株接种到检测管内,37℃厌氧培养12 h,测定检测管液体A值.结果 筛选一株甲胺磷敏感的短双歧杆菌菌株LJM-006,微生物抑制法的最佳接种浓度107 CFU/mL,最低检测限0.5 mg/L.结论 筛选一株甲胺磷敏感的双歧杆菌LJM-006菌株,并可作为微生物法检测甲胺磷残留的菌株.

Objective: To investigate distribution status of CYP2C19 and CYP2C9 genotype in Wenzhou Han population.Methods: The CYP2C19 and CYP2C9 genotype of 287 Wenzhou Han Chinese were deter-mined by sequencing.Results: In Wenzhou Han population, CYP2C19*1, CYP2C19*2 and CYP2C19*3 allele frequencies were 65.16%, 29.27% and 13.6%, respectively; CYP2C19*1/*1, CYP2C19*1/*2, CYP2C19*1/*3, CYP2C19*2/*3, CYP2C19*2/*2 and CYP2C19*3/*3 genotype frequencies were 44.95%, 33.45%, 6.97%, 4.18% and 0.00%, respectively. The frequency of CYP2C19 poor metabolisms was 14.63%; CYP2C9*1 and CYP2C9*3 allele frequencies were 97.74% and 2.26%, respectively; CYP2C9*1/*1 and CYP2C9*1/*3 genotype frequencies were 95.47% and 4.53%, respectively, CYP2C9*3/*3 homozygous genotype was not found in this study.Con-clusion: The frequencie of CYP2C19 and CYP2C9 poor metabolisms are similar to those of Japanese population, whereas they are signiifcantly different from America-European populations.

The genetic diversity and genetic structure of Wenzhou buffaloes distributed in Pingyang,Ruian,Yongjia,Yueqing and Wenzhou in Zhejiang province were analyzed by ISSR-PCR.A total of 154 polymorphic loci of 186 were amplified using 9 primers selected from 22 primers.Percentage of polymorphism loci(PPL) was 82.80%,observed number of alleles was 1.8420,effective number of alleles was 1.5702,Nei's gene diversity was 0.3108,Shannon's information index was 0.4687,all of the genetic parameters in population level were higher than individual level.Genetic differentiation level(Gst) calculated according to Nei's gene diversity was 0.1840,which suggested that 18.40% genetic variation was present in populations.UPMGA cluster analysis in the strains of Wenzhou buffaloes also demonstrated that Wenzhou buffaloes has lower genetic diversity among the populations and the genetic variation mainly exists in the individual ones.

音乐理论界对于歌剧《江姐》的研究成果,大多集中于音乐创作和人物形象塑造等方面,对于歌剧音乐的矛盾冲突、戏剧性等方面很少涉及。创作过程研究方面,侧重于记录该歌剧作品诞生的原委;排练演出纪实方面,侧重于记述该歌剧作品排练、演出的实况;人物形象研究方面,主要侧重于对于该歌剧作品中的主人公江姐形象的分析;音乐特征分析,侧重于研究该歌剧作品的音乐创作手法特点和主要唱段的特色;演唱要领研究方面,侧重于分析该歌剧作品中主人公江姐唱段的演唱要领。