目的:探究医学高校夏令营活动对医学生专业选择的影响.方法:通过对2016年8月至2018年8月来我院参加招生夏令营的学生,进行自评式问卷调查,来研究夏令营活动前后专业选择变化情况和学生对夏令营的了解情况及评价.结论:推荐免试学生在报考院校时存在一定的盲目性,夏令营活动有利于推荐免试学生更理性地选择院校与专业、规划硕士研究生生涯.

目的 分析囊性纤维化跨膜传导调节因子(cystic fibrosis transmembrane conductance regulator,CFTR)在人肿瘤组织中的表达特征,探讨其临床应用价值.方法 采用甲基化特异性PCR检测上皮性卵巢癌组织中CFTR甲基化发生率;采用免疫组织化学法检测卵巢癌组织标本及良性肿瘤组织中CFTR蛋白的表达,分析其表达与临床病理分级的关系及CFTR对卵巢癌细胞侵袭能力的影响.结果 卵巢癌中CFTR甲基化发生率大于50％,同时其蛋白表达明显降低,且卵巢癌中CFTR的表达与卵巢癌的病理分级呈正相关性.CFTR去甲基化处理后,细胞凋亡增加.结论 CFTR在卵巢癌中发挥抑癌作用,可能成为新的肿瘤标志物用于卵巢癌的早期诊断及对病情进展的检测.

目的:构建一种受人端粒酶逆转录酶(hTERT)启动子和miR-145双调控的更加安全的条件复制型腺病毒.方法:在已构建的hTERT启动子调控早期复制必需基因E1A的条件复制型腺病毒Adzq 11基础上,再将四个拷贝的miR-145的靶序列加入到E1A的3’非翻译区,构建新型溶瘤腺病毒Adzq11-145T.应用Real-time PCR测定人非小细胞肺癌细胞A549和人胚肺成纤维细胞HLF中miR-145的表达量;然后在这两株细胞中,分别转染Adzq11和Adzq11-145T对应的穿梭质粒及感染病毒本身,用Real-time PCR检测E1A表达量以测定调控效果;用病毒空斑单位法检测两种病毒的复制情况;用CCK8检测对A549和HLF细胞的杀伤作用.结果:构建的新型溶瘤腺病毒Adzq11-145T在HLF中复制量明显低于Adzq11,其E1A mRNA水平和杀伤效应也显著降低.而在A549中,Adzq11-145T在E1A表达、病毒复制和溶瘤能力方面较Adzq11均无显著降低.结论:成功构建出一种新的受转录和转录后水平双重调控的条件复制型腺病毒Adzq11-145T,它比仅加入hTERT启动子调控早期复制必需基因E1A的腺病毒Adzq11更加安全.

目的:研究胚胎时期表达部位广泛、丰度高,而成年后分化表达的印记基因Neuronatin(Nnat)的两种剪接形式Nnatα和Nnatβ对人视网膜色素上皮细胞(RPE)增殖、迁移的影响.方法:构建Nnatα、β两种剪接形式的表达质粒,转染RPE获得表达该基因的稳定表达细胞株;CCK-8实验检测稳定表达细胞株的增殖能力,流式细胞仪分析细胞周期,细胞划痕实验检测其迁移能力.结果:成功构建了Nnatα和Nnatβ表达质粒,并获得了Nnatα和Nnatβ基因稳定表达PRE细胞株.CCK-8实验结果显示cNNATα组与对照组相比较,增值率为23.33 ％(P＜0.05),cNNATβ组相较于对照组无显著性差异,细胞周期分析cNNATα组和cNNATβ组细胞在G2-S期的百分率分别为18.60％、11.11％,对照组细胞的为9.94％;相较于对照组,cNNATα组的细胞迁移能力显著增强,cNNATβ组的细胞迁移能力微弱增强.结论:Nnatα对RPE有一定的增殖作用,其影响主要在S期;同时,Nnatα显著促进RPE细胞的迁移能力.

目的 构建能自动分泌性表达单核细胞趋化因子-1(MCP-1)的重组卡介苗.方法 分别以卡介苗(BCG)和人MCP-1cDNA为模板,通过PCR扩增得到120 bp的BCG Ag85B信号肽基因序列和300 bp的MCP-1基因序列.将上述两片段基因序列插入大肠杆菌-卡介苗穿梭质粒pMV261,得到重组质粒pMV261-Ag85B-MCP-1.测序鉴定完毕后,用电穿孔法将该重组质粒导入BCG中,构建重组卡介苗rBCG MCP-1.分别应用PCR扩增和Western blot检测rBCG MCP-1中MCP-1的基因和蛋白的表达.结果 质粒pMV261-Ag85B MCP-1用双酶切和PCR扩增及测序鉴定证实克隆基因BCGAg85B信号肽和MCP-1正确插入载体pMV261.Western blot显示rBCG MCP-1的培养上清和菌体中均可检测到MCP-1蛋白的表达.ELISA法可检测到培养上清中有高表达的MCP-1蛋白(340 pg/mL).结论 成功构建了能分泌MCP-1的新型重组卡介苗rBCG MCP-1,为进一步研究其抗膀胱肿瘤的疗效打下了基础.

Objective: To investigate the Neuronatin(NNAT) expression in varied common human tumor tissue,and the effect of α and βfragments of NNAT gene on proliferation of tumor cell.Methods: Issue microarray technology and immunohistochemistry were used.And adenoviral vector carrying α and βfragments of NNAT gene were employed with clone formation experiment and real time cell analyser.Results: NNAT express in 11 kinds of tumor,which include esophageal squamous carcinoma,colon adenocarcinoma,rectal adenocarcinoma,pancreatic adenocarcinoma,specificity of infiltrating ductal carcinoma,cervical squamous cell carcinoma,endometrial carcinoma,transitional cell carcinoma of bladder,lung squamous carcinoma,skin squamous carcinoma and prostate adenocarcinoma,6 kinds of normal tissue include adrenal,skin,testis,brain and skeletal muscle,pancreas and 2 kinds of chronic inflammatory tissue,colitis and hepatitis tissue.In vitro,experiment results show that α fragment of NNAT suppress slightly the proliferation of tumor cell.Conclusion: NNAT express in many humor,and NNATα suppress the colon tumor cell line SW620 cell's proliferation slightly.

Objective To investigate the practical efficacy and safety of ultrasound with microbubbles mediated rAAV2-EGFP to retina of rat after intravitreal and subretinal injection. Methods Gene transfer was examined by rAAV2-EGFP intravitreal and subretinal injection into the Wistar rats with or without microbubbles. The eyes were exposed to US (1 MHz,2 W/cm2, duration 5 minutes,duty cycle 50%,pulse recurrent frequency 100 Hz). The onset of EGFP gene expression, lightness of fluorescence, area of fluorescence and its distribution in the fundus in vivo via fluorescence stereosocope were investigated on the 4th,7th, 35th,49th and 120th day respectively. The value of gene transfer was quantified through the EGFP fluorescence quantitative methods by Axiovision 3. 1 software. HE staining was used to observe tissue damage. Results There was no fluorescence observed by fluorescence stereosocope after intravitreal injection after two-month study. After subretinal injection, ultrasound-targeted microbubbles destruction (UTMD) strongly increased gene transfer efficiency. UTMD used in the experiment did no harm to the rat retina structure. Conclusions UTMD could not enhance rAAV2-EGFP transfecion efficiency to rat retina after intravitreal injection but the transduction could be enhanced significantly after subretinal injection.

Objective To investigate the effects of siRNA inhibiting androgen receptor (AR) gene expression on proliferation and apoptosis of human bladder cancer T24 cell line. Methods Three small interfering RNAs targeting human AR mRNA were designed and synthesized, and then transfected in-to T24 cell line by lipofectamineTM 2000. The expression levels of AR mRNA and protein in the cells were tested by real time RT-PCR and Western blot, respectively. MTT and flow cytometry (FCM) were em-ployed to observe the proliferation and apoptosis of T24 cells after transfection. Results One siRNA suc-cessfully suppressed AR expression in T24 cells,as revealed by real time RT-PCR and Western blot analy-ses. MTT showed that siRNA could significantly inhibit the cell proliferation compared with control group (P<0.05). The apoptosis rate of T24 cells in the absence of AR was increased by 21.61%. Conclusion siRNA targeting AR mRNA can effectively silence AR gene expression,inhibit proliferation,and induce apoptosis of T24 cells significantly.

A fusion gene called Ig-BDNF, in which brain-derived neurotrophic factor cDNA fused to the 3' end of signal peptide of Ig coding sequence, was constructed by PCR, digested and subcloned into shuttle plasmid pSNAV to obtain a recombinant plasmid pSNAV-Ig-BDNF. Then the plasmid encoding fusion protein was transfected into 293 cell lines and the stably transfected clones were selected with neomycin. AAV1 containing Ig-BDNF fusion gene vectors were obtained by super-infection by Herpes virus. The resultant adeno-associated virus vectors AAV-Ig-BDNF were confirmed by PCR, Western blotting and a sandwich enzyme-linked immunosorbent assay (ELISA) after infection of 293 cell lines. The results indicated that AAV-Ig-BDNF contained the target gene, and infected cells and produced the fusion protein into the supernatant. The content of BDNF in medium per 5x104 cells over a 24 h incubation period reached 1000 pg/mL. With the help of non-replicative adenovirus during AAV-Ig-BDNF infection, the expression of BDNF increased 7-8 fold, and the enhancement of BDNF gene expression was observed in a concentration-dependent manner. These results suggested that a functional AAV-Ig-BDNF was successfully constructed and it offers basis for further study for gene therapy of neural degeneration diseases.

Background Retina is important in converting light into neural signals, but little is known about the regulatory genes essential for the retinal morphological formation, development and functional differentiation. This study aimed to investigate the mRNA expression patterns and cellular or subcellular distribution of 33 differentially expressed genes in the retina belonging to the early and middle-late embryogenesis stages as well as the early adult stage during human development. Methods In situ hybridization and real-time fluorescent quantitative reverse transcription polymerase chain reaction (FQ-RT-PCR) were used to assay 33 differentially expressed genes which were screened out using microarray analysis and were not present in the retinal cDNA or the Expressed Sequence Tags (EST) database of the National Eye Institute (NEI) Genebank. Results Nine of the 33 genes belonged to EST or the unknown cDNA fragments, and the remaining belonged to the novel genes in the retina. During the human retinal development 17 genes were down-regulated, 6 were up-regulated and the remaining 10 were relatively unchanged. Most of the genes expressed in all layers of the retina at the gestation stage, and in the fully developed retina some genes examined did show higher expression level in certain specific cells and structures such as retinal ganglion cells or the outer segment of photoreceptor cells. Conclusion The gene expression profile during retinal development possesses temporal and spatial distribution features, which can provide experimental evidence for further research of the functions of those genes.

Objective To observe the morphological changes and gene expression during the transdifferentiation of adult retinal pigment epithelial(RPE) cells into neuronal phenotype in vitro induced by retrovirus and ciliary neurotrophic factor (CNTF). Meothds The adult RPE cells derived from CRL-2302 were infected by retrovirus with green fluoresence protein (GFP) and then were transfected further by liposome-mediated CNTF expressing plasmid. The cellular ability of producing CNTG, and the expression of CNTF, CNTF receptor (CNTFR), and signal transduction molecule janus tyrosine kinases (JAK) were detected by enzyme linked immunosorbent assay, immunohistochemical staining and Western blotting method. Results After infected by retrovirus, the configuration of adult RPE cells didn′t change much, but expressions of neurons and some glial cells markers like neurofilament (NF) protein and glial fibraillary acidic protein (GFAP) were detected. After further transfected by CNTF expressing plasmid, RPE cells which expressed CNTF highly and continuously had differential neurocytes; the expression of CNTFR didn′t change, but the distribution position changed to the cell membrane; expression of signal transduction molecule JAK increased obviously. Conclusion The adult RPE cells may transdifferentiate into neurons induced by retrvirus and CNTF. The transdifferentiation may relate to CNTF-CNTFR-JAK signal transduction pathway.

腺相关病毒(AAV)能感染非分裂期的细胞,对宿主几无致病性及能使宿主长期稳定的表达外源基因,是神经系统疾病基因治疗的常用载体。不同血清型的AAV,由于分子结构与细胞结合机制的差异,在组织细胞中常表现出不同的转导效率。为了寻求更有效的中枢神经系统疾病基因治疗的方法,有必要对各型AAV在神经系统组织细胞内的转导特性进行深入的研究。

OBJECTIVETo investigate the efficacy of subretinal transplantation of CNTF gene transfected fibroblasts for preventing photoreceptor degeneration in RCS.METHODSThe human fetal lung fibroblasts with high level expression of CNTF were established by liposome mediated gene transfer and MTX selection. A 5 microl of cell suspension, containing 1 x 10(5) cells, was injected through pars plana of ciliary body into the subretinal space of the right eye at postnatal 4-5 weeks, the left eye was left without injection or injected with PBS as controls. The both eyes were enucleated for histopathological examinations at 2, 4, 6, 8, 10, 12 and 15 weeks following transplantation.RESULTSThe level of CNTF protein (91,046.15 pg/ml) expressed in the transfected cells was determined by sandwich enzyme-linked immunosorbent assay (ELISA). The four of seven eyes examined by light microscopy and the ten of 14 eyes examined by electro microscopy showed rescue effect. The prolonged photoreceptor survival, reduction of apoptotic cells and debris were observed in transplanted eyes in comparison with untreated or sham-injected eyes.CONCLUSIONThis study provides the first indication that transplanted human fibroblasts with high level expression of CNTF are able to rescue photoreceptor degeneration in RCS dystrophic rat retina.

视网膜是视觉的基础,结构十分精细复杂,视网膜的功能完全依赖于视网膜的结构.鉴于目前人们对视网膜发生的调控基因和机理了解很少,利用含16361个基因的芯片分别检测了12～16周、22～26周胎儿及20～40岁成人视网膜中基因的表达水平,发现814个基因在1或2个时间点表达水平相差3倍以上,其中表达强度值在1个以上的时间点超过100的差异表达基因共106个,依次是发育、分化、信号传导、蛋白质合成翻译、代谢、DNA合成修复重组等相关基因.基因表达模式和聚类分析揭示随视网膜发育成熟呈下调趋势的基因最多,而呈上调趋势的基因较少.在上述106个差异表达基因中,有46个目前在美国国立卫生院(NIH)眼科研究所(NEI)的视网膜cDNA或EST数据库中尚找不到,它们在视网膜中的作用和功能也不清楚.为了进一步确定基因芯片结果的可靠性,采用荧光定量RT-PCR和常规RT-PCR检测分析了上述46个差异表达基因及另外6个已知的视网膜特异表达基因的表达量,其中27个基因的表达谱与芯片检测结果完全一致.另外,还采用原位杂交技术检测了NNAT基因在视网膜内的表达量及表达产物的细胞和亚细胞分布.此外,对106个差异表达基因的染色体定位进行检索揭示,其中1个基因位于已知的视网膜锥体或锥-杆体营养不良基因位点处,并与该病的发病有关.此结果为阐明视网膜发育的调控机理、为视网膜疾病候选基因的确定提供了实验证据.