二氯三嗪基氨基荧光素标记胶联羊膜移植后的转归及生物学行为

目的观察二氯三嗪基氨基荧光素(dichlorotrazinylamino-fluorescein,DTAF)标记胶联羊膜(fibrin glue-amniotic membrane,FG-AM)移植的转归及生物学行为,探讨AM移植转归的影响因素及在临床治疗中的意义。方法建立角膜深层损伤和碱烧伤动物模型,用5g.L-1DTAF荧光标记FG-AM并分组行嵌合法和覆盖法移植,每组大白兔各5只,术后每天观察AM及角膜大体情况,第7天、第15天、第30天、第60天、第90天取角膜组织行组织病理、透射电镜及激光共聚焦显微镜观察。结果FG-AM嵌合移植于角膜创面后,随时间推移,FG逐渐降解,AM被新生角膜上皮细胞及基质胶原覆盖包裹,术后第30天角膜创面厚度已完全恢复正常。虽然肉眼及裂隙灯检查无法准确判断AM情况,但组织病理、透射电镜及激光共聚焦显微镜检查均证实术后第90天角膜基质层中残留有清晰的AM结构,并且其周围无明显炎症及排斥反应。FG-AM覆盖移植后第15天,FG-AM开始出现溶解,至术后第30天时已完全溶解脱落,角膜表面无AM及FG成分残留,角膜上皮细胞完整连续,基质胶原排列整齐。结论FG-AM嵌合移植后,AM可较长时间整合于角膜基质,不被宿主溶解吸收。FG-AM有可能成为角膜基质重建修复的有效材料。

基于荧光响应的氨传感膜研究

利用有机改性溶胶-凝胶的包埋方式，以氨基荧光素（AF）为荧光指示探针制备氨传感敏感膜，考察了AF与γ-[（2，3）-环氧丙氧基]丙基三甲氧基硅烷（GOPS）作用后的膜泄漏度及荧光光谱行为的变化，对氨传感敏感膜的配比进行了一系列的优化，获得了对氨具有高响应灵敏度而对pH无响应的氨光传感敏感膜。利用自行构建的氨传感检测装置对水体中氨进行了检测，传感膜对水溶液NH4的最低检出限为0．3μg·mL^-1，测定的相对标准偏差为4％，线性范围为1～80μg·mL^-1，响应时间（t95）为4min，敏感膜使用寿命大于6个月。

荧光素-Fe(Ⅲ)配合物与BSA的相互作用的光谱学研究

双[N,N-双(羧甲基)氨甲基]荧光素(荧光素-甲胺基二羧酸)是一种含有三环平面结构的荧光素衍生物.研究采用荧光素-甲胺基二羧酸为配体,采用铁离子(Fe(Ⅲ))为中心金属离子,通过直接反应的合成方法,在水溶液中合成了荧光素-甲胺基二羧酸-Fe(Ⅲ)配合物.并采用紫外-可见(UV-vis)光谱和荧光(FL)光谱来研究荧光素-甲胺基二羧酸-Fe(Ⅲ)配合物与牛血清白蛋白(BSA)的相互作用.相应的研究数据和计算结果表明荧光素-甲胺基二羧酸-Fe(Ⅲ)配合物对BSA分子荧光的猝灭机理是静态猝灭过程.同时采用三维荧光光谱和三维等高线剖面图谱进一步考察了荧光素-甲胺基二羧酸-Fe(Ⅲ)配合物存在时BSA分子的荧光猝灭过程和混合前后的构象变化.通过同步荧光光谱研究了荧光素-甲胺基二羧酸-Fe(Ⅲ)配合物与BSA分子相互作用的结合方式和结合位点.研究为荧光素及其衍生物在医疗领域中的进一步应用奠定了基础.

一种荧光素衍生物的表征及活细胞荧光示踪应用

【目的】通过研究荧光素二叔丁基二甲基硅醚(FTBS)的聚集诱导发光效应,将FTBS用于活细胞的长期荧光示踪。【方法】采用荧光素与叔丁基二甲基氯硅烷发生亲核取代,合成荧光素二叔丁基二甲基硅醚;通过测试其荧光发射强度和粒子直径,首次证实其具有聚集诱导发光效应;通过对人宫颈癌细胞株HeLa的染色试验,确定细胞染色时间和浓度。【结果】通过细胞染色,证实FTBS是具有良好细胞通透性的荧光探针,实现基于FTBS的活细胞长期荧光示踪。【结论】基于FTBS的聚集诱导发光效应和良好的细胞通透性,可将其用于对癌细胞及分裂产生的子细胞进行长时间跟踪监测,对于标记癌细胞和监测癌细胞增殖具有一定意义。

微乳电动色谱-激光诱导荧光检测法快速分离测定麻黄碱和伪麻黄碱

以5-（4,6-三氯-s-三嗪基-2-氨基）荧光素为衍生试剂,建立了一种微乳电动色谱-激光诱导荧光检测法分析麻黄碱和伪麻黄碱的新方法。经过实验条件的优化,使用50mmol/L硼砂,5%微乳液（V/V）（微乳液组成（W/W）3.24%正己烷,3.24%SDS,26.44%正丁醇,57.08%水）,14%乙腈（V/V）作为运行缓冲溶液,电压22.5kV,温度为25℃条件下,11min内可实现基线分离。分析物的峰高与浓度（2.5-75μg/L）之间存在较好的线性关系,相关系数为0.9915（麻黄碱）和0.9981（伪麻黄碱）;检出限分别为2.39×10^-3和3.85×10^-3μg/L。将该法成功用于麻黄及其制剂中两种生物碱的分析,回收率在92.0%-108.4%之间。

Polypyrimidine Tract-Binding Protein Enhances Zika Virus Translation by Binding to the 5'UTR of Internal Ribosomal Entry Site

Objectives To identify the 5'untranslated region of Zika virus(ZIKV 5'UTR)RNA-binding proteins and to investigate the impact of the binding protein on the activity of internal ribosomal entry site(IRES)located in ZIKV 5'UTR and virus production.Methods Interacting proteins in U251 cells were captured using tRSA-tagged ZIKV 5'UTR RNA and tRSA-ZIKV 5'UTR RNA-binding proteins were visualized by SDS-PAGE silver staining,Subsequently,liquid chromatographytandem mass spectrometry(LC-MS/MS),bioinformatics analysis,and Western blot were used to identify the candidate proteins binding to ZIKV 5'UTR.Dicistronic expression assay and plaque forming assay were performed to analyze the effect of the binding protein on ZIKV IRES activity and ZIKV production,respecitvely.Results tRSA RNA pull-down assay,LC-MS/MS,and Western blot analysis showed that polypyrimidine tractbinding protein(PTB)bound to the ZIKV 5'UTR.Furthermore,dual luciferase reporter assay revealed that overexpression of PTB significantly enhanced the IRES activity of ZIKV(t=10.220,P<0.001),while PTB knockdown had the opposite effect(t=4.897,P<0.01).Additionally,virus plaque forming assay demonstrated that up-regulation of PTB expression significantly enhanced viral titer(t=6.400,P<0.01),whereas reducing PTB expression level weakened virus infectivity(t=5.055,P<0.01).Conclusion PTB positively interacts with the ZIKV 5'UTR and enhances IRES activity and virus production.

miRNA-181b在氧-糖剥夺致N2A细胞缺血损伤中的作用及对热休克蛋白A5表达的调节

目的探讨miR-181b在氧糖剥夺(OGD)致N2As神经瘤细胞缺血损伤中的作用,及其对热休克蛋白(HSP)A5表达的调节。方法应用N2A细胞OGD模型模拟神经细胞缺血损伤,MTT比色法检测N2A细胞生存率,免疫印迹法检测HSPA5蛋白表达水平,实时定量PCR法检测miR-181b和HSPA5 mRNA表达水平,荧光素酶报告基因技术检测miR-181b对HSPA5 mRNA的直接调控作用。结果 miR-181b在OGD致N2A细胞缺血损伤中表达水平明显降低(n=5);在OGD致N2A细胞缺血损伤过程中,通过上调或抑制miR-181b的表达水平可以显著影响N2A细胞的生存率(n=6);而在非OGD条件下,miR-181b表达水平的改变对N2A细胞活力无影响;miR-181b表达水平的改变可显著影响HSPA5蛋白表达水平(n=3),而非HSPA5的mRNA水平;共转染miR-181b前体(premiR-181b)或miR-181b抑制剂(anti-miR-181b)可显著抑制或增高含有HSPA5 mRNA 3’-UTR的荧光素酶报告基因的活性(n=5)。结论 miR-181b通过负性调节HSPA5的蛋白表达水平,在OGD致N2A神经细胞缺血性损伤中发挥重要作用。

Establishment and Application of a Real-time PCR Method for Detecting stx2 Gene in Shiga Toxin-producing Escherichia coli(STEC)

[Objective] This study aimed to establish a real-time PCR method for de- tecting stx2 gene in Shiga toxin-producing E. coli （STEC）. [Method] According to the known STEC stx2 gene sequences published in GenBank, PCR primers and probes were designed based on the conserved region to construct recombinant plasmid as a positive template, thus optimizing the reaction conditions and establishing the real- time PCR method. [Result] A standard curve was established based on the opti- mized real-time PCR system, indicting a good linear correlation between the initial template concentration and Ct value, with the correlation coefficient F^e of above 0.995. The established method had a good specificity, without non-specific amplifica- tion for 10 non-STEC intestinal bacterial strains; the detection limit of initial template was 1.0x102 copies/μI, indicating a high sensitivity; furthermore, the coefficients of variation within and among batches were lower than 1% and 5% respectively, sug- gesting a good repeatability. [Conclusion] In this study, a real-time PCR method was successfully established for detecting STEC stx2 gene, which provided technical means for rapid detection of STEC in samples.

Establishment of an orthotopic transplantation tumor model of hepatocellular carcinoma in mice

AIM:To improve the outcome of orthotopic transplantation in a mouse model,we used an absorbable gelatin sponge(AGS) in nude mice to establish an orthotopic implantation tumor model.METHODS:MHCC-97L hepatocellular carcinoma(HCC)cells stably expressing the luciferase gene were injected into the subcutaneous region of nude mice.One week later,the ectopic tumors were harvested and transplanted into the left liver lobe of nude mice.The AGS was used to establish the nude mouse orthotopic implantation tumor model.The tumor suppressor gene,paired box gene 5(PAX5),which is a tumor suppressor in HCC,was transfected into HCC cells to validate the model.Tumor growth was measured by bioluminescence imaging technology.Semi-quantitative reverse transcription polymerase chain reaction(RT-PCR) and histopathology were used to confirm the tumorigenicity of the implanted tumor from the MHCC-97L cell line.RESULTS:We successfully developed an orthotopic transplantation tumor model in nude mice with the use of an AGS.The success rate of tumor transplantation was improved from 60% in the control group to 100% in the experimental group using AGS.The detection of fluorescent signals showed that tumors grew in all live nude mice.The mice were divided into 3 groups:AGS-,AGS+/PAX5-and AGS+/PAX5 +.Tumor size was significantly smaller in PAX5 transfected nude mice compared to control mice(P < 0.0001).These fluorescent signal results were consistent with observations made during surgery.Pathologic examination further confirmed that the tissues from the ectopic tumor were HCC.Results from RT-PCR proved that the HCC originated from MHCC-97L cells.CONCLUSION:Using an AGS is a convenient and efficient way of establishing an indirect orthotopic liver transplantation tumor model with a high success rate.

PTEN and PDCD4 are Bona Fide Targets of microRNA-21 in Human Cholangiocarcinoma

Objective To investigate the expression profile of microRNA-21 in human cholangiocarcinoma tis- sues and to validate its bona fide targets in human cholangiocarcinoma cells. Methods The expression profile of microRNA-21 in human cholangiocarcinoma tissues and cholan- giocarcinoma cell line, QBC939, was evaluated by using real-time PCR analysis. The bona fide targets of microRNA-21 were analyzed and confirmed by dual luciferase reporter gene assay and western blot, respec- tively. The expressional correlation of microRNA-21 and its targets was probed in human cholangiocarci- noma tissues by using real-time PCR, locked nucleic acid in situ hybridization （LNA-ISH）, and immunohis- tochemistry analysis. Results Real-time PCR analysis revealed that microRNA-21 expression depicted a significant up-regulation in human cholangiocarcinoma tissues about 5.6-fold as compared to the matched normal bileduct tissues （P〈0.05）. The dual luciferase reporter gene assay revealed endogenous microRNA-21 in cholan- giocarcinoma cell line, QBC939, inhibited the luciferase reporter activities of wild-type PTEN （P〈0.01） and PDCD4 （P〈0.05） and had no this effect on mutated PTEN and PDCD4. Moreover, loss of microRNA-21 function led to a significant increase of PTEN and PDCD4 protein levels in QBC939 cells. Elevated microRNA-21 levels were accompanied by marked reductions of PTEN and PDCD4 expression in the same cholangiocarcinoma tissue. Conclusion microRNA-21 expression is up PDCD4 are direct effectors of microRNA-21. regulated in human cholangiocarcinoma and PTEN,

Molecular mechanism and functional consequences of lansoprazole-mediated heme oxygenase-1 induction

AIM： To investigate the molecular mechanism and functional consequences of heme oxygenase-1 （HO-1） activation by lansoprazole in endothelial cells and macrophages. METHODS： Expression of HO-1 mRNA was analyzed by Northern blotting. Western blotting was used to determine the HO-1 and ferritin protein levels. NADPH-dependent reactive oxygen species （ROS） formation was measured with lucigenin-enhanced chemiluminescence. HO-1 promoter activity in mouse fibroblasts, stably transfected with a 15-kb HO-1 gene that drives expression of the reporter gene luciferase, was assessed usingin vivo bioluminescence imaging. RESULTS： Lansoprazole levels in endothelial cells increased HO-1 mRNA and HO-1 protein levels in macrophages. In addition, lansoprazole-induced ferritin protein levels in both cell systems. Moreover, induction of the antioxidant proteins HO-1 and ferritin by lansoprazole was followed by a decrease in NADPH- mediated ROS formation. The radical scavenging properties of lansoprazole were diminished in the presence of the HO inhibitor, chromium mesoporphyrin IX. Induction of HO-1 gene expression by lansoprazole was not related to oxidative stress or to the activation of the mitogen-activated protein kinase pathway. However, the phosphatidylinositol 3-kinase inhibitor LY294002 showed a concentration-dependent inhibition of HO-1 mRNA and promoter activity.CONCLUSION： Activation of HO-1 and ferritin may account for the gastric protection of lansoprazole and is dependent on a pathway blocked by LY294002.

Gene therapy targeted to telomerase in HCC by AF-hTERT-TK/GCV

Objective： The aim of this study was to observe the affection of targeted therapy to plasmid AF-pGL3-hTERT-TK in HCC cell line HepG2. Methods： We constructed therapeutic plasmid pGL3-hTERT-TK （containing suicide gene TK that promoted by promoter of hTERT） and was conjugated with AF-liposome （a protein that can combine with the receptor ASPGR on HCC cell surface）. Then we transfected HCC cell line HepG2 and hepatic cell L02 with AF-pGL3-hTERT-TK, observed the effects of therapeutic plasmid AF-pGL3-hTERT-TK for HCC cell line growth and apoptosis in vitro by Flow cytometry and Tun- nel method. Results： Our results showed that TK gene was 1100 bp in plasmid pGL3-hTERT-TK. Plasmid pGL3-hTERT-TK can effectively transfect HCC cell HepG2 and the transfection rate was 8.91%. By recognizing and combining effects of recep- tor protein ASPGR on HCC cell surface the therapeutic plasmid AF-pGL3-hTERT-TK could easily enter into HCC cell HepG2 and induce its apeptosis. The apoptosis rate was 85.87% while only 8.65% in L02 cell. Four days after AF-pGL3-hTERT-TK transfected HepG2 was intervention by ganciclovir （GCV）, a lot of apeptotic bodies were found by Tunnel analysis, while little apoptotic body was found in hepatic cell L02. Conclusion： AF-pGL3-hTERT-TK can target to HCC cell line and induce it to apoptesis, almost has no influence on hepatic cell L02. AF-pGL3-hTERT-TK has the potential therapeutic effects for HCC.

Pharmacokinetics and Biodegradation Performance of a Hydroxypropyl Chitosan Derivative

Hydroxypropyl chitosan(HP-chitosan) has been shown to have promising applications in a wide range of areas due to its biocompatibility, biodegradability and various biological activities, especially in the biomedical and pharmaceutical fields. However, it is not yet known about its pharmacokinetics and biodegradation performance, which are crucial for its clinical applications. In order to lay a foundation for its further applications and exploitations, here we carried out fluorescence intensity and GPC analyses to determine the pharmacokinetics mode of fluorescein isothiocyanate-labeled HP-chitosan(FITC-HP-chitosan) and its biodegradability. The results showed that after intraperitoneal administration at a dose of 10 mg per rat, FITC-HP-chitosan could be absorbed rapidly and distributed to liver, kidney and spleen through blood. It was indicated that FITC-HP-chitosan could be utilized effectively, and 88.47% of the FITC-HP-chitosan could be excreted by urine within 11 days with a molecular weight less than 10 k Da. Moreover, our data indicated that there was an obvious degradation process occurred in liver(< 10 k Da at 24 h). In summary, HP-chitosan has excellent bioavailability and biodegradability, suggesting the potential applications of hydroxypropyl-modified chitosan as materials in drug delivery, tissue engineering and biomedical area.

Transactivation of the TIEG1 confers growth inhibition of transforming growth factor-β-susceptible hepatocellular carcinoma cells

AIM:To investigate the role of transforming growth factor(TGF)-β-inducible early gene 1(TIEG1) in TGF-β-induced growth inhibition in hepatocellular carcinoma(HCC) cells.METHODS:Human hepatocyte and HCC cell lines with varied susceptibilities to TGF-β1 were tested by methylthiazoletetrazolium(MTT) assay.The expression changes of Smad2,Smad3,Smad4,Smad7,TIEG1 and TIEG2 gene following treatment with TGF-β1 in a TGF-β-sensitive hepatocyte cell line(MIHA),a TGF-β-sensitive hepatoma cell line(Hep3B) and two TGF-β-insensitive hepatoma cell lines(HepG2 and Bel7404) were examined.SiRNA targeting TIEG1 was transfected into Hep3B cells and the sensitivity of cells to TGF-β1 was examined.Overexpression of TIEG1 was induced by lentiviral-mediated transduction in TGF-β1-resistant hepatoma cell lines(Bel7404 and HepG2).MTT assay and 4',6-Diamidino-2-phenylindole staining were used to identify cell viability and apoptosis,respectively.The expression level of stathmin was measured by reverse transcriptase polymerase chain reaction and Western-blotting analysis,and stathmin promoter activity by TIEG1 was monitored by a luciferase reporter gene system.RESULTS:TIEG1 was significantly upregulated by TGF-β1 in the TGF-β1-sensitive HCC cell line,Hep3B,but not in the resistant cell lines.The suppression of TIEG1 by siRNAs decreased the sensitivity of Hep3B cells to TGF-β1,whereas the overexpression of TIEG1 mediated growth inhibition and apoptosis in TGF-β1-resistant HCC cell lines,which resembled those of TGF-β1-sensitive HCC cells treated with TGF-β1.Our data further suggested that stathmin was a direct target of TIEG1,as stathmin was signif icantly downregulated by TIEG1 overexpression,and stathmin promoter activity was inhibited by TIEG1 in a dose-dependent manner.CONCLUSION:Our data suggest that transactivation of TIEG1 conferred growth inhibition of TGF-β-susceptible human HCC cells.

CLONING PROMOTER OF HUMAN SATBl GENE AND EFFECT OF ATRA AND CoCl_2 ON ITS ACTIVITY

Objective To explore the structure and activity of SATB1 promoter in different cells, ATRA and CoCl2 effect on its activity. Methods Using luciferase system to assay the promoter activity of human SATB1 gene, three luciferase reporter vectors were constructed which driven by different regions of 5＇ untranslated sequence from human SATB1 gene , called pGL3-SP2946-luc , pGL3-SP1718-luc and pGL3-SP751-luc , and transfected into Jurkat T, K562, U937 and Hela cells transiently using lipofectinamine, the expression activity was detected at different dosage of A TRA and CoCl2treatment for different time course. Results The reporter gene expression from SATB1 promoter were high activity in U937 cell, moderate in Jurkat T cell, low activity in K562 cell and showed no obvious activity in Hela cell, the reporter gene expression from pGL3-SP751-1uc kept on the higher lever in Jurkat T, K562 and U937 cells than the other two vectors. We also found that the repressive effect of CoCI2 on SATBI＇s mRNA expression and the relative luciferase expression from pGL3-SP751-luc in U937 cell was down-regu- lated obviously by ATRA and CoCI2 in the concentration- and time-dependent manners. Conclusion SATB1 pro- rooter drives gene expression with cell-specificity and its core promoter region maybe exist in the - 751 - - 9bp of 5＇ untranslated region of human SATB1 gene. Combined with the experiment result we found before that SATB1 was down-regulated by ATRA in U937, the results imply that STAB1 maybe is down-regulated by ATRA and CoCl2 through its promoter in the differentiation of myeloid cell line-U937.

Gaussia Luciferase Reporter Assay for Assessment of Gene Delivery Systems in Vivo

Objective To develop an alternative method for assessment of gene delivery systems in vivo.Methods Mouse primary spleen lymphocytes were genetically modified in vitro by a retroviral vector harboring a Gaussia luciferase(Gluc) expression cassette.After implantation of these cells into recipient mice,the expression of Gluc was detected in whole blood or plasma collected.Results As little as 10 μL whole blood drawn from the recipient mice could guarantee prompt reading of Gluc activity with a luminometer.And the reading was found in good correlation with the number of genetically modified spleen lymphocytes implanted to the mice.Conclusions Gluc may be useful as an in vivo reporter for gene therapy researches,and Gluc blood assay could provide an alternative method for assessment of gene delivery systems in vivo.

A Quantitative Assay for Measuring of Bovine Immunodeficiency Virus Using a Luciferase-based Indicator Cell Line

In order to quantitate the bovine immunodeficiency virus line （BIVL） was established by transfecting baby hamster kidney （BIV） cells infection in vitro, a BIV indicator cell with reporter plasmids containing the firefly luciferase gene driven by a BIV long terminal repeat promoter. The BIV activates promoter activity of the LTR to express luciferase upon infection. BIV infection could therefore by quantified by detection of luciferase activity. Compared to standard assays used to detect BIV infection, the BIVL-based assay is 10 times more sensitive than the the CPE-based assay, and has similar sensitivity with the viral capsid protein Western blot assay BIV indicator cell line could detect BIV infection specifically. Luciferase activity of BIV infected BIVL cells showed a time dependent manner, and 60 h post infection is the optimal time to detect BIV infection. Luciferase activity of BIVL cells correlates with the BIV capsid protein expression. Moreover, a linear relationship was found between MOI and the activated intensity of luciferase expression. In brief, the BIV indicator cell line is an easy, robust and quantitive method for monitoring BIV infection.

An Accurate Model for the Expression of the Antisense RNA Block Gene:the Firefly Luciferase Gene-Xenopus Oocyte System

The plasmid p^(SV-Luc20)or the mRNA of luciferase gene transcribed from p^(SP64-Luc12)was introduced intothe nucleus or cytoplasm of Xenopus oocytes at stages 5-6 by microinjection.Then the injectedoocytes were incubated in MB medium at 18℃ for definite periods,and the crude enzyme ofluciferase was prepared.Results indicated that the luciferase gent and its mRNA could be transcribedand translated into the enzymatic protein of luciferase with high biological activity,and could alsocatalyze the substrates to emit light.If different ratios of firefly lucifcrasc gene and its antisense RNA were introduced together into thenucleus or cytoplasm of Xenopus oocytes,then the expression of firefly luciferase gone was severelyblocked.Since the lucifcrasc activity can be measured rapidly and quantitatively and the Xenopusoocytes obtained easily,the firefly luciferase gene-Xenopus oocyte system is an excellent model forrevealing quantitatively how the antisense RNA can block gene expression.

应用改构的炭疽毒素作为靶向肿瘤细胞的药物递送系统及其效果评价

目的:通过改造炭疽毒素保护性抗原Protective Antigen(PA)及致死因子Lethal Factor(LF),尝试建立更加广谱的新型炭疽毒素靶向给药系统并对其递送效率进行定量评价。方法:采用基因工程手段,分别构建了3种改构的天然炭疽毒素保护性抗原PA及炭疽毒素的LF N端融合海肾荧光素酶(Luciferase)的LFn-linker-Luc的大肠杆菌重组表达体系。利用CCK-8法评价改构PA和LF共同作用肿瘤细胞后的细胞存活率;利用改构PA和LFn-linker-Luc与肿瘤细胞共孵育,通过测定细胞内荧光素酶活性,评价改构PA靶向肿瘤细胞的效果。结果:体外酶解实验证明构建的改构PA蛋白能够被正确地酶解成目的大小的片段;改构PA和LF共同作用肿瘤细胞能够显著降低细胞存活率;利用LFn-linker-Luc能够评价改构PA的靶向效率,PA蛋白的改构方式与其递送效率相关。结论:设计并改构的炭疽毒素药物递送系统,能够实现特异性靶向肿瘤细胞的效果,并具有更广谱的作用效果,为研制新型广谱抗肿瘤药物提供了新的思路和方法。

Genomic structure and evolution of beta-defensin genes in the golden pheasant and hwamei

Defensins play a vital role in the early stage of infection before adaptive immune responses are generated. Thus far, only limited detailed genomic data for avian defensin genes have been described. Here, using bacterial ar- tificial chromosome libraries, we found that a 95 kb and 177 kb sequences in the golden pheasant （Chrysolophus pictus, Galliformes） and hwamei （Garrulax canorus, Passeriformes） corresponds to 16 and 30 defensin genes, respectively. Fluorescence in situ hybridization assigned defensin gene clusters in the golden pheasant and hwamei to chromosomes 2q and 3q, respectively. In combination with the previous chicken （Gallus gallus, Galliformes） and zebra finch （Taeniopygia guttata, Passeriformes） defensin clusters, the comparative genomic analysis revealed that lineage- specific duplications and deletions have given rise to clearly different genomic structures. On the basis of genomic char- acteristics, we further found that transposable elements act as agents of evolution, causing direct and indirect copy number variations in defensin genes via duplications. Tissue ex- pression analysis showed that the Passeriformes-specific duplicated AvBD1 and AvBD3 genes are mainly expressed in the upper and lower respiratory tract, tongue, and spleen. Our analyses indicate that the duplication-and-deletion of defensin genes conformed to the birth-and-death evolutionary process and that transposable elements induced theduplication of defensin genes. Moreover, the respiratory system-specific expression pattern of Passeriformes-specific expanded AvBD1 and AvBD3 genes suggests their important role in maintaining the singing trait of songbirds. The understanding of the genomic structure, expression, and evolution of defensin genes can provide a crucial immunological foundation to study and prevent avian diseases.