Hepatitis B virus envelope as a targeting gene transfer vector for hepatic cancer cells

Objective： The aim of the study was to observe the transfection efficacy of hepatitis B virus envelope （HBVE） and evaluate its ability as a gene transfer vector for liver cancer cells. Methods： To obtain HBVE, the supematant fluid of HepG 2.2.15 cells was mixed with a PEG8000 solution for concentration and was inactivated by β-propiolactone. The acquired HBVE was used to pack plRES2-EGFP to test its package ability. Then, we examined its quantity and quality with ELISA, PCR, SDS-PAGE and electron microscopy. The plRES2-EGFP was packed with HBVE and obtained the product HBVE-GFP. The plRES2-EGFP was packed with liposome and obtained the product liposome-GFP. HBVE-GFP and liposome-GFP were used to transfer HepG 2 cells to study the transfection efficiency. HBVE-GFP was used to transfer HepG 2, A549, HeLa and FB cells to study the targeting ability. The green fluorescent protein （GFP） expression was observed under a fluorescent microscope. The rate of GFP positive cells was determined by flow cytometry. Results： 1. The acquired HBVE could retain the surface protein HBsAg ＋ pre S1 ＋ pre S2 and had no virus DNA. It had good package ability for plRES2-EGFP. 2. Transfection efficiency： The GFP could be observed in both the liposome group and HBVE group under the fluorescent microscope. But the HBVE group had a higher fluorescent intensity than liposome group. The transfection rate of liposome group was 49.97% ＋ 2.37% while the HBVE group was 70.65% ＋ 3.15% and the fluorescent intensity of the HBVE group was 3-4 times （P = 0.000） for liposome group with the determination of flow cytometry. 3. Targeting ability： The GFP could be observed in the four groups under the fluorescent microscope. The HepG 2 group had the highest fluorescent intensity among the four groups. The transfection rate of HepG 2 group was 71.35% ＋ 0.03% which was highly expressed than other groups （P = 0.000） and the fluorescent intensity of the HepG 2 group was 2-3 times （P = 0.000） for the other 3 groups with the determination of flow cytometry. Conclusion： HBVE can be constructed successfully with the methods of PEG8000 and β-propiolactone from the supernatant fluid of HepG 22.15 cells. The HBVE can be a candidate gene transfer vector for liver cancer cells.

TP53基因在弥漫性大B细胞淋巴瘤中作用的研究进展

弥漫性大B细胞淋巴瘤(DLBCL)是全世界范围内发病率和病死率较高的恶性肿瘤之一。目前以药物治疗为主的综合治疗可有效改善DLBCL患者的转归,但仍有部分患者疗效不佳。抑癌基因TP53在肿瘤发生、发展过程中发挥着至关重要的作用,其表达变化与肿瘤细胞增殖、侵袭及迁移密切相关。TP53基因突变对DLBCL的发生、发展及预后分层的影响是近年研究的热点。深入研究TP53基因突变对DLBCL预后的影响,可为其靶向治疗及预后判断提供信息参考,且具有重要的临床应用价值。

基于捕获法的靶向测序技术在弥漫大B细胞淋巴瘤IgH基因重排检测中的应用价值

目的探讨基于捕获法的靶向测序技术在弥漫大B细胞淋巴瘤IgH基因克隆性重排检测中的应用价值。方法收集2020年3月~2022年1月苏州大学附属第一医院送检的DLBCL标本99例,提取患者基因组DNA,使用基于捕获法的靶向测序技术进行IgH基因克隆性重排检测。结果27.3%(27/99)患者检测到IgH基因完全IgHV-IgHD-IgHJ重排,50.5%(50/99)患者检测到IgH基因不完全IgHD-IgHJ重排,IgH基因克隆性重排联合检出率为66.7%(66/99)。27例患者检测到32种IgHV-IgHD-IgHJ重排,50例患者检测到52种IgHD-IgHJ重排,在IgHV-IgHD-IgHJ重排中,IgHV3、IgHD3和IgHJ4家族基因的取用频率最高,在IgHD-IgHJ重排中,IgHD3和IgHJ4家族基因的取用频率最高。3例患者捕获到未知IgH基因融合伴侣,其中2例为MIR4507-IgH融合,1例为IRF8-IgH融合。结论基于捕获法的靶向测序技术不仅可以检测IgH基因克隆性重排具体方式,还能发现IgH基因未知融合伴侣,对DLBCL诊断及个体化治疗策略制定具有重要意义。

AEG1 induces COX-2 expression via activation of NF-κB and AP-1 in hepatoma HepG2 cells

Objective： The aim of this study was to investigate whether astrecyte elevated gene 1 （AEG1） regulates COX-2 expression in human hepatoma HepG2 cells and related pathways involved in this process. Methods： Human hepatoma HepG2 cells were transfected with pcDNA3.1（-）-AEG1 plasmid or psilencer2.0-AEGl-shRNA1 plasmid to up/down-regulate AEG1 expression, pcDNA3.1（-） and psilencer 2.0 empty vector plasmids were transfected respectively as control. Real-time RT-PCR was carried out to measure the expression levels of AEG1 and COX-2 mRNA. The expression levels of AEG1 and COX-2 protein were detected by Western blot. NF-KB signaling was blocked by PDTC, and AP-1 signaling was blocked by curcumin. Results： AEG1 mRNA and protein levels were increased after pcDNA3.1（-）-AEG1 transfection, and decreased after psilencer2.0-AEGl-shRNAs transfection. COX-2 mRNA and protein levels were increased in AEGl-overexpressing cells and decreased in AEGl-knockdown cells. Phosphorylations of p65 and c-jun were up-regulated in AEGl-overexpressing cells. Both PDTC and curoumin reduced COX-2 expression in HepG2 cells with AEG1 overexpression. Conclusion： AEG1- overexpressing and -knockdown HepG2 cells are established successfully. AEG1 could induce COX-2 expression though activating NF-KB and AP-1 in human hepatoma HepG2 cells.

Cell Signaling in Viral and Oncogenic Pathogenesis and Its Implications in Disease Diagnosis and Prognosis

Both viral diseases and cancer account for a large proportion of serious health problems. Viral infection and cancer are biologically and medically correlated and in many ways share common cellular pathways that lead to disease development or progression. Better understanding how these signaling events are specifically activated by different pathogenic stimuli and how they activate different downstream transcriptions in response to these stimuli at high specificity and efficiency will provide a new molecular basis for the development of novel disease biomarkers and therapeutic or preventive targets against both classes of diseases. Research in our laboratory has been prompted to investigate the regulation and modes of action of these pathways, with a more intensive focus on the NF-κB signaling, in the settings of severe or oncogenic viral infection as well as cancer development. It is hoped that our research will lead to eventual clinical application of biomarkers derived from these signaling pathways.

Neuroprotective effect of Angiopep-2 peptide modified scutellarin-loaded PEGylated PAMAM dendrimer nanoparticles on ischemic stroke by modulating the Toll-like receptors-dependent MyD88/IKK/NF-κB signaling pathway

OBJECTIVE The greatest challenge in chemotherapy of ischemic stroke is the construction a suitable delivery system to overcome the poor physicochemical properties of drug and its low permeability across the blood brain barrier(BBB).METHODS In the present study,dendrimer,polyamidoamine(PAMAM),was synthesized as the nano-drug carriers.Angiopep-2,which has been proved excellent ability to cross the BBB,was exploited as the targeting ligand to conjugate PAMAM via bifunctional polyethylene glycol(PEG).Then scutellarin(STA)was encapsulated into the functionalized nanoparticles(NPs)to formulate Angiopep-2 modified STA-loaded PEG-PAMAM NPs.Ischemic stroke model was established to evaluate the treatment efficacy and protective mechanism of Angiopep-2-STA-PEG-PAMAM NPs.RESULTS The pharmacokinetics and biodistribu-tion demonstrated that Angiopep-2-STA-PEG-PAMAM NPs exhibited significantly higher plasma concentration from 1 h to 10 h after intravenous administration and improve accumulation in brain(4.7-fold)compared with STA solution.Moreover,prolonged elimination half-life(4.8-fold)and lower clearance(3.4-fold)were observed.The brain uptake study of 6-coumarin confirmed that Angiopep-2-PEG-PAMAM NPs possessed better brain targeting efficacy(3.2-fold)than PEG-PAMAM NPs.Angiopep-2-STA-PEG-PAMAM NPs obviously ameliorated infarct volume,neurological deficit,histopathological severity and neuronal apoptosis.In addition,Angiopep-2-STA-PEG-PAMAM NPs markedly inhibited the calcium content and the levels of IL-12p40,IL-13,IL-17 and IL-23.Furthermore,Angiopep-2-STA-PEG-PAMAM NPs significantly decreased the m RNA and protein expressions of HMGB1,TLR2,TLR4,TLR5,My D88,TRIF,TRAM,IRAK-4,TRAF6,IкBα,IKKβand NF-кBp65.CONCLUSION The results suggested that Angiopep-2modified scutellarin-loaded PEG-PAMAM nanocarriers possessed remarkable neuroprotective effects on ischemic stroke through modulation of inflammatory cascades and HMGB1/TLRs/MyD 88-induced NF-κB activation pathways.

Construction and application of gene targeting replacement vector formouse coagulation factor IX gene

Objective: To construct and apply a replacement targeting vector for mouse coagulation factor IX(mFIX) gene in embryonic stem (ES) cells. Methods: Based on the cloning and structural analysis of the genomic DNA fragment of coagulation factor IX gene from 129Sv mouse genomic DNA A phage library, PMFIXDEL plasmid was designed with positive-negative-selection (PNS) strategy, and constructed with commonmolecular cloning techniques. Structure of PMFIXDEL was identified by PCR and restriction analysis. Afterelectroporation with the linearized PMFIXDEL DNA, transfected ES cells were cultured in G418/GANC drugselection medium. The recombination efficacy of this vector was tested. Results: The main components ofPMFIXDEL were two copies of negative selection gene (HSV-tk expression cassette), a positive selectiongene (Neo expression cassette), long and short homologous fragments and plasmid backbone. The introduction of negative selection gene (HSV-tk ) into the construct resulted in 24-fold increase of selection.Conclusion: An effective replacement vector for mFIX gene targeting was constructed and applied in ES cell.

EGFR、蛋白激酶B在宫颈癌中表达及EGFR胞外区基因突变的研究

目的:探讨宫颈癌中表皮生长因子受体(EGFR)、蛋白激酶B(PKB/Akt)的表达及EGFR胞外区基因突变的规律和临床意义,为宫颈癌的发生发展机制和靶向个性化治疗提供实验依据。方法:采用免疫组化SP法检测EGFR、Akt蛋白在16例正常宫颈组织、40例宫颈上皮内瘤变(CIN)组织和60例宫颈癌组织中的表达,并分析其与宫颈癌临床病理的关系。采用酚氯仿法抽提60例宫颈癌组织中DNA,PCR+DNA双向测序法检测EGFR胞外IV亚区基因突变。结果:正常宫颈组、CIN组及宫颈癌组中EGFR蛋白的阳性表达率分别为13.33%、43.33%及76.67%,Akt蛋白的阳性表达率分别为6.25%、35.00%及68.33%,差异有统计学意义(P<0.05)。宫颈癌组织中,EGFR表达水平与病理类型、细胞分化程度和临床分期有关(P<0.05),但与患者年龄和淋巴结转移无关(P>0.05);Akt表达水平与细胞分化程度和临床分期有关(P<0.05),但与患者年龄、病理类型和淋巴结转移无关(P>0.05)。60例宫颈癌中EGFR基因外显子15突变率为3.33%(2/60),突变类型G→A,未发现外显子14突变。宫颈癌组织中EGFR、Akt表达呈正相关性(r=0.556,P<0.05)。结论:EGFR、Akt可作为检测宫颈癌及癌前病变的重要参考指标,也为宫颈癌的多靶点联合治疗提供新思路;宫颈癌中EGFR胞外区可有基因突变,但突变率很低,关于其突变的规律和意义有待进一步研究。

真核表达载体pcDNA3.1-myc-his(-)B的改构与应用

目的:对真核表达载体pcDNA3.1-myc-his(-)B进行改构,以简化目的基因克隆的步骤。方法:用事先设计好的2条寡核苷酸链互相退火,替换pcDNA3.1-myc-his(-)B的多克隆位点中NotⅠ与HindⅢ间的部分;利用改构的真核表达载体,分别构建亲环蛋白A、胆绿素还原酶B和过氧化氢酶基因的重组真核表达载体,瞬时转染293T细胞后,用His标签抗体验证其表达。结果:测序结果证实已将预想的序列替换至pcDNA3.1-myc-his(-)B的多克隆位点中相应位置,改构为质粒pcDNA3.1(-)reconstruct;利用改构的真核表达载体表达了相应的目的基因。结论:改构的真核表达载体pcDNA3.1(-)reconstruct可以简化融合myc和His标签的目的基因克隆的过程,并且增加了限制性位点的可选择性。

miR-21在弥漫大B细胞淋巴瘤肿瘤组织中的表达及临床意义

本研究旨在检测弥漫大B细胞淋巴瘤(DLBCL)患者肿瘤石蜡组织中miR-21的差异性表达与PTEN相关性及其临床意义。采用Real-time-PCR方法检测26例DLBCL患者及10名正常对照的miR-21表达,应用聚合物免疫组化检测系统检测PTEN蛋白水平。结果表明,26例DLBCL石蜡组织中miR-21的表达量为6.586(1.10,38.22),显著高于正常对照[0.791(0.35,2.87)](P<0.05),在26例DLBCL中有6例PTEN蛋白(23%)阳性,而在20例(77%)中为阴性。DLBCL中miR-21表达水平与PTEN蛋白水平呈负相关,其高表达与DLBCL血清LDH水平呈正相关、Ⅲ/Ⅳ期患者miR-21表达水平明显高于Ⅰ/Ⅱ期患者(P<0.05),与临床亚型无关(P>0.05)。结论:在DLBCL中miR-21表达升高可能提示肿瘤恶性程度高,预后不良,PTEN可能是miR-21的靶基因。

miR-181b-5p靶向调控Nr6a1抑制GC-1spg细胞的增殖与迁移

Micro RNA(mi RNA)是一段长约为22 nt的内源性非编码单链RNA,研究表明mi RNA对正常精子发生和雄性发育必不可少。为了探究mi R-181b-5p在雄性生殖细胞发育中的功能以及寻找其靶基因,首先采用流式细胞术、噻唑蓝检测以及划痕实验分析了mi R-181b-5p在小鼠GC-1spg精原细胞系中的生物学效应,随后利用Targetscan和GO数据库等生物信息学软件预测发现生殖核受体Nr6a1可能为其靶基因,进而通过双荧光素酶报告基因实验以及q PCR证实了mi R-181b-5p与Nr6a1的靶向识别关系,最后采用细胞功能回补实验明确了mi R-181b-5p通过靶向调控Nr6a1来抑制GC-1spg细胞的增殖和迁移。结果表明mi R-181b-5p通过其靶基因Nr6a1在精子发生中起到负调控的作用。

类转录激活因子效应物核酸酶介导的E-cad和Bmi-1基因联合干预鼻咽癌的体外研究

目的:检测类转录激活因子效应物核酸酶(transcription activator-like effector nuclease,TALEN)介导的E-钙黏蛋白(E-cadherin,E-cad)、B细胞特异性莫洛尼氏白血病毒插入位点1(B-lymphoma Moloney murine leukemia virus insertion region-1,Bmi-1)基因联合干预对鼻咽癌细胞生物学行为的影响。方法:构建多位点基因打靶载体p UC-DS1-E-cad-2ANeo-DS2(以下简称E-cad打靶载体)和p UC-DS1-Bmi-1 sh RNA-Zeo-DS2(以下简称Bmi-1打靶载体),将E-cad打靶载体、Bmi-1打靶载体及佛山科学技术学院分子医学研究院前期构建的TALEN载体以不同组合转染鼻咽癌CNE-2细胞。采用PCR检测靶基因的整合,Western印迹检测靶基因蛋白表达水平变化,细胞计数试剂盒(cell counting kit-8,CCK-8)检测细胞增殖能力变化,流式细胞仪检测细胞周期和细胞凋亡变化,Transwell实验检测细胞迁移和侵袭能力变化。结果:靶基因E-cad和Bmi-1 sh RNA表达元件成功整合到鼻咽癌CNE-2细胞的基因组中;转染后鼻咽癌CNE-2细胞中E-cad的蛋白表达水平明显上升,Bmi-1的蛋白表达水平明显下降;干预E-cad及Bmi-1不影响细胞的增殖、周期和凋亡,但显著抑制细胞的迁移和侵袭能力,且E-cad和Bmi-1联合干预比单独干预更能显著抑制鼻咽癌CNE-2细胞体外迁移能力(均P<0.01)。结论:TALEN介导的E-cad和Bmi-1联合干预能有效抑制鼻咽癌CNE-2细胞的体外迁移和侵袭,可为人类癌症的基因治疗建立前期的实验基础。

Effect of mutated IKBa transfection on multidrug resistance in hilar cholangiocarcinoma cell lines

AIM: To explore the expression effect of mutated IκBα transfection on multidrug resistance gene (MDR-1) in hilar cholangiocarcinoma cells by inhibiting the activity of nuclear transcription factor-κB (NF-κB). METHODS: We used the mutated IicBa plasmid to transfect QBC939HCVC+ cells and QBC939 cells, and electrophoretic gel mobility shift assay (EMSA) to detect the binding activity of NF-κB DNA and the effect of the transfrecting mutated IκBα plasmid on multidrug resistance gene (MDR-1) in hilar cholangiocarcinoma cells and its expression protein (P-GP). RESULTS: Plasmid DNA was digested by restriction enzymes Xbal and Hand III, and its product after electrophoresis showed two bands with a big difference in molecular weight, with a size of 4.9 kb and 1.55 kb respectively, which indicated that the carrier was successfully constructed and digested with enzymes. The radioactivity accumulation of QBC939HCVC+ and QBC939 cells transfected with mutated IκBα plasmid was significantly lower than that of the control group not transfected with mutated IκBα plasmid. Double densimeter scanning showed that the relative signal density between the tansfection group and non-transfection group was significantly different, which proved that the mutated IκBα plasmid could inhibit the binding activity of NF-KB DNA in hilar cholangiocarcinoma cells. Compared to control group not transfected with m IκBα plasmid, the expression level of MDR-1mRNA in the QBC939 and QBC939HCVC+ cells transfected with mutated IκBα plasmid was lower. The expression intensity of P-GP protein in QBC939 and QBC939HCVC+ cells transfected with mutated IκBα was significantly lower than that of the control group not transfected with mutated IκBα plasmid. CONCLUSION: The mutated IκBα plasmid transfection can markedly reverse the multidrug resistance of hilar cholangiocarcinoma cells. Interruption of NF-κB activity may become a new target in gene therapy for hilar cholangiocar-cinogenesic carcinoma.

B-Raf基因在非小细胞肺癌中的表达及其临床意义

非小细胞肺癌(NSCLC)是肺癌的一种,它具有极高的病死率。在NSCLC的发生、发展中丝裂原活化蛋白激酶(MAPK)通路异常激活起重要作用,而B-Raf基因是MAPK通路中MEK/ERK最为关键的激活因子,进而对肿瘤细胞的增殖、分化和凋亡等方面起作用。B-Raf基因表达异常存在于NSCLC中并影响它的发生及预后,针对B-Raf基因的靶向治疗是一个新的研究热点。

bta-miR-34b/c和bta-miR-449a/b/c靶基因预测及生物信息学分析

【目的】试验旨在对bta-miR-34b/c和bta-miR-449a/b/c的序列保守性、转录因子和靶基因进行生物信息学预测和分析,探究其影响精子发生的作用机制,为研究其生物学功能及作用机制提供指导和思路。【方法】利用miRBase数据库获取不同物种的bta-miR-34b/c和bta-miR-449a/b/c序列并进行相似性分析;通过TargetScan、miRWalk、miRDB等方法预测bta-miR-34b/c和bta-miR-449a/b/c的靶基因,对获得的靶基因集合分别进行GO功能和KEGG通路富集分析;通过AnimalTFDB获取共同调控bta-miR-34b/c和bta-miR-449a/b/c的转录因子,并构建TF-miRNA-mRNA作用网络。【结果】bta-miR-34b/c和bta-miR-449a/b/c在不同物种间高度保守。预测得到2个转录因子和49个候选靶基因,这些靶基因主要富集在精子发生、钙离子依赖性胞吐的调节、胰岛素分泌的调节等GO条目,以及HIF-1信号通路、甲状腺激素信号通路、Wnt信号通路等KEGG通路。bta-miR-34b/c、bta-miR-449a/b/c与上游转录因子NR2C2和HIC1,下游靶基因AXL、E2F3、DAAM1等共同构成的作用网络通过调控减数分裂、细胞周期、细胞凋亡、精子细胞能量代谢等生物过程影响精子发生。【结论】bta-miR-34b/c、bta-miR-449a/b/c通过上游转录因子和下游靶基因组成的分子调控网络共同调节精子发生过程。

MiR30b与小鼠Bach2相互作用的研究

目的研究B细胞末端分化过程中miR30b新的作用靶点。方法经生物信息学分析,利用特异性引物以及定点突变引物从小鼠c DNA中分别扩增大小为530、361和189bp三个目的片段,胶回收并对361和189bp进行拼接,获得野生型、突变型小鼠Bach2 mRNA特异性片段,分别与pmir GLO载体连接,构建野生型、突变型重组荧光素酶报告基因质粒pmir GLO-m Bach2、pmir GLO-m Bach2 mt;与miR30b共转染至HEK293 T细胞,分析其荧光素酶活性。结果重组荧光素酶报告基因质粒经双酶切及测序证实构建正确;共转染后,与pmir GLO+miR30b组相比,pmir GLO-m Bach2+miR30b组的荧光素酶活性明显降低(P<0.05),而pmir GLO-m Bach2 mt+miR30b组的荧光素酶活性则无明显改变(P>0.05)。结论小鼠Bach2 mRNA是miR30b的靶点。

Reversion of Multidrug-Resistance by Proteasome Inhibitor Bortezomib in K562/DNR Cell Line

Objective：To observe the reversion of multi-drug resistance by proteasome inhibitor bortezomib in K562/DNR cell line and to analyze the possible mechanism of reversion of multidrug-resistance.Methods：MTT method was used to determine the drug resistance of K562/DNR cells and the cellular toxicity of bortezomib.K562/DNR cells were cultured for 12 hours,24 hours and 36 hours with 100 μg/ml DNR only or plus 4 μg/L bortezomib.The expressions of NF-κB,IκB and P-gp of K562/DNR were detected with Western blot method,the activity of NF-κB was tested by ELISA method and the apoptosis rate was observed in each group respectively.Results：The IC50 of DNR on cells of K562/S and K562/DNR groups were 1.16 μg/ml and 50.43 μg/mL,respectively.The drug-resistant fold was 43.47.The IC10 of PS-341 on Cell strain K562/DNR was 4 μg/L.Therefore,4 μg/L was selected as the concentration for PS-341 to reverse drug-resistance in this study.DNR induced down-regulation of IκB expression,up-regulation of NF-κB and P-gp expression.After treatment with PS-341,a proteasome inhibitor,the IκB degradation was inhibited,IκB expression increased,NF-κB and P-gp expression decreased in a time dependent manner.Compared to DNR group,the NF-κB p65 activity of DNR＋PS-341 group was decreased.Compared to corresponding DNR group,DNR induced apoptosis rate increases after addition of PS-341 in a time dependent manner.Conclusion：Proteasome inhibitor bortezomib can convert the leukemia cell drug resistance.The mechanism may be that bortezomib decreases the degradation of IκB and the expression of NF-κB and P-gp,therefore induces the apoptosis of multi-drug resistant cells.

细胞死亡诱导p53靶蛋白1—一种新型凋亡蛋白的功能研究进展

细胞死亡诱导p53靶蛋白1(CDIP1)是一种新型凋亡蛋白,参与外源性凋亡通路、内源性凋亡通路及细胞焦亡相关通路,是多通路介导细胞凋亡的关键调节因子。近年来研究发现,CDIP1通过与凋亡因子相结合,参与多种肿瘤、心血管疾病的发生发展。然而,目前CDIP1调控细胞凋亡的分子机制、上下游调控因子及在各类肿瘤及疾病的作用机理亟待深入研究和挖掘。该文就CDIP1在细胞凋亡通路中的功能研究进展,不同凋亡通路中与B细胞受体相关蛋白31(BAP31)、凋亡相关基因-2(ALG2)等蛋白分子的作用机制及其在疾病中的作用进行综述。

鳖甲煎丸对CCl4致大鼠肝纤维化模型中NF-κB信号通路的影响

目的：通过研究鳖甲煎丸对四氯化碳（CCl4）致大鼠肝纤维化的抑制作用及其对核转录因子-κB（NF-κB）信号通路信号分子及靶基因的影响,探讨鳖甲煎丸抗肝纤维化的作用机制。方法：使用CCl4复制肝纤维化大鼠模型,鳖甲煎丸药物溶液灌胃,8周末腹主动脉采血,留肝组织检测指标。苏木素-伊红（HE）染色观察肝脏病理形态学改变;酶联免疫吸附法（ELISA）检测各组大鼠血清中肝纤维化4项,基质金属蛋白酶（MMP）-2,MMP-9,组织基质金属蛋白酶抑制剂-1（TIMP-1）的表达;免疫组化法检测大鼠肝组织中p65,转化生长因子-β1（TGF-β1）的表达;蛋白免疫印迹法（Western blot）检测大鼠肝组织中p65,NF-κB抑制蛋白（IκB）α,IκB激酶（IKK）α/β,TGF-β1的表达。结果：病理学结果显示模型组肝小叶被破坏,纤维组织增生严重,而鳖甲煎丸治疗组肝细胞坏死减少,纤维组织增生明显减少,纤维间隔薄;与模型组比较,鳖甲煎丸治疗组大鼠肝纤维化4项指标,TIMP-1含量明显降低（P〈0.05）,而MMP-2,MMP-9含量明显上升（P〈0.05）;免疫组化结果显示鳖甲煎丸治疗组肝组织p65,TGF-β1表达较模型组明显降低,Western blot检测结果显示与模型组比较,鳖甲煎丸治疗组肝组织中p65,TGF-β1蛋白表达明显减少（P〈0.05）,IκBα蛋白表达明显增加（P〈0.05）,而IKKα/β则无显著变化。结论：鳖甲煎丸能够显著减轻四氯化碳致大鼠肝纤维化的程度,减缓其发展,主要与鳖甲煎丸能够抑制p65的表达,从而阻断NF-κB信号通路,减少其下游靶基因TIMP-1,TGF-β1的合成,上调MMP-2,MMP-9的表达,从而加快细胞外基质的降解有关。

大肠杆菌-长双歧杆菌穿梭载体的构建及PTEN在长双歧杆菌中的表达

长双歧杆菌可特异地定植于实体瘤低氧区,可用做肿瘤靶向性基因治疗的载体,而构建大肠杆菌-长双歧杆菌穿梭质粒则被证明是外源基因在长双歧杆菌中稳定表达的有效途径。为了构建能在长双歧杆菌中稳定表达外源基因的穿梭质粒并检测携带抑癌基因的工程菌对小鼠实体瘤的抑制效果,利用软件设计并合成了48条部分序列相互重叠的引物,通过PCR合成了长双歧杆菌质粒pMB1序列及长双歧杆菌HU启动子区序列,插入克隆载体pMD18-T,构建穿梭载体pMB-HU,该载体可在大肠杆菌DH5α及长双歧杆菌L17中稳定复制。PTEN基因编码具有蛋白质和酯类双重特异性磷酸酶活性的抑癌因子。将PTEN基因cDNA序列插入载体pMB-HU中HU启动子下游,构建重组质粒pMB-HU-PTEN,电击转化长双歧杆菌后,Western blot检测表明,表达产物中存在55kDa的PTEN蛋白特异条带。抑癌试验表明:与对照组相比,携带PTEN基因的长双歧杆菌可显著抑制小鼠实体瘤的生长。上述结果为以长双歧杆菌为载体的实体瘤靶向性基因治疗研究奠定了基础。