Suppression of human papillomavirus type 16 E5 oncoprotein:A promising step in fostering the treatment of cervical cancer

Cervical cancer is a growing global disease in developing countries.Persistent infection with human papillomaviruses(HPV)is an essential causative agent in this type of cancer.Several studies demonstrate HPV E5 oncoprotein can impress the normal life cycle of HPV-infected cells by targeting some pivotal cellular signaling pathways,such as the epidermal growth factor receptor(EGFR)signaling pathway.In this study,we used E5-siRNA to knockdown that essential oncogene and considered the effect of E5 silencing on proliferation,apoptosis,cell cycle,apoptosis-related gene expression,and the initiator of the EGFR signaling pathway in cervical cancer cells.The results demonstrate that E5 plays an essential role in the proliferation and inhibited apoptosis in cervical cancer.Furthermore,silencing E5 reduces proliferation,increases apoptosis,and elevates related-genes expression of these malignant cells.Overall,E5 suppression may be appropriate for ameliorating cervical cancer progression.

Transplantation of human placental chorionic plate-derived mesenchymal stem cells for repair of neurological damage in neonatal hypoxic-ischemic encephalopathy

Neonatal hypoxic-ischemic encephalopathy is often associated with permanent cerebral palsy,neurosensory impairments,and cognitive deficits,and there is no effective treatment for complications related to hypoxic-ischemic encephalopathy.The therapeutic potential of human placental chorionic plate-derived mesenchymal stem cells for various diseases has been explored.However,the potential use of human placental chorionic plate-derived mesenchymal stem cells for the treatment of neonatal hypoxic-ischemic encephalopathy has not yet been investigated.In this study,we injected human placental chorionic plate-derived mesenchymal stem cells into the lateral ventricle of a neonatal hypoxic-ischemic encephalopathy rat model and observed significant improvements in both cognitive and motor function.Protein chip analysis showed that interleukin-3 expression was significantly elevated in neonatal hypoxic-ischemic encephalopathy model rats.Following transplantation of human placental chorionic plate-derived mesenchymal stem cells,interleukin-3 expression was downregulated.To further investigate the role of interleukin-3 in neonatal hypoxic-ischemic encephalopathy,we established an in vitro SH-SY5Y cell model of hypoxic-ischemic injury through oxygen-glucose deprivation and silenced interleukin-3 expression using small interfering RNA.We found that the activity and proliferation of SH-SY5Y cells subjected to oxygen-glucose deprivation were further suppressed by interleukin-3 knockdown.Furthermore,interleukin-3 knockout exacerbated neuronal damage and cognitive and motor function impairment in rat models of hypoxic-ischemic encephalopathy.The findings suggest that transplantation of hpcMSCs ameliorated behavioral impairments in a rat model of hypoxic-ischemic encephalopathy,and this effect was mediated by interleukin-3-dependent neurological function.

New categorization of human vascular endothelial cells by pro-vs anti-proliferative phenotypes

AIM: To integrally understand the effects of human vascular endothelial cells(VECs) on the proliferation of vascular smooth muscle cells(VSMCs).METHODS: Various kinds of human VECs of different origins were co-cultured with human aortic smooth muscle cells, a representative of human VSMCs. To exclude the irrelevant effects due to growth competition between VECs and VSMCs, the proliferation of VECs had previously been arrested via a low-dose gamma rayirradiation. To discriminately analyze the proliferation of VSMCs from that of VECs, the former cells were labeled with red fluorescent dye while the latter cells were labeled with green fluorescent dye before performing coculture experiments. After 4 d, total cells were harvested and subjected to flow cytometric analyses. Decrements in red fluorescence intensities due to proliferationmediated dilutions were measured and mathematically processed using a specific software to quantitatively evaluate the proliferation of VSMCs. The findings obtained from the flow cytometry-based analyses were further validated by microscopic observations. RESULTS: Commercially available primary cultured human VECs exclusively promoted VSMC proliferation regardless of their tissue origins and we termed these pro-proliferative VECs as "typeⅠ". By contrast, VECs freshly generated from human bone marrow-derived endothelial progenitors cells or human pluripotent stem cells including embryonic stem cells and induced pluripotent stem cells suppressed VSMC proliferation and we termed these anti-proliferative VECs as "typeⅡ". Repetitive subcultures as well as oxidative stress induced "type Ⅱ VECs to typeⅠ" conversion along with an induction of Regulator of G-protein signaling 5(RGS5)Compatibly, anti-oxidant treatments suppressed both the subculture-dependent "typeⅡ to typeⅠ" conversion and an induction of RGS5 gene. Immunostaining studies of clinical specimens indicated that RGS5 protein expressions in endothelial layers were low in norma arteries but they were up-regulated in pathologica arteries including hypertension, atherosclerosis and autoimmune vasculitis in a dose-dependent manner Overexpression and knockdown of RGS5 caused that"typeⅡ to typeⅠ" and "typeⅠ to type Ⅱ" phenotype conversions of VECs, respectively. CONCLUSION: Human VECs are categorized into two types: pro-proliferative RGS5^(high) VECs(typeⅠ) and antiproliferative RGS5 ^(low) VECs(typeⅡ).

The Apoptotic Effect of the Methanol Extract of <i>Polygonum cuspidatum</i>through Up-Regulation Death Receptor 5 and CHOP in HSC-2 Human Oral Cancer Cells

Polygonum cuspidatum is used as a traditional medicinal herb for the therapy of various diseases including several types of cancers. In the present study, we focused on addressing the anti-cancer activity and molecular mechanism of methanol extract of Polygonum cuspidatum (MEPC) in HSC-2 human oral cancer cells. The effect of MEPC on oral cancer cells was estimated by 3-(4,5-dimethylthiazol-20yl)-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium (MTS) assay, 4’-6-diamidino-2-phenylindole (DAPI) staining and Western blot analysis. MEPC inhibited the cell viability and induced apoptosis through the induction of death receptor (DR) 5. MEPC also increased the expression of C/EBP homologous protein/growth arrest and the DNA damage-inducible gene 153 (CHOP), a transcription factor induced by ER stress. Thus, we concluded that the induction of CHOP leading to DR5 up-regulation is required for the anti-cancer activity of MEPC in HSC-2 cells and MEPC may be a promising drug candidate for oral cancer.

Non-Structural Protein 5 of Zika Virus Interacts with p53 in Human Neural Progenitor Cells and Induces p53-Mediated Apoptosis

Zika virus(ZIKV) infection could disrupt neurogenesis and cause microcephaly in neonates by targeting neural progenitor cells(NPCs). The tumor suppressor p53-mediated cell cycle arrest and apoptotic cell death have been suggested to be activated upon ZIKV infection, yet the detailed mechanism is not well understood. In the present study, we investigated the effects of ZIKV-encoded proteins in the activation of p53 signaling pathway and found that, among the ten viral proteins,the nonstructural protein 5(NS5) of ZIKV most significantly activated the transcription of p53 target genes. Using the immunoprecipitation-coupled mass spectrometry approach, we identified that ZIKV-NS5 interacted with p53 protein. The NS5-p53 interaction was further confirmed by co-immunoprecipitation and GST pull-down assays. In addition, the MTase domain of NS5 and the C-terminal domain of p53 were mapped to be responsible for the interaction between these two proteins. We further showed that ZIKV-NS5 was colocalized with p53 and increased its protein level in the nuclei and able to prolong the half-life of p53. Furthermore, lentivirus-mediated expression of ZIKV-NS5 in hNPCs led to an apparent cell death phenotype. ZIKV-NS5 promoted the cleavage of PARP1 and significantly increased the cell apoptosis of h NPCs.Taken together, these findings revealed that ZIKV-NS5 is a previously undiscovered regulator of p53-mediated apoptosis in hNPCs, which may contribute to the ZIKV-caused abnormal neurodevelopment.

Effects of Qingguang'an(青光安)containing serum on the expression levels of autophagy-related genes in human Tenon's fibroblasts induced by transforming growth factor beta 1

OBJECTIVE:To explore the effects of Qingguang'an(青光安)containing serum on the expression levels of autophagy related genes in the transforming growth factor beta 1(TGF-β1)-activated human Tenon's fibroblasts(HTFs).METHODS:(a)Primary HTFs were stimulated by TGF-β1 and underwent immunohistochemistry,which established a cell model after Glaucoma filtration surgery(GFS).(b)The cell models were divided into 4 group:normal group(normal cells),model group(+TGF-β1),treatment group(+TGF-β1+medicated serum),and positive control group(TGF-β1+rapamycin).Then,Qingguang'an medicated serum with optimum concentration was added to the corresponding group.The autophagy positive cells were identified by the Cyto-ID autophagy detection kits under fluorescent microscope and Cytation 5 multifunctional instrument for cell imaging.And the mean fluorescence intensity of autophagy positive cells was determined by flow cytometry.The expression levels of autophagy related genes—Beclin-1,autophagy related gene 5(ATG-5),and microtubule-associated protein 1 light chain 3(LC-3Ⅱ)were detected by quantitative reverse transcription-polymerase chain reaction and Western blot analysis.RESULTS:Compared with the normal group and the model group,the relative mRNA expression levels of autophagy-related genes(Beclin-1,ATG-5 and LC-3Ⅱ)in the experimental group were notably increased(P<0.05,P<0.01),and with the extension of treatment time,it had an increasing trend(48 h was more obvious),which showed a certain time dependency;the protein expression levels of autophagy-related genes(Beclin-1,ATG-5,and LC-3Ⅱ)were significantly increased in the experimental group(P<0.05,P<0.01).With the prolongation of treatment time,there was an increasing trend(48 h was relatively obvious),and it revealed a certain time dependency CONCLUSION:The Qingguang'an medicated serum could up-regulate autophagy related genes(Beclin1,ATG5,and LC3Ⅱ)in the TGF-β1-activated HTFs.

Effects of Site-directed Mutagenesis of L469 in Helix-5 of Human Papillomavirus 16 L1 on Pentamer Formation

Located at the carboxyl terminal of the human papillomavirus major capsid protein L1, helix-5（h5） is cru-cial to L1 folding and pentamer formation. Site-directed mutagenesis of the leucine residue on site 469 into lysine, alanine, serine and glycine was performed to explore the effect of the resultant mutations on L 1 pentamer formation. The soluble yields of the L1 pentamers of the L469A and L469K mutants were nearly two fold higher than that of the wild type. Molecular dynamics simulation was then performed to reveal the intrinsic mechanisms involved in the improvement of L 1 pentamer yield. Accordingly, the secondary structures of h5, β-G2, β-B1, β-C, β-D, and β-F were altered. The altered structures improved the hydrophobic interaction between h5 and fl-core ＂jelly＂ and the stability of h5. The hydrophobic surface area of residue 469 was reduced by 50% relative to that of the wild type. The C--O group of residue 469 and C--N group of L470 were both exposed to the solvent in the L469A mutant. These modifications may account for the increased solubility and stability and the promotion of pentamer formation induced by the point mutation. Therefore, the changes in the hydrophobic properties of h5 and the core structure determined the pentamer formation and solubility. This study may assist the development of a cost-effective platform for the production of prophylactic virus-like particle vaccines.

糖宁孜亚比土斯片基于高糖人结直肠腺癌细胞模型对小克里斯滕森菌-TαMCA-FXR/TGR5轴的调控作用

目的探讨糖宁孜亚比土斯片(TZT)基于高糖人结直肠腺癌细胞模型对小克里斯滕森菌科-牛磺-α鼠胆酸钠盐(TαMCA)-法尼醇X受体(FXR)/G蛋白偶联受体5轴的调控作用。方法配制菌株液体培养基、高糖培养基、TZT溶液、TαMCA溶液,培养菌株,制备灭活小克里斯滕森菌及其发酵液,常规培养人结直肠腺癌细胞(Caco-2细胞)。取部分细胞随机分为对照组、灭活菌体组、106 CFU/mL活菌组、10^(7)CFU/mL活菌组、10^(8)CFU/mL活菌组、10^(9)CFU/mL活菌组,对照组用无菌Caco-2专用培养基培养,灭活菌体组用灭活小克里斯滕森菌菌体悬液干预,106 CFU/mL活菌组、10^(7)CFU/mL活菌组、10^(8)CFU/mL活菌组、10^(9)CFU/mL活菌组分别在含有完全分化的Caco-2细胞培养板孔中加入2 mL 10^(9)CFU、10^(8)CFU、10^(7)CFU、10^(6)CFU的小克里斯滕森菌活菌干预。取部分细胞随机分为对照组、发酵培养液组,对照组用无菌Caco-2专用培养基培养,发酵培养液组用小克里斯滕森菌发酵液干预。取部分细胞随机分为对照组、高糖组及TZT低、中、中高、高剂量组,除对照组外其他各组加入8 g/L高糖培养基干预24 h,TZT低、中、中高、高剂量组分别加入10、25、50、100μg/mL的TZT含药培养基干预24 h。取部分细胞随机分为对照组、25μmol/L TαMCA组、50μmol/L TαMCA组,后两组换入25、50μmol/L的含TαMCA培养基干预24 h。实时荧光定量PCR法检测FXR、TGR5、IL-8、IL-10 mRNA,Western blotting法检测FXR、TGR5蛋白。结果与对照组比较,10^(6)CFU/mL活菌组、10^(7)CFU/mL活菌组、10^(8)CFU/mL活菌组、10^(9)CFU/mL活菌组TGR5 mRNA表达高(P均<0.05),FXR、IL-8、IL-10 mRNA表达差异无统计学意义(P均>0.05)。与对照组比较,菌发酵液组FXR mRNA表达高(P均<0.05),TGR5 mRNA表达差异无统计学意义(P均>0.05)。与对照组比较,高糖组FXR mRNA表达高(P<0.05),TGR5 mRNA表达低(P<0.05),FXR、TGR5蛋白表达差异无统计学意义(P均>0.05)。与高糖组比较,各TZT组FXR mRNA表达低(P均<0.05),TGR5 mRNA表达高(P均<0.05),FXR、TGR5蛋白表达差异无统计学意义(P均>0.05)。与对照组比较,25μmol/L TαMCA组FXR mRNA、蛋白表达低(P均<0.05),TGR5 mRNA、蛋白表达高(P均<0.05);50μmol/L TαMCA组FXR蛋白表达低(P<0.05)。结论小克里斯滕森菌具有一定的抗炎效果,TZT可能通过促进小克里斯滕森菌的生长,产生代谢产物影响胆汁酸代谢,促进TαMCA肠道内累积,进一步抑制肠FXR表达,促进TGR5表达。

带电多囊体蛋白5在调控血管内皮细胞焦亡中的作用研究

目的:探究带电多囊体蛋白5(charged multivesicular body protein 5,CHMP5)在人脐静脉内皮细胞(hu-man umbilical vein endothelial cell,HUVEC)焦亡中的表达以及敲低CHMP5对HUVEC焦亡的影响。方法:采用聚肌胞苷酸(polyinosinic-polycytidylic acid,Poly I:C)刺激HUVEC建立病毒性脓毒症中双链RNA(double-stranded RNA,dsRNA)诱导血管内皮细胞损伤的体外模型。将HUVEC随机分为对照组、Lipo组、Poly I:C组、siNC组和siCHMP5+Poly I:C组,通过实时荧光定量PCR(real-time fluorescent quantitative PCR,qRT-PCR)检测各组CHMP5的表达。采用乳酸脱氢酶(lactate dehydrogenase,LDH)细胞毒性检测试剂盒检测LDH释放率、ELISA检测白介素-1β(interleukin-1β,IL-1β)分泌及透射电子显微镜(transmission electron microscopy,TEM)检测HUVEC的超微结构以观察细胞膜的完整性,蛋白质印迹(Western blotting)检测相关蛋白胱天蛋白酶-3活性剪切体(cleaved caspase-3,活化Casp-3)、gas-dermin E蛋白N端(gasdermin E N-terminal,GSDME-N)的表达水平,免疫荧光染色检测各组细胞相关蛋白的表达和定位。结果:与对照组相比,Poly I:C组HUVEC细胞肿胀,多处细胞膜破裂,同时活化Casp-3、GSDME-N和CHMP5蛋白表达上调(P<0.05)。RNAi技术敲低CHMP5后,与对照组和siNC组比较,siCHMP5+Poly I:C组LDH和IL-1β释放增加,焦亡相关蛋白表达水平上调以及GSDME蛋白募集分布改变,内皮细胞焦亡明显加重(均P<0.05)。结论:CHMP5在HUVEC细胞焦亡中呈现高表达。敲低CHMP5可增强HUVEC细胞焦亡,可能与抑制膜修复促进GSDME和GSDMD蛋白剪切引起的继发性细胞焦亡有关。

人5-脂氧合酶重组蛋白的纯化及其多克隆抗体的制备

目的:原核表达并纯化人5-脂氧合酶(5LO)△112蛋白,将其免疫新西兰大白兔制备5-LO多克隆抗体。方法:使用DNAMAN设计重组引物,PCR扩增N端缺失112个氨基酸的5-LO截短DNA片段,将其重组到KpnⅠ和EcoRⅠ酶切的pET30a(+)质粒中,重组质粒pET30a-5LO△112转化大肠杆菌Rosetta(DE3),培养至OD_(600)为0.4时加入IPTG以诱导5LO△112重组蛋白表达,目的蛋白经纯化后免疫新西兰大白兔制备其多克隆抗体。结果:使用BugBuster蛋白提取试剂裂解细菌并获得纯度较高的包涵体,包涵体用含6mol/L盐酸胍的溶液变性溶解,再经Ni-NTA agarose亲和纯化得到高纯度的5LO△112重组蛋白。用该蛋白免疫新西兰大白兔获得5-LO的特异性抗体,该抗体的效价为1∶51200。结论:使用6mol/L盐酸胍溶解包涵体蛋白结合Ni-NTA agarose亲和纯化的方法成功获得了纯度较高的5LO△112重组蛋白,并用此蛋白制备了高效价的多克隆抗体。

脑星形细胞瘤磁共振波谱与肿瘤细胞的Survivin、PCNA相关性研究

目的探讨脑星形细胞瘤磁共振波谱(MRS)与肿瘤细胞的Survivin蛋白、细胞核增殖抗原(PCNA)蛋白表达的相关性。方法对照观察50例脑星形细胞瘤MRS检查结果与免疫组化方法测定术后标本的Survivin、PCNA蛋白表达,分析肿瘤实质内最大Cho/NAA、Cho/Cr和最小NAA/Cr值与肿瘤级别的关系,并分别与PCNA增殖指数(PI)、Survivin免疫活性指数(IRS)进行相关性分析。结果高级别星形细胞瘤的Cho/Cr和Cho/NAA高于低级别星形细胞瘤和对照组,NAA/Cr低于低级别星形细胞瘤和对照组。Survivin的IRS和PCNA的PI随着肿瘤恶性度增加而升高。星形细胞瘤Cho/Cr与IRS显著正相关(r=0.745,P<0.01);星形细胞瘤Cho/NAA与IRS显著正相关(r=0.753,P<0.01)。星形细胞瘤Cho/Cr与PI显著正相关(r=0.818,P<0.01);星形细胞瘤Cho/NAA与PI显著正相关(r=0.760,P<0.01)。星形细胞瘤NAA/Cr与IRS呈负相关(r=—0.374,P<0.01),星形细胞瘤NAA/Cr与PI呈负相关(r=-0.436,P<0.01)。结论MRS基本反映着肿瘤细胞PCNA及Survivn表达情况,可在术前预测肿瘤凋亡抑制情况、增殖潜能、侵袭能力和预后,其中Cho/Cr,Cho/NAA较NAA/Cr更有意义。

人纤溶酶原Kringle5区的基因克隆的表达及纯化

目的克隆人纤溶酶原Kringle5 (K5 )区基因 ,并进行重组蛋白的表达纯化及活性鉴定。方法用PCR方法从正常成人肝cDNA库扩增出人纤溶酶原K5区基因 ,构建K5的原核表达载体pET 2 2b(+) K5 6×His,IPTG诱导蛋白表达后经Ni+ -树脂亲和层析进行纯化 ,通过血管内皮细胞增殖抑制试验检测蛋白活性。结果经PCR成功扩增出了 2 4 3bp的K5区基因 ,测序正确后克隆进大肠杆菌分泌型表达载体pET 2 2b(+) ,重组质粒在BL2 1中成功表达出分子量为 14 0 0 0的蛋白质 ,纯化后的蛋白纯度达95 % ,具有抑制血管内皮细胞增殖活性的功能 ,K5蛋白浓度为 4 μg/mL时抑制率达 5 0 %。 结论纤溶酶原K5的成功克隆。

基于数据挖掘分析GPRC5A在胰腺癌中的表达及意义

目的:基于生物信息数据挖掘阐明G蛋白偶联受体C家族5A基因(G protein-coupled receptor familyC, m e m b e r5, g r o u pA, G P R C 5 A)在胰腺癌中的表达及临床意义。方法:检索O n c o m i n e,TCGA,HumanProteinAtlas等基因数据库,分析GPRC5A在胰腺癌中的表达差异,并分析其在不同肿瘤中的表达。采用Western印迹技术在武汉大学人民医院小样本队列中验证GPRC5A在胰腺癌及癌旁组织中的蛋白表达水平,采用Kaplan-Meier进行患者生存分析,并对GPRC5A与靶向药物敏感性关系进行分析。结果:对Oncomine数据库中有差异表达的295项研究数据进行差异性分析,发现胰腺癌组织GPRC5A基因的表达明显高于正常胰腺组织。对GPRC5A在不同肿瘤组织中表达的4184项研究进行荟萃分析,发现GPRC5A在胰腺癌组织中显著表达增高。通过生存分析发现高表达GPRC5A胰腺癌患者生存期明显低于低表达者,高表达患者预后更差。此外,GPRC5A表达与靶向药物厄洛替尼的药物敏感性有一定关联。结论:GPRC5A在胰腺癌组织中呈现高表达,与患者预后显著相关,其有可能成为胰腺癌诊断及药物治疗的新靶点。

表达鸡传染性支气管炎病毒S1蛋白重组腺病毒的构建及免疫原性的初步分析

拟构建表达鸡传染性支气管炎病毒(IBV)QS10株S1蛋白的重组复制缺陷型人5型腺病毒,并在本动物上进行初步的免疫学试验。通过RT-PCR获得S1基因,插入腺病毒表达系统穿梭质粒,构建重组穿梭质粒pac-Ad5CMV-S1,转染293AD细胞获得表达S1蛋白的重组腺病毒。PCR检测显示,S1基因已重组到腺病毒基因组;间接免疫荧光和Western blot检测证明该重组病毒在293AD细胞中真实表达了具有免疫反应性的IBV S1糖蛋白。重组病毒培养滴度可达到107 TCID50.mL-1。免疫接种SPF鸡后,通过ELISA检测,免疫鸡产生了针对IBV的特异性抗体。作者成功构建了表达IBV S1蛋白的重组腺病毒,该重组病毒可在SPF鸡体内诱导产生IBV特异性抗体。

毕赤酵母表达重组hPK-5蛋白不均一性的鉴定

对毕赤酵母表达重组人纤溶酶原Kringle 5（hPK-5）蛋白的不均一性进行了鉴定。应用十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）法及分子排阻高效液相色谱（SEC-HPLC）和反相高效液相色谱（RP-HPLC）进行纯度分析。采用糖蛋白染色法鉴定是否为糖蛋白，以Edman降解法测定N端氨基酸，采用高效液相色谱-电喷雾四极杆飞行时间质谱（HPLC-ESI-QTOF-MS）联用技术测定蛋白质精确分子质量。结果表明：SDS-PAGE测定为单-条带，纯度大于95％，SEC-HPLC测定为单一色谱峰，纯度大于95％，RP-HPLC测定为几个紧密相连的峰。糖蛋白染色为阴性。N端氨基酸测定结果表明含多个N端氨基酸。液质联用测定精确分子质量的结果表明hPK-5蛋白是相对分子质量分别为10744．88，10646．00，10533．00和10419．63的混合物。以上结果准确鉴定出毕赤酵母表达的该重组hPK-5蛋白是C端完整而N端依次缺失0～3个氨基酸的不均一蛋白。

rhPDCD5促进子宫内膜癌KLE细胞对紫杉醇的药物敏感性

目的:探讨在子宫内膜癌细胞中重组人程序性细胞死亡蛋白5(recombinant human programmed cell death protein 5,rhPDCD5)对紫杉醇化疗的促进作用。方法:子宫内膜癌KLE细胞培养完成后,通过重组人rh PDCD5(20μg/ml)处理KLE细胞,再分别以0、1.0、5.0、10.0、50μmol/L紫杉醇(paclitaxel,PTX)处理24 h或以10μmol/L PTX处理0、12、24、48 h,提取细胞总RNA及蛋白后,CCK法检测KLE细胞的增殖情况,流式细胞术检测KLE细胞凋亡情况,实时定量PCR检测KLE细胞中PDCD5mRNA的表达量,实时定量PCR或Western blotting测定凋亡相关基因的Bax、Bcl2、caspase-3 mRNA或蛋白水平的变化。结果:PTX对PDCD5表达的促进作用具有剂量依赖性和时间依赖性;PTX的最佳作用浓度为10μmol/L,最佳作用时间为24 h。rh PDCD5明显增强紫杉醇对KLE细胞的抑制作用。CCK实验、流式细胞术及Western blotting检测显示:PTX+rhPDCD5联合处理组KLE细胞的增殖抑制率和凋亡率均较PTX组明显增加、pro-caspase 3的表达量明显增加(均P<0.01)。促进凋亡蛋白Bax和抑制凋亡蛋白Bcl2的比值亦较明显增加(P<0.01)。结论:rhPDCD5可协同PTX抑制子宫内膜癌KLE细胞的增殖、促进细胞的凋亡,可明显增强KLE细胞对PTX的药物敏感性。

重组人可溶性DR5蛋白的分离纯化及其生物活性检测

目的:提纯毕赤酵母GS115菌株表达的重组人可溶性死亡受体5(sDR5)蛋白,并研究其对肿瘤坏死因子相关凋亡诱导配体(TRAIL)所诱导细胞的阻断效应。方法:大量扩增可溶性DR5酵母表达载体转化阳性的毕赤酵母GS115菌株,收集上清,利用Ni-NTA Agarose亲和层析柱纯化获得重组可溶性DR5,用SDS-PAGE检测纯化产物的纯度。用流式细胞仪检测纯化的可溶性DR5对TRAIL诱导Jurkat细胞凋亡作用的阻断效应。结果:转化阳性的毕赤酵母GS115菌株可以成功表达分子量为23kD的可溶性DR5蛋白,经过Ni-NTAAgarose亲和层析柱纯化,SDS-PAGE鉴定显示获得了高纯度的重组人可溶性DR5蛋白,培养上清产量达28.69mg/L;体外活性检测结果显示,纯化的可溶性DR5蛋白几乎完全可以阻断TRAIL诱导的Jurkat细胞凋亡作用。结论:经纯化后的重组人可溶性DR5蛋白可以有效阻断TRAIL诱导的细胞凋亡作用,显示DR5在TRAIL诱导Jurkat细胞凋亡作用中起着十分关键的作用。

重组人血管生成素-1与肺间质纤维化血管正常化时相的关系

目的探讨重组人血管生成素1（Human Angiopoietin-1,rHuANG-1）对小鼠模型肺间质纤维化微血管正常化的最佳时相及其分子机制。方法将40只C57/BL6小鼠按掷硬币法随机分成对照组和实验组,每组各20只。对照组腹腔注射生理盐水（NS）（0.2ml/d）;实验组腹腔注射rHuANG-1（5mg·kg^-1·d^-1）。每组分别于治疗后的第2、4、6、9天处死5只小鼠留取标本。测量小鼠肺组织羟脯氨酸含量变化。酶联免疫吸附测定法（ELISA）检测肺组织中G蛋白调节信号5（regulator of G-protein signaling 5,RGS5）蛋白浓度,苏木精-伊红（HE）染色检测肺组织病理变化,免疫组织化学形态学分析血管内皮生长因子（vascular endothelial growth factor,VEGF）的变化。结果1成功建立C57/BL6小鼠肺间质纤维化模型。2实验组接受rHuANG-1注射后,第6天,第9天肺羟脯氨酸含量降低（P〈0.01）。3 ELISA结果显示,实验组肺组织中RGS5的表达在治疗第4天和第6天与对照组比较明显增高（P〈0.05）。4HE染色结果显示,实验组和对照组不同时间点均可见不同程度的肺纤维化。对照组坏死程度高于实验组。其中,实验组第4天和第6天组织修复最明显,而对照组不同时点肺间质纤维化比较明显。5免疫组织化学结果表明,与对照组相比,实验组第4、6天VEGF在肺间质纤维化中的表达率升高明显减少（P〈0.05）。结论rHuANG-1作用于小鼠肺间质纤维化后第4~6天可作为血管正常化时间窗,可能与VEGF相关。

人胚胎结肠肠神经系统发育的观察

目的 研究人胚胎结肠肠神经系统的发育过程 ,为进一步研究先天性巨结肠的发病机制提供参考。 方法 采用一抗为蛋白基因产物 (protein gene product9.5 ,PGP9.5 )和 S- 10 0蛋白抗体的免疫组织化学 PAP法 ,显示结肠肠神经系统中的神经元和神经胶质。 结果 人胚胎结肠肠神经系统发育有明显的阶段性。在胚胎发育早期 (胎龄 2～ 3月 ) ,肠管壁发育差 ,以后出现菲薄的平滑肌层和低平的肠粘膜 ,此期偶在原始肌间神经丛位置见S- 10 0蛋白免疫反应性神经 ;至发育中期 (胎龄 4～ 5月 ) ,肠壁分化出 4层结构 ,出现相当发达的绒毛 ,肌间神经丛中细胞明显增多 ,呈弥散分布于整个肌层间并逐渐迁移到粘膜下层和粘膜层 ,由初级和次级突起构成复杂的神经网络 ;至晚期 (6～ 9月 ) ,肠壁各层均增厚 ,肌间神经丛成簇分布 ,神经纤维构成的网络出现更为细小的 3级突起 ,粘膜下神经丛分化形成浅丛和深丛。 结论 结肠神经系的发育具有明显的阶段性。发育早期神经开始在肠壁肌间丛位置出现 ,发育中期神经成分在肠壁各层中出现并发育增生 ,晚期肠壁各层神经已分化和成熟 ,而在不同的发育阶段 。