关于奇异非线性两点边值问题的一点注记

在k（x）具有较强奇性的情况下证明了奇异非线性两点边值问题正解的存在性．同时提出了一个C1正解存在的充要条件

Effect of silencing LC3 on apoptosis of mouse hepatocellular carcinoma Heap1-6 cells

目的建立小鼠LC3基因沉默的Heap1-6稳定表达细胞系，探讨其对衣霉素诱导的肝癌细胞凋亡的影响。方法设计并合成一段针对小鼠LC3基因的shRNA及不针对任何基因的shRNA作为阴性对照，将它们退火后连接构建重组载体，转化扩增及酶切鉴定之后将重组质粒与病毒包装、包膜质粒共同转染293T，收集病毒上清转染Heap1-6细胞，嘌呤霉素筛选10 d，获得稳定的细胞株。以导入LC3基因shRNA的Heap1-6为实验组（Heap1-6 shLC3），以导入shcoo2的Heap1-6为对照组（Heap1-6 shctrl）。衣霉素处理实验组和对照组的细胞，Western Blot检测LC3Ⅱ、c-Caspase3、Caspase9蛋白的表达，流式细胞术检测细胞凋亡。Western Blot 条带灰度值之间两组比较采用独立样本的t检验。结果成功构建了pLKO.1-shLC3重组慢病毒载体，与野生型的Heap1-6相比，LC3基因沉默之后，LC3Ⅱ蛋白表达水平降低了62.9﹪（P 〈 0.01）；野生型的Heap1-6和LC3基因沉默之后的Heap1-6 shLC3都经Tm处理12 h之后，后者LC3Ⅱ蛋白表达水平降低了58.6﹪（P 〈 0.01）。与对照组Heap1-6 shctrl相比，衣霉素作用12 h后实验组Heap1-6 shLC3 c-Caspase3增加了37.7﹪（P = 0.007），Caspase9增加了37.1﹪（P = 0.023））；衣霉素作用24 h后shLC3组c-Caspase3增加了12.6﹪（P = 0.04）， Caspase9增加了14.3﹪（P = 0.043）。药物干预12 h和24 h后，Heap1-6 shLC3组比对照组Heap1-6 shcoo2凋亡比例分别增加22.8﹪和18.6﹪。结论成功建立小鼠LC3基因沉默的Heap1-6稳定表达细胞系，LC3基因沉默促进衣霉素诱导的小鼠肝癌细胞Heap1-6的凋亡。Objective To establish a stable hepatocellular carcinoma Heap1-6 cell line expressing shRNA against mouse LC3 and to study apoptosis of Heap1-6 cells treated with tunicamycin. Methods shRNA targeting LC3 gene and negative shRNA were designed and synthesized, pLKO.1-TRC-shRNA LC3 vector and negative vector were constructed. After amplification and identification, the recombinant lentivirus vectors were transfected into 293T cells with packaging and envelope plasmids. The supernatant of 293T ceils transfected with recombinant vector was collected, and Heap1-6 cells were transfected with plasmids, and treated with puromycin for ten days to acquire a cell line with stable expression of shRNA against mouse LC3. Western blot analysis was used to detect the expression level of LC3 Ⅱ, cleaved-caspase3, and caspase9 protein respectively. Apoptotic cells were measured by flow cytometry. Results We successfully constructed pLKO. 1-shLC3 lentivirus vector. Before treated by tunicamycin, the level of LC3 Ⅱ in the Heap1-6 shLC3 cells was decreased by 62.9 % compared with that in the WT Heap1-6 cells （P = 0.0001 ）. After being treated by tunicamycin for 12 h, the level of LC3 Ⅱ in Heap1-6 shLC3 cells was decreased by 58.6 % compared with that in the WT Heapl-6 cells P = 0.0003 ）. Compared with the control group （Heap1-6 cells transfected with negative shRNA vector）, the level of cleaved-caspase3 and caspase9 in the shLC3 group was increased by 37.7 % and 37.1% respectively under tunicamycin for 12 h （P = 0.007, 0.023 ）. And the level of the same two proteins in the shRNA group was elevated by 12.6 % and 14.3 % compared with those of Heap1-6 shctrl cells respectively （P = 0.040, 0.043）. The ratio of apoptotic cells of the experiment group was increased by 22.8 % and 18.6 % compared with that of the control treated with ttmicamycin for 12 h and 24 h, respectively. Conclusion LC3 knockdown could promote apoptosis of mouse hepatocellular carcinoma Heap 1-6 cell line induced by tunicamycin.国家自然科学基金面上项目（81270431

糖尿病视网膜病变UWFA量化分析及其与糖尿病黄斑水肿的关联研究

目的量化糖尿病视网膜病变（DR）患者超广角荧光素眼底血管造影（UWFA）图像中不同视网膜区域的病灶参数，探讨其与中心凹受累的糖尿病黄斑水肿（CI-DME）的相关性。方法回顾性分析2022年6月至2024年6月于广东省人民医院初诊的76例DR患者（共101眼）在同一天拍摄的UWFA及光学相干断层扫描（OCT）图像。根据OCT测量的中央视网膜厚度（CST）将患眼分为CI-DME组与非CI-DME组。手动标注UWFA图像中微血管瘤、无灌注区、渗漏区、视盘和中心凹，量化计算黄斑区、后极部、中周部、周边部及全UWFA区域的微血管瘤计数、缺血指数和渗漏指数。比较两组病灶量化参数的差异，评估其与CI-DME的关联性及判别效能。结果CI-DME组患眼在黄斑区的微血管瘤计数、缺血指数和渗漏指数均显著高于非CI-DME组（P均＜0.05）。而周边部这些病灶参数在CI-DME组和非CI-DME组中无显著差异（P均＞0.05）。ROC曲线分析显示，渗漏指数对CI-DME的判别效能优于微血管瘤计数和缺血指数，其中黄斑区渗漏指数的区分能力最佳，AUC为0.80（95%CI：0.71，0.89）。结论本研究未发现UWFA周边部病灶参数与CI-DME显著关联，但黄斑区病灶参数（尤其是渗漏指数）与CI-DME的病理进程密切相关。因此，在DME患者的监测与治疗过程中，需关注黄斑区血管稳定性以优化DME管理策略

H_2S gas sensitivity of UV light activated WO_3 gas sensors

以热氧化钨丝法制备的WO3纳米材料为基材制备了厚膜气敏元件,在常温、紫外光激发条件下实验测试了所制纯WO3气敏元件对不同体积分数的H2S气体的气敏特性曲线,探讨了元件对H2S的灵敏度与紫外光的辐射通量密度的依赖关系。结果表明,常温、无紫外光照下WO3气敏元件对H2S不敏感,而在常温及紫外光激发下WO3气敏元件对H2S的灵敏度显著增大,且随着紫外光辐射通量密度增加,元件对H2S的灵敏度先增大而后减小。The thick film gas sensors were prepared by using WO3 nano-materials that prepared by the method of thermal oxidation of tungsten.Under the condition of room temperature and UV excitation,gas sensing characteristic curves of the prepared pure WO3 gas sensors to H2S gas with different volume fractions were tested.Dependencies between sensitivity of WO3 gas sensors to H2S and UV radiant flux density were investigated.The results show that under the room temperature condition,the WO3 gas sensors are not sensitive to H2S without UV irradiation,while the WO3 gas sensors are significantly sensitive to H2S with UV excitation,and with the increase of UV radiant flux density,the sensitivity of gas sensors to H2S first increases and then decreases.福建省省属高校科研专项资助项目(No.JK2011039); 福建省自然科学基金资助项目(No.2012D110;No.2013J05014); 国家级大学生创新项目资助(No.201310395003

间充质细胞外泌体促进小鼠胰岛内皮细胞血管生成的研究

目的探讨间充质细胞（MSC）外泌体对低氧条件下胰岛内皮细胞（MS-1）血管生成的影响。方法 MSC无血清低氧条件培养48 h,超滤离心法富集条件培养基中的外泌体,采用电镜和Western Blot的方法进行鉴定;通过血管形成试验比较分析不同条件下:常氧培养组（NOR组,21%O2、5%CO2）、低浓度氧培养组（HYP组,2%O2、5%CO2）、外泌体+低浓度氧共培养组（HYP+EXO组,2%O2、5%CO2）,MS-1细胞的血管形成能力;image J软件分析血管形成长度;PCR、Q-PCR检测血管内皮生长因子（VEGF） RNA水平的表达,Western Blot检测VEGF、HIF1α蛋白水平表达以及mTOR信号通路激活情况。采用单因素方差分析和SNK-q检验统计学分析。结果超滤离心法富集的MSC条件培养基中的外泌体,大小为30～100 nm,表达CD9,CD63,CD81等外泌体表面标志物;血管形成试验结果显示,低氧促进MS-1血管生成,HYP+EXO组形成明显的血管网状结构;HYP+EXO组血管形成相对长度（2386.0±137.7）像素与NOR组（393.3±174.2）像素和HYP组（1467.0±230.0）像素相比增强,差异有统计学意义（t=12.30,P=0.0065;t=15.74,P=0.0040）; PCR结果显示,HYP+EXO组VEGF相对表达量（20.26±9.972）较常氧对照组（1.000）和低氧组（6.521±3.501）均增强,差异有统计学意义（t=5.462,P=0.0009;t=4.238,P=0.0038）;同时,Western Blot结果显示VEGF蛋白水平表达升高,HIF1-α表达上调,mTOR发生磷酸化。结论 MSC外泌体可促进低氧条件下的小鼠胰岛内皮细胞血管生成。MSC外泌体可能通过上调HIF1-α,调节VEGF表达,激活mTOR信号通路,促进胰岛内皮细胞血管生成。国家自然科学基金青年项目(81601618);;福建省自然科学基金面上项目(2016J01582、2016J01580、2018J01349);;福建省科技创新联合资金重大项目(2017Y9127

转染survivin对HLF细胞增殖及凋亡的影响

【【目的】 探讨survivin基因表达对人肺纤维母细胞（humanlungfibroblast，HLF）增殖、凋亡的影响。【方法】 构建survivin基因真核表达质粒peDNA3．1／SVN；利用脂质体转染法将其转入不表达survivin mRNA的HLF细胞系。连续培养10周。筛选出稳定表达的阳性克隆（HLF／SVN）及转空质粒对照（HLF／K）。用RT-PCR、免疫组化、Western blot检测细胞中survivin、bel-2和caspase3mRNA表达：通过观察细胞生长曲线、细胞凋亡指数。探讨转染survivin基因对HLF细胞生长、凋亡的影响。【结果】转染peDNA3．1／SVN后。在4、6、8和10周HLF／SVN细胞查见survivinmRNA表达；免疫组化和Westernblot见HLF／SVN细胞survivin阳性表达；转染后caspase3mRNA受到抑制，表达减弱或消失。HLF／SVN细胞凋亡指数（0．3％、1．0％、1．5％）皆低于HLF（0．9％、4．3％、2．1％）及HLF／K（0．8％、1．4％、2．8％）。细胞生长曲线示HLF／SVN细胞生长快于HLF和HLF／K，差异有统计学意义（P〈0．05）。【结论】survivin基因能促进HLF细胞生长。可能通过抑制凋亡和促进细胞分裂而发挥作用

戊型肝炎病毒ORF2片段在毕赤酵母中的分泌表达及活性鉴定

　为了寻求新型表达系统来研制戊型肝炎病毒基因工程疫苗,利用Pichiapastoris表达系统表达戊型肝炎病毒(HEV)结构区ORF2基因.利用PCR扩增HEVORF2基因,然后将ORF2基因按正确的阅读框融合到Pichiapastoris分泌型表达载体pPIC9K的а因子信号肽编码序列3′端,重组表达载体在电击转化毕赤酵母,经过含G418的营养缺陷型培养基(RDB)筛选、重组酵母基因组总DNA进行PCR鉴定后,证实HEVORF2基因已经整合到酵母基因组中并得到重组转化子.表型鉴定后对G418具有不抗性的重组菌株诱导表达,用双抗体夹心法ELISA筛选了表达外源蛋白的重组菌株,再经SDS PAGE与Westernblot证实ORF2基因在Pichiapastoris中实现了分泌表达,而且重组蛋白具有较强的特异性与生物学活性;同时用双抗体夹心法证实在Pichiapastoris表达的上清中存在衣壳蛋白聚体

Promotion of proliferation of luminal B breast cancer cells by mesenchymal stem cells and its underlying molecular mechanisms

目的分析人脐带间充质干细胞(hUC-MSCs)对Luminal B型乳腺癌细胞生长增殖的影响,并初步探讨其可能的分子机理。方法绿色荧光蛋白(GFP)和荧光素酶共表达慢病毒感染人Luminal B型乳腺癌细胞BT474,并经嘌呤霉素筛选两周后,于荧光显微镜下观察GFP的表达情况,IVIS Kinetic成像系统拍照以观察和记录慢病毒感染后BT474细胞荧光素酶的表达情况;荧光显微镜下直接观察,结合MTS实验分析hUC-MSCs共培养或其浓缩上清处理对GFP和荧光素酶共表达BT474细胞生长增殖的影响;Western blot法检测hUC-MSCs浓缩上清处理对BT474细胞Akt和MAPK信号通路激活情况以及下游细胞周期调控蛋白Cyclin D1表达的影响;常规RT-PCR法检测hUC-MSCs中NRG-1、NRG-2、IGF-Ⅰ、IGF-Ⅱ和EGF等配体的表达。荧光素酶表达强度与细胞数量的相关性经由Excel软件行统计学分析,MTS实验数据则经由SPSS13.0统计软件行统计学分析。结果荧光显微镜和IVIS Kinetic成像系统的观察结果分别证实,GFP和荧光素酶经慢病毒载体系统的介导可在BT474细胞中成功地共表达,且荧光素酶的表达强度与细胞数量呈直线相关。MSCs共培养或其浓缩上清处理均可显著促进Luminal B型乳腺癌细胞BT474的生长增殖,其细胞存活比例分别为各自对照组的148.06%(P<0.005)和147.99%(P<0.001);MSCs浓缩上清处理同时激活BT474细胞内Akt和MAPK信号通路,并上调细胞周期调控蛋白Cyclin D1表达。此外,RT-PCR结果显示,hUC-MSCs中NRG-1和EGF的mRNAs水平呈高表达,而NRG-2、IGF-Ⅰ和IGF-Ⅱ等配体的mRNAs表达也可见。结论 MSCs可通过表达并分泌NRG-1等配体,从而激活Luminal B型乳腺癌细胞BT474的下游Akt和MAPK信号转导通路以上调细胞周期调控蛋白Cyclin D1的表达,进而促进其生长增殖。Objective To investigate the effect of human umbilical cord mesenchymal stem cells(hUC-MSCs) on proliferation of luminal B breast cancer cells and its underlying molecular mechanisms. Methods Human luminal B breast cancer cells BT474 were infected with GFP and luciferase co-expressing lentiviruses and then subjected for selection with Puromycin for 2 weeks. The expression of GFP and luciferase was detected by fluorescent microscopy and IVIS Kinetic image system, respectively. The effect of coculture or treatment with conditioned medium of hUCMSCs on proliferation of BT474 was analyzed with MTS assay. Western blot was carried out to detect the effect of treatment with conditioned medium of hUC-MSCs on the activation of both Akt and MAPK signalings in BT474, as well as the expression of downstream cell cycle regulator Cyclin D1. Regular RT-PCR was applied to analyze the mRNAs expression of ligands such as NRG-1, NRG-2, IGF-Ⅰ, IGF-Ⅱ, and EGF in hUC-MSCs. The correlation between relative luciferase activity and cell number was analyzed with Excel software, while MTS assay data was statistically analyzed with SPSS 13.0 software. Results The co-expression of GFP and luciferase in BT474 via lentiviral expression system was visualized by fluorescent microscopy and IVIS Kinetic image system. The linear correlation between relative luciferase activity and cell number was determined by curve fitting analysis. Coculture or treatment with conditioned medium of hUC-MSC significantly promoted the proliferation of BT474, with survival rates being 148.06 %(P < 0.005)and 147.99 %(P < 0.001)of control, respectively. In addition, treatment with conditioned medium of hUC-MSC was shown to induce activation of both Akt and MAPK signalings, which further upregulated the expression of Cyclin D1. Moreover, high mRNAs expression levels of both NRG-1 and EGF, as well as moderate mRNAs expression levels NRG-2, IGF-Ⅰ, and IGF-Ⅱ were showed by RT-PCR. Conclusion Our results here demonstrated that MSCs may promote the proliferation of luminal B breast cancer cells through paracrine of ligands such as NRG-1, which in turn results in the activation of both Akt and MAPK signalings and upregulation of the expression of Cyclin D1.国家自然科学基金面上项目(81272922);; 福建省自然科学基金面上项目(2016J01577);; 福州总医院院内课题国际合作研究专项(2015G01

基于磷酸盐浓度和起始细胞密度的沃氏藻与塔玛亚历山大藻种间竞争研究

以分离自厦门港船舶压舱水箱及沉积底泥中的一种潜在入侵藻类—沃氏藻(Woloszynskiasp.)MMDL3013和一种常见有害赤潮藻—塔玛亚历山大藻(Alexandrium tamarense(Lebour)Balech)MMDL3041为研究对象,采用半连续培养的方法研究了不同磷酸盐浓度和藻类不同起始细胞密度两因子对这两种藻种间竞争的影响.结果表明,在磷酸盐限制条件下,沃氏藻为竞争的优势种,沃氏藻和塔玛亚历山大藻表现为资源利用性竞争;而在磷酸盐丰富的环境中种间竞争具有藻类细胞密度依赖性,起始细胞密度的不同可以影响竞争的结果

早期宫颈癌部分子宫照射的物理剂量学研究

目的探索宫颈癌根治性同期放化疗中部分子宫照射对减少盆腔正常脏器剂量的意义。方法选取2014年10月至2016年10月在汕头大学医学院附属肿瘤医院接受根治性放化疗的20例宫颈癌患者,为每个患者设计2套放疗计划:(1)全子宫照射:临床靶区(CTV)包括完整子宫;(2)部分子宫照射:CTV包括肿瘤靶区(GTV)以外10mm的子宫组织。比较每个患者2组放疗计划的参数和盆腔正常器官受照射剂量。结果部分子宫照射和全子宫照射适形度和均匀度差异无统计学意义;部分子宫照射的计划靶区(PTV)体积为(1 065±134)cm~3,小于全子宫照射的PTV体积(1 141±133)cm~3(P=0.00);部分子宫照射能减少部分盆腔脏器受照剂量:部分子宫照射膀胱V45=(27±9)%,全子宫照射V45=(32±11)%(P=0.01);部分子宫照射直肠V40=(84±9)%,全子宫照射直肠V40=(86±9)%(P=0.01);部分子宫照射小肠V45=(76±53)cm~3,全子宫照射小肠V45=(79±56)cm~3(P=0.03)。结论部分子宫照射能减少PTV体积,并减少部分盆腔正常器官受照射剂量,但所减少的绝对体积较小,临床意义不大。广东省科技厅科技计划项目(2016A020215188);;汕头市医疗科技计划项目(汕府科[2015]123号