生物强度电场对人表皮细胞株HaCaT微管乙酰化的调节作用

目的探讨生物强度电场对人表皮细胞株HaCaT方向性迁移及微管乙酰化水平的调节作用, 以期为临床创面修复提供分子理论依据。方法采用实验研究方法。取HaCaT细胞, 分为置于电场装置中不通电处理3 h的模拟电场组和用强度200 mV/mm电场处理3 h的电场处理组(处理方法下同, 样本数分别为54、52), 在活细胞工作站中观察处理3 h内细胞运动的方向并计算细胞运动的位移速度、轨迹速度及运动方向性cosθ。取2批HaCaT细胞, 分为模拟电场组和用强度200 mV/mm的电场处理相应时间的电场处理1 h组、电场处理2 h组、电场处理3 h组及模拟电场组和用相应强度的电场处理3 h的100 mV/mm电场组、200 mV/mm电场组、300 mV/mm电场组, 采用蛋白质印迹法检测乙酰化α-微管蛋白的表达(样本数均为3)。取HaCaT细胞, 分为模拟电场组和电场处理组, 采用免疫荧光法观测乙酰化α-微管蛋白的表达及定位(样本数为3)。对数据行Kruskal-WallisH检验、Mann-WhitneyU检验、Bonferroni校正、单因素方差分析、LSD检验及独立样本t检验。结果处理3 ...     

龙血素B对角质形成细胞增殖和迁移的影响

目的:观察龙血素B(Loureirin B,LB)对体外培养人永生化角质形成细胞(Immortalized human keratinocytes,HaCaT)增殖和迁移的影响,探究LB是否促进创面愈合再上皮化过程。方法:噻唑蓝(MTT)法检测不同浓度LB对体外培养HaCaT细胞增殖的影响,细胞划痕实验分析LB对HaCaT细胞迁移的影响。结果:5、10、25、50μg/mlLB呈剂量依赖性抑制HaCaT细胞增殖(P0.05),10、25μg/mlLB呈剂量依赖性抑制划痕愈合及HaCaT细胞迁移(P0.01)。结论:LB具有显著抑制HaCaT细胞增殖和迁移的作用,提示LB可能抑制创面愈合再上皮化过程。     

通过抑制c-Myc 过多的氧连接氮乙酰葡萄胺糖基化修饰缓解角质形成细胞功能障碍加速糖尿病创面愈合

糖尿病足溃疡是糖尿病的主要并发症之一, 其主要特征是延迟愈合。研究表明表皮KC功能障碍在糖尿病创面愈合延迟中起关键作用, 而c-Myc蛋白及其氧连接氮乙酰葡萄胺糖基化修饰(O-GlcNAc)与KC功能障碍的关系尚不清楚。上海交通大学医学院附属瑞金医院刘琰教授团队近期于《Burns & Trauma》发文《Inhibiting Hyper-O-GlcNAcylation of c-Myc accelerate diabetic wound healing by alleviating keratinocyte dysfunction》, 该研究观察到糖尿病患者创面边缘KC的增殖和分裂活跃, 迁移和分化降低;在糖尿病大鼠创面边缘KC和30 mmol/L葡萄糖培养中的人永生化表皮细胞HaCaT中也观察到前述同样的现象。进一步研究显示KC中c-Myc的表达增加, c-Myc的高表达促进了HaCaT的增殖, 抑制了其迁移和分化, 通过抑制c-Myc促进了大鼠糖尿病创面愈合。30 mmol/L葡萄糖通过增加c-Myc的O-GlcNAc使c-Myc蛋白更稳定, 抑制O-GlcNAc缓解了KC...     

降真香水提取物对光老化角质形成细胞模型的保护作用及其机制研究

目的探讨降真香水提取物对中波紫外线诱导的人角质形成细胞(HaCaT)光损伤的保护作用及机制。方法 2018年12月至2020年4月, 在广西医科大学基础医学院遗传学实验室进行实验。用前期构建的光老化角质形成细胞模型, 用不同浓度降真香水提取物与细胞共培养, 用CCK-8法检测细胞增殖率, 用试剂盒检测细胞氧化指标活性氧(ROS)、丙二醛(MDA)、超氧化物岐化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GPx)、总抗氧化能力(T-AOC)水平, 用酶联免疫吸附(ELISA)法检测细胞中白细胞介素(IL)-1β、IL-6、ɑ-肿瘤坏死因子(TNF-α)含量。结果浓度为0.5～2.0 mg/L降真香水提取物对HaCaT细胞增殖有促进作用, 与对照组比较, 实验组HaCaT细胞增殖率明显升高(P<0.05)。与对照组比较, 实验组细胞ROS含量显著降低(F=214.67, P<0.05), MDA含量显著降低(F=811.88, P<0.05), SOD含量升高(F=28.95, P<0.05), CAT含量升高(F=213.31, P<0.05),...     

Radioprotective effects of the expression of FLT3 ligand regulated by Egr-1 regulated element on radiation injury of SCID mice

Objective:In order to explore the radioprotective effects of the expression of hematopoietic growth factors regulated by radio-inducible promoter on radiation injury. Methods:The human FL (Flt3 ligand) cDNA and EGFP (enhanced green fluorescent protein) cDNA were linked together with IRES and then inserted into the eukaryotic expression vector pCI-Egr, which was constructed by substituting CMV promoter in pCIneo with the Egr-1 promoter (Egr-EF). The vector was transferred into human bone marrow stromal cell line HFCL by lipofectin. The transduced cell clones (HFCL/EF) had been selected by the addition of G418. The cells were exposed to γ-radiation by 60 Co source for 0.5-20Gy. The expressions of transduced cells were detected with FACS, Northern blot ELISA and CFU assay. The HFCL/EF and CD34+ cells from human umbilical cord blood were one after the other transplanted i.v. into sublethally irradiated severe combined immunodeficient (SCID) mice. The white blood cell amount in peripheral blood and human cell engrafted in recipent mice were detected by flow cytometry and CFU-GM etc. Results:The activity of EGFP in transduced cells increased by 3.1 fold as compared to non-transduced cells at 18h after exposure to 2.5Gy. The amounts of secreted FL in serum-free supernatants of Egr-EF increased by 605.46±107.21pg/ml, which were significantly higher than the control group (214.45±35.61pg/ml). The effects of FL in HFCL/EF cultural supernatants on expansion of CD34+ cells derived from cord blood in the presence of SCF, IL-6 and IL-3 were also studied. The results showed that at day 10 of culture the number of CD34+ cells increased by 173. 09±11.58×103/ml, which was significantly higher than that of non-radiation group(68. 04± 13. 73 × 103/ml). It showed that radiation can enhance the ability of the supernatants containing FL of HFCL/EF to expand early hematopoietic progenitor cells and protect hematopoietic cells from radiation-injury effects. The HFCL/EF and CD34+cells from human umbilical cord blood were one after the other transplanted i. v. into sublethally irradiated severe combined immunodeficient (SCID) mice. In contrast to two control groups (HFCL and HFCL/F), HFCL/EF (the Egr-1 regulatory element-drived expression of FL gene therapy) resulted in a proportionally obvious increase in the number of the white blood cell at early stage after radiation, while no significant differences were found for CD45+ 、CD34+ cells in bone marrow cells. In contrast to two control groups (HFCL and HFCL/F), HFCL/EF (the Egr1 regulatory element-drived expression of FL gene therapy) resulted in a proportionally obvious increase in the number of the white blood cell at early stage after radiation, without significant differences being found for CD45+、CD34+ 、CFU-GM and marrow nucleared cells in bone marrow cells. Conclusions:The results suggested both in vivo and in vitro use of the gene therapy of FL gene regulated by Egr-1 promoter could protect hematopoiesis from irradiation-induced damage.     

肿瘤坏死因子α/胞外信号调节激酶途径对HaCaT细胞迁移能力及小鼠全层皮肤缺损的影响

目的探讨肿瘤坏死因子α(TNF-α)/胞外信号调节激酶(ERK)途径对HaCaT细胞迁移能力及小鼠全层皮肤缺损的影响。方法采用实验研究方法。按照随机数字表法(下同)将HaCaT细胞分为常氧组和低氧(氧气体积分数为1%, 下同)条件下培养的低氧组。培养24 h后, 采用微阵列置信度分析软件SAM 4.01筛选出2组细胞的显著差异表达基因, 通过京都基因和基因组百科全书对信号通路中每条基因数目的显著性进行分析以筛选差异显著的信号通路, 样本数为3。另取HaCaT细胞, 在低氧条件下培养0(即刻)、3、6、12、24 h, 采用酶联免疫吸附测定(ELISA)法检测细胞分泌TNF-α的水平, 样本数为5。另取HaCaT细胞, 分为常氧组、单纯低氧组和加入FR180204(一种ERK抑制剂)并置于低氧条件下培养的低氧+抑制剂组, 培养3、6、12、24 h, 采用划痕试验检测细胞的迁移能力, 样本数为12。另取HaCaT细胞, 在低氧条件下培养0、3、6、12、24 h, 采用蛋白质印迹法检测细胞中磷酸化核因子κB(p-NF-κB)、磷酸化p38(p-p38)、磷酸化ERK1/2(p-ERK1/...     

YAP调控肾癌细胞迁移的机制研究

目的探究YAP对肾癌细胞迁移的影响及其调控机制。方法查找TCGA数据库进行YAP表达与肾癌发生发展相关性分析;慢病毒感染建立YAP敲低稳克隆细胞系,并通过Western blot和RT-PCR方法检测YAP蛋白和mRNA的表达;Transwell实验研究YAP敲低对肾癌细胞迁移的影响;鬼笔环肽免疫荧光染色法检测YAP敲低对细胞伪足F-actin的影响。结果分析TCGA数据库资料得到,YAP mRNA表达量升高与肾癌T4期存在显著相关且具有统计学意义(P0.05);敲低YAP显著降低肾癌细胞伪足的形成,从而影响细胞的迁移能力。结论 YAP通过影响细胞F-actin形成以调控肾癌细胞的迁移能力。     

BMSCs来源外泌体调节表皮HaCaT细胞及对烧伤创面愈合的促进作用

目的:探讨骨髓间充质干细胞(Bone mesenchymal stem cells,BMSCs)的外泌体对表皮细胞(HaCaT细胞)增殖、侵袭与迁移的影响,以及对小鼠烧伤创面愈合的作用。方法:分离培养小鼠的BMSCs,超速离心法收集BMSCs来源的外泌体,采用BMSCs来源的外泌体培养HaCaT细胞,CCK-8法检测BMSCs来源的外泌体对HaCaT细胞增殖的影响,细胞划痕实验和Transwell实验检测BMSCs来源的外泌体对HaCaT细胞迁移与侵袭的影响。选取25只C57BL/6小鼠构建烧伤模型,22只成功建模,将存活小鼠分为对照组和外泌体组,每组11只,外泌体组烫伤周围注射BMSCs来源的外泌体,对照组小鼠同时注射等体积PBS溶液。采用ImageJ软件分析小鼠创面愈合率,通过HE染色观察创面组织学变化,采用酶联免疫吸附法(ELISA)检测创面组织中炎性因子肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、白细胞介素-10(IL-10)的水平,免疫组织化学染色检测创面组织Ⅰ型胶原(ColⅠ)和Ⅲ型胶原(ColⅢ)表达。结果:成功分离培养出小鼠BMSCs,并得到BMSCs来源的外泌体。与对照组比较,实验组HaCaT细胞增殖活性明显升高;细胞的划痕愈合率较对照组显著升高,细胞侵袭数目较对照组也显著增加,差异均具有统计学意义(P0.05)。与对照组小鼠比较,经BMSCs来源的外泌体处理的小鼠,第3、7、14、21、28天的创面愈合率显著升高,创面组织结构得到明显改善,且组织中促炎因子TNF-α、IL-1β的含量均显著降低,而抗炎因子IL-10的含量显著升高,Ⅲ型胶原表达明显,差异均具有统计学意义(P0.05)。结论:BMSCs外泌体可促进HaCaT细胞的增殖、侵袭与迁移能力,并减轻小鼠烧伤创面组织的炎症程度,促进创面愈合。     

巨噬细胞对皮肤创面愈合的调控机制研究进展

巨噬细胞是参与皮肤创面愈合的重要细胞类型。该文对近年巨噬细胞调控皮肤创面愈合的机制以及糖尿病难愈性创面中巨噬细胞极化异常的研究进展进行综述。研究表明, 皮肤创面巨噬细胞表型分为M1和M2型, 具有抗炎、促血管新生、促纤维化和组织塑形的作用。糖尿病时巨噬细胞功能障碍、细胞因子分泌时空紊乱、表观修饰异常都导致巨噬细胞极化异常。靶向巨噬细胞极化调控可能是解决皮肤难愈性创面的有效途径。     

Electrotaxis of alveolar epithelial cells in direct-current electric fields

Purpose: This study aims to elucidate the electrotaxis response of alveolar epithelial cells (AECs) in direct-current electric fields (EFs), explore the impact of EFs on the cell fate of AECs, and lay the foundation for future exploitation of EFs for the treatment of acute lung injury. Methods: AECs were extracted from rat lung tissues using magnetic-activated cell sorting. To elucidate the electrotaxis responses of AECs, d...     

两栖动物来源的肽同源二聚体OA-GL17d 通过微小RNA-663a/转化生长因子-β1/Smad 轴促进皮肤创面再生

两栖动物来源的多肽以及微小RNA(miR)在促进慢性创面的愈合方面具有很大的研发潜能, 发现新的多肽药物以及其相关miR, 并开发相应的miR靶向药物对于促进创面愈合具有重要的价值。昆明医科大学基础医学院杨新旺教授团队近期于《Burns & Trauma》发文《Amphibian-derived peptide homodimer OA-GL17d promotes skin wound regeneration through the miR-663a/TGF-β1/Smad axis》, 该研究采用凝胶过滤层析及反相高效液相色谱法从青蛙皮肤分泌物中分离出来一种新的多肽(命名为OA-GL17d), 通过Edman降解、质谱法和与互补DNA克隆相结合的方法对其氨基酸序列进行了测定, 通过钙黄绿素乙酰氧基甲酯/碘化丙啶对人永生化表皮细胞HaCaT的双重染色实验、小鼠血细胞的溶血活性实验和小鼠对急性毒性反应实验评估了该多肽的毒性, 采用淋巴细胞增殖检测实验、细胞划痕试验、与HaCaT细胞进行Transwell共培养实验、小鼠全层皮肤损伤及烫伤模型等实验观察了OA-GL17d的促愈合...     

肺癌相关基因SLC35F2 RNA干扰重组慢病毒载体的构建和鉴定

目的 针对肺癌相关基因SLC35F2构建RNA干扰(RNAi)重组慢病毒质粒并进行慢病毒包装,建立SLC35F2表达稳定抑制的H1299肺癌细胞株,并探讨SLC35F2基因的功能.方法 应用pGCSIL-PUR慢病毒载体构建针对SLC35F2的ShRNA载体,转染包装293T细胞,收集病毒上清转染肺癌细胞株H1299,经嘌呤霉素(puromycin)筛选并扩大培养得到稳定克隆;实时荧光定量聚合酶链反应(Real-time PCR)和Western blot检测癌细胞内SLC35F2的表达;CCK-8比色法检测细胞增殖;Annexin V-FITC/PI双染法检测细胞凋亡.结果 成功构建SLC35F2-ShRNA慢病毒载体(SLC-siRNA)及SLC35F2表达稳定抑制的肺癌细胞株,经过检测证实SLC-siRNA慢病毒载体对SLC35F2的抑制效率达81.8%;CCK-8检测显示SLC-siRNA稳定转染的H1299细胞株生长抑制率达16.3%,稳定转染株凋亡细胞百分比较阴性对照组明显升高(14.88%比3.16%,P＜0.05).结论 慢病毒载体介导的靶向SLC35F2的RNAi可有效抑制SLC35F2表达,降低肺癌细胞的增殖能力,增加其凋亡比例.

Abstract：

Objective To investigate the possibility of SLC35F2 inhibition by lentiviral vector-mediated RNA interference (RNAi) and the influence on cell proliferation and apoptosis in lung cancer cell line,and to set up a lung cancer cell line in which SLC35F2 is stably suppressed.Methods The lentiviral vector of siRNA targeted against SLC35F2 (SLC-siRNA) was constructed and transfected into the packaging cells 293T,and the viral supernatant was collected to transfect H1299 cells.After selection by puromycin and culture expansion,the stable cell clones were attained.Quantitative real-time fluorescent polymerase chain reaction (PCR) and Western blotting were used to detect the expression of SLC35F2.The effect of SLC35F2 silencing by RNAi on cell proliferation was quantified by CCK-8 assay.Annexin V-FITC/PI staining was employed to examine the apoptosis.Results Lentiviral vector SLC35F2-shRNA was constructed successfully.As compared with control group,the SLC35F2 expression was decreased to 81.8% in RNA and protein levels.CCK-8 revealed that the inhibition rate of H1299 cells transfected with SLC35F2 was 16.3%,and the apoptosis rate was significantly increased as compared with negative control group ( 14.88% vs 3.16% ,P ＜0.05 ).Conclusion Lentiviral vector-mediated RNA interference targeting against SLC35F2 can effectively inhibit SLC35F2 expression and cell proliferation.The lung cell line in which SLC35F2 gene was stably suppressed was successfully established.     

Rac1蛋白调控表皮干细胞迁移促进创面愈合的研究

目的 观察Rac1蛋白对表皮干细胞(ESC)迁移运动的调控作用,为完善创面愈合的基础理论以及指导临床应用提供参考.方法 将慢病毒空载体FUGW单独和分别与Rac1蛋白抑制型突变体Rac1T17N、Rac1蛋白活化型突变体Rac1Q61L融合后转染入ESC,按照随机数字表法分为3部分进行如下实验.(1)将ESC分别接种于Ⅰ型胶原溶液(20 μg/mL)、Ⅳ胶原溶液(20 μg/mL)或纤连蛋白溶液(10 μg/mL)包被的24孔细胞培养板,采用CytoTox 96 比色试剂盒检测ESC对不同基质的黏附率.(2)选取上述黏附于Ⅳ型胶原的1000个ESC,四甲基异硫氰酸罗丹明标记的鬼笔环肽染色,激光扫描共聚焦显微镜下观察黏附细胞的形态延展并比较面积大小.(3)选用Transwell小室,上室加入ESC、下室中加入含基质细胞衍生因子1(SDF-1)的限定性角质形成细胞无血清培养液(以不加SDF-1的培养液为对照),倒置相差显微镜下观察ESC的趋化能力,结果以细胞迁移变化率表示.(4)将ESC接种于6孔细胞培养板孵育12 h,加入含4 μg/mL丝裂霉素C的培养液继续孵育2 h,于单层贴壁细胞划痕,6、12 h后统计剩余划痕宽度百分率.对数据进行t检验.结果 与转染FUGW的ESC比较,转染Rac1Q61L的ESC对Ⅰ型胶原的黏附率明显增加(t＝5.302,P<0.05),转染Rac1T17N的ESC对Ⅰ型胶原(t＝13.741,P<0.05)、Ⅳ型胶原(t＝15.676,P<0.05)及纤连蛋白(t＝8.256,P<0.05)的黏附率均明显下降.激光扫描共聚焦显微镜下观察,与转染FUGW的ESC比较,转染Rac1Q61L的ESC面积明显增大,细胞边缘有层板状伪足伸出;转染Rac1T17N的ESC面积显著缩小.在趋化因子SDF-1作用下,与转染FUGW的ESC比较,转染Rac1Q61L的ESC迁移变化率升高43.4%,转染Rac1T17N的ESC迁移变化率下降78.0%;无SDF-1作用时,与转染FUGW的ESC比较,转染Rac1T17N的ESC迁移变化率下降55.2%,转染Rac1Q61L的ESC迁移变化率未见明显变化(升高1.7%).划痕后6、12 h,转染Rac1Q61L的ESC剩余划痕宽度百分率分别为(39±9)%、(6±5)%,低于转染FUGW的ESC[(43±5)%,t=1.027,P>0.05;(18±7)%,t=4.389,P<0.05];划痕后6、12 h,转染Rac1T17N的ESC剩余划痕宽度百分率分别为(81±9)%、(71±11)%,明显高于转染FUGW的ESC(t值分别为11.386、11.726,P值均小于0.05).结论 Rac1蛋白可通过影响ESC的黏附、延展以及趋化能力调控细胞迁移,并可能因此参与ESC促进创面愈合的进程.

Abstract：

Objective To investigate modulatory role of Rac1 protein in epidermal stem cell (ESC) migration during wound healing,in order to provide a reference for enriching basic theory of wound healing and guiding clinical application. Methods Constitutively active mutant of Rac1 protein (Rac1Q61L) or dominant negative isoform of Rac1 protein (Rac1T17N) was transfected into ESC using a retroviral vector FUGW,and retroviral vector FUGW transfected into ESC in singles was used as blank control. The cells were divided into 3 parts according to the random number table and treated as follows. First,equal numbers of cells were inoculated into 24-well plates coated with collagen Ⅰ (20 μg/mL),collagen Ⅳ (20 μg/mL) or fibronectin (10 μg/mL). Cells adhered to above matrices were quantitated using CytoTox 96 colorimetric kit. Second,1000 cells adhered to collagen Ⅳ,after being stained with tetramethyl rhodamine isothiocyanate-phalloidin,were collected for observation of cell morphology and comparison of spreading area under confocal laser scanning microscope. Third,ESC with density of 2×105 cells per well were placed in upper compartment of Transwell chamber,DK-SFM culture medium alone or that containing stromal cell derived factor 1 (SDF-1) was added into lower compartment of Transwell chamber. Migration of ESC was observed using inverted phase contrast microscope,and the result was denoted as migration rate. Lastly,ESC with density of 7.5×105 cells per well was inoculated into 6-well plates for 12 hours,and treated with 4 μg/mL mitomycin C for 2 hours. The remaining scratch width of monolayer was respectively measured 6 hours or 12 hours after scratching to calculate the percentage of remaining scratch width. Data were processed with t test.Results Compared with that of blank control,the number of Rac1Q61L-transfected cells adhered to collagen Ⅰ was significantly increased (t＝5.302,P<0.05),while the number of Rac1T17N-transfected cells adhered to collagen Ⅰ,Ⅳ,and fibronectin were all obviously decreased (with t value respectively 13.741,15.676,8.256,P values all below 0.05). Confocal laser scanning microscope showed that spreading area of Rac1Q61L-transfected ESC (with laminate pseudopodia on edge) and Rac1T17N-transfected ESC was respectively larger and smaller as compared with that of blank control. With SDF-1 effect,the migration rate of Rac1T17N-transfected ESC was decreased by 78.0% and Rac1Q61L-transfected ESC was increased by 43.4% as compared with that of blank control. Without SDF-1 effect,the migration rate of Rac1T17N-transfected ESC was decreased by 55.2%,while the migration rate of Rac1Q61L-transfected ESC was close to that of blank control. Six or 12 hours after scratching,the percentage of remaining scratch width in Rac1Q61L-transfected ESC was lower as compared with that in blank control[(39±9)% vs. (43±5)%,(6±5)% vs. (18±7)%,with t value respectively 1.027,4.389,with P value respectively above and below 0.05],while that in Rac1T17N-transfected ESC[(81±9)%,(71±11)%,respectively]was obviously higher as compared with that in blank control (with t value respectively 11.386,11.726,P values all below 0.05). Conclusions Rac1 protein may control the migration of ESC by regulating its adhesion,spreading,and chemotaxis,and it plays an active role in wound healing accelerated by ESC.     

硅酸钙通过激活表皮生长因子/表皮生长因子受体/胞外信号调节激酶信号通路改善人表皮干细胞的功能以加速皮肤创面愈合

人表皮干细胞(hESC)在促进创面愈合及再上皮化过程中发挥着重要作用, 但激活hESC的有效方法仍有待探索。2021年9月解放军总医院第四医学中心(医学创新研究部创伤修复与组织再生研究中心)付小兵院士和张翠萍教授团队在《Burns & Trauma》发文《Calcium silicate accelerates cutaneous wound healing with enhanced re-epithelialization through EGF/EGFR/ERK-mediated promotion of epidermal stem cell functions》, 该文探讨了硅酸钙对hESC 的作用及其可能机制。该研究观察到, 通过稀释硅酸钙离子提取物来培养hESC, 可改善其增殖能力、迁移能力和干性等生物学特性, 同时证实了硅酸钙可激活EGF/EGF 受体(EGFR)/胞外信号调节激酶(ERK)信号通路。该研究还采用小鼠全层皮肤缺损模型探讨了硅酸钙在创面愈合和再上皮化中的作用, 结果显示硅酸钙上调了细胞角蛋白19和整合素β1 的表达, 提示硅酸钙改善了hESC 的干...     

1, 4, 5-三磷酸肌醇3激酶A调控脑胶质瘤细胞增殖、迁移和上皮间质化

目的探讨1, 4, 5-三磷酸肌醇3激酶A(ITPKA)在脑胶质瘤细胞的表达和功能。方法利用实时定量聚合酶链反应(PCR)和蛋白质印迹法(Western blot)比较SVGP12、U118和U87细胞系(购自中国科学院生物化学与细胞生物学研究所)ITPKA mRNA和蛋白表达水平。将U87细胞分为对照组、对照短发卡RNA(shRNA)组和sh-ITPKA组, 噻唑蓝法检测转染细胞培养24、48、72、96 h后细胞活性, 平板克隆和小室迁移(Transwell)法检测克隆形成能力与细胞迁移能力, Western blot检测ITPKA、波形蛋白、N-钙黏蛋白和E-钙黏蛋白表达水平。多组间比较采用单因素方差分析或双因素方差分析。结果 U118和U87细胞中ITPKA mRNA和蛋白表达水平明显高于SVGP12细胞(mRNA水平:2.81±0.40、3.92±0.19比1.06±0.06, F=94.48, P<0.01;蛋白水平:0.57±0.05、0.66±0.03比0.20±0.01, F=161.30, P<0.01)。培养24、48、72、96 h后, sh-ITP...     

脂肪干细胞外泌体在创面修复中的研究进展

脂肪干细胞外泌体(adipose-derived stem cell derived exosomes,ADSC-Exos)是一种大小为30～150 nm的脂质双分子层囊泡,参与细胞间信息交换,并在组织修复与再生,以及疾病诊断等方面发挥着重要作用。ADSC-Exos可以调节炎症反应,加速细胞增殖、分化,促进细胞迁移,调控血管生成速度,改变细胞外基质重建程度,抑制瘢痕形成等,促进创面修复过程的同时抑制异常修复。现对其在各类创面修复中的作用进行总结,并结合临床实践应用,为临床医师提供参考。     

诱导型一氧化氮合酶与转化生长因子-β1的相互调控:诱导型一氧化氮合酶参与创面愈合的分子和细胞机制

创面愈合异常和瘢痕影响健康, 并对社会经济和患者心理造成影响。慢性创面和瘢痕的形成与诱导型NOS(iNOS)的上调有关。为了进一步明确iNOS在创面愈合中的生理调控作用, 研究人员通过生物化学和免疫组织化学分析研究了iNOS在创面愈合中的作用机制, 研究显示:(1)iNOS是创面一氧化氮的主要来源;(2)在创面中抑制NOS的表达, iNOS、mRNA和酶活性下调, 创面一氧化氮水平降低;(3)在创面形成早期抑制iNOS会导致愈合延迟, 在晚期抑制iNOS会导致瘢痕加重。对创面的分子和细胞进行分析表明, 抑制iNOS 可显著增加TGF?β1 mRNA 及其蛋白水平, 增加Fb 和胶原沉积, 提示iNOS 可能通过TGF?β1信号通路在创面中发挥作用, TGF?β1激活创面Fb产生过多的胶原蛋白。该研究结果证明iNOS对生理性创面愈合至关重要, 炎症期iNOS的失调会损害愈合, 并导致愈合后瘢痕;iNOS与TGF?β1之间在基因、蛋白和功能水平上相互反馈调节, 可能是iNOS调节愈合的机制;监测和维持创面一氧化氮水平对易感人群(包括糖尿病创面、静脉溃疡或瘢痕疙瘩易感人群)创面的愈合和避免长...     

维持表皮内树突状T细胞稳态及活化的分子机制及其在创面愈合中的作用

表皮是皮肤的最外层结构, 是保护机体的第1道屏障, 表皮内树突状γδT细胞(DETC)是表皮免疫微环境的重要组成部分, 在皮肤创伤愈合、恶性肿瘤和自身免疫性疾病等方面发挥重要的调控作用。陆军军医大学(第三军医大学)第一附属医院贺伟峰教授团队近期于《Burns & Trauma》发文《The molecular mechanisms supporting the homeostasis and activation of dendritic epidermal T cell and its role in promoting wound healing》, 对DETC的起源、发育、定植、活化及其对皮肤稳态维持和创面愈合调控的作用进行了总结。该文提到DETC在胚胎期产生并定植在表皮, 之后依靠自身的增殖维持更新, 其主要功能包括通过分泌胰岛素样生长因子1和角质细胞生长因子1/2促进皮肤稳态和再上皮化, 释放促炎因子和趋化因子调节创面愈合的炎症微环境。充分认识DETC起源、发育、激活和相关的信号通路, 对于探索促进创面愈合的新方法具有重要的参考价值。     

缓激肽促进人肺腺癌细胞株A549的侵袭及其机制

目的 观察缓激肽(BK)对人肺癌细胞株A549迁移、侵袭能力的影响.方法 应用划痕愈合实验和Transwell实验检测BK对40例人肺癌细胞株A549的划痕愈合和运动侵袭能力.通过Western blot分析刺激和抑制缓激肽各20例后基质金属蛋白酶(MMP)-2、MMP-9和E-钙黏素(E-Cadherin)的变化并研究其机制.结果 划痕实验后分别测量愈合距离,结果 示BK组24h和48h后迁移细胞数分别为69.2±3.3、94.1±2.9,而阻断其受体或者ERK1/2通路则迁移细胞数分别为51.2±2.1、73.2±2.7和47.5±3.4、77.6±3.8.Transwell实验检测BK对A549侵袭能力的影响结果 显示BK可以明显促进A549的侵袭能力.阻断BK受体或ERK1/2通路则可以抑制其侵袭能力.结论 缓激肽促进人肺腺癌细胞株A549的迁移和侵袭能力.

Abstract：

Objective To investigate the effect of bradykinin (BK) on human lung cancer cell line A549 migration,invasion ability. Methods Scratch Healing and Transwell assay of bradykinin were used to detect scratch healing and movement on A549 human lung cancer cells. Analysis the changes of matrix metalloproteinase (MMP)-2, MMP-9 and E-Cadherin after stimulate and suppress 20 cases respectively in the aim to describe the mechanism by Western blotting. Results In the former, BK group promote cell migration significantly,migrated cell was 69.2±3.3, 94.1±2.9 respectively after 24 h and 48 h,blocking its receptor or ERK1/2 pathway inhibit migration,migrated cell was 51.2±2.1, 73.2±2.7 and 47.5±3.4,77.6±3.8 after 24 h and 48 h. In the latter,Transwell assay of A549 BK showed invasion of A549 BK can significantly promote the invasion ability of A549 to penetrate the basement membrane of the cell. And blocking BK receptor or ERK1/2 pathway can inhibit the invasion. Conclusion Bradykinin promote human lung adenocarcinoma cell line A549 migration and invasion.