脐血内皮细胞对脐血早期造血细胞的体外扩增作用

目的:观察脐血内皮细胞联合细胞因子组合通过非接触方式对脐血早期造血细胞的扩增作用。方法:采用优化的内皮细胞培养基培养脐血内皮细胞;实验组分为细胞因子组合组(SCF+IL-3+IL-6+GM-CSF,CKs组)和脐血内皮细胞联合CKs非接触脐血CD34+细胞培养组(noncontact组)。Min-iMACS磁珠分选脐血CD34+细胞;检测CKs组与nocontact组培养7d后脐血早期造血细胞的扩增倍数。结果:noncontact组和CKs组都能扩增早期造血细胞,noncontact组对早期造血细胞的扩增倍数显著优于CKs组的扩增倍数。结论:脐血内皮细胞联合CKs非接触培养对脐血早期造血细胞的扩增作用显著优于CKs组。

脐血内皮祖细胞尾静脉与局部注射治疗糖尿病下肢缺血

背景:内皮祖细胞治疗糖尿病下肢缺血临床及动物实验多采用局部肌肉注射。目的:比较脐血内皮祖细胞鼠尾静脉与局部注射治疗糖尿病下肢缺血效果的差异。方法:取Wistar雄性大鼠分成5组:①糖尿病射线照射后结扎双后肢股动脉,尾静脉注射内皮祖细胞(DLV)。②糖尿病结扎双后肢股动脉左后肢局部肌肉注射PBS(DLC),右后肢局部肌肉注射内皮祖细胞(DLM)。③正常大鼠射线照射后结扎双后肢,尾静脉注射内皮祖细胞(NLV)。④糖尿病不结扎不注射内皮祖细胞(DC)。用绿色荧光示踪内皮祖细胞,苏木精-伊红染色检测肌纤维间毛细血管数,RT-PCR检测双后肢肌肉血管内皮生长因子mRNA水平。结果与结论:DLV组与DLM组比较,右后肢腓肠肌溃疡及缺血好转明显,二者无明显区别;有明显荧光,差别不明显,Ⅷ因子免疫组织化学染色肌纤维间毛细血管数多,相互间无明显差别;腓肠肌血管内皮生长因子表达差异无显著性意义(P>0.05)。提示脐血内皮祖细胞治疗糖尿病大鼠下肢缺血尾静脉注射与局部肌肉注射效果相当。

一种从脐血培养高增殖潜能内皮祖细胞的新方法

【目的】建立一种培养脐血高增殖潜能内皮祖细胞(High proliferative potential-endothelial progenitorcells,HPP-EPCs)的稳定经济的新方法,并将由HPP-EPCs增殖而来的内皮祖细胞(Endothelial progenitor cells,EPCs)与脐静脉内皮细胞(Human umbilical vein endothelial cells,HUVECs)进行体外比较。【方法】分离脐血单个核细胞,接种到纤连蛋白(Fibronectin,FN)预处理的器皿中,用含有血管内皮生长因子(VEGF)和内皮细胞生长添加物(ECGS)等的MCDB131培养基培养,4d后去除未贴壁细胞,继续培养10～21d后,可以得到HPP-EPCs来源的EPC克隆;从人脐带中分离HUVECs,用含有EGF的MCDB131培养基扩增培养;同时分离培养人成纤维细胞(Humanembryonic fibroblast cells,hEFs)。通过免疫表型、UEA1结合试验、Matrigel试验、DiI-Ac-LDL吞噬试验等对分离的脐血EPCs进行鉴定,以HUVECs为阳性对照,hEFs为阴性对照。【结果】1个HPP-EPC可在2个月中扩增出108～1010个EPCs细胞,在长期体外培养中可以保持正常核型。脐血EPCs和HUVECs均可表达CD31、CD144、vWF,结合UEA1,并能在Matrigel上形成毛细血管样结构,也能吞噬DiI-Ac-LDL。【结论】本研究所建立的新方法能从脐血中高效经济地获得较原始的EPCs,并能在体外长期扩增培养,有望成为缺血性疾病细胞替代治疗有效的种子细胞。

莱菔硫烷对过氧化氢诱导人脐静脉血内皮细胞损伤的保护作用及机制研究

目的探讨莱菔硫烷(Sulforaphane,SFN)对过氧化氢(H_2O_2)诱导的人脐静脉血内皮细胞(HUVEC)氧化损伤的保护作用,并进一步研究莱菔硫烷在保护内皮细胞损伤中的机制。方法复苏由中国科学院上海细胞库提供的人脐静脉内皮细胞株,37℃、5%CO2条件下培养细胞至80%以上融合度时,分组进行干预。实验分组:(1)空白对照组(对照组):细胞正常培养2.5 h;(2)过氧化氢组(H_2O_2组):分别给予100、200、400、600、800μmol/L浓度培养细胞2 h,选择400μmol/L为其最佳干预浓度(后续各组如未特殊注明,默认H_2O_2浓度为400μmol/L);(3)莱菔硫烷+过氧化氢组(SFN+H_2O_2组):不同浓度SFN(2.5、5、10μmol/L)预孵育30 min后,加入400μmol/L的H_2O_2继续培养2 h。收集干预后的各组细胞,用MTT法比较细胞活性,流式细胞技术测定细胞内ROS水平,逆转录聚合酶链反应法(RT-PCR法)测定细胞内转录因子核因子E2(Nrf2)m RNA和血红素加氧酶-1(HO-1) mRNA表达情况。结果与对照组相比,H_2O_2组MTT法测定的细胞吸光光度值(A值)降低(P<0.05),并浓度依赖性的使A值下降,SFN+H_2O_2组细胞吸光光度值高于H_2O_2组(P<0.05)。H_2O_2组ROS水平高于对照组(P<0.05),SFN+H_2O_2组ROS水平低于H_2O_2组(P<0.05)。H_2O_2组Nrf2 mRNA、HO-1 mRNA表达水平低于对照组(P<0.05),SFN+H_2O_2组Nrf2 mRNA、HO-1 mRNA表达水平高于H_2O_2组(P<0.05)。结论莱菔硫烷对过氧化氢致HUVEC的氧化损伤有保护作用,其机制可能与莱菔硫烷在转录水平激活Nrf2/HO-1表达有关。

连接蛋白Lnk在人脐静脉内皮细胞中的表达

目的:证实体外培养的人脐静脉内皮细胞(HUVEC)存在Lnk分子的表达。方法:体外培养HUVEC系用逆转录聚合酶链反应(RT-PCR)和W estern印迹技术,检测HUVEC细胞中的Lnk mRNA及蛋白表达。结果:连接蛋白Lnk mRNA及蛋白在HUVEC中均有表达。结论:HUVEC组成性地表达Lnk,可作为体外研究Lnk的细胞系。

脐血内皮祖细胞移植改善肢体缺血的研究

目的 研究脐血来源血管内皮祖细胞 (endothelialprogenitorcells,EPC)的移植对局部缺血的恢复作用。方法 脐血CD133+ 细胞在体外诱导扩增 10～ 14d后 ,收集贴壁梭形细胞 ,检测其内皮细胞特异标志的表达。将荧光标记的贴壁细胞从尾静脉移植到后肢单侧缺血的裸鼠体内。结果从脐血CD133+ 细胞中可诱导出EPC ,表现为表达内皮细胞标志的贴壁细胞。移植后 ,可整合到缺血部位新生血管内。移植EPC后裸鼠缺血后肢的毛细血管密度、血流灌注及坏死程度均较对照组明显改善 :缺血手术后 2周EPC组的缺血肢 /正常肢的血流比由 19 1%± 3 1%恢复为 77 3%± 5 6 % ,而对照组仅恢复为 4 0 6 %± 3 4 % (P <0 0 0 1) ;缺血后肢的完全恢复率由对照组的 1/10上升至 7/12 (P <0 0 5 )。缺血局部VEGFmRNA上调 ,体外VEGF对EPC具有趋化作用。 4h内趋化到下腔的细胞数为817个± 33个 ,而对照组为 4 74个± 6 2个 (P <0 0 5 )。结论 脐血CD133+ 细胞能诱导EPC ,体内移植后能促进肢体缺血的恢复 ,而缺血局部上调的VEGF表达可能是EPC定向整合到缺血局部的关键因素。

泰山白花丹参提取物对H_2O_2诱导的人脐血内皮祖细胞损伤的保护作用及机制

目的:研究泰山白花丹参提取物对过氧化氢(H2O2)诱导的人脐血内皮祖细胞(EPCs)氧化应激损伤的保护作用及机制。方法:通过密度梯度离心分离人脐血单个核细胞,EBM-2完全培养基诱导分化培养EPCs。以0.001%H2O2诱导EPCs损伤,以泰山白花丹参提取物干预,以eNOS抑制剂L-NMMA定位泰山白花丹参对EPCs的作用靶位。通过光镜观察细胞形态,以四甲基偶氮噻唑蓝比色法检测细胞活力,用流式细胞仪AnnexinV/PI双染法检测细胞凋亡变化,取各实验组上清行丙二醛(MDA)含量检测。结果:EPCs受到氧化损伤后,细胞活力明显降低,细胞凋亡明显增加,上清MDA含量明显增加,加入白花丹参提取物可明显改善上述结果,而1 mmol.L-1L-NMMA+0.6 g.L-1白花丹参制剂+H2O2处理组与H2O2组比较细胞活力和细胞凋亡率都没有显著差异。结论:泰山白花丹参提取物对H2O2损伤后内皮祖细胞有显著保护作用,eNOS抑制剂L-NMMA能抵消这种保护作用,说明泰山白花丹参可能是通过eNOS途径对EPCs氧化应激损伤起保护作用(eNOS是白花丹参的作用靶点之一)。

脐血内皮祖细胞治疗糖尿病大鼠下肢缺血的实验研究

目的研究脐血内皮祖细胞(EPC)治疗糖尿病下肢缺血的有效性及机制,为临床治疗糖尿病足病(DF)提供理论依据。方法取产妇足月产脐血分离单核细胞,培养7d,流式细胞仪鉴定细胞,并计数活细胞数。将Wistar雄性大鼠分成3组:(1)糖尿病大鼠射线照射后结扎双后肢股动脉,尾静脉注射血管EPC(DE组)。(2)糖尿病大鼠结扎双后肢股动脉,尾静脉注射缓冲液(DP组)。(3)正常大鼠射线照射后结扎双后肢,尾静脉注射EPC(NC组)。用绿色荧光(GFP)示踪EPC,Ⅷ因子免疫组化检测肌纤维间毛细血管数,RT-PCR检测双后肢肌肉血管内皮生长因子(VEGF)mRNA水平。结果 DE、NC组右后肢腓肠肌溃疡及缺血明显好转,有明显荧光,肌纤维间毛细血管数多,VEGF表达高,DP组好转不明显,无荧光,毛细血管数少,VEGF表达少,其差异有统计学意义(P<0.05)。结论脐血EPC治疗糖尿病大鼠下肢缺血有效。

CD4^+CD25^+调节性T细胞对氧化型低密度脂蛋白致内皮祖细胞损伤的保护作用

目的:探讨CD4+CD25+调节性T细胞(CD4+CD25+ regulatory T cells,Tregs)对氧化型低密度脂蛋白(ox-LDL)致人外周血内皮祖细胞(EPCs)损伤的保护机制。方法:密度梯度离心法分离单个核细胞,培养7d后收获EPCs;磁性细胞分离器(MACS)分离获得Tregs。流式细胞仪鉴定EPCs及Tregs纯度。实验随机分为对照组,ox-LDL组(50mg/L)及ox-LDL+Tregs组,干预24h后,取各组细胞上清液行超氧化物歧化酶(SOD)、丙二醛(MDA)含量检测。收获各组细胞行凋亡相关基因Bcl-2及一氧化氮(NO)的检测。结果:与对照组比较,ox-LDL作用于EPCs后,其SOD,Bcl-2及NO含量显著下降、MDA含量显著升高;Tregs干预24h后,显著改善了EPCs的功能,各组SOD,Bcl-2及NO含量显著升高,MDA含量显著减少。结论:Tregs对ox-LDL致EPCs损伤有显著保护作用,其机制可能与其抗氧化损伤、抗凋亡及促进EPCs的动员与分化有关。

Protective effects of icariin on human umbilical vein endothelial cell injured by angiotensin Ⅱ

To investigate the effects of icariin （ICA） on angiotensin Ⅱ（Ang Ⅱ）-induced injury in human umbilical vein endothelial cells line （ECV-304）. The ECV-304 cells were cultured in vitro. After 24 h incubating with icariin, the model of AngⅡ-induced injury in ECV-304 was established. The cell viability （MTT method）, Lactate dehydrogenase （LDH） release and Nitric oxide （NO） production in the medium, the capacity of scavenging superoxide anion radicals （O2^-） and hydroxyl radicals （.OH） were measured. The activities of superoxide dismutase （SOD）, total nitric oxide synthase （T-NOS）, inducible nitric oxide synthase （iNOS） and constitutive nitric oxide synthase （cNOS） in the cells were determined. Compared with the Ang Ⅱ-treated group, ICA can significantly raise the viability of EC, increase the activities of SOD, T-NOS and cNOS, increase the production of NO, enhance the capacity of scavenging superoxide anion radicals （ O2^- ） and hydroxyl radicals（.OH）, and lower LDH leakage and iNOS activity. The results suggest that ICA can protect endothelial cells （ECV-304） from Ang II-induced injury.

Immortalization of human umbilical vein endothelial cells with telomerase reverse transcriptase and simian virus 40 large T antigen

Objective: To establish normally conditionally-immortalized human umbilical vein endothelial cells (HUVECs) by ectopic expression of the human telomerase catalytic enzyme (hTERT) and simian virus 40 large T (SV40 LT) antigen. Methods:Primary HUVECs were transfected with recombinant retrovirus containing hTERT or SV40 LT respectively. Subsequently drug resistant cell clones were screened and expanded for further studies. Endothelial cell biomarkers were confirmed by examination.Results: The morphological phenotype of the transfected cells was similar to the non-transfected cells. Von Willebrand factor,hTERT and SV40 LT could be detected in transfected HUVECs. Moreover, higher telomerase activity in transfected cells was maintained for over 50 population doublings compared with only low level of endogenous telomerase transiently at early population doublings in primary HUVECs. When exposed to TNF-α (tumor necrosis factor-α), the expression of E-selectin in transfected cells was significantly up-regulated, but no alteration of endothelial lipase was found. Conclusion: Ectopic coexpression of hTERT and SV40 LT can effectively immortalize HUVECs without tumorigenicity in vitro. Immortalized HUVECs may be an ideal target of further molecular function studies.

Kinase domain insert containing receptor promoter controlled suicide gene system selectively kills human umbilical vein endothelial cells

AIM： To study the selective killing of human umbilical vein endothelial cells （HUVECs） by a double suicide gene under the regulation of a kinase domain insert containing receptor （KDR） promoter and mediated by an adenoviral gene vector. METHODS： Human KDR promoter was cloned by polymerase chain reaction （PCR）, and two recombinant adenoviral plasmids pAdKDR-CdgIyTK, pAdCMV-CDglyTK were constructed according to a two-step transformation protocol. These two newly constructed plasmids were then transfected into 293 packaging cells to grow adenovirus, which were further multiplied and purified. HUVECs and LoVo cells were infected with either of the two resultant recombinant adenoviruses （AdKDR-CDglyTK and AdCMV-CDglyTK） respectively, and the infection rates were estimated by detection of green fluorescent protein （GFP） expression. Infected cells were cultured in culture media containing different concentrations of 5-fiuoroo/tosine （5-FC） and ganciclovir （GCV）, and the killing effects were measured. RESULTS： The two recombinant adenoviral plasmids pAdKDR-CdglyTK, pAdCMV-CDglyTK were successfully constructed and transfected into 293 cells. The resultant recombinant adenoviruses infected cells caused similar infection rates; and the infected cells exhibited different sensitivity to the prodrugs： HUVECs infected with AdCMV-CDglyTK and LoVo cells infected with AdCMVo CDglyTK were highly sensitive to the prodrugs, and HUVECs infected with AdKDR-CDglyTK were similarly sensitive but significantly more sensitive than the LoVo cells infected with AdKDR-CdglyTK （P 〈 0.001）. CONCLUSION： Selective killing of HUVECs may be achieved by gene transfer of double suicide gene under the regulation of the KDR promoter. This finding may provide an optional way to target gene therapy of malignant tumors by abrogation of tumor blood vessels.

Effects of static magnetic field on human umbilical vessel endothelial cell

Objective: To investigate the effects of static magnetic field(SMF) on the viability, adhesion molecule expression of human umbilical vessel endothelial cell. Methods: Magnetic flux intensity was 0. 1 mT, 1 mT, 10 mT. Cell viability and proliferation were measured with 3H-TdR and MTT methods; and apoptosis of human umbilical vein endothelial cell (HUVEC) was studied by flow cytometry and transmission electric microscopy. ELISA was used to measure the expression of ICAM-1 and VCAM-1 on endothelium. Results: 0. 1 mT SMF had no effects on the growth of HUVEC. however,SMF of 1 mT, 10 mT attenuated growth of HUVEC. 10 mT static magnetic field could induce apoptosis and necrosis of HUVEC. 10 mT SMF enhanced the expression of ICAM-1 and VCAM-1 on endothelium. Conclusion: The effect of SMF depends on the intensity of SMF. 10 mT SMF has adverse effects on human umbilical vessel endothelial cell.

Extract of Meretrix meretrix Linnaeus induces angiogenesis in vitro and activates endothelial nitric oxide synthase

Meretrix meretrix Linnaeus has long been used as traditional Chinese medicine in oriental medicine.The angiogentic activity of the extract of M.meretrix was investigated in this study,using human umbilical vein endothelial cells(HUVECs).Extract of M.meretrix Linnaeus(AFG-25) was prepared with acetone and ethanol precipitation,and further separated by Sephadex G-25 column.The results show that AFG-25 promoted proliferation,migration,and capillary-like tube formation in HUVECs,and in the presence of eNOS inhibitor NMA,the tube formation induced by AFG-25 is inhibited significantly.Moreover,AFG25 could also promote the activation of endothelial nitric oxide synthase(eNOS) and the resultant elevation of nitric oxide(NO) production.The results suggested that M.meretrix contains active ingredients with angiogentic activity and eNOS/NO signal pathway is in part involved in the proangiogenesis effect induced by AFG-25.

The Inhibitory Effects of Arresten Protein on Tumor Formation

Objective To examine the inhibitory effects of recombinant purified arresten on tumor formation. Methods Purified arresten protein was incubated with human umbilical vein endothelial cells (HUVECs) and HeLa cells in vitro. The effect on proliferation of HUVECs and HeLa cells was examined using 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide assay, and apoptosis of these cells monitored by flow cytometry. The effect on migration of HUVECs and HeLa cells was examined by Boyden chamber. Twenty colon carcinoma-bearing C67BL/6 mice were used to investigate the antitumor effects of arresten protein. The mice were randomly divided into arresten treatment group (n=10) and control group (n=10). The microvessel densities of the tumors were measured by immunohistochemical staining with anti-CD31 monoclonal antibody. Results Arresten inhibited the proliferation and migration of HUVECs in a dose-dependent manner while promoting apoptosis. However, arresten had no significant effects on the proliferation and apoptosis of HeLa cells. The migration of HeLa cells was modestly inhibited by arresten. The arresten treatment group of mice showed no weight loss or unusual behavior during the course of treatment, and the tumor growth was significantly decreased; in contrast, the control group of mice exhibited rapidly growing tumors and cachexia. A dramatically decreased microvessel density in tumor tissues was found in arresten-treated mice compared with that in the control mice. Conclusion Arresten can inhibit tumor growth through inhibition of tumor angiogenesis.

ANTI-INFLAMMATORY EFFECTS OF MAGNOLIAE FARGESII VOLATILE OIL

Objective To explore the anti-inflammatory effects of magnoliae fargesii volatile oil.Methods Human umbilical vein endothelial cells (HUVECs) were stimulated by TNF-α to express the adhesion molecules. Then the anti-adhesion effects of magnoliae fargesii volatile oil between HUVECs and human peripheral neutrophils were observed. The ischemia-reperfusion animal models were established by 60min renal ischemia followed by 1, 3, 6 and 24h reperfusion. Rats were randomly divided into the following groups: the sham-operation controls, ischemic group only treated with normal saline, and treated group infused magnoliae fargesii volatile oil before reperfusion. Then the renal injury of rats was detected. Results High rate of cell adhesion between HUVECs and neutrophils was observed. Magnoliae fargesii volatile oil could inhibit the adhesion process at the concentration of 0.5μL/mL (191.6±8.6), 1.0μL/mL (158.2±9.0) and 2.0μL/mL (155.2±9.7) (P<0.05). The anti-adhesion effects were strengthened with the increase of volatile oil concentration. Blood urea nitrogen and creatinine levels of the animal models were significantly increased after 24h reperfusion while the increase was remarkably attenuated by the treatment with magnoliae fargesii volatile oil. The renal injury was severe after 1h reperfusion, which was significantly attenuated by the treatment of magnoliae fargesii volatile oil. Conclusion Magnoliae fargesii volatile oil has anti-inflammatory effects.

Inhibition of angiogenesis by DADAG in vivo and in vitro

1,2,5,6-Dianhydro-3,4-diacetyl-galactitol （DADAG）, an alkylating sugar alcohol derivative, has been shown effective against tumor growth. In this research, we explored the effect of DADAG on angiogenesis in chick chorioallantoic membrane （CAM） model and on the proliferation and migration of human umbilical vein endothelial cells （HUVECs）. We also studied the possible mechanism of the anti-angiogenesis effect of DADAG. The results showed that DADAG （100, 500 and 1000μnol/L） inhibited angiogenesis in CAM model dose-dependently. Sulforhodamine B （SRB） assay indicated that DADAG （45, 90, 135, 180 and 225 μmol/L） suppressed HUVECs proliferation in a dose-dependent and time-dependent manner. High Content Screening （HCS, Cellomics） assay, in which the influence of cell proliferation on migration could be excluded, indicated that DADAG （45, 135 and 225 ~xmol/L） directly inhibited the motility ofHUVECs. Immunofiuorescence assay suggested that DADAG inhibited angiogenesis possibly by decreasing vascular endothelial growth factor （VEGF） expression in HUVECs. Our findings reveal that DADAG show anti-angiogenic activity in vivo and in vitro, which is related to the downregulation of VEGF expression in endothelial cells.

葛根芩连汤对高糖诱导HUVECs线粒体影响研究

目的观察葛根芩连汤(GQD)对于高糖处理的人脐静脉血内皮细胞(HUVECs)线粒体功能、结构、线粒体动力学相关蛋白的影响,探讨GQD对血管内皮的保护作用及机制。方法制备GQD含药血清,体外培养HUVECs,高糖诱导HUVECs损伤模型,以CCK8法筛选出最佳给药浓度及时间并将细胞分为对照组、模型组、含药血清组和阳性对照组。以磷钼酸比色法检测HUVECs三磷酸腺苷(ATP)含量,乳酸脱氢酶比色法检测HUVECs乳酸(LD)含量,葡萄糖氧化酶-过氧化物酶法测定HUVECs葡萄糖(Glu)含量,以流式法检测线粒体膜电位,透射电镜观察线粒体结构,Wstern Blot法检测线粒体融合蛋白Mfn1、Mfn2和分裂蛋白Drp1表达。结果最佳给药浓度为5%,最佳作用时间为48 h。高糖处理的HUVECs细胞Glu及LD含量增加,ATP含量下降,线粒体膜电位降低,Mfn1、Mfn2表达下降,Drp1表达增加(P<0.05)膜结构被破坏。与模型组相比,GQD含药血清组及阳性对照组HUVECs Glu及LD含量下降,ATP含量增加,线粒体膜电位增加,Mfn1、Mfn2表达显著上调,Drp1表达下降(P<0.05)膜结构恢复。结论GQD含药血清能够改善高糖诱导的HUVECs细胞线粒体结构与功能,其机制可能与GQD能够调节线粒体动力学有关。

Cloning of differentially expressed genes following lipopolysaccharide stimulation in human umbilical vein endothelial cells

Objective: To clone the differentially expressed genes in human umbilical vein endothelial cells (HUVEC) stimulated by lipopolysaccharide (LPS). Methods: Two-directional (forward and backward) suppression subtractive hybridization (SSH) was performed on HUVEC cultured in either standard media or treated for 6 hours with LPS (100 ng/ml). To restrict the number of false-positive clones, colony dot hybridization was used to further verify the differentially expressed cDNA clones. Positive clones were sequenced. Results: These analyses have identified both novel and known genes whose expression is influenced by LPS. The known genes include a group related to proinflammatory events, a group related to cellular apoptosis and proliferation, a group related to protein synthesis and cytoskeletal rearrangment, and a group related to energy metabolism and signal transduction. Conclusions: SSH is a powerful technique of high sensitivity for the detection of differential gene expression in HUVEC stimulated by LPS.

Transcriptome analysis of blood stasis syndrome in subjects with hypertension

OBJECTIVE:To screen for m RNAs associated with blood stasis syndrome and to explore the genetic mechanisms of blood stasis syndrome in hypertension.METHODS:This study involved groups of patients with hypertension and blood stasis,including those with Qi deficiency,Qi stagnation,cold retention and heat retention;as well as hypertensive patients without blood stasis and healthy individuals.Human umbilical vein endothelial cells were co-cultured with the sera of these healthy individuals and patients with blood stasis syndrome.Total RNA was extracted from these cells and assessed by a high-throughput sequencing method(Solexa)and digital gene expression.Differentially expressed genes among these six groups were compared using whole genome sequences,and m RNAs associated with blood stasis syndrome identified.Differences in gene use and gene ontology function were an-alyzed.Genes enriched significantly and their pathways were determined,as were network interactions,and encoded proteins.Gene identities were confirmed by real-time polymerase chain reactions.RESULTS:Compared with cells cultured in sera of the blood stasis groups,those culture in sera of healthy individuals and of the non-blood stasis group showed 11 and 301 differences,respectively in stasis-related genes.Genes identified as differing between the blood stasis and healthy groups included activating transcription factor 4,activating transcription factor 3,DNA-damage inducible transcription factor 3,Tribbles homolog 3,CCAAT/enhancer binding protein-β,and Jun proto-oncogene(JUN).Pathway and protein interaction network analyses showed that these genes were associated with endoplasmic reticulum stress.Cells cultured in sera of patients with blood stasis and Qi deficiency,Qi stagnation,heat retention,and cold retention were compared with cells cultured in sera of patients with the other types blood stasis syndrome.The comparison showed differences in expression of 28,28,34,and 32 specific genes,respectively.CONCLUSION:The pathogenesis of blood stasis syndrome in hypertension is related to endoplasmic reticulum stress and involves the differential expression of the activating transcription factor 4,activating transcription factor 3,DNA-damage inducible transcription factor 3,Tribbles homolog 3,CCAAT/enhancer binding protein-β,and JUN genes.