Subcellular Localization of Small GTP-binding Protein DsRab in Dunaliella salina

With the total RNA of Dunaliella salina as a template,the cD NA sequence of D. salina small GTP-binding protein gene was amplified by RT-PCR technique,and cloned onto pM Dl8-T simple vector,the recon was subjected to PCR detection and restriction endonuclease analysis,and the total sequence of DNA was determined. The results showed that the cloned fragment was 612 bp,and shared 100% homology with reported D. salina DsRab gene（ GenB ank： JN989548）. The target gene fragment was inserted downstream of pM DCG 35 S promoter,constructing subcellular localization recombinant vector pM DCG-DsRab. The successfully constructed subcellular localization recombinant vector pM DCG-DsRab was transformed into Agrobacterium tumefaciens LBA4404,and positive single clones were screened and used for transinfection of onion epidemical cells by Agrobacterium-mediated method,and the instant expression of DsRab was observed under fluorescence microscope. The results showed that the fusion protein GFP-DsRab was successfully expressed in onion epidemical cells,and mainly distributed on cytomembrane. This study will provide reference for further illumination of the function and action mechanism of D. salina small GTP-binding protein DsRab.

肺炎衣原体感染通过PI3K激活Rac1而诱导血管平滑肌细胞迁移

目的:研究Rac1活化在肺炎衣原体(C.pn)感染诱导血管平滑肌细胞(VSMCs)迁移中的作用及磷脂酰肌醇3-激酶(PI3K)对其活化的影响。方法:谷胱甘肽巯基转移酶(GST)-p21活化激酶1 p21结合结构域(PBD)即GST-PBD重组质粒转化感受态细菌,诱导融合蛋白表达并纯化;GST-pull down实验检测C.pn感染VSMCs后Rac1活性的变化;PI3K特异性抑制剂LY294002(25μmol/L)预处理VSMCs,GST-pull down实验检测Rac1活性的变化;Rac1特异性抑制剂NSC23766(50μmol/L)预处理VSMCs,wound-healing实验和Transwell实验观察VSMCs迁移能力的变化。结果:重组质粒转化感受态细菌后,经诱导表达和纯化,得到足量有效的GST-PBD融合蛋白;GST-pull down实验结果显示,C.pn感染VSMCs后Rac1活性增强且显著高于正常对照组(P<0.05);LY294002预处理VSMC后,C.pn感染诱导的Rac1活性明显下降(P<0.05);细胞迁移实验结果显示,NSC23766预处理的C.pn感染组细胞迁移能力明显低于单纯感染组(P<0.05)。结论:C.pn感染可能通过PI3K激活Rac1,从而诱导VSMCs迁移。

小G蛋白Rac1在马兜铃酸诱导肾小管上皮细胞损伤中的作用及意义

目的 :探讨马兜铃酸(AA)致肾小管上皮细胞损伤的机制及小G蛋白Rac1在此过程中发挥的作用。方法:以溶剂作为对照组,AA以浓度10μg/mL作用于大鼠肾小管上皮细胞NRK-52E,培养24 h后,WST-1法检测细胞增殖的抑制率;Hoechst 33258染色观察细胞核的形态变化;细胞免疫荧光染色检测Ⅲ型胶原和Rac1蛋白的表达;real-time RT-PCR检测TGF-β1 mRNA的表达;ELISA法检测细胞培养上清液中Rac1和TGF-β1含量,并分析两者相关性。结果:AA可明显抑制NRK-52E细胞增殖,并诱导细胞凋亡。AA以10μg/mL处理NRK-52E细胞24 h后,TGF-β1 mRNA的表达明显升高(P<0.05),并且细胞外基质成分Ⅲ型胶原的表达明显增加(P<0.05)。此外,AA也可诱导小G蛋白Rac1的表达(P<0.05)。相关分析显示,AA诱导NRK-52E细胞损伤过程中,Rac1的表达量与TGF-β1的分泌水平呈现明显正相关(r=0.967,P<0.01)。结论:AA诱导肾小管上皮细胞出现纤维化样改变,同时伴有Rac1蛋白的高表达;Rac1及其介导的信号通路可能被TGF-β1的高表达所活化,进而参与AA所致的胶原累积过程。

Rac1抑制剂联合替莫唑胺协同抑制胶质瘤细胞增殖活性和侵袭能力的体外研究

目的探讨Rac1抑制剂联合替莫唑胺对胶质瘤细胞增殖活性、迁移能力和侵袭能力的协同抑制作用。方法分别以Rac1抑制剂、替莫唑胺、Rac1抑制剂联合替莫唑胺于体外培养人胶质瘤细胞系U87和U251,噻唑蓝法、细胞迁移实验和细胞侵袭实验检测胶质瘤细胞增殖活性、迁移能力和侵袭能力。结果经Rac1抑制剂、替莫唑胺、Rac1抑制剂联合替莫唑胺培养后,U87和U251细胞增殖活性均降低(均P<0.05),且替莫唑胺的抑制作用强于Rac1抑制剂(均P<0.05),Rac1抑制剂联合替莫唑胺的抑制作用更强(均P<0.05);U87和U251细胞迁移能力[U87:空白对照(78.00±11.53)细胞数/低倍视野、Rac1抑制剂(39.00±9.53)细胞数/低倍视野、替莫唑胺(42.00±8.54)细胞数/低倍视野、Rac1抑制剂联合替莫唑胺(18.67±10.54)细胞数/低倍视野,P=0.001,0.001,0.000;U251:空白对照(75.00±4.00)细胞数/低倍视野、Rac1抑制剂(37.00±5.56)细胞数/低倍视野、替莫唑胺(36.00±9.00)细胞数/低倍视野、Rac1抑制剂联合替莫唑胺(14.33±5.50)细胞数/低倍视野,均P=0.000]和侵袭能力[U87:空白对照(64.33±4.04)细胞数/低倍视野、Rac1抑制剂(30.33±3.51)细胞数/低倍视野、替莫唑胺(24.00±2.64)细胞数/低倍视野、Rac1抑制剂联合替莫唑胺(11.00±2.00)细胞数/低倍视野,均P=0.000;U251:空白对照(77.33±3.06)细胞数/低倍视野、Rac1抑制剂(40.67±4.04)细胞数/低倍视野、替莫唑胺(37.33±4.51)细胞数/低倍视野、Rac1抑制剂联合替莫唑胺(15.33±2.52)细胞数/低倍视野,均P=0.000]均降低,尤以二者联合应用降低更明显(迁移能力U87:P=0.021,0.011;迁移能力U251:P=0.002,0.003;侵袭能力U87:P=0.000,0.001;侵袭能力U251:均P=0.000)。结论 Rac1抑制剂和替莫唑胺均可抑制胶质瘤细胞的增殖活性、迁移能力和侵袭能力,二者联合应用更具协同抑制作用。

稻瘟菌Rac1蛋白的原核表达与纯化

Ras相关的C3肉毒素底物1(Rac1)是Rho族蛋白主要成员,在稻瘟菌(Magnaporthe oryzae)的侵染致病过程中发挥重要作用。本研究目的是采用结构生物学的方法进一步探究Rac1结构与功能以及与其他蛋白互作的机制,进一步阐释其致病机制。试验以稻瘟菌的cDNA为模板,根据Ras相关的C3肉毒素底物1基因(MoRac1)序列设计特异性引物进行PCR扩增,克隆了MoRac1基因并构建原核表达载体pHAT2-Rac1,异丙基硫代半乳糖苷(IPTG)诱导表达。SDS-PAGE检测与Western blot分析表明,pHAT2-Rac1在BL21(DE3)中表达,大小为25kD。通过亲和层析、离子交换与分子筛对重组蛋白进行纯化,获得了高纯度的目的蛋白。该蛋白的表达与纯化对其结构功能的研究奠定了基础。

Upregulation of Phagocytic Clearance of Apoptotic Cells by Autoimmune Regulator

To investigate the effect of autoimmune regulator （AIRE） on phagocytic clearance of apoptotic cells, a recombinant expression vector containing full-length human AIRE cDNA was transfected into 16HBE cells. After incubation with transfected 16HBE cells, engulfment of apoptotic HL-60 cells induced by camptothecin was detected by myeloperoxidase （MPO） staining. The change in the expression of Rac 1 in transfected 16HBE cells was determined by RT-PCR and Western blotting. The results showed that the phagocytosis percentage of the experimental group, the mock trans- fection group and the negative control group （non-apoptotic cells） was （25.50±3.67）%, （6.25±1.58）% and （1.0±0.67）%, respectively. Moreover, the expressions of Rac 1 mRNA and protein were up-regulated in AIRE-transfected 16HBE cells, suggesting that AIRE may function as a regulator in the phagocytic clearance of apoptotic cells by promoting the expression of Rac 1.

Mechanisms mediating cholinergic antral circular smooth muscle contraction in rats

AIM:To investigate the pathway(s)mediating rat antral circular smooth muscle contractile responses to the cholinomimetic agent,bethanechol and the subtypes of muscarinic receptors mediating the cholinergic contraction. METHODS:Circular smooth muscle strips from the antrum of Sprague-Dawley rats were mounted in muscle baths in Krebs buffer.Isometric tension was recorded.Cumulative concentration-response curves were obtained for(+)-cis- dioxolane(cD),a nonspecific muscarinic agonist,at 10^(-8)- 10^(-4)mol/L,in the presence of tetrodotoxin(TTX,10^(-7)mol/L). Results were normalized to cross sectional area.A repeat concentration-response curve was obtained after incubation of the muscle for 90 min with antagonists for M1(pirenzepine), M2(methoctramine)and M3(darifenadn)muscarinic receptor subtypes.The sensitivity to PTX was tested by the ip injection of 100 mg/kg of PTX 5 d before the experiment.The antral circular smooth muscles were removed from PTX-treated and non-treated rats as strips and dispersed smooth muscle cells to identify whether PTX-linked pathway mediated the contractility to bethanechol. RESULTS:A dose-dependent contractile response observed with bethanechol,was not affected by TTx.The pretreatment of rats with pertussis toxin decreased the contraction induced by bethanechol.Lack of calcium as well as the presence of the L-type calcium channel blocker,nifedipine,also inhibited the cholinergic contraction,with a reduction in response from 2.5±0.4 g/mm^2 to 1.2±0.4 g/mm^2(P<0.05).The dose- response curves were shifted to the right by muscarinic antagonists in the following order of affinity:darifenacin (M_3)>methocramine(M_2)>pirenzepine(M_1). CONCLUSION:The muscarinic receptors-dependent contraction of rat antral circular smooth muscles was linked to the signal transduction pathway(s)involving pertussis-toxin sensitive GTP-binding proteins and to extracellular calcium via L-type voltage gated calcium channels.The presence of the residual contractile response after the treatment with nifedipine,suggests that an additional pathway could mediate the cholinergic contraction.The involvement of more than one muscarinic receptor(functionally predominant type 3 over type 2)also suggests more than one pathway mediating the cholinergic contraction in rat antrum.

Study on Lymphocyte Activation and Proliferation Induced by Anti-CD3 McAb

T cell activation and proliferation via CD3-TCR complex were investigated by lymphocyte DNA synthesis in vitro.Several interfering factors were also discussed.The result indicated that lymphocyte activation and proliferation are calciumdependent.A rise of cytoplasmic free Ca2+ quickly following activation with CD3 McAb is mainly due to intracellular mobilization of Ca2+,while lymphocyte proliferation needs both intracellular mobilization of Ca2+ as well as influx of extracellular Ca2+, It was confirmed that CTX sensitive G protein plays a role in regulating T cell proliferation by pretreatment with CTX suppressing lymphocyte H-TdR incorporation obviously.PLC and PKC inhibitor neomycin and P.S.S could also decrease T cell proliferation.

Racl在正常及庆大霉素耳毒性损伤豚鼠耳蜗中的表达

目的研究庆大霉素所致耳毒性损伤及应用抗氧化剂水杨酸钠保护时Racl在豚鼠耳蜗中的表达，探讨RacI在庆大霉素耳毒性机制中的作用。方法健康雄性白色红目豚鼠30只，按随机数字表法分成3组。对照组：腹腔内注射生理盐水，连续7d；庆大霉素组：腹腔内注射硫酸庆大霉素，连续7d；庆大霉素＋水杨酸钠组：腹腔内注射硫酸庆大霉素和水杨酸钠，连续7d。7d后处死大鼠，采用石蜡切片免疫组织化学染色技术检测各组豚鼠耳蜗Racl蛋白的表达与分布情况；提取耳蜗蛋白，采用Westernblot检测各组Racl蛋白表达水平。结果对照组耳蜗螺旋器和螺旋神经节细胞Racl呈弱阳性表达，主要表达于细胞浆和细胞膜；庆大霉素组耳蜗Racl表达增强；庆大霉素＋水杨酸钠组呈阳性表达，其表达程度介于对照组和庆大霉素组之间；各组灰度值比较差异有统计学意义（P〈0．05）。对照组耳蜗Racl蛋白有低强度表达；庆大霉素组耳蜗Racl蛋白表达最强；庆大霉素＋水杨酸钠组耳蜗Racl蛋白表达程度介于对照组和庆大霉素组之间；各组间蛋白电泳积分光密度值比较差异有统计学意义（P〈0．05）。结论Racl在正常豚鼠耳蜗螺旋器和螺旋神经节细胞的胞浆及胞膜弱表达，庆大霉素给药后耳蜗中Racl表达增强，同时给予抗氧化剂水杨酸钠后可使RacI表达较庆大霉素组减弱。提示Racl可能在庆大霉素导致耳蜗损伤过程中起作用。

微小RNA-182通过靶基因Rac1调控高糖诱导的心肌细胞肥大

目的探讨微小RNA（miR）-182调控高糖诱导的心肌细胞肥大的机制。方法利用生物信息学方法预测调控Ras相关的c3肉毒素底物1（Racl）的候选miR。在糖尿病组小鼠（db／db小鼠）和对照组小鼠（db／m小鼠）心肌组织中验证所有候选miR的表达，并分别与RaclmRNA做Pearson相关性分析。体外实验，将原代培养的乳鼠心肌细胞分为对照组（葡萄糖浓5mmol／L）和高糖组（葡萄糖浓度33mmol／L，HG组）。光镜下观察各组乳鼠心肌细胞的形态学变化。采用实时逆转录聚合酶链反应（RT-PCR）检测两组乳鼠心肌细胞中miR-182和RaclmRNA表达水平。再将原代乳鼠心肌细胞分为4组，分别为正常糖组（葡萄糖浓度5mmol／L，对照组）、高糖组（葡萄糖浓度33mmol／L，HG组）、正常糖＋miR-182模拟物组（miR-182组）和高糖＋miR-182模拟物组（HG＋miR-182组）。采用Lipofectamine2000转染miR-182模拟物（终浓度为100nmol/L）。分别采用RT．PCR和蛋白免疫印迹法检测乳鼠心肌细胞中Racl、3-肌球蛋白重链（B—MHC）、α-平滑肌肌动蛋白（α-SMA）的mRNA和蛋白表达水平。结果生物信息学方法预测到参与调控Racl的候选miR共6个，分别为miR-182、miR-142—3p、miR-140、miR-101a、miR-429和miR-200b。糖尿病组小鼠心肌组织中miR-182和miR-142—3pmRNA表达水平均明显低于对照组（P均〈0．05），且miR-182、miR-142—3pmRNA均与RaclmRNA表达水平呈负相关（相关系数分别为r=-0．89102，r=-0．83719），miR．182与Racl负相关性最为显著。体外实验结果显示HG组乳鼠心肌细胞中miR-182mRNA表达水平明显低于对照组（P〈0．05），RaclmRNA表达水平则明显高于对照组（P〈0．05）；光镜下观察HG＋miR．182组乳鼠心肌细胞肥大程度轻于HG组；HG＋miR-182组乳鼠心肌细胞Racl和B—MHCmRNA及蛋白表达水平均明显高于HG组（P均〈0．05）。结论miR-182可能通过其靶基因Racl对高糖诱导的心肌细胞肥大进行调控。

NSC23766对糖尿病大鼠局灶性脑缺血再灌注损伤的影响

目的评价NSC23766对糖尿病大鼠局灶性脑缺血再灌注损伤的影响。方法健康成年雄性SD大鼠，2～3月龄，体重250—300g，采用腹腔注射链脲佐菌素60mg／kg的方法制备糖尿病模型。取糖尿病模型制备成功的大鼠51只，采用随机数字表法，将其随机分为3组（n=17）：假手术组（S组）、缺血再灌注组（I／R组）和Racl特异性抑制剂NSC23766组（N组）。I／R组和N组采用线栓法制备局灶性脑缺血再灌注损伤模型，N组于缺血前15min经侧脑室注射NSC2376650μg，S组与I／R组给予等容量生理盐水。于再灌注24h时进行神经功能缺陷评分，然后处死大鼠，取脑组织，测定脑梗死体积，HE及Nissl染色，光镜下观察病理学结果，并测定细胞凋亡指数和p38丝裂原活化蛋白激酶（p38MAPK）磷酸化水平。结果与S组比较，I／R组和N组神经功能缺陷评分和细胞凋亡指数升高，脑梗死体积增大，p38MAPK磷酸化水平上调（P〈0．05）；与I／R组比较，N组神经功能缺陷评分和细胞凋亡指数降低，脑梗死体积缩小，p38MAPK磷酸化水平下调（P〈0．05）。结论NSC23766可减轻糖尿病大鼠局灶性脑缺血再灌注损伤。

血管内皮细胞生长因子激活Rac1引起肾小球内皮细胞通透性增高的机制

目的探讨细胞内Racl信号激活是否在血管内皮细胞生长因子（VEGF）增加肾小球内皮细胞的通透性和导致紧密连接酪氨酸磷酸化中起作用。方法采用原代培养的大鼠肾小球内皮细胞作为实验对象。通过体外研究，检测跨内皮细胞电阻抗观察不同浓度VEGF（5和50μg／L）对内皮细胞通透性的影响。内皮细胞转染野生型Racl和显性负性Racl质粒后，采用免疫沉淀和免疫印迹等方法观察上述效应是否源自Racl信号的激活，并观察紧密连接occludin蛋白酪氨酸磷酸化状态的改变。结果高浓度的VEGF（50μg／L）刺激可引起大鼠肾小球内皮细胞单层通透性显著增高（P〈0．05），并引起肾小球内皮细胞中GTP结合的Racl和膜结合的Hacl显著增加（P〈0．01），同时紧密连接蛋白occludin的酪氨酸磷酸化也增加（P〈0．05）。用Racl显性负性突变体转染肾小球内皮细胞能显著减弱VEGF对occludin蛋白酪氨酸磷酸化和内皮细胞通透性的影响（P〈0．05）。结论高浓度的VEGF可导致肾小球内皮细胞通透性增高，其与紧密连接蛋白occludin的酪氨酸磷酸化相关，这一作用需要Racl信号途径的激活。糖尿病中增高的VEGF可能通过Racl激活-occludin磷酸化导致肾小球内皮通透性增高，这可能是糖尿病肾病的发病机制之一。

Rac1蛋白表达与神经前体细胞增殖分化的关系

目的探讨Rac1蛋白表达与神经前体细胞增殖分化的关系。方法取新生大鼠大脑皮层,原代培养获得混合细胞培养体系,在含10%血清的培养基中培养6 d细胞汇合后更换无血清培养基继续培养。实验中设计4个时间点,用倒置相差显微镜观察细胞增殖分化的变化,免疫荧光染色技术鉴定细胞种类以及Rac1蛋白在细胞内的表达,通过Western blot检测Rac1蛋白的表达变化。结果建立了新生SD大鼠大脑皮层原代混合细胞培养体系,此体系底层主要是由Ⅰ型胶质细胞组成的单层细胞,GFAP染色呈阳性;在单层细胞上面有一些小圆形强折光性神经前体细胞生长,βⅢ-Tubulin染色呈阳性。更换无血清培养基后上层神经前体细胞大量增殖,βⅢ-Tubulin、Rac1染色呈阳性。Western blot定量分析结果显示,神经前体细胞增殖过程中Rac1蛋白表达上升,而分化过程中Rac1蛋白的表达下降。结论Rac1蛋白表达与神经前体细胞的增殖分化过程密切相关,可能参与了神经前体细胞的增殖分化过程。

LMP1瞬时过表达对鼻咽癌CNE2细胞迁移的影响

目的 :探讨潜伏膜蛋白1(latent membrane protein 1,LMP1)瞬时过表达对鼻咽癌CNE2细胞迁移的影响及其可能的作用机制。方法 :成功构建重组真核表达载体pEGFP-C1-LMP1,并将其瞬时转染至CNE2细胞,激光共聚焦显微镜下观察LMP1在CNE2细胞中的定位,Transwell法检测LMP1对CNE2细胞迁移和侵袭能力的影响,实时荧光定量PCR和蛋白质印迹法检测LMP1和Ras相关C3肉毒素底物1(Ras-related C3 botulinum toxin substrate 1,Rac1)mRNA及蛋白的表达。结果 :pEGFP-C1-LMP1成功转染至CNE2细胞后,可见LMP1主要表达于细胞膜和部分细胞质中,CNE2细胞的侵袭和迁移能力明显低于转染空载体的对照组(P<0.05),CNE2细胞中LMP1 mRNA及蛋白的表达水平明显高于转染空载体的对照组(P<0.01),而Rac1 mRNA及蛋白的表达水平明显低于转染空载体的对照组(P<0.05)。结论 :LMP1瞬时过表达可抑制鼻咽癌CNE2细胞的迁移,这一作用可能与Rac1表达下调有关。

RhoA、Rac1及Cdc42在胆道闭锁肝纤维化中的作用

目的研究胆道闭锁（biliary atresia，BA）患儿肝组织中RhoA、Rac1及Cdc42表达情况与肝纤维化的关系，探讨其在BA发病中可能的作用机制。 方法选取胆管扩张症患儿肝组织（胆扩组）15例，BA患儿肝组织（BA组）15例，BA发展至肝硬化接受肝移植患儿自体肝活检组织（移植组）10例，采用HE观察肝组织标本纤维化程度；免疫组化染色检测RhoA、Rac1及Cdc42在肝组织中的蛋白表达情况；实时荧光定量聚合酶链式反应（qRT-PCR）方法检测肝组织中RhoA、Rac1及Cdc42基因表达情况。结果①HE染色：胆扩组偶见少许纤维组织增生；BA组汇管区增宽，纤维组织增生、桥接纤维化现象普遍，可见假小叶形成；移植组假小叶显著；②免疫组化：胆扩组RhoA、Rac1及Cdc42均表达为弱阳性，BA组及肝移植组患儿RhoA、Rac1及Cdc42蛋白在肝细胞、汇管区胆管上皮细胞中均为阳性表达；③半定量分析：RhoA（0.1419±0.0683、0.2203±0.0476和0.2295±0.0623），Rac1（0.1587±0.0072、0.1970±0.0242和0.1993±0.0270）和Cdc42（0.887±0.0270、0.1397±0.0360和0.1519±0.0168）在三组蛋白表达水平之间比较差异有统计学意义（P〈0.05），且三组中BA组及肝移植组RhoA、Rac1及Cdc42蛋白表达水平明显高于胆扩组（P〈0.05）；移植组RhoA、Rac1及Cdc42蛋白含量与BA组，表达差异无统计学意义（P〉0.05）；④qRT-PCR：RhoA[0.3350（0.2525～0.6350）、1.5200（1.4375～2.1700）和1.9350（1.7950～2.3925）]，Rac1[0.2800（0.1450～0.3025）、0.8200（0.7275～0.9300）和0.8000（0.7725～0.9825）]，Cdc42[0.5950（0.4375～0.7125）、1.8950（1.8675～2.1925）和1.9900（1.8675～2.2050）]，三组中mRNA表达水平比较差异有统计学意义（P〈0.017），同时在这三组中，BA组及肝移植组RhoA、Rac1及Cdc42含量表达明显高于胆扩组（P〈0.017）。结论RhoA、Rac1及Cdc42在BA患儿肝组织呈高水平表达，表明其可能参与并促进BA肝纤维进程。

RNA干扰Ras相关C3肉毒素底物1抑制大鼠视网膜新生血管生成

目的观察RNA干扰Ras相关C3肉毒素底物1（Racl—siRNA）重组载体对视网膜新生血管生成的抑制作用。方法25只成年Sprague-Dawley（SD）大鼠，采用光动力疗法诱导双眼视网膜静脉阻塞后，1只眼玻璃体腔转染Racl-siRNA重组载体作为基因干预组，另1只眼注射空白载体作为空白对照组。25只SD大鼠单眼玻璃体腔内转染Racl—siRNA重组载体作为空白干预组。2周后对3组大鼠进行心内灌注右旋异硫氰酸荧光素，视网膜铺片，检测视网膜新生血管生成情况。摘除3组大鼠眼球，苏木素-伊红染色后计数突破内界膜的内皮细胞数。结果视网膜铺片发现，空白对照组视网膜产生大片新生血管，大量荧光素渗漏；基因干预组仅见小片状新生血管网，少量荧光素渗漏；空白干预组呈正常血管形态。基因干预组大鼠视网膜切片中，突破内界膜的血管内皮细胞平均数与空白对照组和空白干预组比较，两两间差异具有统计学意义（F=47．168，P=0．000）。结论Racl-siRNA重组载体能有效抑制体内视网膜新生血管的生成。

Gene Expression Profiling of the Proliferative Effect of Periplocin on Mouse Cardiac Microvascular Endothelial Cells

Objective： Periplocin is an active digitalis-like component from Cortex Periplocae, which has been widely used in the treatment of heart diseases in China for many years. According to the recommendations on the cardiovascular effect of periplocin from in vivo experiments, subsequent in vitro experiments are greatly needed for the global assessment of periplocin. The objective of this study is to investigate the cell proliferation effect and the mechanism of periplocin on endothelial cells. Methods： The proliferative activity of periplocin （0.4, 2, 10, 50, 250 pmol/L; 6, 12, 24, 48, 72 h） was investigated by a comparison with the well-reported cardiac glycoside, ouabain, on mouse cardiac microvascular endothelial cells （CMEC）. 3-（4,5-dimethylthiazolyl）- 2,5-diphenyltetrazolium bromide （MTT）, lactate dehydrogenase （LDH） and 5-bromo-2-deoxyuridine （BrdU） assays were used to evaluate cell proliferation and viability. Subsequently, cDNA microarray experiments were performed on periplocin- （50 pmol/L） and ouabain- （50 pmol/L） treated cells, and data was analyzed by ArrayTrack software. Results： Periplocin could increase cell viability to a level lower than ouabain in the MIF analysis, but decrease LDH release simultaneously. The BrdU incorporation assay showed an increase in cell proliferation with 2-50 μmol/L periplocin. Genes related to protein serine/threonine kinase were the most significantly enriched in the 160 genes identified in periplocin versus the control. In the 165 genes regulated by periplocin versus ouabain, GTP-binding was the most altered term. Conclusions： The results demonstrated the proliferation action of periplocin on CMEC. Meanwhile, its lower cytotoxicity compared to ouabain provides a new insight into the treatment of heart failure.

Maxingxiongting mixture attenuates hypoxia pulmonary arterial hypertension to improve right ventricular hypertrophy by inhibiting the rho-kinase signaling pathway

OBJECTIVE:To explore the mechanism of Maxingxiongting mixture(MXXTM)on pulmonary hypertension in a rat model established by intraperitoneal injection of monocrotaline solution,smoking and forced swimming.METHODS:A total of 30 male Sprague-Dawley rats were randomly divided into five groups:control group,model group,high-dose of MXXTM group(HM),low-dose of MXXTM group(LM),and fasudil group.The mean pulmonary artery pressure(m PAP)was measured by using a miniature catheter.Lung tissue and right ventricular tissue sections were stained with hematoxylin-eosin.The right ventricle(RV)and left ventricle+septum(LV+S)were weighted.RV/(LV+S)was calculated to reflect the degree of right ventricular hypertrophy.Rho/Rho-kinase signaling pathway key proteins(Rho A,ROCKⅠand ROCKⅡ)in rat right ventricular tissue were measured by Western blot analysis.The levels of serum hypoxia-inducible factor-1α(HIF-1α),vascular endothelial growth factor(VEGF)and the levels of plasma renin activity(PRA),angiotensinⅡ(ANG-Ⅱ),aldosterone(ALD)in rat anticoagulated plasma were all measured by enzyme-linked immunosorbent assay.RESULTS:Compared with the control group,the m PAP and RV/(LV+S)in the model group were significantly increased.Administration of fasudil resulted in a significant decrease of m PAP and RV/(LV+S).In the HM group and LM group,m PAP and RV/(LV+S)were significantly lower than the model group.Compared with the control group,the contents of HIF-1α,VEGF,PRA,ANG-Ⅱand ALD in the model group were significantly increased.The administration of fasudil and high-dose MXXTM significantly reduced the contents of HIF-1α,VEGF,PRA,ANG-II and ALD.Compared with the control group,the expression of Rho A,ROCKⅠand ROCKⅡin the right ventricle of the model group were significantly increased.The administration of fasudil and high-dose MXXTM significantly reduced the expression of Rho A and RockⅡproteins.Our results indicated that high-dose of MXXTM had similar effects on reducing pulmonary artery pressure and improving right ventricular remodeling to fasudil.However,MXXTM was unable to restore parameters above to control levels.CONCLUSIONS:MXXTM attenuates hypoxia pulmonary arterial hypertension to improve right ventricular hypertrophy by inhibiting the Rho-kinase signaling pathway.

Wnt5A基因过表达对黑素细胞骨架蛋白的影响

目的 探讨携带Wnt5A全基因序列的质粒转染原代黑素细胞后，Wnt5A表达升高对黑素细胞骨架蛋白的影响。方法 培养原代人表皮黑素细胞，并分为3组：①空白对照组：除细胞外不加任何试剂；②阴性对照组：加入空白的去内毒素pcDNA3.1（＋）质粒、Opti-MEM培养基及Lipo3000；③Wnt5A质粒组：加入Wnt5A去内毒素pcDNA3.1（＋）质粒、Opti-MEM培养基及Lipo3000。Wnt5A质粒转染黑素细胞，采用qPCR法检测各组Wnt5A、Ras相关C3肉毒素底物1（Rac1）、丝状肌动蛋白（F肌动蛋白）和β微管蛋白基因表达，Western印迹法测定Wnt5A、酪氨酸激酶受体2（ROR2）、Rac1、F肌动蛋白和β微管蛋白表达，并通过细胞免疫荧光试验观察细胞骨架蛋白表达情况。结果 qPCR法显示，Wnt5A质粒组、阴性对照组和空白对照组Wnt5A mRNA及其下游基因Rac1和F肌动蛋白mRNA表达量3组间差异均有统计学意义（F = 1 374.179、112.576、66.458，均P 〈 0.01），但β微管蛋白mRNA表达差异无统计学意义（P 〉 0.05）。Wnt5A质粒组Wnt5A、Rac1和F肌动蛋白mRNA表达量均显著高于空白对照组及阴性对照组（P 〈 0.05）。Western印迹法显示，Wnt5A质粒组Wnt5A蛋白表达较空白对照及阴性对照明显升高，差异均有统计学意义（P 〈 0.05）。Wnt5A质粒组Rac1、ROR2、F肌动蛋白表达均明显低于空白对照组和阴性对照组（P 〈 0.05），但β微管蛋白表达在3组间差异无统计学意义（P 〉 0.05）。细胞免疫荧光实验显示，Wnt5A质粒组绿色（β微管蛋白）、红色（F肌动蛋白）荧光强度与两个对照组相比均无明显差别，但黑素细胞体积明显增大，树突数目明显增多，细胞骨架显著改变，表现为F肌动蛋白强弱不一，纹理模糊，张力丝断裂，局部呈团块状。结论 黑素细胞Wnt5A基因表达升高可调控细胞骨架相关基因及蛋白表达，使黑素细胞体积增大、树突增多、骨架改变，可能有利于黑素小体的输送传递，参与色素沉着性疾病的发病。

Cloning of the Full-length cDNA of the Wheat Involved in Salt Stress：Root Hair Defective 3 Gene （RHD3）

: The full-length cDNA of the wheat (Triticum aestivum L.) root hair defective 3 gene (RHD3) has been cloned from the salt-tolerant hybrid wheat variety Shanrong No. 3 (Za3) using the mRNA differential display and 5’rapid amplification of cDNA ends (RACE) methods. Analysis of the amino acid sequence deduced from the wheat RHD3, gene shows that two conservative GTP-binding motifs, namely GXXXXGKS and DXXG, in eukaryotes also exist at the N-terminal of wheat RHD3. In addition, an 18 amino acid residue transmembrane domain, namely FYLAVMFVVFLVGKAIWV, exists at positions 701—718 of the C-terminal of the deduced protein of wheat RHD3 obtained, but this domain is absent in another three proteins aligned, including rice RHD3, Arabidopsis RHD3, and yeast homologue SEY1. Northern blot revealed that transcription of the wheat RHD3, gene is down-regulated in both the salt-tolerant line and in JN177 under saline stress. A possible stress-responsive mechanism for this gene is discussed.