Protective effect of non-mitogenic human acidic fibroblast growth factor from renal ischemia-reperfusion injury

AIM: To investigate the effect of non-mitogenic human acidic fibroblast growth factor (nm-haFGF) on renal ischemia-reperfusion injury in rats. METHODS: Rat renal ischemia-reperfusion (I/R) injury was produced by removing the left kidney and subsequently clamping the right renal artery for 60 min followed by reperfusion for 24 h. 5 min after reperfusion, different doses of nm-haFGF and haFGF (as positive control) were injected by lingual vein. 24 h later, the samples of blood, urine and kidney were collected and the contents of malondialdehyde (MDA),blood urea nitrogen (BUN), creatinine (Cr) and superoxide dismutase (SOD) activity were detected. Histopathological changes were also observed. RESULTS: In the serum, SOD activity of all the nm-haFGF groups and the haFGF group increased significantly while the content of MDA decreased dramatically compared with the model group; The content of BUN and Cr also decreased wherever in serum or in urine; In renal tissue, SOD activity in nm-haFGF 20 μg/kg group, 40 μg/kg group and haFGF group rose significantly compared with the model group, while MDA decreased dramatically. Histological examination showed that nm-haFGF markedly attenuates the renal edema, brush border’s defluvium and cell necrosis induced by ischemia-reperfusion. CONCLUSION: nm-haFDF could resist the renal injury induced by ischemia- reperfusion in rats.     

Effect of Sonic Hedgehog signaling blockade on growth of hepatocarcinoma cells

AIM: To investigate the effect of Sonic Hedgehog (Shh) signaling blockade on the growth of hematocarcinoma cells and underlying mechanisms. METHODS: The expression of Shh signaling molecules in hematocarcinoma cell lines BEL-7402, Huh7 and HepG2 was detected by RT-PCR. The cell viability was detected by MTT assay. The cell cycle and apoptosis were analyzed by flow cytometry. The expression of apoptosis-related proteins was determined by Western blot. RESULTS: Shh signaling molecules were all expressed in BEL-7402, Huh7 and HepG2 cells. The mRNA expression of Patched (Ptch), Gli1 and Gli2 was down-regulated by anti-Shh antibody. Blockade of Shh signaling pathway inhibited the proliferation of hepatocarcinoma cells with increasing cells in G₀/G₁ phase and induced the apoptosis of hepatocarcinoma cells. Treatment with anti-Shh antibody down-regulated the protein expression of pro-caspase-3, pro-caspase-8 and pro-caspase-9, while up-regulated the protein levels of cleaved caspase-3, cleaved caspase-8 and cleaved caspase-9 in BEL-7402 cells. CONCLUSION: Blockade of Shh signaling pathway inhibits the growth of hepatocarcinoma at different levels by cell cycle arrest and inducing apoptosis of hematocarcinoma cells.     

Erk1/2 MAP kinase mediates function of FGF21 to regulate hepatic lipid metabolism

AIM: To investigate whether attenuation of extracellular signal-regulated kinase 1/2 (Erk1/2), an MAP kinase, will affect the regulatory function of fibroblast growth factor 21 (FGF21) on hepatic lipid metabolism. METHODS: Male C57BL/6J wild-type (WT) mice (8 weeks old) were fed with high-fat diet (HFD) for 4 weeks, and then treated with U0126 (an MEK inhibitor, inactivating Erk1/2) and/or adeno-associated virus-FGF21 (AAV-FGF21) for 4 weeks. The change of hepatic lipid profiles and its relevant regulatory genes were evaluated. Meanwhile, the molecular mechanism was explored by in vitro study. RESULTS: Treatment with AAV-FGF21 significantly reduced serum trigly-ceride and total cholesterol levels in the HFD-fed WT mice. Moreover, AAV-FGF21 administration significantly decreased the expression of lipogenic regulatory gene Srebp-2, largely increased the expression of relevant genes including Acacb, ABCG5 and Cyp7a1, which were involved in fatty acid oxidation, cholesterol transport and bile acid production, and strongly inhibited the expression of hepatic inflammatory factors such as IL-1β, MCP-1 and TNF-α in the mice. However, these beneficial effects caused by AAV-FGF21 were strongly attenuated by U0126. On the other hand, in vitro experiments indicated that treatment with recombinant human FGF21 protein significantly down-regulated Srebp-2 expression at mRNA and protein levels in a dose- and time-dependent manner. These effects were abolished after U0126 administration. CONCLUSION: FGF21 improves the lipid metabolism in the liver, and Erk1/2 signaling partially regulates these biological effects of FGF21.     

Pharmaceutical inactivation of Erk1/2 does not affect function of FGF21 to regulate glucose and lipid metabolic hemostasis

AIM: To investigate whether inactivation of extracellular signal-regulated kinase 1/2 (Erk1/2) will affect the function of fibroblast growth factor 21 (FGF21) to regulate glucose and lipid metabolism. METHODS: Male db/db mice (8 weeks old) were treated with U0126 (an inhibitor of Erk1/2 kinase) for 1 week, and then treated with recombinant human FGF21 protein and adenovirus-mediated FGF21 (Ad-FGF21). The profile changes of blood glucose and blood lipid were evaluated at 120 min or 4 weeks after FGF21 administration. Meanwhile, the molecular mechanism was explored by in vitro study. RESULTS: Treatment of db/db mice with recombinant human FGF21 protein significantly reduced blood glucose and triglyceride levels at 120 min after FGF21 administration, but these changes were comparable in U0126-treated mice. Furthermore, abnormal glucose and triglyceride levels, and glucose and insulin tolerance were strongly improved in db/db mice as accompanied with decreasing body fat content after 4 weeks of ad-FGF21 administration. Interestingly, treatment with or without U0126 did not influence these effects of FGF21. Mechanically, treatment with Ad-FGF21 significantly upregulated the protein levels of p-Erk1/2 and peroxisome proliferator-activated receptor γ (PPARγ) as well as the expression of adiponectin at mRNA and protein levels in adipose tissues. However, treatment with or without U0126 did not change the profiles. On the other hand, in vitro experiments also indicated that treatment of adipocytes with recombinant human FGF21 protein significantly activated Erk1/2 phosphorylation, and upregulated the expression levels of PPARγ and adiponectin (P<0.05). However, pre-administration of U0126 did not affect the profiles. CONCLUSION: Pharmaceutical inactivation of Erk1/2 by U0216 does not affect the biological function of FGF21 to regulate blood glucose balance and improve abnormal blood lipids in vivo.     

Effects of epithelial cells treated with poly (I:C) on activation of fibroblasts and roles of EMMPRIN

AIM: To investigate the effects of epithelial cells treated with polyinosinic-polycytidylic acid [poly(I:C)] on the proliferation, transdifferentiation and signaling mechanisms of airway fibroblasts.METHODS:Human alveolar epithelial cells were treated with poly(I:C). The cell culture supernatants were used to stimulate the airway fibroblasts or the fibroblasts growing in collagen gels. The proliferation of the fibroblasts, the expression of a-smooth muscle actin (α-SMA) in the fibroblasts and the contractility of the collagen gels containing fibroblasts, as well as the expression of extracellular matrix metalloproteinase inducer (EMMPRIN) were observed. The proliferation of the fibroblasts and the expression levels of α-SMA, MMP-2 and MMP-9 in the fibroblasts stimulated with EMMPRIN were detected. The inhibitors specific for p38 MAPK or ERK1/2 were used to explore the effects on α-SMA expression and EMMPRIN secretion. RESULTS:The culture supernatants of the epithelial cells treated with poly(I:C) induced the proliferation, α-SMA expression and gel contraction as well as EMMPRIN secretion in the fibroblasts. EMMPRIN dose-dependently enhanced fibroblast proliferation, α-SMA expression and activity of MMP-2 and MMP-9. The supernatants of epithelial cells treated with poly(I:C) activated p38 MAPK and ERK1/2 signaling in the fibroblasts, as the specific inhibitors of MAPK or ERK1/2 signaling attenuated the expression of α-SMA and EMMPRIN in the fibroblasts. CONCLUSION: Poly(I:C) induces fibroblast proliferation, α-SMA expression and gel contraction by affecting the process mediated by p38 MAPK and ERK1/2 signaling pathways in epithelial cells. EMMPRIN may be the important media involved in this event.     

Fasudil attenuates brain white matter injury in premature rats

AIM To study the effect of fasudil on white matter injury （WMI） in preterm rats. METHODS Intrauterine inflammation induced by intraperitoneal injection of lipopolysaccharide （LPS） in Wistar pregnant rats was used to establish the WMI model of preterm rats， and the preterm rats with WMI were treated with fasudil. The effect of fasudil on lateral ventricular dilatation in WMI preterm rats was evaluated by HE staining. The effects of fasudil on oligodendrocyte precursor cells and activated glia cells in WMI preterm rats were detected by immunohistochemistry. The effects of fasudil on the mRNA levels of myelin formation-related genes in WMI preterm rats were detected by RT-qPCR. The effects of fasudil on the expression of Rho/ROCK pathway-related proteins in preterm WMI rats were determined by Western blot. RESULTS Fasudil significantly reduced lateral ventricular dilatation， astrocyte and microglia activation levels， and Rho/ROCK signaling activity in WMI preterm rats， and significantly increased the number of oligodendrocyte precursor cells and expression levels of myelin formation-related genes （P<0.01）. CONCLUSION Fasudil attenuates WMI in premature rats， which may be though reducing Rho/ROCK signaling activity， inhibiting glial cell activation and increasing the number of oligodendrocyte precursor cells.     

Protection of maFGF against anoxia/reperfusion injury in isolated rat hearts

AIM: To investigate the protective effects and mechanisms of modified acidic fibroblast growth factor (maFGF) in anoxic reperfusion of rat hearts. METHODS: Using Langendorff apparatus, we established the model of anoxia/reperfusion of isolated hearts to compare the protective effects of maFGF and acidic fibroblast growth factor (aFGF). The changes of left ventricle development pressure (LVDP) and maximal rates of rise of ventricular pressure(dp/dtmax), maximal rates of decline of ventricular pressure (dp/dtmin) were determined, changes of LDH and MDA levels，SOD activity in efflux from coronary artery were also detected at different time point. RESULTS: Pretreatment with maFGF and aFGF produced a similar protective effect on myocardium during anoxia /reperfusion, including promoting obviously heart functional recovery after myocardial anoxia/reperfusion and reversing changes of LDH, MDA contents and SOD activity induced by anoxic/reperfusion. CONCLUSION: maFGF has a protective effect on anoxia/reperfusion heart, and the mechanism of this effect may be related to suppression of lipid peroxidation.     

Effects of miR-92a and miR-19b on insulin gene expression in mouse pancreatic β-cells

AIM To investigate the effect of microRNA-92a （miR-92a） and microRNA-19b （miR-19b） on the insulin expression in mouse pancreatic β-cells. METHODS The relative expression levels of endogenous miR-92a and miR-19b in mouse insulinoma MIN6 cells were detected by qPCR. The MIN6 cells were divided into control group， and experimental groups I and II， with 3 samples in each group， and transfected with negative control miRNA （NC）， miR-92a and miR-19b， respectively. The over-expression of the miRNAs was detected by qPCR. The morphological changes and viability of the cells were detected by optical microscopy and CCK8 assay， respectively. The expression of insulin was detected by qPCR， Western blot and immunofluorescence. The possible mechanisms of miR-92a and miR-19b regulating insulin expression were analyzed by bioinformatics prediction， dual-luciferase reporter assay， and Western blot. RESULTS Compared with the adult pancreatic progenitor cells， the expression of endogenous miR-92a and miR-19b in the MIN6 cells was decreased significantly （P<0.05）. Over-expression of miR-92a and miR-19b had no effect on the viability of MIN6 cells， but inhibited the expression of insulin at mRNA and protein levels. miR-19b significantly inhibited the luciferase activity of NeuroD1 3′UTR and the protein expression of NeuroD1 （P<0.05）. miR-92a had a fine-tuning effect on the luciferase activity of NeuroD1 3′UTR and the protein expression of NeuroD1. CONCLUSION miR-92a and miR-19b inhibit the insulin expression in mouse pancreatic β-cells.     

Effect of TNF-related apoptosis inducing ligand on the biological activity of hepatocarcinoma cell line

AIM: To explore the effect of TNF-related apoptosis inducing ligand (TRAIL), a new apoptotic inducing molecule on the biological activity of hepatocarcinoma cell line. METHODS: The expression of membrane binding TRAIL on HepG2 cells was detected by immuno-cytochemistry. Quantity of secretory TRAIL was assayed by ELISA method. The cytotoxicity and apoptosis induced by TRAIL was detected by MTT and TUNEL method, respectively. The telomerase activity of HepG2 cells was detected by TRAP-PCR assay kit. The expression of hTERT, the catalytic subunit of telomerase, was detected by FCM. RESULTS: TRAIL was constitutively expressed on the membrane of HepG2 cell line. Soluble TRAIL was also expressed to a certain degree. Cytotoxicity assay showed that TRAIL significantly inhibited the growth of hepatocarcinoma cells. TUNEL assay indicated that TRAIL induced apoptosis in hepatocarcinoma cells. Detection of telomerase activity showed that TRAIL inhibited telomerase activity and the expression of telomerase catalytic subunit. CONCLUSION: TRAIL is an effective molecule to inhibit the growth of hepatocarcinoma through multiple pathways, such as inducing apoptosis and inhibiting the activity of telomerase.     

Establishment of a cellular model of hypoxic acclimatization in human HepG2 cells

AIM: To establish a cellular model of hypoxic acclimatization using HepG2 cells to explore the mechanism of cellular hypoxia acclimatization. METHODS: HepG2 cells were cultured in 1% O2 for 24 hours, then in 21% O2 for another 24 hours，which composed a hypoxic treating cycle. After 6 cycles, the activity of cell proliferation was estimated with MTT method. The morphologic features of HepG2 cells were observed with optical microscope and transmission electron microscope. EPO gene expression was detected by Northern blotting technique. RESULTS: Acute hypoxia inhibited cellular mitosis, impaired cellular ultrastructure and induced EPO gene expression. After 6 cycles of hypoxic treatment, proliferation ability of HepG2 treated with acute hypoxia for 48 h was resumed to the level of control cells cultured in 21% O2. The ultrastructure of HepG2 cells injured by hypoxia recovered and the level of EPO gene expression returned to that in control cells. CONCLUSION: After 6 cycles of hypoxic treatment, the ability of HepG2 to endure hypoxia is obviously enhanced and HepG2 cells might reach the status of hypoxic acclimatization.     

Establishment and application of cell model for P2ry2 modulators screening based on CaCC

AIM To construct a high-throughput screening cell model for P2Y₂ purinergic receptor （P2ry2） modulators based on calcium-activated chloride channel （CaCC）. METHODS The mRNA expression of P2ry2 in Fischer rat thyroid （FRT） cells was detected by RT-PCR， and the PCR products were collected and sequenced. The protein expression of P2ry2 in the FRT cells was also detected by Western blot. The eukaryotic expression vectors ANO1 and YFP-H148Q/I152L were constructed. The FRT cells co-expressing ANO1 and YFP-H148Q/I152L were obtained by liposome transfection， antibiotic screening and limited dilution. The expression of ANO1 and YFP-H148Q / I152L in the cells was observed under fluorescent inverted microscope. The validation of the cell model for screening P2ry2 modulators was verified by the fluorescence quenching kinetics tests， and intracellular free calcium was analyzed by Fura-2 staining to investigate the dose-dependent relationship between intracellular calcium concentration and P2ry2 modulators. Z' factor was applied to evaluate the stability and repeatability of the cell model. RESULTS P2ry2 were endogenously expressed in the FRT cells. The expression of ANO1 on the cell membrane and the expression of YFP-H148Q/I152L in the cytoplasm were observed under fluorescent inverted microscope. The cell model was successfully constructed. The fluorescence quenching kinetics test confirmed the cell model for screening P2ry2 modulators was constructed successfully， and the calcium concentration in cytoplasm was increased rapidly after the addition of a small amount of P2ry2 agonist， indicating that the cell model was sensitive for detecting the calcium concentration in cytoplasm. The calcium concentration in cytoplasm， P2ry2 modulators and the slope of fluorescence change were in a dose-dependent manner， respectively. The Z' factor was 0.75， indicating that the established cell model was able to use for high-throughput screening of P2ry2 modulators with excellent stability and repeatability. CONCLUSION A simple， economical， and efficient cell screening model of P2ry2 modulators is successfully constructed， which is suitable for the detection and evaluation of P2ry2 modulators.     

乙酰苯胺和2-氯苯磺酰胺诱导IN2-2启动子在芥菜中的表达

分别用不同浓度(50、100、200mg·L-1)的除草剂安全剂类似化合物乙酰苯胺和2-氯苯磺酰胺处理IN2-2::GUS转基因芥菜。结果显示:乙酰苯胺和2-氯苯磺酰胺处理后,IN2-2启动子在芥菜根、叶、花器官的花萼、花瓣、雄蕊及花粉中表达,但不在胚珠中表达。100mg·L-1乙酰苯胺以及50mg·L-12-氯苯磺酰胺适合用于在芥菜中调控IN2-2启动子的表达,乙酰苯胺较2-氯苯磺酰胺诱导表达所需时间更短。高浓度的2-氯苯磺酰胺影响芥菜种子发芽及生长发育,并且明显抑制IN2-2启动子的表达活性。4℃低温胁迫诱导IN2-2启动子在幼苗叶片中表达,IN2-2启动子轻微受150mmol·L-1Na Cl胁迫表达,但不受重金属Gu~(2+)离子的诱导表达。     

月季花瓣特异表达启动子的筛选和鉴定

以月季品种‘萨曼莎’（Rosa hybrida‘Samantha’）为试验材料，选取了13个花色、花香相关的基因，研究其在根、茎、叶和花等器官中的表达规律及其启动子活性。结果表明，RhOOMT2（Rosa hybrida orcinol o-methyltransferase 2）和RhCCD4（Rosa hybrida carotenoid cleavage dioxygenase 4）在花瓣中具有相对较高的表达量，RhNUDX1（Rosa hybrida nudix hydrolase 1）在根部表达量最高，花瓣中其次，在茎和叶中几乎没有表达，另外10个类黄酮代谢途径相关基因在各器官中表达差异较小。使用FPNI-PCR的方法克隆到ATG上游1 601 bp的RhOOMT2启动子、1 539 bp的RhCCD4启动子和1 433 bp的RhNUDX1启动子。将携带RhOOMT2、RhCCD4和RhNUDX1启动子的PBI121载体通过农杆菌的介导，在月季‘Samantha’、洋桔梗（Eustoma grandiflorum‘Green Pelleted’）和百合（Lillium Oriental hybrida‘Siberia’）花瓣中进行瞬时表达，GUS染色结果表明，与阳性对照35S启动子相比，RhOOMT2启动子在月季、洋桔梗和百合花瓣中具有更强的活性，RhCCD4启动子在百合花瓣中具有较弱的活性，而在月季和洋桔梗中活性很弱，RhNUDX1启动子则在3种花中均无活性。进一步在4个月季品种‘萨曼莎’、‘戴安娜’、‘香槟月季’和‘雪山’中比较了RhOOMT2和35S的启动子活性，证实了在花瓣中RhOOMT2启动子具有比35S更高的活性。     

Protective effects of nmhaFGF on NRK52E cell apoptosis induced by H2O2

AIM: The present study was designed to establish H2O2-induced NRK52E cell apoptotic model and to investigate the effects and the mechanism of nmhaFGF on the NRK52E cell apoptosis induced by H2O2. METHODS: In vitro experiment, a apoptotic model of normal rat kidney epithelium (NRK52E) was made by MTT method, Hoechst33342 dyeing and flow cytosorting (FCR). NRK52E cells were cultured with different concentrations of nmhaFGF and haFGF for 24 h before H2O2 was added. The apoptotic rate was detected with FCR method. RESULTS: H2O2 at concentration of 0.4 mmol/L, the optimal condition to establish the apoptotic model, was used to treat NRK52E cells for 18h. Different doses of nmhaFGF (0.01, 0.03, 0.10, 0.30, 1.00 mg/L) reduced the apoptotic rates with the dose rising. However, the decreasing tends of apoptotic rates with dose increasing for haFGF was not so obvious. CONCLUSION: nmhaFGF protects the NRK52E cells against apoptosis. The mechanism might be connected with its non-mitogenic property.     

Effects of human recombinant acidic fibroblast growth factor on cell proliferation and apoptosis in NIH3T3 cells

AIM:Through studying the differences betwe en wild-type acidic fibroblast growth factor (waFGF) with recombinant aFGF (raFG F ),the biological effect of raFGF concerned with mitogenic activity was evaluate d.METHODS:NIH3T3 cell line was used.Cell proliferation with MTT method was used to study the mitogenic activity.Flow cytometry was also used fo r detection of apoptosis,cell membrane permeability and mitochondria potential.The role of heparin sulfate (HS) on aFGF biological effect was studied at the s ame time in this research.RESULTS:The enhancement of raFGF on cell proliferation was sign ificantly lower than that of waFGF.The restriction of raFGF on apoptosis and th e enhancement of it on cell membrane permeability were all lower than those of w aFGF significantly.The enhancement of raFGF on mitochondria potential was lower than that of waFGF significantly.The HS improved the biological effect of aFGF .CONCLUSION:The mitogenic activity of raFGF is lower than that of waFGF and raFGF has little effect on apoptosis.     

Analysis of expressive proteome in human hepatocarcinoma cell HepG2 transfected with miR-26a mimics

AIM: To determine whether microRNA-26a(miR-26a) is involved in development of liver cancer by analysis of proteomic expression profile of human hepatocarcinoma cell HepG2 transfected with miR-26a mimics.METHODS: HepG2 cells were cultured by a routine method and transfected with miR-26a mimics for 48 h for cell cycle analysis. The expressive proteome profiles of HepG2 cells with or without miR-26a mimics treatment were established by the methods of two-dimensional electrophoresis separation following lysis of the cells and extraction of the proteins. The proteomic expression profiles were analyzed by comparative proteomics technique to discover the important protein spots with differential expression. The identification of the proteins was conducted by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF-MS). RESULTS: miR-26a brought down the proliferation of HepG2 cells. Total 11 protein spots with alteration of expressive amounts more than 2 times were successfully identified in the proteomic expression profile of HepG2 cells treated with miR-26a mimics, including annexin A1, peroxiredoxin 4, proliferating cell nuclear antigen, apolipoprotein A1, cytochrome C oxidase subunit 5A, cyclin E2, ribose-phosphate pyrophosphokinase 3, cyclin-dependent kinase 1 and phosphatidylethanolamine-binding protein 1. Among these, the expression of 3 protein spots was up-regulated and 8 of them was down-regulated.CONCLUSION: miR-26a contributes to the anti-cancer effect by expressive regulation of the proteins mentioned above, or directly or indirectly controls the proliferation, differentiation and death of hepatocarcinoma cells.     

Dengue virus type 2 induces apoptosis and expression of death receptor in hepatic veno-endotheliocyte ED25

AIM: To investigate apoptosis and the expression of death receptors of TRAIL,TNF and Fas on hepatic veno-endotheliocyte ED25 cell strain induced by dengue virus type 2(DV2).METHODS: Flow cytometric analysis was used to detect the number of apoptotic cells and the expression levels of TRAILR1-4 ，TNFR1-2，Fas on ED25 cells before/after DV2 infection.RESULTS: The numbers of apoptotic cells of ED25 increased after DV2 infection,there were only about 5.7%±1.2% of apoptotic cells before virus infection while there were approximately 27.3%±1.6% of apoptotic cells after virus infection.At the same time the expression level of Fas also increased,before virus infection about 44.3%±2.2% of ED25 cells expressed Fas while 63.0%±2.3% of ED25 cells expressed Fas after virus infection.CONCLUSION: DV2 infection can induce apoptosis of ED25 cells,and it suggests strongly that Fas/FasL may be involved in the apoptotic signal transduction.     

Effect of microRNA-100 on proliferation activity and cell cycle of hepatocarcinoma cells

AIM：To investigate the effect of microRNA-100 (miR-100) on the proliferation activity and cell cycle of hepatocarcinoma cells. METHODS：Synthetic miR-100 mimic and its negative control were transfected into human hepatocarcinoma HepG2 cells by liposome method. After transfection, the cell counting kit-8 (CCK-8) was used to measure the cell proliferation activity. The cell cycle distribution was determined by flow cytometry. The expression of Polo-like kinase 1 (Plk1) at mRNA and protein levels was detected by quantitative real-time PCR (qRT-PCR) and Western blotting. RESULTS：The transfection efficiency mediated by cationic liposome was greater than 85%. The inhibitory rates of cell proliferation in HepG2 cells were (43.5±12.2)%, (46.5±3.7)% and (52.1±0.2)% at 24 h, 48 h and 72 h after transfected with miR-100 mimic, respectively, which were significantly increased as compared with the control cells. Moreover, the cell proliferation index in experimental group (35.8 ± 1.4) was higher than that in negative control group (39.2 ± 1.0) and simple liposome group (40.7 ± 2.0) at 72 h. At the same time, the mRNA and protein expression levels of Plk1 obviously decreased in HepG2 cells transfected with miR-100 at 72 h after transfection. CONCLUSION：miR-100 suppresses the proliferation activity of hepatocarcinoma cells by down-regulating Plk1 gene expression.     

CsWRKY22启动子的克隆及响应柑橘溃疡病菌侵染的表达特征

由柑橘黄单胞杆菌致病变种(Xanthomonas citri subsp. citri,Xcc)引起的柑橘溃疡病严重危害着柑橘产业的健康可持续发展。前期研究表明,CsWRKY22可能是溃疡病感病基因。从‘晚锦橙’(Citrus sinensis Osbeck)中克隆了CsWRKY22的两个等位启动子pCsWRKY22-1和pCsWRKY22-2,长度分别为1 428和1406bp。序列分析表明两个等位启动子序列一致性为91.76%。两个等位启动子含有多种参与植物防御反应和生长发育的顺式作用元件,相同元件的数量和位置基本一致,不同的是,在TATA-box下游,pCsWRKY22-1含有2个22 bp长的TA-rich转录增强序列,而pCsWRKY22-2只含有1个。构建CsWRKY22启动子控制GUS报告基因的植物表达载体pC sW RKY22::GUS,并用农杆菌介导法转化‘晚锦橙’,经GFP活性检测和PCR鉴定获得pCsWRKY22-1::GUS和pCsWRKY22-2::GUS转基因植株各8株和4株。GUS组织化学染色、GUS酶活性及定量PCR分析表明,CsWRKY22启动子具有较强的机械伤诱导活性和柑橘溃疡病病原菌诱导活性,同时具有一定水平的基础表达特性。pCsWRKY22-1机械伤诱导活性高于pCsWRKY22-2,而pCsWRKY22-2溃疡病菌诱导活性高于pCsWRKY22-1。