基于CRISPR/Cas9技术的全基因组基因编辑MDCK细胞文库的构建

本研究利用CRISPR/Cas9基因编辑技术,首先靶向犬全基因组设计合成转录sgRNA的DNA文库,并将DNA文库全部克隆于lentiCRISPR v2慢病毒转移载体上,高通量测序结果显示文库覆盖度高、均一性良好。将质粒文库和慢病毒包装系统质粒共转染293T细胞,收取含慢病毒样病毒的上清液感染MDCK细胞,在最适浓度嘌呤霉素筛选下,收集嘌呤霉素抗性细胞,冻存于-80℃,提取一部分细胞的基因组,通过PCR扩增转录sgRNA的DNA,将PCR产物插入到T载体后,挑取单克隆菌落,测序表明细胞文库中转录的sgRNA具有较好的覆盖度。本实验成功构建了犬全基因组范围转录sgRNA的质粒文库,并成功构建了MDCK细胞全基因组范围的基因编辑细胞库,该文库可作为后续筛选病毒复制关键宿主因子的细胞平台。

Urotensin Ⅱ-induced insulin resistance is mediated by NADPH oxidase-derived reactive oxygen species in Hep G2 cells

AIM: To investigated the effects of urotensin Ⅱ(UII) on hepatic insulin resistance in Hep G2 cells and the potential mechanisms involved.METHODS: Human hepatoma Hep G2 cells were cultured with or without exogenous UII for 24 h, in the presence or absence of 100 nmol/L insulin for the last 30 min. Glucose levels were detected by the glucoseoxidase method and glycogen synthesis was analyzed by glycogen colorimetric/fluorometric assay. Reactive oxygen species(ROS) levels were detected with a multimode reader using a 2′,7′-dichlorofluorescein diacetate probe. The protein expression and phosphorylation levels of c-Jun N-terminal kinase(JNK), insulin signal essential molecules such as insulin receptor substrate-1(IRS-1), protein kinase B(Akt), glycogen synthase kinase-3β(GSK-3β), and glucose transporter-2(Glut 2), and NADPH oxidase subunits such as gp91 phox, p67 phox, p47 phox, p40 phox, and p22 phox were evaluated by Western blot.RESULTS: Exposure to 100 nmol/L UII reduced the insulin-induced glucose consumption(P < 0.05)and glycogen content(P < 0.01) in Hep G2 cells compared with cells without UII. UII also abolished insulin-stimulated protein expression(P < 0.01) and phosphorylation of IRS-1(P < 0.05), associated with down-regulation of Akt(P < 0.05) and GSK-3β(P < 0.05) phosphorylation levels, and the expression of Glut 2(P < 0.001), indicating an insulin-resistance state in Hep G2 cells. Furthermore, UII enhanced the phosphorylation of JNK(P < 0.05), while the activity of JNK, insulin signaling, such as total protein of IRS-1(P < 0.001), phosphorylation of IRS-1(P < 0.001) and GSK-3β(P < 0.05), and glycogen synthesis(P < 0.001) could be reversed by pretreatment with the JNK inhibitor SP600125. Besides, UII markedly improved ROS generation(P < 0.05) and NADPH oxidase subunit expression(P < 0.05). However, the antioxidant/NADPH oxidase inhibitor apocynin could decrease UII-induced ROS production(P < 0.05), JNK phosphorylation(P < 0.05), and insulin resistance(P < 0.05) in HepG 2 cells. CONCLUSION: UII induces insulin resistance, and this can be reversed by JNK inhibitor SP600125 and antioxidant/NADPH oxidase inhibitor apocynin targeting the insulin signaling pathway in HepG 2 cells.

Growth-inhibiting and Apoptosis-inducing Effects of Tanshinone ⅡA on Human Gastric Carcinoma Cells

To explore the effects of Tanshinone Ⅱ A on the proliferation, apoptosis and gene expression of p53 and bcl-2 in human gastric carcinoma MKN-45 cells. Cell count and MTT assay were used to study the proliferation-inhibiting effect of Tanshinone Ⅱ A on MKN-45 cells. The effect of Tanshinone Ⅱ A on the cell cycle and apoptosis of MKN-45 cells were examined by propidium iodide （PI） staining and flow cytometry. Semi-quantitative RT-PCR was used to further verify the ex- pression of p53 and bcl-2 gene after exposure to Tanshinone Ⅱ A in MKN-45 cells. The results showed that Tanshinone Ⅱ A significantly inhibited the growth and proliferation of MKN-45 cells in a dose- and time-dependent manner （P〈0.05）. Tanshinone Ⅱ A arrested MKN-45 cells in G2/M phase which led to an obvious accumulation of G2/M phase cells while decreased number of Go/G1 phase cells. This resulted in apoptosis of MKN-45 cells and the apoptosis rate was as high as 43.91% after treatment with 2.0 lag/mL Tanshinone Ⅱ A for 96 h. It was also found that Tanshinone Ⅱ A up-regulated expression of p53 gene and down-regulated expression of bcl-2 gene. The cytostatic and antiproliferative effect of Tanshinone Ⅱ A makes it a promising anticancer agent for the treatment of gastric carcinoma.

MDCK细胞无血清悬浮培养技术在流感疫苗研究与生产中的应用进展

流感是一种常见的呼吸道疾病,由流感病毒引起,接种疫苗是预防流感的有效手段。MDCK细胞(Madin-Darby Canine Kidney Cells)具有易于培养和高产量的特点,可以支持流感病毒的复制和增殖,被广泛用于流感疫苗的研究和生产。随着对流感病毒研究的不断深入,为了进一步拓展MDCK细胞用于流感病毒研究和工业应用的能力,研究人员开始发展MDCK细胞无血清全悬浮培养技术。通过长期的培养和优化,驯化的MDCK悬浮细胞株可以更好地适应流感病毒的生长环境,提高流感病毒的产量和感染性。总之,MDCK细胞无血清悬浮培养技术在流感病毒研究和工业应用中发挥着重要作用,为流感疫苗生产和抗流感药物研发提供更好的工具。同时也必须重视MDCK细胞的安全性,采取合适的措施来确保其应用的安全性和可靠性。

Valsartan Inhibits Angiotensin Ⅱ-induced Proliferation of Vascular Smooth Muscle Cells via Regulating the Expression of Mitofusin 2

Angiotensin Ⅱ (ANGⅡ) plays an important role in the pathogenesis of atherosclerosis by inducing proliferation of vascular smooth muscle cells (VSMCs).In our study,we observed the effects of valsartan on proliferation of cultured VSMCs treated with or without ANGⅡ by cell counting and methyl thiazolyl tetrazolium (MTT) assay,and detected the expression of mitofusin 2 (Mfn2),a newly discovered cell proliferation inhibitor and a related cell proliferation signaling pathway pro-tein by Western blotting.ANGⅡ at a concentration of 10-6 mol/L significantly stimulated VSMCs proliferation,down-regulated the expression of Mfn2 and upregulated the expression of Raf and ERK1/2.Valsartan inhibited such effects of ANGⅡ at concentrations of 10-5 and 10-6 mol/L,but not at 10-7 mol/L.Valsartan had no significant effect on the proliferation of untreated VSMCs.These results suggest that valsartan inhibits ANGⅡ-induced proliferation of VSMCs in vitro via Mfn2-Ras-Raf-ERK/MAPK signaling pathway.

地榆皂苷Ⅱ抑制结肠癌细胞增殖、迁移、侵袭和诱导凋亡的体外实验研究

目的探讨地榆皂苷Ⅱ对结肠癌细胞HT-29增殖、迁移、侵袭和凋亡的作用并探讨其作用机制。方法采用CCK-8法检测地榆皂苷Ⅱ对细胞增殖的影响,采用划痕试验检测地榆皂苷Ⅱ对细胞迁移能力的影响,采用Transwell小室实验检测地榆皂苷Ⅱ对细胞侵袭能力的影响,采用流式细胞术测定地榆皂苷Ⅱ对细胞凋亡的影响,分别采用实时定量反转录聚合酶链反应(qRT-PCR)和Western blot法测定地榆皂苷Ⅱ对细胞中蛋白激酶B(AKT)/磷脂酰肌醇-3-激酶(PI3K)信号通路mRNA和蛋白表达的影响。结果地榆皂苷Ⅱ(0、1、5、10、20、40、60和80μmol/mL)可剂量依赖性抑制结肠癌细胞HT-29的增殖;地榆皂苷Ⅱ(5、10和20μmol/mL)可剂量依赖性抑制结肠癌细胞HT-29的迁移能力;地榆皂苷Ⅱ(5、10和20μmol/mL)可剂量依赖性抑制结肠癌细胞HT-29的侵袭能力;地榆皂苷Ⅱ(5、10和20μmol/mL)可剂量依赖性促进结肠癌细胞HT-29的凋亡;地榆皂苷Ⅱ(5、10和20μmol/mL)可剂量依赖性降低结肠癌细胞HT-29中AKT和PI3K mRNA的表达,增加Caspase-3和Caspase-9 mRNA的表达;地榆皂苷Ⅱ(5、10和20μmol/mL)可剂量依赖性地降低结肠癌细胞HT-29中磷酸化蛋白激酶B(p-AKT)和磷酸化磷脂酰肌醇-3-激酶(p-PI3K)蛋白的表达,增加Cleaved-Caspase-3和Cleaved-Caspase-9蛋白的表达,差异有统计学意义(P<0.05)。结论地榆皂苷Ⅱ具有抑制结肠癌细胞HT-29的增殖、迁移、侵袭,促进细胞凋亡作用,可能与其促进AKT和PI3K蛋白磷酸化、Caspase-3和Caspase-9蛋白活化而调节AKT/PI3K信号通路有关。

Nonmuscle myosin Ⅱ regulates migration but not contraction in rat hepatic stellate cells

AIM: To identify and characterize the function of non-mu-scle myosin Ⅱ (NMM Ⅱ) isoforms in primary rat hepatic stellate cells (HSCs).METHODS: Primary HSCs were isolated from male Spra-gue-Dawley rats by pronase/collagenase digestion. Total RNA and protein were harvested from quiescent and culture-activated HSCs. NMM Ⅱ isoform (Ⅱ-A, Ⅱ-B and Ⅱ-C) gene and protein expression were measured by RealTime polymerase chain reaction and Western blot analyses respectively. NMM Ⅱ protein localization was visualized in vitro using immunocytochemical analysis. For in vivo assessment, liver tissue was harvested from bile duct-ligated (BDL) rats and NMM Ⅱisoform expression determined by immunohistochemistry. Using a selective myosin Ⅱ inhibitor and siRNA-mediated knockdown of each isoform, NMM Ⅱ functionality inprimary rat HSCs was determined by contraction and migration assays.RESULTS: NMM Ⅱ-A and Ⅱ-B mRNA expression was increased in culture-activated HSCs (Day 14) with sig-niflicant increases seen in all pairwise comparisons (Ⅱ-A: 12.67 ± 0.99 (quiescent) vs 17.36 ± 0.78 (Day 14), P < 0.05; Ⅱ-B: 4.94 ± 0.62 (quiescent) vs 13.90 ±0.85 (Day 14), P < 0.001). Protein expression exhibited similar expression patterns (Ⅱ-A: 1.87 ± 2.50 (quiescent) vs 58.64 ± 8.76 (Day 14), P < 0.05; Ⅱ-B: 1.17 ± 1.93 (quiescent) vs 103.71 ± 21.73 (Day 14), P < 0.05). No signif icant differences were observed in NMM Ⅱ-C mRNA and protein expression between quiescent and activated HSCs. In culture-activated HSCs, NMM Ⅱ-A and Ⅱ-B merged with F-actin at the cellular periphery and throughout cytoplasm respectively. In vitro stud-ies showed increased expression of NMM Ⅱ-B in HSCs activated by BDL compared to sham-operated animals. There were no apparent increases of NMM Ⅱ-A and Ⅱ-C protein expression in HSCs during hepatic BDL injury. To determine the contribution of NMM Ⅱ-A and Ⅱ-B to migration and contraction, NMM Ⅱ-A and Ⅱ-B expres-sion were downregulated with siRNA. NMM Ⅱ-A and/or Ⅱ-B siRNA inhibited HSC migration by approximately 25% compared to scramble siRNA-treated cells. Conversely, siRNA-mediated NMM Ⅱ-A and Ⅱ-B inhibition had no signif icant effect on HSC contraction; however, contraction was inhibited with the myosin Ⅱ inhibitor, blebbistatin (38.7% ± 1.9%).CONCLUSION: Increased expression of NMM Ⅱ-A and Ⅱ-B regulates HSC migration, while other myosin Ⅱclasses likely modulate contraction, contributing to development and severity of liver f ibrosis.

Astilbin Inhibits Proliferation of Rat Aortic Smooth Muscle Cells Induced by Angiotensin Ⅱ and Down-regulates Expression of Protooncogene

This study examined the effect of astilbin on the proliferation of rat aortic smooth muscle cells (RASMCs) induced by angiotensin Ⅱ (AngⅡ) and explored the possible mechanisms. Cell proliferation model of RASMCs was induced by treatmente with AngⅡ. Cells were randomly divided to 8 groups. Normally cultured VSMCs serves as blank control group; in AngⅡ model group, cells were treated with AngⅡ at 10–7 mol/L; in three astilbin groups, cells were treated with 10, 15, 30 mg/L of astilbin; in three AngⅡ+astilbin groups, cells were treated with AngⅡ (at 10–7 mol/L) and astilbin at 10, 15, 30 mg/L. Cell proliferation ability was detected by MTT method and the cell cycles and proliferation index were flow cytometrically determined. The expression of c-myc mRNA was assessed by using reverse transcription polymerase chain reaction (RT-PCR), and the expression of NF-κB in RASMCs was immunocytochemically observed. Our results showed that MTT metabo-lism in RASMCs in the basic and AngII stimulated situation was inhibited by astilbin, and the cells numbers of G0/G1 phase were increased and that of G2/S phase were decreased markedly. Not only highly expression of c-myc gene stimulated by AngⅡ could be inhibited by Astilbin significantly, but also the expression of NF-κB protein can be down regulated by Astilbin. We are led to conclude that astilbin astilbin can inhibit the AngⅡ-mediated proliferation of RASMCs by blocking the transition of RASMCs from G0/G1 phase to S phase and by down-regulating the expression of NF-κB, c-myc gene.

Primary Culture of Alveolar Epithelial Type Ⅱ Cells and Its Bionomic Study

To establish a better method of primary culture for alveolar epithelial type Ⅱ cells （AEC Ⅱ ） and to study its bionomics, alveolar epithelial type Ⅱ cells were isolated by digestion with trypsin and collagenase, which were then purified by plated into culture flask coated with rat immunoglobulin （i The purified AEC Ⅱ were identified by alkaline phosphatase staining, electron mi- croscopy, immunocytochemical staining of pulmonary surfactant protein A （SPA）. The SPA expression and transfection characteristics were compared with those of A549 cell line. The results showed that AEC Ⅱ could be isolated by digestion with trysin and collagenase and purified by adhesive purification by using IgG, with a yield of about 2-3 × 10^7, and a purity of about 75%-84 %. Cells could be quickly identified with AKP staining. AEC Ⅱ were different from A549 cell line in terms of SPA expression and transfection characteristics. It is concluded that adhesive purification with IgG can improve the purity of AEC Ⅱ, and AKP staining is simple in cell identification. AEC Ⅱ can not be completely replaced by A549 cells in some studies because the differences between them, such as SPA expression.

Immunohistochemical Studies of Macrophages and MHC Class Ⅱ -positive Cells in the Iris and Ciliary Body of Lewis Rats

Purpose:To investigate the density, distribution and morphology of macrophages and MHC class II -positive dendritic cells in the iris and ciliary body of lewis rats. Methods:Immunohistochemistry was performed using monoclonal antibodies specific to monocytes and macrophages (ED1,ED2) and MHC class II -positive cells (OX6) on wholemounts of the iris-ciliary body complex isolated form normal lewis rats.Results:A well developed network of macrophages was present in the iris and ciliary body of normal lewis rats. These cells, morphologically displaying dendriti-form or pleiomorphic appearance, were more densely arranged in mid-iris (950 + 189 cells/mm2) than in iris base (482 ± 78 cells/mm2) and pupil margin (595 ± 92 cells/mm2). A similar network of MHC class II -positive cells with a cell density 452 ± 78 cells/mm2 was almost uniformly distributed in the iris of normal lewis rats.Conclusions : A network of macrophages and MHC class II -positive cells was established in the iris and ciliary body of

Multi-objective optimization of the cathode catalyst layer micro-composition of polymer electrolyte membrane fuel cells using a multi-scale,two-phase fuel cell model and data-driven surrogates

Polymer electrolyte membrane fuel cells(PEMFCs)are considered a promising alternative to internal combustion engines in the automotive sector.Their commercialization is mainly hindered due to the cost and effectiveness of using platinum(Pt)in them.The cathode catalyst layer(CL)is considered a core component in PEMFCs,and its composition often considerably affects the cell performance(V_(cell))also PEMFC fabrication and production(C_(stack))costs.In this study,a data-driven multi-objective optimization analysis is conducted to effectively evaluate the effects of various cathode CL compositions on Vcelland Cstack.Four essential cathode CL parameters,i.e.,platinum loading(L_(Pt)),weight ratio of ionomer to carbon(wt_(I/C)),weight ratio of Pt to carbon(wt_(Pt/c)),and porosity of cathode CL(ε_(cCL)),are considered as the design variables.The simulation results of a three-dimensional,multi-scale,two-phase comprehensive PEMFC model are used to train and test two famous surrogates:multi-layer perceptron(MLP)and response surface analysis(RSA).Their accuracies are verified using root mean square error and adjusted R^(2).MLP which outperforms RSA in terms of prediction capability is then linked to a multi-objective non-dominated sorting genetic algorithmⅡ.Compared to a typical PEMFC stack,the results of the optimal study show that the single-cell voltage,Vcellis improved by 28 m V for the same stack price and the stack cost evaluated through the U.S department of energy cost model is reduced by$5.86/k W for the same stack performance.

Organic photovoltaic cells with copper(Ⅱ) tetra-methyl substituted phthalocyanine

Efficient heterojunction organic photovoltaic （OPV） cells are fabricated based on copper tetra-methyl phthalocyanine （CuMePc） as donor and fullerene （C60） as acceptor. The power conversion efficiency of CuMePc/C60 OPV cell （2.52%） is increased by 88% compared with that of the non-peripheral substituted copper phthalocyanine （CuPc）/C60 OPV cell （1.34%）. The introduction of methyl substituent leads to stronger π–π interaction of CuMePc （～ 3.5 ？） than that of CuPc （～ 3.8 ？）. The efficiency improvement is attributed to the enhanced carrier mobility of CuMePc thin film （1.1×10-3 cm2/V·s） and better film morphology by introducing methyl groups into the periphery of CuPc molecule.

Growth Inhibition and Apoptosis Induction in Human Hepatoma Cells by Tanshinone Ⅱ_A

In order to .study the effect of tanshinone ⅡA on growth and apoptosis in human hepatoma cell line BEL-7402 in vitro, the human hepatoma cell line BEL-7402 was treated with tanshinone ⅡA at various concentrations for 72 h. Growth suppression was evaluated by MTT assay; apoptosis-relat-ed alterations in morphology and biochemistry were ascertained under cytochemical staining (Hoechst 33258), transmission electron microscopy (TEM), and DNA agarose gel electrophoresis. Apoptotic rate was quantified by flow cytometry (FCM). The results showed that Tanshinone ⅡA could inhibit the growth of hepatoma cells in a dose-dependent manner, with IC50 value being 6. 28μg/ml. After treatment with 1-10μg/ml tanshinone ⅡA for 72 h, BEL-7402 cells apoptosis with nuclear chro-matin condensation and fragmentation as well as cell shrinkage and the formation of apoptotic bodies were observed. DNA ladder could be demonstrated on DNA electrophoresis. FCM analysis showed hypodiploid peaks on histogram, and the apoptotic rates at μg/ml concentration for 12 h> 24 h, 36 h, 48 h and 72 h were (2. 32±0. 16)%, (3. 01±0. 35) %, (3. 87±0. 43)%, (6. 73±0. 58)% and (20. 85 ± 1. 74) % respectively, which were all significantly higher than those in the control group (1. 07±0. 13) %. It is concluded that Tanshinone ⅡA could induce human hepatoma cell line BEL-7402 apoptosis, which may be related to the mechanism of growth inhibition.

FOLLICULO-STELLATE CELLS OF THE RAT ANTERIOR PITUITARY RESPONDED TO ANGIOTENSIN Ⅱ BY INCREASING INTRACELLULAR Ca^(2+) CONCENTRATION

Objective The main purpose of this study was to investigate whether the folliculo stellate cells (FSC) respond to angiotensin(Ang)Ⅱ by increasing intracellular free Ca 2+ concentration ([Ca 2+ ]i),and where the origin of Ca 2+ mobilization is if that has occurred.Methods Pituitary cells in primary culture were prepared from male Wister rats(250g) by a conventional method and cultured in MEM supplemented with 4% normal rat serum.After 2 days in culutre,cells were loaded with 1 μmol/L fura PE3/AM for 1 h and subjected to a Ca 2+ imaging experiment with Quanti Cell 700 system.Excitation wavelengths of 340 and 380 nm were selected by means of a computer controlled filterwheel.Results The [Ca 2+ ]i of FSC in the rat anterior pituitary was elevated by Ang Ⅱ.The elevation of [Ca 2+ ]i of FSC induced by 0.1,1.0,10 and 100 nmol/L Ang Ⅱ was (56.33±6.18)( ±s ),(117.07±36.07),(175.59±40.01) and (216.02±11.52) nmol/L,respectively.The increase of [Ca 2+ ]i of FSC induced by 100 nmol/L Ang Ⅱ was not influenced by the medium without Ca 2+ (0Ca),but significantly suppressed by thapsigargin(TG),an inhibitor of ATPase.The rate of responsive FSC to Ang Ⅱ (100 nmol/L) was 61.84% which was obviously higher than that of pituitary endocrine cells(43.49%).Conclusion The present experiment demonstrates that the FSC in the rat anterior pituitary responds to Ang Ⅱ by increasing [Ca 2+ ]i,which raises the possibility that Ang Ⅱ released from either lactotrophs or gonadotrophs affects FSC through paracrine mechanism.The elevation of [Ca 2+ ]i induced by Ang Ⅱ presents a dosage dependent relation, and is possibly because of the release of Ca 2+ from an intracellular Ca 2+ pool(s).Fashions of Ca 2+ release are relative to the concentration of Ang Ⅱ.The results indicate that Ang Ⅱ functions as a paracrine factor among pituitary cells including FSC.

Effects of angiotensin Ⅱ receptor antagonist, Losartan on the apoptosis, proliferation and migration of the human pancreatic stellate cells

AIM: To investigate the effects of AT1 (Type 1 angiotensin Ⅱ receptor) antagonist (Losartan) on the apoptosis,proliferation and migration of the human pancreaticstellate cells (hPSCs).METHODS: hPSCs were isolated from pancreatic sample of patients with pancreatic carcinoma using radioimmunoassay (RIA) technique to detect the concentration of AngⅡ in culture media and cell homogenate. Immunocytochemistry (ICC) and in situ hybridization (ISH) methods were utilized to test AT1 expression in hPSCs. Effects of Losartan on hPSCs proliferation, apoptosis and migration were investigated using BrdU incorporation, TUNEL, flow cytometry (FCM),and phase-contrast microscope separately when cells treated with Losartan. Immunofluorescence and Western blot were applied to quantify the expression of type Ⅰ collagen in hPSCs.RESULTS: There exists AT1 expression in hPSCs, while no AngⅡ was detected in culture media and cell homogenate. Losartan induces cell apoptosis in a doseand time-dependent manner (apparently at 10-5 mol/L),no pro-proliferative effect was observed in the same condition.Corresponding dosage of Losartan can also alleviate the motion capability and type Ⅰ collagen content of hPSCs compared with AngⅡ treatment and non-treatment control groups.CONCLUSION: These findings suggest that paracrine not autocrine functions of AngⅡ may have effects on hPSCs,which was mediated by AT1 expressed on cells, while Losartan may exert anti-fibrotic effects by inhibiting hPSCs motion and partly by inducing apoptosis.

Transforming growth factor β receptor Ⅱ expression inexperimental cryptorchidism and apoptosisin spermatogenic cells in rats

Objective: To study the transforming growth factor β receptor Ⅱ (TGFβ-R Ⅱ) expression in experimental cryptorchidism and apoptosis in spermatogenic cells in rats. Methods: The apoptosis of spermatogenic cells was detected by means of the terminal deoxynucleotldyl transferase mediated dUTP nick end lableling method (TUNEL) and the TGFβ-R Ⅱ expression was observed with the immunohistochemistry SABC methods. Results: There was a significant increase in the TGFβ-R Ⅱ expression in unilateral undescended testes (UUTs) compared with that in contralateral descended testes (CDTs, P<0.01). However, there was a significant and time-dependent increase in the mean apoptotic index in UUTs than in CDTs. Conclusion: TGFβ-R Ⅱ may play an important role in spermatogenic cell apoptosis.

Effect of Different Culture Media on the Proliferation of Avian Influenza Virus H9 Subtypes in MDCK Cells

[Objective] To screen the best culture media for the proliferation of avian influenza virus （AIV） H9 subtypes in MDCK cells. [Method] The DMEM containing 10% （V/V） newborn calf serum, low-serum containing medium （ MEM-MD-611 ） and serum-free medium （SFE4Mega） were used to culture the MDCK monolayer ceils, which were then inoculated with different dilutions of AIV H9 subtypes, and the 3 kinds of media were al- so used as the maintenance solution to culture the virus. The cytopathic changes were observed at every 24 h, and the HA titers of the culture su- pernatants were also determined. [ Result] After culturing for 72 -96 h, the HA titers of the serum-free media were higher than that of low-serum culture media, while the HA titers were higher in the low-serum media than in the serum containing media. [ Conclusion] The 3 kinds of media can all used for the proliferation of AIV_ but the low-serum culture medium （MEM-MD-611 ） and serum-free medium （SFE4Meaa3 are preferred.

Multiplication of the Recombinant Strain Re-7 of Avian Influenza Virus Subtype H5 in MDCK Cells

This study was conducted to explore the multiplication pattern of the recombinant strain Re-7 of avian influenza virus subtype H5 in Madin Darby Canine Kidney （MDCK） cells and to determine the optimal multiplicity of infection （MOI） and the optimal time for virus harvest. The recombinant strain Re-7 was inoculated at different MOIs into MDCK cells grown in serum-free medium in 100 L bioreactors for replication. Then, the hemagglutination（HA） titer, 50% tissue culture infectious dose （TCID50） and 50% embryo infectious dose （EID50） of culture medium were measured once every 12 h from 24 h after virus inoculation to determine the optimal MOI. After that, virus was inoculated at the optimal MOI determined above into MDCK cells for large-scale virus replication to determine the optimal time for virus harvest. The results showed that the optimal MOI was 10 2, and the optimal time for virus harvest was 60 h after inoculation. Under these conditions, the HA titer, TCIDso per 1 mL and EIDso per 0.1 mL were increased to 1：102 4, 10^7.33 and 10^6.83, respectively. This study provides relatively stable parameters for large-scale production of the recombinant strain Re-7 of avian influenza virus subtype H5.

Effect of Angiotensin Ⅱ on Cord Blood CD^(34+) Cells Expansion in vitro

In order to investigate the influence of angiotensin Ⅱ on hematopoietic system, CD34 + cells in cord blood were purified, and the effects of angiotensin Ⅱ in combination with various cytokines on their growth and differentiation were studied by cell culture in vitro. It was found that angiotensin Ⅱ in suspending medium could stimulate both BFU-E and CFU-GM expansion. The number of BFU-E and CFU-GM was increased with the increases of angiotensin Ⅱ concentrations during a certain range. In addition, the expansion fold of CFU-GM was increased from 2.3±0.8 times to 7.8±2.3 times when angiotensin Ⅱ was added in the presence of SCF+G-CSF+GM-CSF+IL-3 cytokines mixture. Similarly, the expansion fold of BFU-E was increased from 3.1±1.8 times to 9 2±2.3 times with angiotensin Ⅱ in the presence of SCF+EPO+TPO+IL-3. In the semi-solid medium, angiotensin Ⅱ could stimulate CFU-GM expansion but had no effect on the growth of BFU-E. In conclusion, angiotensin Ⅱ had some stimulating effects on cord blood hematopoietic progenitors expansion in vitro in the presence of other cytokines.

Induction of interleukin-8 production by angiotensin Ⅱ in rat vascular smooth muscle cells

Objective： Interleukin-8（IL-8） represents the prototypical chemokine that is made by a wide variety of cell types. Previously studies have suggested that angiotensin Ⅱ（Ang Ⅱ） is involved in atherogenesis through induction ofproinflammatory cytokines such as interleukin- 6 or monocyte chemoattractant protein-1（MCP-1） in vascular smooth muscle cells（VSMCs）, while the role of Ang II on IL-8 expression in VSMCs is poorly studied. Methods： In this study, VSMCs were isolated from the thoracic aorta of Sprague-Dawley rats. The expression of smooth muscle α-actin was confirmed by an immunohistochemical method. Semi-quantitative RT-PCR and enzyme-linked immunosorbent assay （ELISA） analyses were conducted to detect IL-8 expression. Results： In the present study we found that Ang Ⅱ significantly increased the expression of IL-8 both at the mRNA and protein levels in rat VSMCs in a dose- and time-dependent manner. Conclusion： These findings suggested that Ang Ⅱ may participate in atherosclerosis through induction of inflammatory mediator in VSMCs.