Molecular evolution of connective tissue growth factor in Cyprinidae (Teleostei: Cypriniformes)

Connective tissue growth factor (CTGF) plays an important role in regulation of cell growth, differentiation, apoptosis and individual development in animals. The study of sequences variation and molecular evolution of CTGF gene across various species of the cyprinid could be helpful for understanding of speciation and gene divergence in this kind of fish. In this study, 19 novel sequences of CTGF gene were obtained from the representative species of the family Cyprinidae using PCR amplification, cloning and sequencing. Phylogenetic relationships of Cyprinidae were reconstructed by neighbor-joining (NJ), maximum parsimony (MP), maximum likelihood (ML) and Bayesian method. Oryzias latipes from the family Cyprinodontidae was assigned to be the outgroup taxon. Leuciscini and Barbini were clustered into the monophyletic lineages respectively with the high nodal supports. The estimation of the ratio of nonsynonymous to synonymous substitution (dN/dS) for the various branches indicated that there stood the different evolution rates between the Leuciscini and the Barbini. With the ratio of dN/dS of the Leuciscini being lower than that of the Barbini, species within the Barbini were demonstrated to be subjected to the relatively less selection pressure and under the relaxable evolution background. A 6 bp indel (insertion/deletion) was found at the 5' end of CTGF gene of Cyprinidae, and this 6 bp deletion only appeared in the Leuciscini, which is a typical characteristic of the Leuciscini and provides evidence for the monophylogeny of the Leuciscini. For the amino acid sequences of CTGF protein, the most variations and Indels were distributed in the signal region and IGFBP region of this protein, implying that these variations were correlated with the regulation of the CTGF gene expression and protein activity.     

Molecular evolution of connective tissue growth factor in Cyprinidae （Teleostei： Cypriniformes）

Connective tissue growth factor （CTGF） plays an important role in regulation of cell growth, differentiation, apoptosis and individual development in animals. The study of sequences variation and molecular evolution of CTGF gene across various species of the cyprinid could be helpful for understanding of speciation and gene divergence in this kind of fish. In this study, 19 novel sequences of CTGF gene were obtained from the representative species of the family Cyprinidae using PCR amplification, cloning and sequencing. Phylogenetic relationships of Cyprinidae were reconstructed by neighbor-joining （NJ）, maximum parsimony （MP）, maximum likelihood （ML） and Bayesian method. Oryzias latipes from the family Cyprinodontidae was assigned to be the outgroup taxon. Leuciscini and Barbini were clustered into the monophyletic lineages, respectively, with the high nodal supports. The estimation of the ratio of non-synonymous to synonymous substitution （dN/dS） for the various branches indicated that there stood the different evolution rates between the Leuciscini and the Barbini. With the ratio of dN/dS of the Leuciscini being lower than that of the Barbini, species within the Barbini were demonstrated to be subjected to the relatively less selection pressure and under the relaxable evolution background. A 6 bp indel （insertion/deletion） was found at the 5＇ end of CTGF gene of Cyprinidae, and this 6 bp deletion only appeared in the Leuciscini, which is a typical characteristic of the Leuciscini and provides evidence for the monophylogeny of the Leuciscini. For the amino acid sequences of CTGF protein, the most variations and indels were distributed in the signal region and IGFBP region of this protein, implying that these variations were correlated with the regulation of the CTGF gene expression and protein activity.     

幼鼠正畸牙移动中牙周组织内b-FGF的动态变化

目的：研究碱性成纤维细胞生长因子（b—FGF）在幼年Wistar大鼠牙周组织内的表达与分布及其在正畸牙移动中的作用。方法：建立幼年Wistar大鼠正畸牙移动的模型,在不同牵引时间段用4％中性甲醛行升主动脉灌注固定、取材、脱钙、制作石蜡标本。应用SP免疫组织化学法和图象分析仪进行半定量分析。结果：正常大鼠牙周组织中b—FGF表达较低。实验组在1d组张力侧的成牙骨质细胞b—FGF表达高于对照组,10、14d组张力侧的成纤维细胞、成骨细胞b—FGF阳性着色较正常对照组及压力侧表达有显著性差异。玻璃样变区无b—FGF表达。结论：b—FGF参与了正畸牙移动过程中牙周组织的改建。     

葫芦巴总皂苷对心肌成纤维细胞转分化的影响及机制的实验研究

目的探讨葫芦巴总皂苷(Trigonella foenum greacum.L Saponin,TFGs)对尾加压素Ⅱ(UrotensinⅡ,UⅡ)诱导体外培养的大鼠心肌间质成纤维细胞(CFs)发生肌成纤维细胞转分化的干预作用及相关分子机制.方法体外培养大鼠心肌间质成纤维细胞,随机分为正常对照组、UⅡ刺激组和TFGs干预组.正常对照组在整个培养过程中未加任何刺激;UⅡ刺激组加入10-8mol/L的尾加压素Ⅱ培养,并分别终止培养于12,24和48 h;TFGs干预组将UⅡ作为刺激因素,分别加入终浓度为0,25,50,100和200 mg/L的TFGs.应用免疫荧光和免疫组化方法检测显示α-SMA的表达,应用RT-PCR和免疫印迹法分别检测结缔组织生长因子(CTGF)的基因及蛋白表达.结果免疫荧光检测显示,培养24 h后,正常对照组细胞胞浆中仅有少量α-SMA表达,而α-SMA表达量在经UⅡ作用后明显增多.尾加压素Ⅱ干预CFs不同时间后(12,24,48 h),免疫组化显示α-SMA表达量逐渐增加.UⅡ的这种诱导CFs发生肌成纤维细胞转分化作用可被含TFGs的培养液所抑制,且呈浓度和时间依赖性.与对照组比较,UⅡ刺激组的CTGF的基因及蛋白表达均明显增高(P<0.01).而TFGs则可以抑制UⅡ诱导的CTGF基因及蛋白表达量的上调(P<0.01).结论尾加压素Ⅱ具有诱导大鼠心肌间质成纤维细胞发生肌成纤维细胞转分化的作用,此作用可能与其促进CTGF的基因及蛋白表达有关.葫芦巴总皂苷能有效地抑制大鼠CFs的转分化,并且下调UⅡ诱导的CTGF的过表达.这进一步明确UⅡ在心肌纤维化发生、发展中的作用,也为临床应用葫芦巴总皂苷防治心肌纤维化提供了实验依据.     

神经生长因子

概述了神经生长因子的分子结构及作用机制，综述了神经生长因子的生理功能，神经生长因子不仅对神经细胞的存活，生长发育，分化，再生和功能维持起调控作用。同时也能诱导细胞凋亡。     

正常人口腔粘膜中表皮生长因子及其受体的表达

目的：研究人口腔粘膜中表皮生长因子（EGF）及其受体（EGF－R）的分布状态,方法：采用免疫组织化学方法,结果：EGF主要分布于口腔上皮底层细胞中,并随细胞向上分化而逐渐减少,其免疫染色出现在细胞浆中,EGF－R也主要在基底层细胞表达,除存在于细胞浆外,在细胞核中亦见表达,结论：表皮生长因子及其受体对口腔上皮细胞生长,分化的调节有重要作用,但EGF－R在细胞核中的表达,其作用尚需深入研究。     

碱性成纤维细胞生长因子对无血清培养大鼠生长板软骨细胞的影响

目的：探讨碱性成纤维细胞生长因子（bFGF）用于无血清培养扩增软骨细胞的可行性及最适质量浓度。方法：分离、培养大鼠肋生长板软骨细胞（rat costochondral growth plate chondrocyte,RGC）。细胞化学和免疫细胞化学染色的方法检测第1代RGC中蛋白多糖和Ⅱ型胶原的表达。观察0．1、1．0、10．0和100．0μg/L bFGF对无血清培养RGC形态的影响,并分别用^3H—TdR和^3H—Proline掺入法检测上述质量浓度bFGF对无血清培养RGC增殖和胶原合成的影响。结果：第1代RGC表达蛋白多糖和Ⅱ型胶原,保持了软骨细胞的分化表型。随着bFGF质量浓度的增加RGC的形态由多角形变为梭形和圆形。与无血清对照组相比,上述4个质量浓度的bFGF均显著促进RGC的增殖和胶原合成（P〈0．05）,其中1．0和10．0μg／L bFGF促RGC增殖的作用与10％胎牛血清（FBS）对照组差异无显著性意义（P〉0．05）,但促胶原合成作用较弱,相当于10％FBS对照组的印％左右（P〈0．05）。结论：bFGF可用于无血清扩增软骨细胞,1．0～10．0μg/L是适宜的质量浓度范围,但其促胶原合成作用较弱,还需添加其他因子以弥补促胶原合成作用的不足。     

Nerve growth factor activity of several immunal related serine proteases

Rabbit was immuned by the previously purified protein with high nerve growth factor (NGF) bioactivity (NGF_like protease) from \%Agkistrodon halys Pallas\% and the antisera were collected. The polyclonal antibodies were tentatively purified and then used as ligands of an affinity column. The \%A.h.Pallas\% crude venom was fractionated by this affinity column and then by Mono Q on fast protein liquid chromatography (FPLC). As a result, fraction Ⅱ and fraction Ⅲ were purified respectively, whose N_terminal amino acid sequences show high homology with the serine proteases in snake venoms, as well as the previous NGF_like protease. However, they possessed different levels of NGF bioactivity. The NGF activity of the previous NGF_like protease is equivalent to that of NGF, while the activity of fraction Ⅱ seems relatively low in contrast to fraction Ⅲ which had no NGF activity.     

亮毛杜鹃组织培养技术探索

亮毛杜鹃组织培养研究过程中，遇到的首要问题是不易成活。因此，在前期工作的基础上，进一步研究了不同激素配比及浓度对亮毛杜鹃的成活率及生长势的影响，通过在两种激素配比下生长索种类及浓度梯度、细胞分裂素种类及浓度梯度，在三种激素配比下BA浓度梯度的实验，得出的结果是1／10MS＋ZT0．5＋IAA0．05培养基比较适合亮毛杜鹃生长。     

温阳化浊通络方对硬皮病小鼠CTGF表达的影响

本文研究温阳化浊通络方对硬皮病小鼠模型皮肤结缔组织生长因子（CTGF）、羟脯氨酸表达的影响。通过对BALB／C小鼠用博莱霉素制作硬皮病模型,分别用积雪苷片、温阳化浊通络方连续治疗4周。采用比色法、RT—PCR法和免疫印迹法分别测定皮肤组织羟脯氨酸含量、CTGFmRNA的表达及蛋白合成。实验结果与PBS组相比,模型组小鼠皮肤羟脯氨酸、CTGFmRNA及蛋白含量明显增加（均P〈0．01）,且模型组羟脯氨酸与CTGF呈直线正相关性（r=0．902,P〈0．01）。羟脯氨酸、CTGFmRNA及蛋白在温阳化浊通络方组含量均低于模型组（均P〈O．01）,且三者表达量在温阳化浊通络方组与积雪苷组问差异具有显著性（P〈0．05或P〈0．01）。结论：羟脯氨酸、CTGFmRNA及蛋白在硬皮病小鼠皮肤组织中具有较高表达;温阳化浊通络方能降低羟脯氨酸、CTGFmRNA及蛋白表达,改善皮肤硬化。     

抗松针褐斑病湿地松组培再生植株生长性状观察

对移栽至南京林业大学南大山苗圃的湿地松组培再生植株进行了苗高、地径、干形的比较。结果表明,各家系及无性系的生长状态存在差异。组培苗与实生苗间苗高差异较大,组培苗在早期生长速度不及实生苗,且抗病的家系不及普通的家系,抗病的8#和32#家系苗高差异不显著;实生苗、组培苗斜生现象严重;继代次数对组培苗的苗高、地径生长影响不大;组培苗年龄越大,苗高生长势越好,地径差异小;8#家系组培苗各无性系间苗高差异显著,地径差异不显著。     

生长调节剂对唐菖蒲茎段培养中器官分化的影响

研究了细胞分裂素和生长素对唐菖蒲器官分化的影响，结果：在MS培养基中单一的加入细胞分裂素以6BA1．0mgL^-1对唐菖蒲愈伤组织诱导和芽的增殖效果较好；生长素与细胞分裂素配合使用的效果优于只加入细胞分裂素。6BA0．5mgL^-1 NAA0．1mgL^-1可使愈伤组织诱导率达到70％，在继代培养中6BA1．0mgL^-1 NAA0．2mgL^-1可使每个转植芽增殖7．6个芽，根系诱导率以IBA0.5mgL^-1效果最好。说明生长调节剂对器官分化的促进效果与其种类和浓度有关。     

Transforming growth factorβ1 and epidermal growth factor decrease the expression of 17β-hydroxysteroid dehydrogenase type 2 in endometrial carcinoma cells

Estradiol (E2) is the major molecular form of estrogens. Its biological effects are determined by estrogen receptors and intracellular E2 concentration in target cells. Regulation of intracellular E2 concentration involves the action of 17β(-hydroxysteroid dehydrogenase (17HSD) type 2, the enzyme inactivating E2 to estrone. It has been demonstrated that 17HSD type 2 is expressed in normal endometrial epithelia and emdometrial carcinoma cells (RL 95-2). However, the regulatory mechanism of 17HSD type 2 expression in emdometrial cancer cells remains unknown. In the present study, the effects of transforming growth factor-(1 (TGF-β1) and epidermal growth factor (EGF) on 17HSD type 2 expression in RL 95-2 cells have been investigated using enzyme activity assay and Northern blot analysis. After stimulation with TGF-β1 or EGF, the in vivo oxidative 17HSD activity in RL 95-2 cells was significantly decreased. It appeared that the inhibitory effect of TGF-β1 and EGF on the enzyme activity of 17HSD type 2 is dose- and time-dependent. Northern blot analysis further revealed that treatment of cells for 48 h with 10 ng/mL TGF-1β And 50 ng/mL EGF reduced the expression 17HSD type 2 mRNA to 30% and 20% of the control level, respectively. The data demonstrate that 17HSD type 2 expression in endometrial carcinoma cells is down-regulated by certain growth factors.     

不同生态因子对蛹虫草菌丝生长影响的研究

本文主要针对蛹虫草菌丝生长对营养元素需求和对生态因子的要求进行试验研究。结果表明：蛹虫草菌丝生长的适宜碳源是葡萄糖，适宜氮源是蛋白胨，适宜C：N为40：1．适宜无机盐是KH2PO4，最适宜温度是20℃，最适宜含水量是70％，最适宜pH值是6．5。     

Expression of vascular endothelial growth factor in rat uterus during peri-implantation

The first distinct mark of rodent implantation is the increased vascular permeability and significant angiogenesis at the sites of blastocyst implantation, but its mechanism is not clearly defined. Vascular endothelial growth factor (VEGF) is the key mediator for angiogenesis during embryogenesis and adult span and also serves as a vascular permeability factor. The aim of this study is to explore VEGF regulation mechanism and the possible role that VEGF plays in implantation by studying the VEGF expression and angiogenesis in the rat uterus during estrous cycle, ovarioectomized and peri-implantation stages usingin situ message RNA hybridization and confocal laser scanning techniques. The results indicated that VEGF was regulated by ovarian steroid hormones. VEGF expression before implantation was localized at luminal epithelium, shifted to stroma as implantation initiated and extensively located at the decidualizing stroma region after implantation. Bandeiraea simplicifolia-1 (BS-1) agglutinin and antibody against von Willebrand factor (vWF) were used to mark the endothelial cells and blood vessels. The results showed that the active angiogenesis occurred during the implantation process and this effect was probably mediated by VEGF. The results suggest that under the regulation of ovarian steroid hormones, VEGF plays an essential role in angiogenesis and increasing vascular permeability in endometrium, which are necessary for successful implantation.     

FGF-21对2型糖尿病大鼠心肌氧化应激影响的研究

目的探讨成纤维细胞生长因子-21（FGF-21）对2型糖尿病大鼠心肌氧化应激的影响。方法用高热量脂肪饮食联合小剂量链脲佐菌素建立2型糖尿病大鼠模型,随机分成2型糖尿病组与FGF-21治疗组,并与正常对照组比较。测定三组问大鼠血浆及心肌组织葡萄糖糖、胰岛素、FGF-21、脂质产物及氧化应激指标的变化。结果2型糖尿病组血糖、胰岛素、游离脂肪酸（FFAs）、甘油三酯、FGF-21及氧化应激指标平均水平较其他两组显著增高（P〈0．05）。FGF-21治疗组血糖、胰岛素、FFAs、甘油三酯及氧化应激指标平均水平较2型糖尿病组显著降低（P〈0．05）,与对照组无差异。结论FGF-21可以改善2型糖尿病大鼠心肌心肌氧化应激状态,并参与调控2型糖尿病代谢紊乱。     

柞蚕核型多角体病毒fgf基因的克隆和分析

为获得柞蚕NPV的全基因组序列,在柞蚕尸体中分离纯化柞蚕NPV,提取其基因组DNA,分别建立ApNPV DNA的HindⅢ和SalⅠ基因文库．对插入片段进行测序,经过同源性分析发现一个与人源fgf基因相似的基因,其读码框含有185aa．核苷酸和氨基酸同源性比较的结果表明：在genebank中没有同源性极高的序列,是个新基因;柞蚕NPV的fgf基因与云杉卷叶蛾NPV的同源性较高,分别为56．1％和45．9％,而与家蚕NPV及AcNPV的同源性相对较低,说明柞蚕NPV与家蚕NPV在进化上亲缘关系较远．该基因的克隆为进一步研究其在病毒感染过程中的作用打下基础．     

糖类对碱性成纤维细胞生长因子稳定性的影响

目的:研究糖类对bFGF(m)在溶液中的保护作用。方法:在相同浓度的bFGF(m)溶液中添加不同浓度的糖类,测定放置一定时间后的溶液中bFGF(m)可溶性浓度的差异和促细胞有丝分裂能力的变化。结果:在相同的条件下,添加蔗糖与海藻糖的bFGF(m)溶液随糖浓度增加,可溶性蛋白质量浓度和比活性增加。在蔗糖与海藻糖为0.8 mol时bFGF(m)可溶蛋白浓度和活性数值最高,所测得的蛋白质量浓度依次为(1.145±0.007 2)mg/mL、(1.125±0.005 6)mg/mL;所测活性数值依次为ED50=0.48 ng/mL、ED50=0.58 ng/mL;而添加葡萄糖、乳糖和麦芽糖的bFGF溶液,实验组中蛋白质量浓度和活性相对于对照组均未见有显著性提高。结论:海藻糖和蔗糖能够减少bFGF(m)聚集和沉淀程度,增加bFGF(m)在溶液中的溶解度,提高单位体积中的bFGF(m)活性单体量。