大鼠妊娠期胰岛适应性增殖的分子机制研究

利用基因芯片技术探讨大鼠妊娠期胰岛β细胞适应性增殖的分子机制.与正常未孕对照组相比,Nupr1、Art3、Btg2、β-catenin及c-Mye等基因在孕期表达明显增强,提示它们可能参与调节孕期胰岛的适应性增殖.     

丝胶对2型糖尿病大鼠胰岛细胞凋亡的保护作用

目的观察丝胶对2型糖尿病大鼠胰岛细胞凋亡的保护作用。方法雄性SD大鼠36随机分为3组:正常对照组、糖尿病模型组和丝胶治疗组。链脲佐菌素(STZ,25 mg/kg,连续3 d)腹腔注射建立2型糖尿病大鼠模型;待模型成功建立后,丝胶治疗组大鼠给予丝胶灌胃(2.4 g.kg-1.d-1)35 d。免疫组织化学染色观察胰岛β细胞Bcl-2和Bax蛋白的表达。结果与正常对照组大鼠相比,模型组大鼠胰岛β细胞Bax蛋白的表达明显升高(P<0.01),Bcl-2蛋白的表达明显降低(P<0.01);与模型大鼠相比,丝胶治疗组大鼠胰岛β细胞Bax蛋白的表达明显降低(P<0.01),Bcl-2蛋白的表达明显升高(P<0.01)。结论丝胶可通过上调胰岛β细胞Bcl-2蛋白的表达、下调胰岛β细胞Bax蛋白的表达,抑制2型糖尿病大鼠胰岛细胞凋亡,对糖尿病时胰岛细胞损伤具有一定的保护作用。     

熊脱氧胆酸减轻内质网应激介导的胰岛β细胞凋亡的机制

目的 探讨熊脱氧胆酸( Ursodeoxycholic acid,UDCA)通过减轻内质网应激保护链脲佐菌素诱导的糖尿病大鼠胰岛β细胞凋亡的作用.方法 链脲佐菌素(50 mg/kg)一次性腹腔注射建立糖尿病大鼠模型(n=40),并将14只造模成功大鼠按随机数字表法随机分为糖尿病组7只,UDCA组7只,另取正常对照组10只.自造模成功第10天开始以UDCA(40 mg·kg-1·d-1)给大鼠灌胃30 d.饲养期间观察大鼠血搪.处死后留取大鼠血清和组织标本,测定空腹胰岛素,TUNEL检测胰岛β细胞凋亡的形态学改变.胰腺组织总RNA采用定制基因芯片对89条凋亡相关基因的表达进行检测,以RT-PCR和Western印迹法验证相关基因的表达.结果 糖尿病大鼠血糖在给予UDCA治疗后逐渐下降,但仍然未降到正常水平.糖尿病大鼠空腹胰岛素降低,给予UDCA治疗后空腹胰岛素水平有所增加.TUNEL显示糖尿病组大鼠予UDCA治疗后减少了由链脲佐菌素引起的胰岛β细胞凋亡(P＜0.01).在89条基因中,糖尿病组上调基因42条,下调基因46条.UDCA可以逆转部分基因的影响.与对照组相比,糖尿病组大鼠Bax、Caspase-3、Bip、CHOPmRNA和CHOP蛋白显著上调(P＜0.05),而抗凋亡蛋白Bcl-2 mRNA显著下调(P＜0.05),给予UDCA治疗后这些参数均有明显改善(P＜0.05).结论 熊脱氧胆酸可能通过减轻内质网应激达到保护链脲佐菌素诱导的糖尿病大鼠胰岛β细胞凋亡的作用.     

丝胶对糖尿病大鼠胰岛细胞神经肽Y表达的影响

目的观察丝胶对2型糖尿病大鼠胰岛细胞神经肽Y(NPY)表达的影响。方法 36只雄性SD大鼠随机分为正常对照组、糖尿病模型组和丝胶治疗组,每组12只。链脲佐菌素腹腔注射建立2型糖尿病大鼠模型并给予丝胶(2.4 g/kg)灌胃。SP免疫组织化学染色法观察大鼠胰岛细胞NPY的表达。结果 NPY蛋白阳性产物位于胰岛α细胞细胞质,呈棕黄色颗粒状。糖尿病模型组大鼠胰岛α细胞NPY蛋白的表达明显高于正常对照组大鼠(P<0.01);丝胶治疗组大鼠胰岛α细胞NPY蛋白的表达明显低于糖尿病模型组大鼠(P<0.01)。结论丝胶可通过降低神经肽Y的表达保护糖尿病时胰岛细胞损伤。     

细胞内基质金属蛋白酶2基因过表达对INS-1细胞功能的影响

目的探讨细胞内基质金属蛋白酶2(MMP-2)基因过表达对大鼠胰岛β细胞株INS-1细胞功能的影响。方法采用基因重组技术将大鼠MMP-2cDNA插入真核表达载体pcDNA 3.1(+)构建大鼠MMP-2真核表达质粒,培养INS-1细胞。随机分为正常对照组,空质粒转染组及MMP-2质粒转染组。脂质体Lipofectmine 2000转染INS-1细胞,观察INS-1细胞内MMP-2基因和蛋白表达量变化,MMP-2酶活性变化,以及INS-1细胞凋亡情况与胰岛素分泌功能的变化。结果与正常对照组、空质粒转染组比较,MMP-2质粒转染组MMP-2mRNA表达增加(P均0.05),蛋白表达水平和酶活性上调(P均0.05);MMP-2质粒转染组细胞凋亡率(56.07±3.68)%高于正常对照组(33.70±6.53)%及空质粒转染组(38.02±5.60)%(P0.05),而IRI(1.30±0.27)低于正常对照组(3.37±0.76)与空质粒转染组(2.90±0.84)(P0.05)。结论细胞内MMP-2基因的过表达可引起胰岛β细胞凋亡增加,胰岛素分泌功能下降。     

大鼠胰岛B细胞长寿基因SIRT1与转录调节因子FOXO1表达及其相关性研究

目的探讨大鼠胰岛B细胞在衰老进程中长寿基因SIRT1与转录调节因子FOXO1表达的相关性。方法2005年10月至2006年10月,在汕头大学医学院第二附属医院将11只18月龄健康雄性SD大鼠随机分为热量限制(CR)组6只和正常喂养对照组5只,饲养6个月后取胰尾组织,应用免疫组化染色分别检测胰岛中SIRT1、FOXO1和胰岛素表达及分布,衰老相关β-半乳糖苷酶(β-Gal)染色以反映胰岛B细胞衰老情况。结果SIRT1与FOXO1均可表达于胰岛细胞胞浆、胞核中;β-Gal和胰岛素表达于细胞浆中;与对照组大鼠相比,CR组大鼠胰岛SIRT1表达量较多(P<0.05),衰老相关β-Gal染色强度较弱(P<0.01),胰岛素表达量较低(P<0.05),但FOXO1表达量差异无显著性意义(P>0.05),伴随着SIRT1表达增加,FOXO1胞核阳性率明显降低(P<0.01)。结论热量限制诱导了大鼠胰岛B细胞SIRT1蛋白高表达;SIRT1可能通过抑制FOXO1活性而调控胰岛B细胞的衰老,有利于2型糖尿病的防治。     

GLP-1对大鼠胰岛细胞增殖及凋亡的影响

目的 观察胰升糖素样肽1（GLP-1）对大鼠胰岛细胞增殖及凋亡的影响，并探讨其作用机制。方法 （1）GLP-1与大鼠胰岛细胞瘤细胞株RINm5f共育，观察其增殖情况；（2）检测GLP-1对IL-1D诱导的RINm5f细胞凋亡的保护作用；（3）GLP-1与原代培养大鼠胰岛细胞共育1、3、5天后，检测BAX及Bcl-2基因的表达。结果（1）随着GLP-1浓度增高及刺激时间延长，RINm5f细胞A值呈剂量及时间依赖性增加；（2）GLP-1组与对照组相比RINm5f细胞凋亡率显著下降（P〈0．05）；（3）与GLP-1共育后，大鼠胰岛BAX基因的表达量无明显变化，对照组BAX基因的表达逐渐增加（P〈0．05）；而Bcl-2基因的表达量随着与GLP-1共育时间的延长呈时间依赖性增加。结论GLP-1对大鼠胰岛细胞增殖有明显促进作用；同时对胰岛细胞的凋亡有保护作用。     

辣椒素对类固醇糖尿病大鼠的影响及其作用机制

目的研究辣椒素对类固醇糖尿病(SDM)大鼠的影响及相关机制。方法 SD大鼠随机分为正常对照组(NC组),SDM组和辣椒素组;检测各组大鼠血糖,胰岛素抵抗,血清中脂联素和降钙素基因相关肽(CGRP)的改变,胰岛细胞凋亡率和胰腺组织细胞凋亡蛋白酶-3(Caspase-3)mRNA变化。结果小剂量腹腔注射辣椒素明显降低SDM大鼠血糖,抑制胰岛素抵抗;增加血清脂联素和CGRP的表达;下调SDM大鼠胰岛细胞凋亡率和胰腺组织Caspase-3 mRNA的表达。结论辣椒素能改善SDM大鼠,与降低SDM大鼠血糖、抑制胰岛素抵抗、增加血清脂联素和CGRP的表达和抑制胰岛细胞凋亡有关。     

Bcl-X基因转录后剪切方式改变在大鼠胰岛细胞凋亡中的作用

目的　探明 Bcl- x基因在大鼠胰岛细胞凋亡时剪切方式的变化及其该基因在胰岛细胞凋亡中的作用。方法　应用 TUNEL法检测长期高糖培养时大鼠胰岛细胞凋亡 ,应用半定量多重 RT- PCR(SQ- RT- PCR)检测了胰岛细胞凋亡时 Bcl- x基因 m RNA的表达变化情况 ;同时观察小剂量 STZ所致的糖尿病大鼠胰岛 Bcl- x基因 m RNA的表达变化。结果　 2 5 .5 m M葡萄糖浓度体外培养 14天较 5 .5 m M和 11.1m M葡萄糖浓度培养 ,胰岛细胞凋亡百分率明显增加 ,分别为 31.5± 4 .5 % ,15 .4± 3.0 % ,17.4± 4 .8% ;P <0 .0 1。细胞凋亡百分率变化的同时伴有 Bcl- x基因转录后剪切方式的改变 ,表现为 Bcl- x S m RNA表达明显增加 ;低剂量 STZ所致的糖尿病大鼠胰岛 Bcl- x S m RNA表达明显增加 ,Bcl-x L与 Bcl- x S比率明显减少。结论　长期高糖可以诱导大鼠胰岛细胞凋亡 ,Bcl- x基因剪切方式的改变所致的 Bcl- x L/Bcl- x S比率的变化在高糖和 STZ所致的胰岛细胞凋亡中起重要作用     

糖耐量受损大鼠胰岛α细胞胰高血糖素及神经肽Y的表达

目的测定并比较胰岛素抵抗(IR)、糖耐量受损(IGT)及2型糖尿病(DM)大鼠模型胰岛α细胞中胰高血糖素(GLC)、神经肽Y(NPY)、β细胞中胰岛素(INS)蛋白表达变化及胰岛中NPY mRNA表达,确定IR、IGT、2型DM大鼠模型胰岛α细胞是否存在IR.方法分别用高脂饮食/缓冲液,高脂饮食/小剂量链脲佐菌素制备IR、IGT与2型DM大鼠模型;采用荧光单标记及双标记法分别对各组大鼠胰岛α细胞中NPY、GLC及β细胞中INS进行蛋白的定位、定量分析.采用原位杂交定位、定量分析胰岛中NPY mRNA.结果实验性IR、IGT及2型DM大鼠模型的生化代谢指标及发病过程与临床相关疾病的表现一致;各组大鼠胰岛α细胞GLC及NPY平均光密度(MOD)值与正常对照组(687±127,275±14)相比,分别为IR组1755±226,646±122;IGT组1720±108,599±63及2型DM组1986±88,585±67,均显著增高(均P＜0.01).与正常对照组(1425±220)比较,上述各组胰岛β细胞INS蛋白表达亦显著增高,3组分别为2073±340(P＜0.01);1976±200(P＜0.01)和1709±184(P＜0.05),表达NPY及GLC的α细胞分布方式由正常的胰岛外周向全胰岛分布改变.正常对照组未发现NPY mRNA表达,但在其余各组是全胰岛表达.结论在IR、IGT及2型DM状态下,升高的GLC及NPY表达未被高胰岛素所抑制.α细胞可能存在内源性的IR.     

Stimulation of anterior pituitary galanin and prolactin gene expression in suckling rats

Recent evidence suggests that galanin, may regulate prolactin (PRL) secretion during lactation. In this article, we describe the regulation of anterior pituitary galanin and PRL gene expression during pregnancy and after parturition in the rat. Expression of galanin and PRL in the anterior pituitary were significantly higher at d 20 of pregnancy compared to diestrus. One day after parturition, galanin mRNA levels increased a further 4.5-fold. This post partum increase in gene expression was not observed for PRL. The increase in galanin gene expression was maintained above the diestrous level for at least 10 d after parturition. PRL mRNA expression, on the other hand, was largely unchanged after parturition. Although the increase in galanin gene expression 1 d after parturition was independent of suckling, subsequently, galanin, gene expression was significantly higher in nursing mothers. Anterior pituitary galanin gene expression was 12-fold higher in nursing mothers compared with those that were not, 3 d after parturition. Similarly, PRL gene expression was significantly lower in mothers who were not suckling their pups 3 d after parturition. Initiation of suckling alone was insufficient to stimulate galanin and PRL expression. Despite suckling for 2 d, removal of the suckling stimulus subsequently resulted in a rapid decrease in galanin gene expression. Hence, the stimulatory effect of suckling on galanin expression requires a sustained suckling stimulus. In conclusion, the data support the hypothesis that anterior pituitary galanin plays an important role during lactation, likely acting to amplify lactotroph stimulation through paracrine and autocrine mechanisms.     

Resistin is expressed in the human placenta

The mechanism for decreased insulin sensitivity in pregnant women is not fully clarified. Resistin, a novel peptide hormone, is specifically expressed in the adipose tissue and decreases insulin sensitivity in rodents. In the present study, we demonstrate resistin gene expression in the human placental tissue, mainly in trophoblastic cells. The resistin gene expression in term placental tissue was more prominent than was seen in the first trimester chorionic tissue. In contrast resistin gene expression in adipose tissue was rather weak and remained unchanged by pregnancy. Thus, resistin is a newly isolated placental hormone in humans which may modulate insulin sensitivity during pregnancy.     

Enhanced expression of uterine stathmin during the process of implantation and decidualization in rats

We used the library subtraction technique to identify genes specifically expressed in the rat uterus during early pregnancy. One such gene was that for stathmin, a factor that is associated with tubulin binding and the destabilization of microtubules. Stathmin was expressed at higher levels in implantation sites than in interimplantation sites on d 6 and 7 of pregnancy; the levels on d 6 and 7 were higher in implantation sites than in the entire uterus on d 3-5 of pregnancy or in nonpregnant uteri. Intense expression of stathmin mRNA was primarily limited to the subluminal stromal cells at the implantation site. Expression was also detected in the decidual zones and was accentuated during the period of decidualization (d 7-12). In the delayed implantation pregnant rat model, uterine stathmin expression was low, but increased after implantation induced by administration of 17beta-estradiol to the progesterone-primed animal. Further, decidualization in the pseudopregnant rat, induced by intrauterine infusion of oil, enhanced stathmin expression. Stathmin expression clearly increases in the uterus when stimulated by embryo implantation and decidualization and may play a role in the early stages of pregnancy.     

Expression of Na+/I- symporter and Pendred syndrome genes in trophoblast cells

Placental iodide transport is critical for the fetal thyroid function, but the molecular mechanisms of this transport are not understood. The expression of two recently identified iodide transporters, namely the sodium/iodide symporter (NIS) and pendrin, the product of the gene responsible for the Pendred syndrome (PDS), was studied using real-time kinetic quantitative PCR and immunohistochemistry 1) in placental tissues collected at different gestational ages and 2) in primary cultures of villous cytotrophoblast cells (VCT) that differentiate and fuse over 2-3 days in vitro to form villous syncytiotrophoblast (VSCT) cells. Both NIS and PDS genes are expressed in placenta, albeit at low levels compared with those in thyroid tissue. NIS gene expression in placental samples from first trimester and term pregnancies was similar. In contrast, the expression of PDS gene was higher in term than in first trimester pregnancy samples. In vitro, NIS gene was expressed at a high level in VCT obtained from first trimester pregnancy, and its expression decreased by 3- to 4-fold during the differentiation of VCT in VSCT. Expression of NIS was lower (up to 30-fold) in VCT obtained in placental samples from third trimester than from first trimester pregnancy. In contrast, the expression of PDS gene was low in VCT and increased by 5- to 10-fold during VSCT formation; this was observed in cells isolated from placental samples of both first trimester and term pregnancies. Immunohistochemical analysis showed that NIS protein was present on the entire membrane of VCT, whereas pendrin was mainly located at the brush border membrane of VSCT, facing the mother. In conclusion, 1) NIS and PDS genes are differently expressed in the placenta during gestation; and 2) whereas pendrin is expressed at the brush border membrane of syncytiotrophoblast cells, NIS protein is mainly located in the cytotrophoblast layer.     

Aldolase C/zebrin gene regulation by prolactin during pregnancy and lactation

Prolactin (PRL) is necessary for the genesis of mammary alveolar buds and for lactation. A cDNA library enriched for PRL-dependent genes was made by suppression subtractive hybridization. Aldolase C/zebrin (AldC/zebrin), a “brain-specific” aldolase, was found to be PRL-dependent in the mouse mammary glands. AldC/zebrin was preferentially expressed in the alveolar buds. Expression of the gene in the ovary was also evident. During pregnancy, mammary AldC/zebrin mRNA levels were elevated beginning at midpregnancy (d 10 of pregnancy) in accordance with the genesis of the lobuloalveolar system, and the expression level was gradually increased through the end of pregnancy. Lactating mammary gland contained a very high level of AldC/zebrin mRNA, and the gene expression decreased during involution. By contrast, levels of aldolase A and B mRNA expression in the mammary glands were less affected by pregnancy and lactation. The selective regulation of AldC/zebrin may contribute to a shift in nutrient metabolism during pregnancy and lactation to facilitate epithelial growth and biosynthesis of milk constituents.     

Hormonal regulation of mitochondrial Tim23 gene expression in the mouse mammary gland

Tim23, a mitochondrial inner membrane protein, is essential for cell viability. Mouse Tim23 cDNA consisted of 1142 nucleotides plus poly(A) at the 3' end. In situ hybridization showed that mammary epithelial cells expressed Tim23 mRNA during pregnancy. In order to examine the hormonal regulation of the Tim23 gene expression at lactogenesis, the quantity of Tim23 mRNA in the mammary gland was determined by the competitive RT-PCR. The level of Tim23 mRNA was low until mid-pregnancy, increased toward the end of pregnancy and was the highest on day 18 of pregnancy. On day 13 of pregnancy, Tim23 mRNA increased 2.7-fold between 8 and 16 h after ovariectomy but this increase was cancelled out by the simultaneous operation of adrenalectomy. In adreno-ovariectomized mice, the administration of cortisol increased Tim23 mRNA 2-fold but with progesterone, the stimulatory action of cortisol was no longer observed. The results indicated that the expression of the Tim23 gene became active in response to glucocorticoid.     

Analysis of lysophosphatidic acid (LPA) receptor and LPA-induced endometrial prostaglandin-endoperoxide synthase 2 expression in the porcine uterus

Lysophosphatidic acid (LPA), a simple phospholipid-derived mediator with diverse biological actions, acts through the specific G protein-coupled receptors endothelial differentiation gene (EDG) 2, EDG4, EDG7, and GPR23. Recent studies indicate a critical role for LPA receptor signaling in embryo implantation. To understand how LPA acts in the uterus during pregnancy in pigs, we evaluated: 1) spatial and temporal expression of LPA receptors in the uterine endometrium during the estrous cycle and pregnancy and in early-stage concepti, 2) LPA levels in uterine luminal fluids from d 12 of the estrous cycle and pregnancy, 3) effects of steroid hormones on EDG7 mRNA levels, and 4) effects of LPA on prostaglandin-endoperoxide synthase 2 (PTGS2) mRNA levels in the uterine endometrium using explant cultures. Of the four receptors, EDG7 was dominant, and its expression was regulated by pregnancy stage and status. EDG7 expression was highest on d 12 pregnancy, and localized to the luminal and glandular epithelium, and EDG7 mRNA levels were elevated by estrogen in the endometrium. EDG7 expression was also detected in concepti of d 12 and 15. LPA with various fatty acyl groups was present in the uterine lumen on d 12 of both the estrous cycle and pregnancy. LPA increased PTGS2 mRNA abundance in the uterine endometrium. These results indicate that LPA produced in the uterine endometrium may play a critical role in uterine endometrial function and conceptus development through EDG7-mediated PTGS2 expression during implantation and establishment of pregnancy in pigs.