大肠癌组织bFGF,PKB,Cyclin A的表达及其与临床的相关性

目的:研究大肠癌组织中碱性成纤维细胞生长因子(bFGF),蛋白激酶B(PKB)及细胞周期蛋白A(Cvclin A)mRNA的表达水平及与临床病理的关系.方法:采用TRIzol法分别提取60例大肠癌组织,癌旁组织和10例正常组织的RNA,应用RT-PCR方法检测bFGF,PKB,Cyclin A mRNA的表达水平,PCR产物经凝胶成像及分析系统扫描后,以β-actin为参照标化bFGF,PKB,Cvclin A的含量.通过统计学分析其与临床病理关系.结果:bFGF,PKB,Cyclin A在大肠癌组织中的表达与癌旁组织相比有显著差异(bFGF:60%vs 10%;PKB:55% vs 35%;CyclinA:70% vs 5%;all P＜0.05),其表达水平与大肠癌Dukes分期有关(bFGF:χ2=4.434,P＜0.05;PKB:χ2=13.549,P＜0.01;CyclinA:χ2=21.210,P＜0.01).PKB在低分化肿瘤中的表达阳性率高于中高分化肿瘤(14/14 vs 29/46,P＜0.05).Cyclin A的表达在高、中分化肿瘤中明显高于低分化肿瘤(37/46 vs 3/14,P＜0.05).结论:bFGF,PKB,Cyclin A的mRNA水平与大肠癌的发生、发展相关.     

补体C7在结直肠癌组织中的表达及其对结肠癌细胞株增殖的影响

目的检测补体C7在结直肠癌组织中的蛋白表达,探讨补体C7对人结肠癌细胞株HCT-116、LoVo增殖的影响及其可能的机制。方法利用免疫组织化学方法分别检测50例结直肠癌患者癌组织及癌旁组织中补体C7蛋白分布与表达水平;将过表达C7质粒和C7-siRNA分别转染人结肠癌细胞株HCT-116和LoVo细胞,通过CCK-8检测C7对这两种细胞增殖的影响;通过Western blotting检测HCT-116在PI3K抑制剂LY294002处理后C7的变化。结果结直肠癌组织中补体C7蛋白表达水平明显高于癌旁组织。与空载质粒组和NC-siRNA组相比,C7过表达组细胞增殖能力明显增强,C7-siRNA组细胞增殖能力明显减弱; LY294002处理结肠癌细胞后C7蛋白表达量明显降低。结论补体C7在结直肠癌组织中明显高表达,抑制结肠癌LoVo细胞中C7基因可降低癌细胞增殖,其机制与PI3K/AKT信号通路的调控有关。     

LY294002对人肺腺癌A549细胞p-Akt、VEGF表达的影响

目的 探讨PI3K抑制剂LY294002对肺腺癌A549细胞p-Akt及血管内皮生长因子(VEGF)表达的影响.方法 不同浓度LY294002(5μmol/L、10 μmol/L、20μmol/L、40 μnol/L)处理肺腺癌A549细胞24h后,分别用免疫细胞化学法和Western印迹法检测p-Akt、VEGF蛋白表达.结果 P13K抑制剂LY294002可抑制肺腺癌A549细胞株细胞中p-Akt蛋白的表达,而且其表达呈浓度依赖型降低(r=-0.913,P＜0.05).LY294002也可抑制该细胞株中VEGF蛋白的表达,其表达也呈浓度依赖型降低(r=-0.969,P＜0.01).结论 LY294002可抑制p-Akt活性及下调VEGF蛋白的表达,该作用可能是PI3K抑制剂LY294002发挥抗癌作用及抗血管生成的机制之一.     

MiR-451作用于MIF对大肠癌细胞株LoVo的影响

目的:研究MicroRNA-451(MiR-451)作用于巨噬细胞迁移抑制因子(macrophage migration inhibitory factor,MIF)对人大肠癌细胞株LoVo的影响.方法:构建包含有MiR-451过表达的慢病毒(hsa-mir-451a),感染LoVo细胞株,采用实时荧光定量PCR及Western blot技术检测MIF mRNA及蛋白在人大肠癌细胞LoVo及人大肠上皮细胞FHC中的表达变化情况,MTT法检测各组细胞的增殖能力.结果:与未感染组及对照组相比,感染组中MIF在mRNA及蛋白水平的表达均显著降低,感染组细胞的增殖活性明显受到抑制.结论:MIF可能是miR-451的直接作用靶点,高表达miR-451可以有效抑制LoVo中MIF的表达,进而抑制了LoVo的增殖.     

LY-294002对人宫颈癌细胞株Bcl-2表达的影响

目的探讨不同浓度磷脂酰肌醇3激酶(PI3K)抑制剂(LY-294002)对人宫颈癌细胞株Bcl-2表达的影响。方法培养人宫颈癌Hela细胞,分为对照组和LY294002干预组,干预组再细分为4个亚组,分别以终浓度为5、10、20、40μmol/L的PI3K抑制剂LY294002处理人宫颈癌Hela细胞,作用48 h。Western印迹方法和RT-PCR方法检测各组人宫颈癌细胞株Bcl-2表达变化。结果 Western印迹方法检测发现,对照组宫颈癌细胞株有大量的Bcl-2阳性表达,与对照组相比在以终浓度为5μmol/L的LY294002处理48 h后,宫颈癌细胞株的Bcl-2阳性表达迅速降低(P<0.05),随着LY294002终浓度的升高,宫颈癌细胞株的Bcl-2阳性表达也随之降低。RT-PCR方法检测Bcl-2 mRNA时发现,Bcl-2 mRNA的表达也随着LY294002的终浓度的升高而降低。结论 LY-294002能够降低人宫颈癌细胞Bcl-2的表达。     

PI3K/Akt抑制剂对肺腺癌A549细胞增殖活性的影响

目的 研究磷脂酰肌醇-激酶特异性抑制剂LY294002对人肺腺癌A549细胞株体内外生长的影响,探讨其可能的作用机制.方法 采用MTT方法检测A549细胞增殖活力的改变,用流式细胞仪进行细胞周期分析;建立裸鼠肺癌移植瘤模型,观察LY294002对荷瘤小鼠肿瘤生长情况的影响及肿瘤抑制率.Western印迹检测小鼠肿瘤组织内p-Akt蛋白及Bcl-2蛋白的表达.结果 ①LY294002对A549细胞增殖有抑制作用,各浓度组与对照组相比差异均有统计学意义(均P＜0.01),并随着剂量的增加及时间的延长,抑制率增加.②经LY294002治疗后,对照组的瘤重为(773.33±32.04)mg,LY294002组的瘤重为(461.67±36.56)mg,抑瘤率达40.3%.③LY294002组的p-Akt蛋白及Bcl-2蛋白表达水平较对照组明显降低.结论 LY294002能抑制肺癌A549细胞的恶性增殖,呈明显的时间及剂量依赖性,并降低p-Akt蛋白及Bcl-2蛋白表达水平,诱导肺癌细胞凋亡可能是LY294002发挥抗癌作用的重要机制.     

丹皮酚通过下调COX-2表达及PGE_2合成抑制大肠癌细胞增殖及诱导细胞凋亡

目的观察丹皮酚对大肠癌细胞增殖活性和细胞凋亡的影响,并探讨丹皮酚对大肠癌的抑制作用是否与其下调COX-2表达及PGE2合成有关。方法以大肠癌LoVo细胞株为研究对象,CCK-8比色法检测不同浓度丹皮酚或PGE2或塞来昔布处理后细胞活力及丹皮酚对PGE2刺激细胞增殖的影响;流式细胞仪检测细胞凋亡情况;Western blotting方法检测COX-2和凋亡相关蛋白Bax、Bcl-2、Caspase-3、Caspase-9的表达情况;ELISA法检测大肠癌细胞上清液中PGE2含量。结果丹皮酚处理后LoVo细胞活力显著降低,呈明显的时间和浓度依赖性;流式细胞仪检测结果显示丹皮酚可诱导LoVo细胞凋亡;丹皮酚可降低LoVo细胞COX-2表达及上清液中PGE2含量;经PGE2处理后LoVo细胞活力明显增强,丹皮酚可抑制PGE2诱导的LoVo细胞活力增加;经塞来昔布处理后,LoVo细胞活力显著降低,且细胞凋亡率增加;经丹皮酚处理后,LoVo细胞Bax蛋白表达上调,Bcl-2蛋白表达下调,Pro-Caspase-3、Pro-Caspase-9表达减弱,而Cleaved-Caspase-3、Cleaved-Caspase-9表达增强,呈浓度依赖性。结论丹皮酚可抑制LoVo细胞增殖并诱导细胞凋亡,可能的机制是通过下调COX-2表达及PGE2合成进而激活线粒体凋亡途径。     

Apelin抑制人成骨细胞的凋亡

目的 研究apelin对人成骨细胞凋亡的作用.方法 用茜素红染色观察原代培养的人成骨细胞矿化结节的形成.碱性磷酸酶(ALP)用ELISA检测;骨钙素(OC)用放射免疫测定法测定;Ⅰ型胶原用ELISA法检测.用ELISA法检测细胞凋亡.Bcl-2,Bax,caspase-3,cytochmme C,P-Akt,Akt用Western blot检测.PI3-K p85α用免疫沉淀法检测.用小分子RNA干扰技术(siRNAs)抑制APJ表达,联合PI3-K信号转导阻断剂LY294002及Akt信号转导阻断剂HIMO干预,以观察Apelin对成骨细胞凋亡的作用及信号转导.结果Apelin能抑制无血清诱导的人成骨细胞凋亡,siRNAs转染阻断APJ表达可消除此效应.Apelin干预可诱导入成骨细胞Bcl-2蛋白质表达,抑制Bax蛋白质表达和Cytochrome C的分泌及caspase-3的裂解活化.Apelin可诱导人成骨细胞PI3-K及Akt磷酸化;siRNAs转染沉默APJ可消除此效应;PI3-K信号转导阻断剂LY294002或Akt阻断剂HIMO能消除Apelin对人成骨细胞Akt的活化作用;LY294002或HIMO也能阻断Apelin对人成骨细胞抑制凋亡作用.结论 Apelin可抑制人成骨细胞的凋亡;Apelin通过APJ/PI3-K/Akt信号通路介导参与对成骨细胞凋亡的调节.     

PUMA在大蒜素诱导肠癌细胞凋亡过程中的作用

目的探讨凋亡相关基因PUMA在大蒜素诱导肠癌细胞凋亡过程中的作用。方法蛋白印迹法检测在大肠癌LoVo细胞株中大蒜素对PUMA蛋白表达的诱导作用,Hoechst33258染色检测细胞凋亡,MTT法测定细胞增殖。结果大蒜素可以诱导大肠癌LoVo细胞的凋亡,抑制其增殖,并且诱导PUMA的表达,在PUMA的表达受抑制后,大蒜素诱导大肠癌细胞凋亡的作用明显减弱。结论 PUMA在大蒜素诱导LoVo细胞凋亡的过程中具有一定作用,诱导PUMA表达可能是大蒜素抗癌作用的机制之一。     

LY294002对人胃腺癌细胞株BGC-823糖酵解水平的影响及其机制探讨

背景:有氧糖酵解是肿瘤细胞的特征性表型之一,靶向糖酵解有望成为一种有效的肿瘤治疗策略。M2型丙酮酸激酶(PKM2)在肿瘤组织中呈高表达,参与肿瘤细胞的有氧糖酵解。缺氧诱导因子-1α(HIF-1α)是调控肿瘤细胞糖酵解相关基因表达的重要转录因子,而PI3K信号在肿瘤细胞中可上调HIF-1α表达。目的:探讨PI3K特异性抑制剂LY294002对人胃腺癌细胞增殖和糖酵解水平的影响及其可能机制。方法:以不同浓度LY294002(10～100μmol/L)在不同条件下(常氧或缺氧)处理人胃腺癌细胞株BGC-823,CCK-8实验和流式细胞分析检测细胞增殖和细胞凋亡,蛋白质印迹法检测p-Akt、p-mTOR、HIF-1α、PKM2表达,比色法检测反映糖酵解水平的胞内乳酸脱氢酶活性和胞外乳酸浓度。结果:LY294002可呈浓度和时间依赖性地抑制BGC-823细胞增殖,在较高浓度时可诱导细胞早期凋亡。LY294002可呈浓度依赖性地抑制p-Akt、p-mTOR、HIF-1α表达,在较高浓度时可抑制PKM2表达,同时降低糖酵解水平。缺氧诱导可消除LY294002对HIF-1α、PKM2表达和糖酵解水平的抑制作用。结论:LY294002可通过阻断PI3K/Akt/mTOR信号通路抑制人胃腺癌细胞增殖及其糖酵解水平,而HIF-1α介导了糖酵解的抑制过程。     

Normal growth and techniques of growth assessment

The shape of the human growth curve is described and illustrated. Growth studies may be longitudinal, cross-sectional, mixed longitudinal or linked-longitudinal; each has advantages and disadvantages, and each requires appropriate statistical methods for handling the data. Standards for height and height velocity for use in a clinical setting wherein follow-up over several years is presumed are described and illustrated. Such standards have to take into account tempo of growth at ages over nine years. Cross-sectionally derived standards do not do this and are not suitable for clinical use. The techniques of measurement of height, sitting height and skinfolds are described and illustrated. Growth and development during puberty is described; there are changes in body composition as well as in body size and shape. Standards for pubertal stages of breasts, pubic hair and genitalia are given and emphasis is laid on the great variation in both the timing and the duration of these pubertal changes. Measurement of developmental age is discussed. The Greulich-Pyle and Tanner-Whitehouse methods for skeletal age are described. These methods can be used for predicting adult height which is useful both in diagnosis and in following the effects of treatment. In diagnosis the predicted adult height is compared to the range of expected heights in the children of the particular pair of parents concerned (the so-called 'target' range of heights) to see if smallness is simply due to delay. Change in Tanner-Whitehouse predicted height occurs on successful treatment of, for example, growth hormone deficient short stature, and is thus a guide to the success of treatment. Standards are also given for height of children from age two to nine inclusive, with allowance for height of their parents.     

肝硬化生长激素-胰岛素样生长因子轴的基础与临床研究

生长激素(GH)是由脑腺垂体前叶嗜酸性粒细胞分泌的一种含191个氨基酸的蛋白质多肽。肝脏是生长激素最主要的靶器官。生长激素可直接与靶组织特异性受体结合发挥其生物学作用；另一方面，生长激素又可通过生长激素-肝-胰岛素样生长因子轴(GH—IGF轴)，刺激肝、肾及其他部位产生胰岛素样生长因子(insulin—like growth factors，IGFs)而间接发挥其生物学效应。     

Dose dependence of growth response to human growth hormone in growth hormone deficiency.

A trial of the relative effect on growth of 20 IU/week and 10 IU/week of human growth hormone has been made in 38 patients with "isolated" growth hormone deficiency over 1 year of treatment, 18 patients over 2 years and 10 over 3 years, and in 17 patients with surgically treated craniopharyngiomata over 1 year. The velocity of height growth in the first year of treatment, compared with a full year of pre-treatment control, was 1.3 times as great in both groups of patients on the larger dose as it was in those on the smaller one. Second-degree equations fitted to the treatment catch-up curve gave estimates of 1.7 cm more height gained on the larger dose by the end of the first year, 2.7 cm by the end of the second, and 3.4 cm by the end of the third. Adjusting treatment increment by covariance for bone age at the beginning of treatment, pre-treatment velocity, and body surface area did not alter these mean differences. Bone age velocity during treatment was the same in both treatment groups (mean 1.09 "years"/year in the first year); thus we anticipate a gain in final adult height of the order of 10 cm from employing the larger dose. The decrease in skin folds occurring on treatment, however, was no different with the larger than with the smaller dose. This reinforces previous observations that the short-term metabolic and longer-term auxologic effects of hGH are not necessarily related.     

Pubertal growth and growth hormone secretion

A dramatic increase in linear growth velocity, often referred to as the pubertal growth spurt, is a central feature of pubertal development. Despite the existence of numerous investigative attempts, a precise understanding of the hormonal events subserving this process has proved elusive. Nevertheless, evidence has gradually accumulated that indicates that sex steroid-induced modulation of growth hormone secretion is a central and critical feature of the pubertal growth spurt. As a result, disorders of either growth hormone or sex steroid hormone production may result in clinical growth disorders during puberty.     

Growth cartilage expression of growth hormone/insulin-like growth factor I axis in spontaneous and growth hormone induced catch-up growth

IntroductionCatch-up growth following the cessation of a growth inhibiting cause occurs in humans and animals. Although its underlying regulatory mechanisms are not well understood, current hypothesis confer an increasing importance to local factors intrinsic to the long bones' growth plate (GP).AimThe present study was designed to analyze the growth-hormone (GH)-insulin-like growth factor I (IGF-I) axis in the epiphyseal cartilage of young rats exhibiting catch-up growth as well as to evaluate the effect of GH treatment on this process.Material and methodsFemale Sprague–Dawley rats were randomly grouped: controls (group C), 50% diet restriction for 3 days + refeeding (group CR); 50% diet restriction for 3 days + refeeding &; GH treatment (group CRGH). Analysis of GH receptor (GHR), IGF-I, IGF-I receptor (IGF-IR) and IGF binding protein 5 (IGFBP5) expressions by real-time PCR was performed in tibial growth plates extracted at the time of catch-up growth, identified by osseous front advance greater than that of C animals.ResultsIn the absence of GH treatment, catch-up growth was associated with increased IGF-I and IGFBP5 mRNA levels, without changes in GHR or IGF-IR. GH treatment maintained the overexpression of IGF-I mRNA and induced an important increase in IGF-IR expression.ConclusionsCatch-up growth that happens after diet restriction might be related with a dual stimulating local effect of IGF-I in growth plate resulting from overexpression and increased bioavailability of IGF-I. GH treatment further enhanced expression of IGF-IR which likely resulted in a potentiation of local IGF-I actions. These findings point out to an important role of growth cartilage GH/IGF-I axis regulation in a rat model of catch-up growth.     

Normal growth hormone secretion in growth hormone insufficient children retested after completion of linear growth

OBJECTIVE The recognition of the syndrome of adult GH deficiency suggests that young GH deficient adults, deprived of GH replacement at completion of linear growth, may suffer effects of GH deficiency. We assessed GH reserve in young adults previously diagnosed as having idiopathic GH insufficiency, who were treated with hGH replacement (14 IU/m²/week) in childhood. DESIGN Eight patients (7 males, 1 female) diagnosed as having GH insufficiency by insulin tolerance test (ITT) in childhood (ages 8.5–15.6 years) were retested by ITT at completion of linear growth (ages 15.1–19.6 years), 3 months after discontinuation of hGH therapy. MEASUREMENTS GH reserve was measured during ITT at diagnosis and at retesting. Height velocity (HV) and HV SDS were calculated before and during GH therapy. RESULTS At diagnosis, the mean peak GH response to ITT was 10.5 ± 2.0 mU/l (range 7.7–13.6). At retesting, mean GH was 52.4 ± 33.2 mU/l (range 10.4–100), 7/8 subjects having peak GH levels greater than 15 mU/l. During hGH therapy mean HV increased from 4.0 ± 1.5 cm/year at diagnosis to 7.3 ± 1.9 cm/year during the 1st year (P = 0.004) and 6.9 ± 2.3 cm/year during the 2nd year (P = 0.02). Mean HVSDS increased from ?1.6 ± 2.1 at diagnosis to 3.1 ± 2.9 during the 1st year (P = 0.004) and 2.2 ± 4.2 during the 2nd year (P = 0.05, NS) of treatment. CONCLUSIONS Seven out of 8 children diagnosed as having idiopathic GH insufficiency had normal GH secretion at completion of linear growth. Children with GH insufficiency cannot be assumed to become GH deficient adults and should not continue on GH therapy into adult life without reinvestigation. All who were GH insufficient children should be retested at completion of linear growth to identify those who are truly GH insufficient adults and may benefit from replacement therapy.