组蛋白去乙酰化酶9对肾小球发育的调控

目的:研究组蛋白去乙酰化酶9(histone deacetylase,hdac9)缺失对肾小球发育的影响。方法:利用全胚胎原位杂交检测斑马鱼hdac9在受精后24h、36h、48h、60h、72h的表达分布;应用基因编辑技术CRISPR/Cas9构建hdac9基因敲除模型;透射电镜观察hdac9基因敲除对肾脏超微结构的影响;利用全胚胎原位杂交检测hdac9敲除后对肾脏祖细胞和足细胞标志物表达的影响;在hdac9基因敲除斑马鱼中,回输hdac9 mRNA以恢复hdac9缺失的表型。结果:全胚胎原位杂交检测发现hdac9在斑马鱼前肾发育阶段高峰度表达,即受精后24h开始表达,直至肾小球发育完成hdac9表达消失;透射电镜观察发现hdac9基因敲除导致肾小球发育障碍,肾脏祖细胞标志物(lhx1a,pax2a,mafba和wt1b)和足细胞标志物(podocin和nephrin)表达均显著抑制;在hdac9基因敲除斑马鱼中回输hdac9 mRNA可明显恢复hdac9基因敲除的表型。结论:hdac9在肾小球发育阶段表达,为肾小球发育所必需的,可能具有调控肾脏祖细胞增殖以及祖细胞向足细胞分化的功能。     

AML1-EVI1转基因斑马鱼模型的建立及对造血转录因子的调控

目的:通过建立表达急性髓系白血病(AML)1-同向性病毒整合位点(EVI)1融合基因斑马鱼模型,研究AML1-EVI1融合基因对造血转录因子的影响。方法:构建携带绿色荧光蛋白的AML1-EVI1质粒,显微注射到斑马鱼胚胎单细胞期,建立生殖系稳定转染的转基因模型,常规养殖到F2代。以F2代转基因斑马鱼作为实验对象,运用反转录(RT)-PCR验证AML1-EVI1融合基因的表达,外周血涂片、肾细胞涂片检测血细胞形态学改变,全胚胎原位杂交及荧光实时定量PCR(RFQ-PCR)检测融合基因对造血转录因子[GATA结合蛋白1(GATA1)、髓过氧化物酶(MPO)]的影响。结果:在性成熟146条嵌合表达的F0代斑马鱼中鉴定得到3条(2.1%)转基因阳性斑马鱼,其中雄鱼2条,雌鱼1条。外周血涂片及肾细胞涂片显示转基因斑马鱼出现造血细胞分化障碍,幼稚细胞增多,全胚胎原位杂交及RFQ-PCR均显示,与野生型相比,转基因型斑马鱼造血转录因子MPO基因表达量无论造血细胞发育早期还是晚期都明显下降,转录因子GATA1则在发育早期上调,晚期呈现明显下调。结论:AML1-EVI1融合基因通过调节转录因子抑制髓系分化,红系发育,促进原始细胞增殖。     

斑马鱼抗podocin抗体的制备、鉴定及在足细胞损伤模型中的应用

目的:利用合成的podocin多肽制备针对斑马鱼podocin的多克隆抗体,鉴定其特异性,并应用于斑马鱼足细胞损伤研究中。方法:(1)设计、合成斑马鱼podocin抗原多肽,将合成后的多肽与钥孔血蓝蛋白(KLH)偶联,免疫新西兰大白兔制备抗血清,protein A亲和纯化得到抗podocin多克隆抗体;(2)ELISA和免疫组化检测抗体效价及组织特异性;(3)构建足细胞特异性表达硝基还原酶(NTR)的转基因斑马鱼Tg(pod:Gal4;UAS:NTR-m Cherry),NTR/MTZ(甲硝唑)系统特异性地诱导足细胞损伤;用podocin反义寡核苷酸阻断正常斑马鱼胚胎podocin mRNA的翻译过程,利用抗podocin抗体对上述两种疾病模型进行免疫荧光染色以验证合成的podocin抗体能否应用于斑马鱼足细胞研究中。结果:(1)化学合成的多肽纯度为92.6%,达到免疫用抗原标准;(2)ELISA和免疫组化染色结果表明抗体效价分别达1∶5.12×105和1∶106;(3)免疫荧光染色表明无论在斑马鱼前肾还是中肾,podocin均特异性地表达于足细胞,且沿着毛细血管袢呈连续、线性分布,表明抗体具有良好的组织特异性;(4)MTZ诱导足细胞损伤24h后见podocin呈粗糙的颗粒状分布,48h后阳性表达面积占整个肾小球面积比值明显下降;(5)显微注射podocin反义寡核苷酸的斑马鱼胚胎出现心包水肿、体轴弯曲的表型,第3天时几乎无Podocin表达,随后逐渐恢复至正常,第5天时表达与正常组无异。结论:所制备的斑马鱼抗podocin抗体效价高、组织特异性强,能够反应足细胞损伤情况,是斑马鱼足细胞研究的有力工具。     

氯硝柳胺对斑马鱼幼鱼甲状腺内分泌系统的影响

目的　研究灭螺药氯硝柳胺对斑马鱼幼鱼甲状腺内分泌系统的干扰效应。方法　将受精2 h内的斑马鱼胚胎分别暴露于浓度为0、5、10、20、40 μg/L和80 μg/L的氯硝柳胺 120 h,观察不同暴露浓度下斑马鱼胚胎/幼鱼体重、胚胎孵化率、畸形率和存活率等指标;同时检测鱼体内四碘甲状腺原氨酸 （T4） 和三碘甲状腺原氨酸 （T3）水平,并采用荧光定量 PCR法检测与甲状腺激素调节相关的两个重要基因tshβ和ttr表达情况。结果　暴露于不同浓度氯硝柳胺后,各组斑马鱼幼鱼孵化率（F = 0.947, P = 0.924）和体重（F = 1.042, P = 0.409）差异无统计学意义,存活率（F = 9.309, P = 0.005）和畸形率（F = 14.900, P = 0.001）差异有统计学意义;与对照组相比,较高浓度的氯硝柳胺暴露(40 μg/L和80 μg/L)可显著降低斑马鱼幼鱼存活率 （P均 < 0.05）,并显著增加斑马鱼幼鱼畸形率 （P均 < 0.05）。 随着氯硝柳胺暴露浓度的增加, 斑马鱼幼鱼体内T4含量增加 （R2 = 0.927, F = 6.858, P = 0.003）、T3含量减少 （R2 = 0.925, F = 8.212, P = 0.001）,tshβ基因表达水平上调 （R2 = 0.840, F = 9.032, P = 0.002）、ttr基因表达水平下调 （R2 = 0.952, F = 9.130, P = 0.002）。结论　环境相关浓度的氯硝柳胺暴露可对斑马鱼幼鱼甲状腺内分泌系统造成干扰。     

Galectin-3在甲状腺结节中的表达

目的研究galectin-3在甲状腺结节中的表达.方法应用免疫组化及RT-PCR方法检测甲状腺标本中galectin-3表达.结果无论采用免疫组化或RT-PCR方法,甲状腺癌组织的galectin-3表达率均较高（阳性率为89.7%和91.7%）,不同类型的癌组织中有较大差异,其中乳头状癌和滤泡状腺癌具有较高水平的表达（阳性率100%）.有淋巴结转移者galectin-3表达水平高于无淋巴结转移者.在良性甲状腺结节及正常甲状腺组织,采用免疫组化方法检测的25份甲状腺腺瘤标本和13份结节性甲状腺肿标本仅3例呈阳性,余均为阴性,10份正常甲状腺组织及25份癌旁组织均未发现galectin-3表达.结论galectin-3可作为鉴别良、恶甲状腺疾病的一个较有价值的肿瘤指标.     

真核表达载体pCMV-(Kozak)TFPI在搭桥模型移植静脉中的表达

目的构建真核表达载体pCMV-（Kozak）TFPI并检测其在移植静脉中的表达，为冠状动脉旁路移植术中转染大隐静脉内皮细胞抗凝治疗的临床试验奠定理论应用基础。方法用逆转录聚合酶链反应（RT-PCR）的方法提取人组织因子途径抑制因子（TFPI）基因，并引入Kozak序列，将（Kozak）TFPI亚克隆入pCMV质粒中。在阳离子脂质体介导下，转染颈总动脉搭桥模型移植静脉内皮细胞。RT-PCR、蛋白印迹法（Western印迹）和免疫组化法检测移植静脉中外源TFPI基因mRNA和蛋白的表达。结果（Kozok）TFPI成功克隆入pCMV中。RT-PCR、Western印迹和免疫组化检测到移植静脉中外源TFPI基因mRNA和蛋白的表达。结论pCMV-（Kozak）TFPI真核表达载体构建成功，移植静脉中检测到其蛋白的表达。     

人朊蛋白相关蛋白Shadoo的表达、纯化及抗体的制备

目的克隆、表达和纯化人朊蛋白相关Shadoo（SHO）蛋白并制备抗Shadoo的多克隆抗体。方法提取仓鼠各组织的mRNA，分析SHO基因在仓鼠各组织中转录水平的差异;提取人DNA，PCR方法获得人SHO基因，克隆至融合表达载体，在大肠埃希菌中表达人SHO蛋白并纯化;以纯化蛋白为抗原免疫家兔制备抗血清，用Western blot方法分别检测与重组及内源性SHO蛋白的免疫反应性。结果SHO基因在仓鼠各组织中的转录水平差异很大，脑组织转录水平高。在大肠埃希菌中表达了分子质量单位为37 ku的人SHO融合蛋白，制备的SHO蛋白抗体可与重组的SHO蛋白反应，可识别内源性的SHO。结论成功表达纯化人SHO融合蛋白并制备了抗SHO抗体，为研究SHO和正常朊蛋白之间的关系打下初步基础。     

胃癌组织中hTERT基因表达及意义

目的探讨hTERT基因在胃癌发生、发展中的作用及机制。方法采用免疫组化SP法及核酸原位杂交法检测胃黏膜肠化生、异型增生及胃癌组织中hTERT蛋白及hTERT mRNA表达情况。结果胃黏膜肠化生、异型增生及胃癌组织中hTERT蛋白及hTERTmRNA阳性表达率均显著高于正常胃黏膜（P〈0.05）。hTERT蛋白表达阳性者hTERTmRNA表达阳性率显著高于hTERT蛋白表达阴性者（P〈0.01）。结论 hTERT基因与癌前病变及胃癌的发生有关,可能是胃癌的生物学标志之一。     

核酸原位杂交检测人淋巴结组织中的弓形虫

目的：探讨弓形虫感染的淋巴结组织中的病原体检测。方法：用聚合酶链反应（ＰＣＲ）和基因工程技术制备弓形虫（ＲＨ株）特异ＤＮＡ克隆片段，以地高辛随机引物法标记克隆的弓形虫ＤＮＡ片段作为探针，与淋巴结组织切片中核酸原位杂交试验（ＩＳＨ）检测病理标本中的弓形虫ＤＮＡ。结果：３２例何杰金淋巴瘤（ＨＤ）和４１例非何杰金淋巴瘤（ＮＨＬ）病理标本中各有一例阳性，４７例慢性淋巴结炎（ＣＬ）病理标本中呈ＩＳＨ阳性者２例，总阳性率为３．３％（４／１２０）。研究证明，所制备的探针能检测１０ｐｇ的ＤＮＡ，且特异性强。结论：核酸原位杂交为弓形虫病的病理标本中病原体检测提供了可行的方法。     

FWA116基因在心衰动物模型心脏血管内皮的表达

建立亚急性和慢性心衰SD大鼠模型，用RNA-RNA原位杂交方法观察新基因FWA116在心脏组织的表达。以重组质粒FWA116／PGEM-T线性cDNA为模板，在T7或SP6RNA聚合酶作用下，体外合成地高辛（DIG）半抗原标记的FWA116cRNA探针。结果表明：与对照组相比，该基因在两种心衰大鼠心脏的中、小动脉内皮组织的mRNA表达均明显增高；与Northern blot及免疫组化的结果相一致，这提示DIG标记cRNA原位杂交方法的敏感和可靠。同时，FWA116基因在心衰缺血缺氧心脏动脉内皮中的转录和翻译水平的高表达，说明该基因可能在心衰的病理过程中起到重要的作用。     

Fluorescence-activated cell sorting (FACS) of whole mount in situ hybridization (WISH) labelled haematopoietic cell populations in the zebrafish

The zebrafish is a robust animal model for studying vertebrate haematopoiesis and immune cell interactions. However, fluorescence activated cell sorting (FACS) has been limited due to a paucity of available functional zebrafish antibodies. We have developed a technique combining FACS with whole mount in situ hybridization (WISH) that enables the sorting and examining of fixed zebrafish blood cell populations at different stages of embryonic development, providing the opportunity to correlate RNA expression data with cellular morphology.     

Zebrafish serotonin N-acetyltransferase-2: marker for development of pineal photoreceptors and circadian clock function.

Serotonin N-acetyltransferase (AANAT), the penultimate enzyme in melatonin synthesis, is typically found only at significant levels in the pineal gland and retina. Large changes in the activity of this enzyme drive the circadian rhythm in circulating melatonin seen in all vertebrates. In this study, we examined the utility of using AANAT messenger RNA (mRNA) as a marker to monitor the very early development of pineal photoreceptors and circadian clock function in zebrafish. Zebrafish AANAT-2 (zfAANAT-2) cDNA was isolated and used for in situ hybridization. In the adult, zfAANAT-2 mRNA is expressed exclusively in pineal cells and retinal photoreceptors. Developmental analysis, using whole mount in situ hybridization, indicated that pineal zfAANAT-2 mRNA expression is first detected at 22 h post fertilization. Retinal zfAANAT-2 mRNA was first detected on day 3 post fertilization and appears to be associated with development of the retinal photoreceptors. Time-of-day analysis of 2- to 5-day-old zebrafish larvae indicated that zfAANAT-2 mRNA abundance exhibits a dramatic 24-h rhythm in a 14-h light, 10-h dark cycle, with high levels at night. This rhythm persists in constant darkness, indicating that the zfAANAT-2 mRNA rhythm is driven by a circadian clock at this stage. The techniques described in this report were also used to determine that zfAANAT-2 expression is altered in two well characterized genetic mutants, mindbomb and floating head. The observations described here suggest that zfAANAT-2 mRNA may be a useful marker to study development of the pineal gland and of circadian clock mechanisms in zebrafish.     

Expression of leptin receptor gene in developing and adult zebrafish

Interactions of leptin and leptin receptors play crucial roles during animal development and regulation of appetite and energy balance. In this study we analyzed expression pattern of a zebrafish leptin receptor gene in both developing and adult zebrafish using in situ hybridization and Q-PCR methods. Zebrafish leptin receptor message (lepr) was detected in all embryonic and larval stages examined, and in adult zebrafish. In embryonic zebrafish, lepr was mainly expressed in the notochord. As development proceeded, lepr expression in the notochord decreased, while its expression in several other tissues, including the trunk muscles and gut, became evident. In both larval and adult brains, large lepr expressing cells were detected in similar regions of the hindbrain. In adult zebrafish, lepr expression was also observed in several other brain regions including the hypothalamic lateral tuberal nucleus, the fish homolog of the arcuate nucleus. Q-PCR experiments confirmed lepr expression in the adult fish brain, and also showed lepr expression in several adult tissues including liver, muscle and gonads. Our results showed that lepr expression was both spatially and temporally regulated.     

Cloning of zebrafish activin type IIB receptor (ActRIIB) cDNA and mRNA expression of ActRIIB in embryos and adult tissues.

A full-length cDNA encoding for activin type IIB receptor (ActRIIB) was cloned from zebrafish embryos. It encodes a protein with 509 amino acids consisting of a signal peptide, an extracellular ligand binding domain, a single transmembrane region, and an intracellular kinase domain with predicted serine/threonine specificity. The extracellular domain shows 74-91% sequence identity to human, bovine, mouse, rat, chicken, Xenopus and goldfish activin type IIB receptors, while the transmembrane region and the kinase domain show 67-78% and 82-88% identity to these known activin IIB receptors, respectively. In adult zebrafish, ActRIIB mRNA was detected by RT-PCR in the gonads, as well as in non-reproductive tissues, including the brain, heart and muscle. In situ hybridization on ovarian sections further localized ActRIIB mRNA to cytoplasm of oocytes at different stages of development. Using whole-mount in situ hybridization, ActRIIB mRNA was found to be expressed at all stages of embryogenesis examined, including the sphere, shield, tail bud, and 6-7 somite. These results provide the first evidence that ActRIIB mRNA is widely distributed in fish embryonic and adult tissues. Cloning of zebrafish ActRIIB demonstrates that this receptor is highly conserved during vertebrate evolution and provides a basis for further studies on the role of activin in reproduction and development in lower vertebrates.     

Molecular cloning, tissue distribution, and ontogenic thyroidal expression of the chicken thyrotropin receptor

TSH and the interaction with its receptor (TSHR) in the thyroid gland play a crucial role in the pituitary-thyroid axis of all vertebrates. Released upon stimulation by TSH, thyroid hormones influence numerous processes in the body and are extremely important during the last week of chicken embryonic development. In this study, we have cloned and functionally characterized the chicken TSHR (cTSHR), which was found to be a G protein-coupled receptor consisting of 10 exons. Besides the full-length cDNA, we detected two splice variants lacking either exon 3, or exons 2 and 3, both part of the extracellular domain of the receptor. Bovine TSH increased intracellular cAMP levels in HEK-239 cells transiently expressing the full-length cTSHR (EC50 = 1.43 nm). In situ hybridization showed the expression of cTSHR mRNA in the thyroidal follicular cells. cTSHR mRNA expression, as determined by real-time PCR, was also found in several other tissues such as brain, pituitary, pineal gland, and retina, suggesting that the TSH-TSHR interaction is not only important in regulating thyroid function. TSHR mRNA expression in the thyroid gland did not change significantly during the last week of embryonic development, which suggests that an increased thyroidal sensitivity is not part of the cause of the concomitant increasing T4 levels.     

Identification and functional characterization of zebrafish solute carrier Slc16a2 (Mct8) as a thyroid hormone membrane transporter

Most components of the thyroid system in bony fish have been described and characterized, with the notable exception of thyroid hormone membrane transporters. We have cloned, sequenced, and expressed the zebrafish solute carrier Slc16a2 (also named monocarboxylate transporter Mct8) cDNA and established its role as a thyroid hormone transport protein. The cloned cDNA shares 56-57% homology with its mammalian orthologs. The 526-amino-acid sequence contains 12 predicted transmembrane domains. An intracellular N-terminal PEST domain, thought to be involved in proteolytic processing of the protein, is present in the zebrafish sequence. Measured at initial rate and at the body/rearing temperature of zebrafish (26 C), T(3) uptake by zebrafish Slc16a2 is a saturable process with a calculated Michaelis-Menten constant of 0.8 μM T(3). The rate of T(3) uptake is temperature dependent and Na(+) independent. Interestingly, at 26 C, zebrafish Slc16a2 does not transport T(4). This implies that at a normal body temperature in zebrafish, Slc16a2 protein is predominantly involved in T(3) uptake. When measured at 37 C, zebrafish Slc16a2 transports T(4) in a Na(+)-independent manner. In adult zebrafish, the Slc16a2 gene is highly expressed in brain, gills, pancreas, liver, pituitary, heart, kidney, and gut. Beginning from the midblastula stage, Slc16a2 is also expressed during zebrafish early development, the highest expression levels occurring 48 h after fertilization. This is the first direct evidence for thyroid hormone membrane transporters in fish. We suggest that Slc16a2 plays a key role in the local availability of T(3) in adult tissues as well as during the completion of morphogenesis of primary organ systems.