人甲胎蛋白基因顺式作用元件的重新组合

本研究采用ＤＮＡ重组技术及ＰＣＲ方法对人甲胎蛋白（ＡＦＰ）基因顺式调控元件＋２９ｂｐ至－５．１ｋｂ区进行改造，将改建的ＤＮＡ片段分别克隆到荧光素酶报告基因载体ｐＧＬ２－Ｂａｓｉｃ中，构建了６种含有ＡＦＰ基因增强子和／或沉寂子不同组合的载体。以期筛选出对人肝癌细胞特异的，具有强启动活民生的ＡＦＰ顺序调控元件组合，为进一步实行肝癌基因治疗提供依据。     

Biliary epithelial expression of MUC1, MUC2, MUC3 and MUC5/6 apomucins during intrahepatic bile duct development and maturation. An immunohistochemical study.

Phenotypic alterations of biliary epithelial cells such as changes in intermediate filament composition and presence of carbohydrate residues, occur during the development of intrahepatic bile ducts. In this study, we examined the expression of MUC1, MUC2, MUC3, and MUC5/6 apomucins (mucin core proteins) by immunohistochemical means in the human intrahepatic bile duct during its development and maturation. In the fetal liver, new bile ducts in the portal tracts, either at the hilar level (corresponding to the large bile ducts) or peripheral level (corresponding to the small bile ducts), frequently expressed MUC1 apomucin at their luminal surface. Ductal plates also focally expressed MUC1 apomucin. By contrast, in the postnatal liver, the biliary epithelial cells of intrahepatic large bile ducts constantly expressed MUC3 apomucin, whereas those of small bile ducts did not. MUC2 and MUC5/6 apomucins were absent in the intrahepatic biliary elements of the fetal as well as postnatal livers. These data suggest that the biliary epithelial cells switch MUC1 apomucin expression before birth to that of MUC3 after birth. This characteristic transition may be similar to the changes in the hepatocellular expression of alpha-fetoprotein and albumin during the perinatal period.     

组织特异性表达基因PLUNC调控元件的生物信息学分析

目的分析组织特异性表达基因PLUNC的调控元件。方法采用进化足迹法,结合生物信息学工具,预测PLUNC基因的顺式作用元件和反式作用因子。结果预测的PLUNC基因的转录起始位点位于-1～＋10bp区域间、-29bp存在TATA盒子,启动子集中在-490bp～＋89bp内,在人和小鼠PLUNC基因的启动子保守区内存在29个共同的转录因子结合部位及5个增强子。结论PLUNC基因调控元件的分析可为探讨上呼吸道特异性表达基因的调控机制提供模型。生物信息学技术结合进化足迹法,在基因表达调控机制的研究中具有重要的应用价值。     

Activation, proliferation, and differentiation of progenitor cells into hepatocytes in the D-galactosamine model of liver regeneration.

Rat liver regeneration was studied from 24 hours to 8 days after a single intraperitoneal injection of D-galactosamine (GalN). Morphological changes in the liver were analyzed in parallel with sequential changes in expression of histone-3 mRNA (a marker of cell proliferation), fetal alpha-fetoprotein (AFP) mRNA and gamma-glutamyl transpeptidase (GGT) (markers of fetal hepatocytes), and albumin mRNA and glucose-6-phosphatase (G6Pase) (markers of adult hepatocytes). Proliferation of nonparenchymal epithelial cells (NPC), detected in situ by [3H]thymidine labeling or histone-3 mRNA expression, began after 24 hours primarily in the portal area around the bile ducts. After 2 days, histone-3 labelling intensity increased in rows and clusters of NPC which expanded from the portal zone and invaded into the parenchyma. On days 3 and 5, NPC expressing his-3 mRNA expanded further, forming pseudo-ducts and islet-like structures (NPC structures). Proliferating NPC were positive for GGT. Some GGT positive cells were also positive for the fetal form of AFP mRNA, which lagged behind GGT by 24 hours and peaked on day 5. On day 3, some cells with the appearance of NPC expressed albumin mRNA. Double label in situ hybridization for fetal AFP and albumin mRNAs and dual histochemistry for GGT and G6Pase showed simultaneous expression of these markers in NPC on day 5. Other cells expressing fetal AFP mRNA or GGT on day 5 had a morphological appearance between NPC and hepatocytes (transitional cells). Proliferation of hepatocytes began on day 2, reached maximum on day 5 and then declined. Proliferating hepatocytes did not express fetal AFP mRNA or GGT. These findings indicate that after GalN injury, the liver responds by activation of progenitor cells that proliferate and then differentiate into mature hepatocytes. Adult hepatocytes can also proliferate after GAlN injury, but these hepatocytes do not undergo dedifferentiation/redifferentiation during regeneration of the hepatic lobule.     

Spatiotemporal expression of flk-1 in pulmonary epithelial cells during lung development

Vascular endothelial growth factor-A (VEGF-A) responsive effects mediated via the receptors fetal liver kinase-1 (flk-1) and fms-like tyrosine kinase (flt-1), are key processes of pulmonary vascular development. Flk-1 has been shown to be involved in early embryonic lung epithelial to endothelial crosstalk and branching morphogenesis. Recent reports suggested a role of VEGF-A in lung epithelial cell function. Based on these observations, we hypothesize that epithelial flk-1 has a unique function in pulmonary development. Thus, the aim of this study is to elucidate spatiotemporal expression of flk-1 during lung development with respect to the epithelial system. Embryonic lungs were screened for flk-1 messenger RNA and protein at daily intervals, including postnatal stages. From Embryonic Day (ED) 12.5 through ED 15.5, flk-1 expression was restricted to the early vascular primitive network, while from ED 16.5 on flk-1 was detectable in the epithelial system and persisted there postnatally. At postnatal stages, flk-1 expression was increasingly restricted to individual cells in the alveolar septa. Isolation and in vitro cultivation of alveolar epithelial cells confirmed flk-1 expression and showed VEGF secretion into the supernatant. To our knowledge, this is the first murine study characterizing epithelial flk-1 expression at different stages throughout lung organogenesis until birth and at postnatal stages. To confirm epithelial flk-1 expression, we performed reporter gene analysis of the flk-1 promoter in vivo. Investigations on transgenic mouse strains, containing either a complete or incomplete flk-1 promoter driving expression of the lacZ reporter gene, suggested differential flk-1 regulation in endothelial and epithelial cells.     

A new mutation in the AFP gene responsible for a total absence of alpha feto-protein on second trimester maternal serum screening for Down syndrome

Alpha feto-protein (AFP) is a major plasma protein produced by the yolk sac and the liver during the fetal period. During the second trimester of pregnancy, APF and betahCG serum concentrations are commonly used for screening Down syndrome. AFP deficiency is rare (estimated to be 1/105,000 newborns) and only one sequence alteration has previously been reported in the AFP gene. We report a new mutation in exon 5 of the AFP gene, leading to a total absence of AFP on 2nd-trimester maternal serum screening for Down syndrome, confirmed on the amniotic fluid. Despite this, fetal development and birth were normal. After PCR-amplification, the whole AFP gene was sequenced. The new mutation was a guanine to adenine transition in position 543 creating a premature stop codon in position 181. In order to search for eventual modifications of the amniotic fluid profile, proteins were separated by electrophoresis and compared with 10 normal amniotic fluids sampled at the same developmental age (18 weeks). In the amniotic fluid of our patient albumin rate was reduced whereas alpha1 and beta protein fractions were increased, suggesting that AFP deficiency may modify the distribution of protein fractions. This observation emphasizes the complex molecular mechanisms of compensation of serum protein deficiency. Studies on other families with AFP deficiency are necessary to confirm this observation.     

靶向腺病毒载体介导的EGFP基因在甲胎蛋白阳性肝癌细胞的特异表达

目的：建立一种在甲胎蛋白(AFP)阳性的肝癌细胞中靶向表达目的基因的重组腺病毒载体。方法： 基于腺病毒载体Adeno-XTM Expression system,以300 bp AFP特异启动子替换穿梭质粒Pshuttle中CMV启动子，将增强型绿色荧光蛋白(EGFP)基因作为报告基因亚克隆至Pshuttle，HEK293细胞包装腺病毒，收集病毒后分别转染人正常肝脏LO2细胞，人肝癌HepG2细胞及HeLa细胞；通过Northern杂交检测EGFP基因在3种细胞中的转录水平，荧光显微镜下观察3种细胞中绿色荧光蛋白的表达。结果：Northern杂交显示，HepG2细胞中有大量EGFP基因的转录，而正常肝细胞LO2和HeLa细胞中仅能检测到微量基因的转录；荧光显微镜检测发现HepG2细胞内有强绿色荧光表达，而在LO2以及HeLa细胞内见极弱绿色荧光。 结论： 在AFP特异启动子作用下，腺病毒携带的目的基因在AFP阳性的肝癌细胞中得到显著转录和表达，而在非AFP阳性细胞仅微量转录，蛋白表达极弱。该腺病毒载体可作为AFP阳性的肝癌基因靶向治疗的适宜载体。     

利用甲胎蛋白启动子活力筛选人胚胎肝脏前体细胞

目的 利用甲胎蛋白启动子活力筛选人胚肝脏前体细胞克隆。 方法 经聚合酶链反应(PCR)扩增及酶切后连接,将甲胎蛋白启动子片段构建于pGL3载体中并测序鉴定,将其与pRL-TK质粒共转染到HepG2、A549和HeLa细胞中,通过相对荧光素酶活力分析甲胎蛋白启动子活力的特异性。采用克隆化培养法获得人胚胎肝脏细胞克隆,检测各克隆的甲胎蛋白启动子活力,并应用间接免疫荧光染色方法检测甲胎蛋白表达情况。 结果 经PCR、酶切分析及DNA序列测定证实pGL3-AFP质粒克隆成功。将其与pRL-TK质粒共转染到表达甲胎蛋白的HepG2细胞和不表达甲胎蛋白的A549、HeLa细胞中,仅在表达甲胎蛋白的HepG2细胞中检测到了较高的甲胎蛋白启动子活力,胚胎肝脏细胞中也检测到了甲胎蛋白启动子活力,表明其中含有表达甲胎蛋白的细胞。利用克隆培养法获得5个胚胎肝脏细胞克隆,将其分别共转染pGL3-AFP和pRL-TK质粒,发现其中1个克隆的甲胎蛋白启动子活力与HepG2细胞接近,且免疫荧光染色结果显示,该克隆细胞甲胎蛋白阳性率为(99.1±0.6)%,表明此克隆为肝脏前体细胞克隆。 结论 利用甲胎蛋白启动子活力结合克隆培养法可以获得肝脏前体细胞克隆。     

Quantitative analysis of cell allocation during liver development, using the spf(ash)-heterozygous female mouse

Mosaicism of ornithine transcarbamylase (OTC) expression in hepatocytes was quantitatively analyzed during liver development of the spf(ash)-heterozygous female mouse. Because the mosaic patterns depend on cell migration and cell mixing, such analysis could give insights on the growth pattern or allocation pattern of hepatocytes during liver development. Complex mosaic patterns of OTC-positive and -negative hepatocytes were observed in sections of fetal and postnatal livers. Sizes of patches, which were aggregates of OTC-positive or -negative hepatocytes, increased during development. Patches were slender and comparatively simple in 15.5- and 17.5-day fetal and neonatal livers. Quantitative analysis of patch shapes demonstrated that undulation of patches was maximal at 7 postnatal days. Patches with nodular shapes also started to increase in number at this stage. Isolated patches in sections of fetal livers and postnatal livers three-dimensionally connected with one another. However, especially in fetal livers, in which OTC-positive patches were minor, due to the presence of abundant hemopoietic cells, isolated three-dimensional patches consisting of approximately 5 to 70 cells were often found. They were shaped like slender branching or zigzag-shaped cords, but no definite orientation such as portal-central was observed in them at any stage. These results suggest that hepatocytes contiguously allocate their daughter cells as zigzag-shaped or branching cords at younger stages. Some hepatocytes grow with nodular formation after 7 postnatal days. Migration and mixing of hepatocytes appear to be more extensive at fetal stages than in the adult liver. Immunohistochemical analysis of intercellular junction proteins (E-cadherin, connexins 26 and 32, occludin, and ZO-1) also revealed that their expression and distribution changed in hepatocytes during development, which may be correlated with the OTC mosaic patterns.