Explant culture of human and rabbit articular chondrocytes.

Copious outgrowth of chondrocytes was obtained by explantation from each of three rabbit and one surgically-resected human articular cartilages pretreated briefly with trypsin. In lapine explants, ascorbate (40 micrograms/ml) increased DNA three-fold over control values and resulted in deposition of a chondroid matrix. It doubled radiosulfate incorporation by the outgrowths. Up to 56% of the sulfated glycosaminoglycan synthesized was located in the trypsin-digestible pericellular coat compared with about 15% in previous monolayer cultures. The collagens synthesized were characterized partially. In rabbit cell cultures, the alpha 1:alpha 2 ratio varied from 2.9 to 3.8. In human cultures, an unusual post-alpha 2 peak was observed. The findings suggest an uncoupling of the phenotypic expression of the major cartilaginous macromolecules in the cultures. There were no distinctive differences between chondrocytes derived from normal and fibrillated human cartilage of the same individual.     

Explant culture of human submandibular gland epithelial cells: evidence for ductal origin

As an approach to investigating the disease cystic fibrosis, attempts were undertaken to culture from human submandibular glands epithelial cells with a potential for manifesting the cystic fibrosis genetic defect. To initiate the culture of submandibular gland epithelial cells, tissue fragments from glands were explanted as a function of both the composition of the serum-free growth medium and of the matrix utilized to coat the culture vessel growth surface. A morphologically homogeneous growth of submandibular gland epithelial cells, uncontaminated by fibroblasts, was obtained, once optimum culture conditions were defined. Light microscopic examination of these explant cultures in a transverse plane of section demonstrated variation in the outgrowth according to distance from the explant. At its outer margin, the outgrowth consisted of one or two layers of viable low cuboidal cells, and more centrally, it was multilayered. Mitotic figures were observed in the periphery of the outgrowth. In the region, a few cells removed from the periphery where the outgrowth consisted of about three to six cell layers, dilated intercellular spaces, indicative of secretion of fluid and ions into the spaces, separated the basal cuboidal cells. Overlying cells were increasingly flattened toward the culture surface and devoid of nuclei. Centrally, near the explant, the multilayer appeared completely involuted throughout. Ultrastructural examination in a transverse plane of the multilayered region with viable basal cells confirmed these observations showing wide spaces separating the cuboidal basal cells, keratinization of midstratum cells, and complete involution of the upper layer of ghost-like cells. These cells cultured from the submandibular gland reacted positively to immunochemical staining for keratin.     

Explant culture of human peripheral lung. I. Metabolism of benzo[alpha]pyrene

Human lung explants have been maintained in vitro for a period of 25 days. Autoradiographic studies indicated that the broncholar epithelial cells, type 2 alveolar epithelial cells, and stromal fibroblasts incorporated 3H-thymidine during the culture. After 7 to 10 days, type 2 cells were the predominant alveolar epithelial cell type. Lamellar inclusion bodies were released from the type 2 cells and accumulated in the alveolar spaces. The metabolism of benzo[alpha]pyrene (BP) in human lung explants cultured for up to 7 days was investigated. Human lung explants had measurable aryl hydrocarbon hydroxylase activity and could metabolize BP into forms that were bound to cellular DNA and protein. Peripheral lung had significantly lower aryl hydrocarbon hydroxylase activity than cultured bronchus but both tissues had similar binding levels of BP to DNA. Radioautographic studies indicated that all cell types in the peripheral lung can metabolize BP. The major ethylacetate extractable metabolites of BP formed by peripheral lung were tetrols and trans-7,8-diol. The primary water-soluble metabolite released with arylsulfatase and beta-glucuronidase was 3-hydroxybenzo[alpha]pyrene.     

常染色体显性多囊肾组织差异表达基因的初步研究

目的应用基因芯片技术及最新公共数据库，筛选常染色体显性多囊肾组织中差异表达的基因，对其进行功能分类，并对其中1条基因利用原位杂交技术进行验证。方法将代表8398条人类基因的PCR产物制成基因芯片。将等量的多囊肾组织和正常肾组织mRNA分别用Cy5、Cy3荧光标记，逆转录合成cDNA探针，混合后与上述基因芯片杂交。扫描杂交信号荧光强度，找出差异表达基因，对获得的基因进行分子生物信息学分析。并对其中的上调表达基因IGF1 mRNA进行原位杂交，验证基因芯片结果的准确性。结果（1）在进入研究的8398条基因中，共发现357条差异表达基因。94条基因在多囊肾组织中低表达，263条基因高表达；（2）上调表达基因主要属于原癌基因，细胞骨架蛋白和运动相关蛋白，凋亡相关蛋白，细胞信号和传递蛋白，细胞因子；下调表达基因主要属于抑癌基因，DNA结合、转录和转录因子，细胞信号和传递蛋白，参与代谢的基因；（3）IGF1 mRNA原位杂交结果与芯片结果一致。结论基因表达谱芯片可快速、高效地筛选差异表达基因；多囊肾病的发生、发展中存在着多种不同功能基因表达调控的改变。     

组织块法培养SD大鼠的成骨细胞及鉴定

背景：获得足够量高纯度的成骨细胞比较困难,因此掌握简单而快速提取成骨细胞并进行培养鉴定势在必行。 目的：应用组织块法对SD大鼠成骨细胞的体外培养与鉴定。 方法：取新生(<24 h)SD大鼠颅骨,去除周围多余的组织,将剔净颅骨剪成1 mm3大小的碎块,分别用常规方法和组织块法(改良)方法进行成骨细胞培养,从形态学、碱性磷酸酶和茜苏红染色等方法鉴定。 结果与结论：利用改良方法培养的细胞具有典型的成骨细胞形态特征,碱性磷酸酶呈阳性染色,茜素红染色后有矿化结节的形成。组织块法培养的成骨细胞具有典型的成骨细胞成分单一、细胞培养时间短、数量、纯度、密度均一致,从而的得到稳定的成骨细胞系,为进行体外实验建立了良好的平台。     

Autosomal recessive polycystic kidney disease

 Autosomal recessive polycystic kidney disease (ARPKD) is a rare inherited disorder which usually becomes clinically manifest in early childhood, although the spectrum of ARPKD is much more variable than generally known. Presentation of ARPKD at later ages and survival into adulthood have been observed in many cases. The responsible gene has been mapped to chromosome 6p. Thus there is no evidence of genetic heterogeneity. The most important indication for DNA diagnosis is the prenatal diagnosis in families with at least one affected child. The critical region has been narrowed with the use of recombinant families of about 4 cM. Several possible candidate genes have been excluded. Received: 23 April 1997 / Accepted: 12 August 1997     

Autosomal recessive polycystic kidney disease

Summary Autosomal recessive polycystic kidney disease is a rare inherited disorder which usually becomes clinically manifest in early childhood, whereas autosomal dominant polycystic kidney disease usually is a disorder of adult onset. With increasing knowledge and improving diagnostic techniques, it becomes evident that the spectrum of both entities is much more variable than generally known. The presentation of autosomal recessive polycystic kidney disease at later ages and survival into adulthood have been reported. The diagnostic criteria, clinical course, genetics and differential diagnosis of autosomal recessive polycystic kidney disease will be presented.Abbreviations ADPKD, ARPKD autosomal dominant/recessive polycystic kidney disease - CHF congenital hepatic fibrosis     

Mutations of the human polycystic kidney disease 2 (PKD2) gene

Autosomal dominant polycystic kidney disease (ADPKD) is an inherited nephropathy, usually of late onset (onset between third to seventh decade), primarily characterized by the formation of fluid‐filled cysts in the kidneys. It is one of the most frequent inherited conditions affecting approximately 1:1,000 Caucasians. Two major genes have been identified and characterized in detail: PKD1 and PKD2, mapping on chromosomes 16p13.3 and 4q21‐23, respectively. A third gene, PKD3, has been implicated in selected families. Polycystic kidney disease of types 1 or 2 follows a very similar course of symptoms, both being multisystem pleiotropic disorders of indistinguishable picture on clinical grounds. The only difference is that patients with PKD2 mutations run a milder course compared to PKD1 carriers, with an average 10–20 years later age of onset and lower probability to reach end‐stage‐renal failure. The proteins polycystin‐1 and ‐2 are trans‐membranous glycoproteins hypothesized to participate in a common signaling pathway, interacting with each other and with other proteins, and coordinately expressed in normal and cystic tissue. Renal cysts most probably arise after a second somatic event, which inactivates the inherited healthy allele of the same gene, or perhaps one of the alleles of the other gene counterpart, generating a trans‐heterozygous state. This article reviews the reported mutations in PKD2. Mutations of all kinds have been reported over the entire sequence of the PKD2 gene, with no apparent significant clustering and with some evidence of genotype/phenotype correlation. Most families harbor their own private mutations but a few recurrent events have been reported in unrelated families. Hum Mutat 18:13–24, 2001. © 2001 Wiley‐Liss, Inc.     

Freeze-fracture observations on human fetal kidney in serum-free organ culture

The current study was undertaken to examine and characterize junctional complexes, through freeze-fracture, in developing human fetal kidney and in cultured renal explants maturing in vitro. Tissue specimens were cultured for 7 days in Leibovitz's L-15 medium in the absence of serum or hormones. In uncultured explants, cells in the different nephron segments were joined by zonulae occludentes which consisted of ridges on the P-face and grooves on the E-face of lateral membranes. Tight junction composition was heterogeneous and complexity increased from proximal to collecting tubules. Proximal tubule cells were also characterized by the presence of gap junctions and a brush border. Podocytes were joined by macular junctions, while zipper-like junctions were observed between collecting duct cells. Intercalated cells were decorated with rod-shaped intramembrane particles on lateral and apical membranes, instead of the usual spherical particles present in other cells. All these structures could be observed at various intervals during tissue culture, indicating the preservation of ultrastructural integrity of the explants. These observations extend and support previous studies made at the light and electron microscopic levels. Thence, the present culture model constitutes a valuable tool to study the direct effect of growth factors on nephrogenesis.     

Characterization of the human homologue of the mouse Tg737 candidate polycystic kidney disease gene

We previously identified a gene from the mutant locus in a newmouse mutation that causes recessive polycystic kidney disease.Here we describe the cloning, characterization and mapping ofthe homologous human gene. The human and mouse genes are 95%identical at the predicted amino acid sequence level, and bothgenes encode a putative protein that contains a tetratricopeptiderepeat motif. The human gene, called hTg737, is expressed witha broad tissue distribution that includes the kidney and liver,and gives rise to a 2.9 kb mRNA. The gene contains 26 exonsand spans a genomic region greater than 100 kb. Chromosome mappingexperiments revealed that the hTg737 gene maps near the centromereon the long arm of human chromosome 13, at position 13q12.1.While this gene does not map to the primary locus that has beenidentified for ARPKD in humans, it may represent a candidategene for other recessive renal disorders that have yet to bemapped.     

Exaggerated natriuresis in adult polycystic kidney disease

Angiotensin II (AII), aldosterone (Aldo) arginine vasopressin (AVP) in plasma, serum osmolality (Sosm), and renal sodium excretion (UNaV) were studied before and after infusion of hypertonic sodium chloride solution in 20 patients with adult polycystic kidney disease (PKD) with normal or moderately reduced creatinine clearance (Ccr) and in 10 healthy control subjects. UNaV increased after sodium loading in all, significantly more in the PKD patients. AII and Aldo were normal before sodium loading and suppressed after saline in PKD patients and controls. The increase in VNaV correlated with Aldo in patients but not in controls. AVP before loading was increased in hypertensive PKD patients with reduced Ccr, but not in normotensive patients with normal Ccr. After hypertonic saline, Sosm increased to the same degree both in PKD and control subjects, but AVP increased more in those with PKD. The exaggerated natriuresis of PKD is probably not explained by a change in the activity of the renin-angiotensin-aldosterone system. The enhanced response of AVP to osmotic stimuli in PKD may be a compensatory reaction to a reduced renal tubular effect of AVP.     

Genetic counseling in adult polycystic kidney disease

We evaluated 22 patients with end-stage renal disease (ESRD) due to adult polycystic kidney disease (APKD) to assess their knowledge of the hereditary nature of the condition and to determine whether they received adequate genetic counseling. Patients were evaluated by means of a questionnaire and a review of their medical records. Only 5 of 22 (23%) knew their disorder was hereditary at the time of diagnosis, and in only 4 (18%) was genetic counseling suggested. In no instance had proband and spouse received genetic counseling together. Diagnostic studies of children at risk were rarely suggested. We also evaluated the children of 9 probands for APKD. Of 26 children evaluated, 17 had APKD (65%). Sixteen had no children at the time of testing. All but two of the 26 were less than 25 years old. Of the probands' children over 15 years of age, 55% knew the name of the condition in the family but only 9% knew they should be tested. Our study demonstrated inadequacy of genetic counseling and follow-up in this group of patients; we suggest that referral for counseling become a routine part of their management. Early diagnosis and effective counseling has the potential benefit for the individuals of making rational reproductive decisions appropriate for their situation. Counseling may have to be repeated during the course of the patients' disease, as their perception of risk may change with time. With advances in dialysis and transplantation, ESRD may not be as devastating in years to come as it is now.     

Seminal megavesicles with adult polycystic kidney disease

Adult polycystic kidney disease has been found in association with pathological dilatation of the seminal vesicles in six patients. These men appeared normal on clinical examination, but had azoospermia or severe oligozoospermia. They were investigated by scrotal exploration with vasography, renal and transrectal ultrasound scans (TRUS), and percutaneous puncture of the seminal vesicles in one case, before and after resection of the ejaculatory ducts. This revealed that the gross dilatation of the seminal vesicles was not caused by obstruction, but appeared to be due to atonicity (megavesicles). These ultrasonic appearances, when described previously, were incorrectly thought to be due to seminal vesicle cysts.