Physical and functional interaction of human nuclear uracil-DNA glycosylase with proliferating cell nuclear antigen

Uracil residues arise in DNA by the misincorporation of dUMP in place of dTMP during DNA replication or by the deamination of cytosine in DNA. Uracil-DNA glycosylase initiates DNA base excision repair of uracil residues by catalyzing the hydrolysis of the N-glycosylic bond linking the uracil base to deoxyribose. In human cells, the nuclear form of uracil-DNA glycosylase (UNG2) contains a conserved PCNA-binding motif located at the N-terminus that has been implicated experimentally in binding PCNA. Here we use purified preparations of UNG2 and PCNA to demonstrate that UNG2 physically associates with PCNA. UNG2 co-eluted with PCNA during size exclusion chromatography and bound to a PCNA affinity column. Association of UNG2 with PCNA was abolished by the addition of 100 mM NaCl, and significantly decreased in the presence of 10 mM MgCl(2). The functional significance of the UNG2.PCNA association was demonstrated by UNG2 activity assays. Addition of PCNA (30-810 pmol) to standard uracil-DNA glycosylase reactions containing linear [uracil-(3)H]DNA stimulated UNG2 catalytic activity up to 2.6-fold. UNG2 activity was also stimulated by 7.5 mM MgCl(2). The stimulatory effect of PCNA was increased by the addition of MgCl(2); however, the dependence on PCNA concentration was the same, indicating that the effects of MgCl(2) and PCNA on UNG2 activity occurred by independent mechanisms. Loading of PCNA onto the DNA substrate was required for stimulation, as the activity of UNG2 on circular DNA substrates was not affected by the addition of PCNA. Addition of replication factor C and ATP to reactions containing 90 pmol of PCNA resulted in two-fold stimulation of UNG2 activity on circular DNA.     

Characterization of plant XRCC1 and its interaction with proliferating cell nuclear antigen

In plants, there are no DNA polymerase β (Pol β) and DNA ligase III (Lig3) genes. Thus, the plant short-patch base excision repair (short-patch BER) pathway must differ considerably from that in mammals. We characterized the rice (Oryza Sativa L. cv. Nipponbare) homologue of the mammalian X-ray repair cross complementing 1 (XRCC1), a well-known BER protein. The plant XRCC1 lacks the N-terminal domain (NTD) which is required for Pol β binding and is essential for mammalian cell survival. The recombinant rice XRCC1 (OsXRCC1) protein binds single-stranded DNA (ssDNA) as well as double-stranded DNA (dsDNA) and also interacts with rice proliferating cell nuclear antigen (OsPCNA) in a pull-down assay. Through immunoprecipitation, we demonstrated that OsXRCC1 forms a complex with PCNA in vivo. OsXRCC1 mRNA was expressed in all rice organs and was induced by application of bleomycin, but not of MMS, H₂O₂ or UV-B. Bleomycin also increased the fraction of OsXRCC1 associated with chromatin. These results suggest that OsXRCC1 contributes to DNA repair pathways that differ from the mammalian BER system.     

Analysis of proliferative activity in colorectal mucosa by immunohistochemical detection of proliferating cell nuclear antigen (PCNA)

Immunohistochemical detection of proliferating cell nuclear antigen (PCNA) has been suggested as a new approach for determinating proliferative activity in paraffin-embedded tissue. In a prospective study PCNA immunostaining was performed in 284 colorectal biopsies using monoclonal antibodies 19F4 (Ogata et al. 1987) and PC10 (Waseem and Lane 1990) and compared with the Ki67 method. From each site three biopsies were taken and a variety of fixation regimens for frozen and paraffin-embedded samples tested. For frozen biopsies methanol fixation at −20° C proved best. In paraffin sections PCNA could be detected after methacarn fixation as well as after controled fixation at 4° C in 4% paraformaldehyde for 1 h and in most biopsies routinely fixed with 10% formalin. However, the latter fixation regimens revealed additional PCNA-positive cells in the normal superficial colonic mucosal epithelium. Although the percentage of cells positive for PCNA was generally lower than for Ki67, the rates correlated in a highly significant fashion, both in frozen methanolfixed biopsies, and in paraformaldehyde-fixed paraffinembedded samples. PCNA immunohistochemistry revealed a similar proliferative activity in different parts of the large bowel. A higher proliferative activity was found in inflamed mucosa, adenomas, carcinomas and even in normal mucosa from patients with colorectal neoplasms. In routinely fixed biopies, the monoclonal antibody PC10 was superior to 19F4 because of considerably less background staining. However, in the routine material only a rough estimate of the proliferative activity was possible by PCNA immunohistochemistry using these antibodies, because unpredictable numbers of non-S-phase cells were also stained. Thus, it was concluded that reliable results are only obtainable after careful control of the fixation conditions. Taking this reservation into account, PCNA immunohistochemistry still represents a convenient method for measurements of proliferative activity in paraffin-embedded colorectal mucosa and can be applied using methanol-containing fixatives as well as after 4% paraformaldehyde fixation. Supported by a grant of the Werner and Klara Kreitz-Stiftung, Kiel to J.D.     

Proliferating cell nuclear antigen (PCNA/cyclin) in plant proliferating cells: immunohistochemical and quantitative analysis using autoantibody and murine monoclonal antibodies to PCNA.

Proliferating-cell nuclear antigen (PCNA), also known as cyclin, is synthesized in proliferative cells and recently was identified as DNA polymerase-delta auxiliary protein. In this paper, the association of PCNA to the proliferative cells of plants was analysed using both autoantibodies to PCNA obtained from a patient with systemic lupus erythematosus (SLE) and murine monoclonal antibodies. By immunohistochemical analysis, nuclei of cells around the growing point in soybean root tips reacted strongly with autoantibodies to PCNA in the serum from a patient with SLE. The plant PCNA in root tip cells was purified by ammonium sulfate fractionation, DEAE chromatography, and affinity chromatography. The partially purified plant PCNA was tested by immunoblotting and a 34 kD polypeptide reacted with both the human anti-PCNA autoantibody and a mouse monoclonal antibody against human PCNA (TOB 7). In addition, the purified plant PCNA reacted with both antibodies in enzyme-linked immunosorbent assay (ELISA). The binding of anti-PCNA serum to the animal PCNA was blocked by the plant PCNA in this ELISA. The association of PCNA with growing cells in plants was further confirmed by quantitative sandwich type ELISA using two murine monoclonal antibodies to PCNA, TOB7 and TO17. Those results suggested that PCNA in both plant and animal cells had the same immunological and biochemical characteristics and the plant PCNA might play an important role in cell growth, existing as it does in proliferating plant cells. The concentration of PCNA in soybean germ extract before germination was less than 5 ng ml-1 (protein concentration, 6.8 mg ml-1), but that of the root tip stem including the growing point increased to 887 ng ml-1 (protein concentration 3.8 mg ml-1) in the second day after germination.     

Induction of proliferating cell nuclear antigen (PCNA)-immunoreactive cells in goldfish retina following intravitreal injection with 6-hydroxydopamine

1. The dopaminergic neurotoxin, 6-hydroxydopamine (6-OHDA), was injected intravitreally into the eyes of juvenile (5- to 6-cm) goldfish. 2. Proliferation of rod neuroblasts caused by 6-OHDA (2 micrograms in 2 microliters saline) was detected in retinal wholemounts by immunofluorescence for proliferating cell nuclear antigen (PCNA) 3, 7, 14, 20, or 30 days after injection. 3. The injected dose of 6-OHDA was sufficient to cause permanent loss of dopaminergic interplexiform and serotonergic amacrine cells in the injected eye but not in the contralateral control eye. 4. 6-OHDA increased the density (mm-2) of PCNA-ir cells in the outer nuclear layer (ONL) of the injected eye to 2.65 times the initial density 20-30 days after injection, and it increased the density of PCNA-ir cells in the ONL of the contralateral, untreated eye, equally but after a delay of less than or equal to 7 days with respect to the injected eye. 5. 6-OHDA also increased the density of PCNA-ir cells in the inner nuclear layer (INL) to greater than 20 times the initial density 7 days after injection, followed by a rapid decline almost to control levels by 14 days after injection. 6. The sequence of responses to 6-OHDA, with PCNA-ir cells first scattered in the ONL and then clustered in the INL, suggests that neuroblasts from the ONL migrate to the INL to compensate for toxin-induced cell loss. 7. Double staining for 5-bromodeoxyuridine (BrUdR; a thymidine analogue) and PCNA, carried out on 7 days after intravitreal injection with 6-OHDA, showed that 77% of all PCNA-ir cells in the outer nuclear layer had been in S phase during the previous 24 hr. 8. Immunoreactivity for PCNA was found to be a valid marker for rod neuroblasts which have entered S phase within 1-2 days before sampling and was shown to be especially convenient for investigating the distribution of proliferating cells in whole mounts. 9. In controls injected unilaterally with saline or saline plus 1% dimethyl sulfoxide (DMSO), the differences in densities of PCNA-ir rod precursor nuclei 2-30 days after injection vs. day 0 (uninjected) were statistically insignificant in both injected and uninjected eyes (Negishi et al., 1991). Therefore the local effect of injecting 6-OHDA was due to 6-OHDA itself, not to mechanical damage or nonspecific actions of foreign substances.(ABSTRACT TRUNCATED AT 400 WORDS)     

A morphometric study of 24-hour variations in subcellular structures of the rat pancreatic acinar cell

Summary Subcellular structures of pancreatic acinar cells were examined at six evenly spaced time points in the 24-h period (light cycle: 06.00 h–18.00 h) in four Wistar male rats at each time point. At each sampling point, the area and circumference of acinar cell bodies and the area, number and circumference of their cytoplasmic organelles were measured using a semiautomatic computer system for morphometry and a point-counting method.The area, number and circumference-area ratio of the cytoplasmic organelles were subject to strong circadian variations, and the cellular area and circumference exhibited weak circadian variations. Variation pattern of the cytoplasmic organelles suggested an intracellular route for secretory proteins during a 24-h span. From the results it was possible to divide the 24-h period into three stages. 1. The resting or protein synthetic stage (00.00 h to 08.00h): the area of the rough surfaced endoplasmic reticulum (rER) was strongly increased, and that of zymogen granules was clearly decreased. 2. The granule accumulation stage (08.00h to 16.00h): the area of the rER was markedly decreased; that of zymogen granules was increased. 3. The secretion stage (16.00 h to 00.00): as a result of the release of zymogen granules from the acinar cell, the area of zymogen granules decreased, and that of the rER increased. The relationship between the area of the rER and zymogen granules varied in a reciprocal manner. Other cytoplasmic organelles, namely the Golgi complex, condensing vacuoles, mitochondria and lysosomes also varied prominently during the 24-h span, corresponding to variations in the rER and zymogen granules.     

A novel proteomic approach identifies new interaction partners for proliferating cell nuclear antigen

During DNA replication and repair, many proteins bind to and dissociate in a highly specific and ordered manner from proliferating cell nuclear antigen (PCNA). We describe a combined approach of in silico searches at the genome level and combinatorial peptide synthesis to investigate the binding properties of hundreds of short PCNA-interacting peptides (PIP-peptides) to archaeal and eukaryal PCNAs. Biological relevance of our combined approach was demonstrated by identification an inactive complex of Pyrococcus abyssi ribonuclease HII with PCNA. Furthermore we show that PIP-peptides interact with PCNA largely in a sequence independent manner. Our experimental approach also identified many so far unidentified PCNA interacting peptides in a number of human proteins.     

Morphometric and fine-structural studies of rat pancreatic acinar cells during early postnatal life

Summary Pancreatic acinar cells of rats obtained at 1,2, 3, 5, 7 and 14 days of age were examined using fine structural and morphometric techniques. From 5 days of age onwards, the acinar cells were analysed twice per day, at 20.00 h and 08.00 h.The present study demonstrates changes in the average volume of the cell, nucleus and cytoplasm, and volume densities of various cytoplasmic organelles during the first two weeks after birth. During early postnatal life, the volume density of rER increases, whereas that of zymogen granules decreases. From 5 days of age onwards, the volume densities of these two organelles differ significantly at 20.00 h and 08.00 h. During the first 2–3 days after birth, inclusion body-like structures appear in the cytoplasm of acinar cells; they contain aggregated zymogen granules and, sometimes, amorphous structures or cytoplasmic organelles. These structures also occur in interstitial cells and cells located in the intercalated region between acinar and ductal epithelial cells. Serum level of -amylase activity is high at birth, compared with other stages during the first three weeks. Degenerating acinar cells and cell debris can be seen in the acinar and ductal lumina during these stages, a feature suggesting holocrine secretion. Cellular polarity appears to be incomplete during the first two or three days after birth.     

Proliferating cell nuclear antigen in normal urothelium and urothelial lesions of the urinary bladder: a quantitative assessment using a true color image analysis system

To evaluate proliferating cell nuclear antigen (PCNA) staining for assessing proliferative activity in routine pathology specimens of urinary bladder, the bladder carcinoma cell line J82 and a total of 122 specimens of normal bladder and urothelial lesions were stained with the antibody clone PC10 against proliferating cell nuclear antigen. In in vitro plateau cultures the proportion of PCNA-positive cells exceeded that of Ki-67-positive cells, and only very few cells were negative. In formalin-fixed tissues, the PCNA staining pattern, which should be confined to replicon units in the nucleus, was optimized by 1 h postfixation in an organic solvent (methacarn). Sections showed positive nuclear staining confined to basal and some suprabasal cells in normal urothelium and grade 1 dysplasias, but more generalized nuclear staining in all other neoplastic lesions. In addition, stromal cells adjacent to invasive tumors showed nuclear positivity in some instances. Using quantitative true color image analysis of sections counterstained with hemalum, the degree of brown staining of the PCNA reaction product is contrasted with the blue staining of the nuclear area. With this method low contrast specific staining not appreciated optically can be reliably detected. Image analysis data confirmed observations made on noncounterstained sections and showed significant differences between grade 1 and 2 dysplasias as well as between grade 1 dysplasia and all grades of papillary tumor. Furthermore, a significant difference in PCNA staining indices was found between grade 1 and 3 bladder carcinomas. The results indicate that PCNA staining using the PC10 antibody is not confined to the proliferative fraction of neoplastic urothelium. In contrast with data from normal tissue and malignant hematological neoplasms, the amount of PCNA is regulated differently in urothelial neoplasms, emphasizing the biological differences between the following two sets: mild dysplasia and moderate dysplasia; mild dysplasia and papillary carcinomas. The use of image analysis to standardize the detection process after controlled staining conditions is advisable in order to provide reliable data. Supported by the DFG project: Knuechel/Urothelcarcinom 263     

A morphometric study of the 24-hour variations in subcellular structures of rat pancreatic acinar cells during the periweaning period

Summary Pancreatic acinar cells of rats obtained before (21 days of age) and after (31 and 42 days) weaning were morphometrically examined at six evenly spaced times during 24 h.Morphometric analysis demonstrated clear-cut variations in the average volume of the cell and nucleus, the incidence of binucleate acinar cells, and the volume and numerical densities of various cytoplasmic organelles during 24 h, at each stage. The daily mean values and variation of these parameters at 21 days clearly differed from those at 31 and 42 days. In particular, changes in the volume densities of rER and zymogen granules were bimodal at 21 days, whereas they were unimodal at 31 and 42 days. Moreoever, some parameters also differed between 31 and 42 days, the main difference being an 8 h time shift in the variation curves of the volume densities of rER and zymogen granules.These findings indicate that morphometric analysis of the subcellular structures of pancreatic acinar cells during 24 h can be used to determine their developmental stage.     

Proliferating cell nuclear antigen (PCNA): a key factor in DNA replication and cell cycle regulation

Background

PCNA (proliferating cell nuclear antigen) has been found in the nuclei of yeast, plant and animal cells that undergo cell division, suggesting a function in cell cycle regulation and/or DNA replication. It subsequently became clear that PCNA also played a role in other processes involving the cell genome.

Scope

This review discusses eukaryotic PCNA, with an emphasis on plant PCNA, in terms of the protein structure and its biochemical properties as well as gene structure, organization, expression and function. PCNA exerts a tripartite function by operating as (1) a sliding clamp during DNA synthesis, (2) a polymerase switch factor and (3) a recruitment factor. Most of its functions are mediated by its interactions with various proteins involved in DNA synthesis, repair and recombination as well as in regulation of the cell cycle and chromatid cohesion. Moreover, post-translational modifications of PCNA play a key role in regulation of its functions. Finally, a phylogenetic comparison of PCNA genes suggests that the multi-functionality observed in most species is a product of evolution.

Conclusions

Most plant PCNAs exhibit features similar to those found for PCNAs of other eukaryotes. Similarities include: (1) a trimeric ring structure of the PCNA sliding clamp, (2) the involvement of PCNA in DNA replication and repair, (3) the ability to stimulate the activity of DNA polymerase δ and (4) the ability to interact with p21, a regulator of the cell cycle. However, many plant genomes seem to contain the second, probably functional, copy of the PCNA gene, in contrast to PCNA pseudogenes that are found in mammalian genomes.     

Molecular characterization and expression of an alternate proliferating cell nuclear antigen homologue,PfPCNA2, in Plasmodium falciparum

The malaria parasite Plasmodium falciparum genome sequencing has revealed the existence of a second gene for proliferating cell nuclear antigen (PCNA), a key factor in a variety of DNA metabolic events. The alternate copy of PCNA (PfPCNA2) shows only 23% identity to an earlier reported P. falciparum PCNA homologue (PfPCNA1). Our analysis indicated structural conservation of PfPCNA2 compared to eukaryotic PCNAs. PfPCNA1 and 2 polypeptides showed differential expression in the intraerythrocytic cell cycle of the malaria parasite. PfPCNA1 expression slowly increases about threefold from the ring to the late schizont stage. In contrast PfPCNA2 showed robust expression in trophozoites and early schizonts with a sudden drop in expression in the late schizont stage, suggesting that the two PfPCNAs may function under different physiological conditions. Chemical cross-linking indicated the presence of a trimeric PfPCNA2 protein, indicating the possible existence of a functional ring-like PfPCNA2 structure.     

A morphometric study of developing pancreatic acinar cells of rats during prenatal life

Summary The pancreatic acinar cells of rat embryos obtained at 15, 17, 19 and 21 days of gestation have been examined using fine-structural and morphometric techniques.Morphometric analysis demonstrates significant variations in the average volume of the cell, nucleus and cytoplasm, and the volume, surface and numerical densities of various cytoplasmic organelles during fetal life. In particular, the volume and surface densities of rER exhibit maximal values at 19 days of gestation, suggesting that secretory proteins are produced most actively at this time. Further-more, membrane continuity between the nuclear envelope and rER is frequently discernible in acinar cells, indicating that at this stage the rER is mainly derived from the nuclear envelope. Zymogen granules first appear at 17 days of gesstation. By 21 days, they occupy the greater part of the cytoplasm of the acinar cells, no polarity being seen in their distribution pattern. No direct evidence for the secretion of zymogen granules has been observed during fetal life.It therefore appears that membrane transport involved with intracellular movement of newly synthesized proteins from rER via the Golgi complex to zymogen granules occurs in one direction and lacks regulation.     

Extracellular-signal regulated kinase 8 of Trypanosoma brucei uniquely phosphorylates its proliferating cell nuclear antigen homolog and reveals exploitable properties

The Trypanosoma brucei subspecies T. brucei gambiense and T. brucei rhodesiense are vector-borne pathogens that cause sleeping sickness also known as Human African Trypanosomiasis (HAT), which is fatal if left untreated. The drugs that treat HAT are ineffective and cause toxic side effects. One strategy for identifying safer and more effective HAT drugs is to therapeutically exploit essential gene targets in T. brucei. Genes that make up a basic mitogen-activated protein kinase (MAPK) network are present in T. brucei. Tb927.10.5140 encodes an essential MAPK that is homologous to the human extracellular-signal regulated kinase 8 (HsERK8) which forms a tight complex with the replication factor proliferating cell nuclear antigen (PCNA) to stabilize intracellular PCNA levels. Here we demonstrate that (TbPCNA) is uniquely phos-phorylated on serine (S) and threonine (T) residues in T. brucei and that TbERK8 phosphorylates TbPCNA at each of these residues. The ability of an ERK8 homolog to phosphorylate a PCNA homolog is a novel biochemical property that is first demonstrated here in T. brucei and may be unique to this pathogen. We demonstrate that the potent HsERK8 inhibitor Ro318220, has an IC₅₀ for TbERK8 that is several hundred times higher than its reported IC₅₀ for HsERK8. This indicated that the active sites of TbERK8 and HsERK8 can be selectively inhibited, which provides a rational basis for discovering inhibitors that specifically target this essential parasite MAPK to kill the parasite.     

The proliferating cell marker monoclonal antibody Ki-67 recognizes specific antigens associated with the nuclear envelope of the early Drosophila embryo

Summary— Immunofluorescence and immunoelectron microscopy indicated that the antibody raised against the nuclear antigen Ki-67 of mammalian cells recognized antigenic determinants of early Drosophila embryos, localized on the outside of the nuclear envelope. Hence, the nuclear envelope of Drosophila appears to share a similar epitope with the chromosome scaffold of mitotic mammalian cells. With the progression of mitosis the antigen persisted around the mitotic spindle region and was also found in the pole regions at metaphase and anaphase. The antibody also stained the equatorial regions of the spindles from anaphase to late telophase. The antibody may therefore be used as a biochemical marker of the nuclear envelope for studying nuclear membrane biogenesis and behavior during the mitotic divisions of the Drosophila embryo.     

Hair cell regeneration in the chick inner ear following acoustic trauma: ultrastructural and immunohistochemical studies

The regeneration of hair cells in the chick inner ear following acoustic trauma was examined using transmission electron microscopy. In addition, the localization of proliferation cell nuclear antigen (PCNA) and basic fibroblast growth factor (b-FGF) was demonstrated immunohistochemically. The auditory sensory epithelium of the normal chick consists of short and tall hair cells and supporting cells. Immediately after noise exposure to a 1500-Hz pure tone at a sound pressure level of 120 decibels for 48 h, all the short hair cells disappeared in the middle region of the auditory epithelium. Twelve hours to 1 day after exposure, mitotic cells, binucleate cells and PCNA-positive supporting cells were observed, and b-FGF immunoreactivity was shown in the supporting cells and glial cells near the habenula perforata. Spindle-shaped hair cells with immature stereocilia and a kinocilium appeared 3 days after exposure; these cells had synaptic connections with the newly developed nerve endings. The spindle-shaped hair cell is considered to be a transitional cell in the lineage of the supporting cell to the mature short hair cell. These results indicate that, after acoustic trauma, the supporting cells divide and differentiate into new short hair cells via spindle-shaped hair cells. Furthermore, it is suggested that b-FGF is related to the proliferation of the supporting cells and the extension of the nerve fibers.     

Interplay between DNA polymerase and proliferating cell nuclear antigen switches off base excision repair of uracil and hypoxanthine during replication in archaea

Archaeal family-B DNA polymerases bind tightly to uracil and hypoxanthine (the deamination products of cytosine and adenine), resulting in profound inhibition of DNA replication. Investigation of the mechanism of inhibition, using single-turnover kinetics with polymerase in excess of DNA, indicated that deoxy-NTPs were efficiently bound to the polymerase-DNA complex but very poorly incorporated into the extending chain. Addition of the processivity factor proliferating cell nuclear antigen (PCNA) resulted in increased affinity of the polymerase for all primer-templates, producing extremely tight complexes when uracil (K_d = 16 pM) or hypoxanthine (K_d = 65 pM) was present. Analytical ultracentrifugation confirmed the stability of these complexes and revealed a polymerase/PCNA/DNA stoichiometry of 1:1:1. However, PCNA had no influence on the ability of the polymerase to read through uracil and hypoxanthine, the same kinetic parameters being observed with or without the processivity factor. The specificity constants determined using single-turnover kinetics showed that uracil and hypoxanthine slowed the polymerase by factors of ∼ 5000 and 3000, respectively. Uracil and hypoxanthine are removed from DNA by base excision repair, initiated by uracil-DNA glycosylase and endonuclease V, respectively. Both enzymes are profoundly inhibited by the simultaneous binding of both PCNA and polymerase to primer-templates, with polymerase alone being much less effective. Thus, when the PCNA-polymerase complex encounters uracil/hypoxanthine in DNA templates, base excision repair is switched off, protecting the complex from a repair pathway that is dangerous in the context of single-stranded DNA formed during replication.     

Expression of proliferationg cell nuclear antigen during the cell cycle of human diploid fibroblasts

Summary Proliferating cell nuclear antigen mRNA levels were determined in human diploid fibroblasts as they progressed through the cell cycle. PCNA message levels were low at G0, gradually increased following entrance into G1, peaked at G1/S, and declined during S phase. PCNA mRNA was determined to have a half life of 12 hours when cells were blocked at the G1/S interface. PCNA protein levels increased two- to three-fold as cells moved from G0 to S phase.     

CD47基因对食道癌细胞增殖的影响

本研究将CD47-si RNA转染至食道癌细胞,采用蛋白免疫印迹检测食道癌细胞中CD47蛋白的表达,MTT法检测CD47-siRNA转染组和空质粒转染组(对照组)食道癌细胞增殖状态,蛋白免疫印迹检测CD47-siRNA转染组和空质粒转染组(对照组)食道癌细胞中PCNA蛋白表达,DCFDA染色流式细胞仪检测食道癌细胞CD47-siRNA转染组和空质粒转染组(对照组)中ROS水平,以探究CD47基因对食道癌发生发展的影响。研究结果表明,CD47-si RNA转染组食道癌细胞中CD47蛋白明显低于对照组;CD47-siRNA转染组食道癌细胞增殖率显著低于对照组(p<0.05);CD47-siRNA转染组细胞增殖相关蛋白PCNA低于对照组(p<0.01);CD47-siRNA转染组食道癌细胞中ROS水平明显高于对照组(p<0.05)。本研究初步认为:CD47-siRNA可降低食道癌细胞中CD47蛋白表达,抑制食道癌细胞的增殖并增加食道癌细胞中ROS水平。