Seventeen-year follow-up study on chromosomal aberrations in five victims accidentally exposed to several Gy of 60Co gamma-rays.

On 25 June 1990, a radiation accident occurred in a (60)Co source radiation unit in Shanghai, due to violations in operation regulations. This accident resulted in the exposure of seven individuals to acute high-dose and dose-rate whole-body external irradiation. Conventional chromosomal aberration analysis, G-banding automatic karyotype analysis and/or fluorescent in situ hybridization (FISH) painting methods were used to analyze chromosomal aberrations in peripheral blood lymphocytes from five of the victims 24 h to 17 years after accidental exposure to 1.9-5.1 Gy of (60)Co gamma-rays. The frequency of unstable chromosomal aberrations (dicentrics and rings) remained at constant levels 1 month after exposure. Three months after exposure, the frequency was reduced by 20-40% in three victims, while no reduction was seen in the other two victims. Twelve years after exposure, the number of dicentrics and rings decreased by more than 90%, and did not reveal a dose-dependent relationship. However, even at 12-17 years after exposure, stable chromosome aberrations, dominated by translocations, remained at a high level in a dose-dependent manner. The frequency of stable chromosomal aberrations detected by FISH showed a similar dose-dependent relationship as that detected by karyotype analysis of G-banding chromosomes. The G-banding analysis also suggested that the pattern of chromosome breakpoints is random. The FISH data showed a decreasing tendency with time for chromosome translocation frequency in the peripheral lymphocytes, and the rate of reduction varied among different individuals. It is likely that the higher dose the victim received, the lesser the translocation frequency decreased with time. The G-banding data also showed that the rate of reduction of translocations is different among individuals. From 5 to 17 years after accidental irradiation, a very small reduction (approximately 10%) of translocation frequency was observed in victims C and D, while there was about a 35% reduction (the highest among the victims) for victim G who received the smallest dose (1.9 Gy). These observations can be used to validate the existence of chromosomal aberrations in peripheral blood lymphocytes as a biological dosimeter for radiation exposures.

Analysis of differentially expressed genes in fetal skin of scarless and scar-forming periods of gestational rats.

OBJECTIVE: To study the differences of gene expression between earlier gestational skin and later gestational skin of rats with the aids of single primer amplification (SPA) and high-density oligonucleotide DNA array to understand the molecular mechanism of scarless healing. METHODS: Total RNAs were isolated from fetal rat skin of the scarless (E15) and scar-forming (E18) periods of gestation (term = 21.5 days). The RNAs from earlier gestational skin (EGS) and later gestational skin (LGS) were both reversely transcribed to cDNAs, then labeled with the incorporation of fluorescent dCTP for preparing the hybridization probes by SPA method. The mixed probes were then hybridized to the oligonucleotide DNA arrays which contained 5,705 probes representing 5,705 rat genes. After highly stringent washing, these DNA arrays were scanned for fluorescent signals to display the differentially expressed genes between the 2 groups of skin. RESULTS: Among 5,705 rat genes, there were 53 genes (0.93 percent) with differentially expressed levels between EGS and LGS groups, 27 genes, including fibroblast growth factor 2 (FGF2) and follistatin were up-regulated (0.47%) and 26 genes were down-regulated (0.46%) in fetal skin during scarless period versus scar-forming period. Higher expressions of FGF2 and follistatin in EGS than those in LGS were also revealed by RT-PCR method. CONCLUSIONS: High-density oligonucleotide DNA array provided a powerful tool for investigating differential gene expression in earlier and later gestational fetal skins. This technology validates that the mechanism of fetal scarless healing is very complicate and the change of many gene expressions is associated with fetal scarless healing.

Acid fibroblast growth factor reduces rat intestinal mucosal damage caused by ischemia-reperfusion insult.

AIM: To detect the effects of acid fibroblast growth factor (aFGF) on apoptosis and proliferation of intestinal epithelial cells in differentiation or proliferation status to explore the protective mechanisms of aFGF. METHODS: Wistar rats were randomly divided into sham-operated control group (C, n=6), intestinal ischemia group (I, n=6), aFGF treatment group (A, n=48) and intestinal ischemia-reperfusion group (R, n=48). Apoptosis of intestinal mucosal cells was determined with terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) technique. Proliferating cell nuclear antigen (PCNA) protein expression and distribution were detected with immunohistochemical method. Plasma levels of D-lactate were determined with modified Brandts method. RESULTS: In A group, administration of exogenous aFGF could improve intestinal histological structure and decrease plasma D-lactate levels at 2-12 h after the reperfusion compared with R group. The apoptotic rates and PCNA protein expressions were not increased until 2 h after reperfusion and were maximal at 12 h. After reperfusion for 2-12 h, the apoptotic rates were gradually augmented along the length of jejunal crypt-villus units. Administration of aFGF could significantly reduce the apoptotic response at 2-12 h after reperfusion (P<0.05). Apoptosis rates in villus and crypt epithelial cells in A group at 12 h after reperfusion were (62.5+/-5.5)% and (73.2+/-18.6)% of those in R group, respectively. Treatment of aFGF could apparently induce protein expression of PCNA in intestinal mucosal cells of A group compared with R group during 2-12 h after reperfusion (P<0.05). There were approximately 1.3- and 1.5-times increments of PCNA expression levels in villus and crypt cells in A group at 12 h after reperfusion compared with R group, respectively. CONCLUSION: Intestinal I/R insult could lead to histological structure change and apoptotic rate increment. The protective effects of aFGF against ischemia/reperfusion in rat intestinal mucosa might be partially due to its ability to inhibit ischemia/reperfusion-induced apoptosis and to promote cell proliferation of crypt cells and villus epithelial cells.

Exogenous acid fibroblast growth factor inhibits ischemia-reperfusion-induced damage in intestinal epithelium via regulating P53 and P21WAF-1 expression.

AIM: To detect the effect of acid fibroblast growth factor (aFGF) on P53 and P21WAF-1 expression in rat intestine after ischemia-reperfusion (I-R) injury in order to explore the protective mechanisms of aFGF. METHODS: Male rats were randomly divided into four groups, namely intestinal ischemia-reperfusion group (R), aFGF treatment group (A), intestinal ischemia group (I), and sham-operated control group (C). In group I, the animals were killed after 45 min of superior mesenteric artery (SMA) occlusion. In groups R and A, the rats sustained for 45 min of SMA occlusion and were treated with normal saline (0.15 mL) and aFGF (20 mug/kg, 0.15 mL), then sustained at various times for up to 48 h after reperfusion. In group C, SMA was separated, but without occlusion. Apoptosis in intestinal villi was determined with terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling technique (TUNEL). Intestinal tissue samples were taken not only for RT-PCR to detect P53 and P21WAF-1 gene expression, but also for immunohistochemical analysis to detect P53 and P21WAF-1 protein expression and distribution. RESULTS: In histopathological study, ameliorated intestinal structures were observed at 2, 6, and 12 h after reperfusion in A group compared to R group. The apoptotic rates were (41.17+/-3.49)%, (42.83+/-5.23)%, and (53.33+/-6.92)% at 2, 6, and 12 h after reperfusion, respectively in A group, which were apparently lower than those in R group at their matched time points (50.67+/-6.95)%, (54.17+/-7.86)%, and (64.33+/-6.47)%, respectively, (P<0.05)). The protein contents of P53 and P21WAF-1 were both significantly decreased in A group compared to R group (P<0.05) at 2-12 h after reperfusion, while the mRNA levels of P53 and P21WAF-1 in A group were obviously lower than those in R group at 6-12 h after reperfusion (P<0.05). CONCLUSION: P53 and P21WAF-1 protein accumulations are associated with intestinal barrier injury induced by I-R insult, while intravenous aFGF can alleviate apoptosis of rat intestinal cells by inhibiting P53 and P21WAF-1 protein expression.

Intravenous acid fibroblast growth factor protects intestinal mucosal cells against ischemia-reperfusion injury via regulating Bcl-2/Bax expression.

AIM: To detect the effect of acid fibroblast growth factor (aFGF) on apoptosis and gene expression of bax and bcl-2 gene in rat intestine after ischemia/reperfusion (I/R) injury, and to explore the protective mechanisms of aFGF. METHODS: One hundred and eight Wistar rats were randomly divided into sham-operated control group (C) (n = 6), intestinal ischemia group (I) (n = 6), aFGF treatment group (A) (n = 48) and intestinal ischemia-reperfusion group (R) (n = 48). In group I, the animals were killed after 45 min of superior mesenteric artery (SMA) occlusion, while in groups R and A, the rats sustained 45 min of SMA occlusion and were then treated with normal saline and aFGF, respectively, sustained 15 min, 30 min, 1, 2, 6, 12, 24, or 48 h of reperfusion, respectively. In group C, SMA was separated, but without occlusion. Apoptosis in intestinal villus was determined with terminal deoxynucleotidyl transferase mediated dUTP-biotin nick-end labeling technique (TUNEL). Intestinal tissue samples were taken not only for detection of bax and bcl-2 gene expression by RT-PCR, but also for detection of bax and bcl-2 protein expression and distribution by immunohistochemical analysis. RESULTS: The rat survival rates in aFGF treated group were higher than group R (P<0.05) and the improvement of intestinal histological structures was observed at 2, 6, and 12 h after the reperfusion in group A compared with group R. The apoptotic rates were (41.17+/-3.49)%, (42.83+/-5.23)% and (53.33+/-6.92)% at 2, 6 and 12 h after reperfusion, respectively in group A, apparently less than those of group R at matched time points (50.67+/-6.95, 54.17+/-7.86, 64.33+/-6.47, respectively) (P<0.05). The bax gene transcription and translation were significantly decreased in group A vs group R, while mRNA and protein contents of Bcl-2 in group A were obviously higher than those in group R during 2-12 h period after reperfusion. CONCLUSION: The changes in histological structure and the increment of apoptotic rate indicated that the intestinal barrier was damaged after intestinal I/R injury, whilst intravenous aFGF could alleviate apoptosis induced by ischemia and reperfusion in rat intestinal tissues, in which genes of bax and bcl-2 might play important roles.

Ontogeny of expression of basic fibroblast growth factor and its receptors in human fetal skin.

OBJECTIVE: To investigate the expression characteristics of basic fibroblast growth factor (bFGF) and its receptors, flg (FGFR1) and bek (FGFR2), in fetal skin at different gestational ages underlying the relevance of these 3 proteins to skin development and the mechanisms underlying the phenotypic transition from scarless to scar-forming healing. METHODS: Eighteen specimens of fetal skin biopsies of human embryo were obtained from spontaneous abortions at different gestational ages of 13-32 weeks. Gene expression of bFGF, bek and flg was examined with reverse transcription-polymerase chain reaction (RT-PCR). The dynamic expression and distribution of these 3 proteins were detected with streptavidin peroxidase (SP) immunohistochemical staining method. RESULTS: In the early gestational fetal skin, genes of bFGF and flg were strongly expressed and more protein contents of these 2 proteins were found as compared with the genes at late gestation fetal skin (2.446+/-0.116 and 2.066+/-0.152 versus 2.157+/-0.101 and 1.818+/-0.086, respectively, P<0.05). On the contrary, the levels of gene expression and protein content of bek were not differently expressed in the early gestational fetal skin versus the late ones. Protein particles of bFGF were mainly distributed in the epidermal cells and some fibroblasts. Bek was mainly located in the cell membrane and cytoplasm of epidermal cells while flg protein was principally located in the epidermal cells, endothelial cells and some fibroblasts. CONCLUSIONS: The endogenous bFGF and their receptors might be involved in the cutaneous development at fetal stage. The differently expressing levels of bFGF and flg during gestation may be related to scarless or scar-forming repair during gestation.

Development of gene microarray in screening differently expressed genes in keloid and normal-control skin.

BACKGROUND: Keloid is an intricate lesion that is probably regulated by many genes. In this study, the authors used the technique of complementary DNA (cDNA) microarray to analyse abnormal gene expression in keloids and normal control skins. METHODS: The polymerase chain reaction (PCR) products of 8400 genes were spotted in an array on chemical-material-coated-glass plates. The DNAs were fixed on the glass plates. The total RNAs were isolated from freshly excised human keloid and normal control skins, and the mRNAs were then purified. The mRNA from both keloid and normal control skins were reversely transcribed to cDNAs, with the incorporation of fluorescent dUTP, for preparing the hybridisation probes. The mixed probes were then hybridised to the cDNA microarray. After thorough washing, the cDNA microarray was scanned for differing fluorescent signals from two types of tissues. Gene expression of tissue growth factor-beta1 (TGF-beta1) and of c-myc was detected with both RT-PCR and Northern blot hybridisation to confirm the effectiveness of cDNA microarray. RESULTS: Among the 8400 human genes, 402 were detected with different expression levels between keloid and normal control skins. Two hundred and fifty genes, including TGF-beta1 and c-myc, were up-regulated and 152 genes were down-regulated. Higher expressions of TGF-beta1 and c-myc in keloid were also revealed using RT-PCR and Northern blot methods. CONCLUSION: cDNA microarray analysis provides a powerful tool for investigating differential gene expression in keloid and normal control skins. Keloid is a complicated lesion with many genes involved.

[Effects of acute hypoxia and intermittent hypoxic acclimatization on erythropoietin and hypoxia-inducible factor-1alpha gene expression in rat hepatic and renal tissues].

OBJECTIVE: To explore the effects of acute hypoxia and intermittent hypoxic acclimatization on gene expression of erythropoietin (EPO) and hypoxia-inducible factor-1alpha (HIF-1alpha) in rat hepatic and renal tissues. METHODS: Twenty-four rats (weight was 180 to 220 g) were divided into three groups: normal control group (NC), intermittent hypoxic acclimatization (IH) and acute hypoxia groups (AH). Gene expression of EPO and HIF-1alpha were examined with Northern dot blot method. RESULTS: As compared with NC group, the levels of EPO gene expression in rat hepatic and renal tissues in AH group were both significantly elevated (P<0.05 or P<0.01). In IH group, the contents of EPO mRNA in hepatic and renal tissues were apparently decreased versus AH group (P<0.01), whilst were not differently elevated in comparison with group NC (P>0.05). In hepatic tissue from AH group, the content of HIF-1alpha mRNA were more than those in NC group and IH group, while the levels of gene expression of HIF-1alpha were no substantial change in renal tissues from different groups (all P>0.05). CONCLUSION: Hypoxic acclimatization can inhibit the increment of EPO gene expression induced by acute hypoxia in rat hepatic and renal tissues, in which HIF-1alpha may play important roles.

[Expression characteristics of transforming growth factor-beta1 in human skin at different development stages of gestation].

OBJECTIVE: To investigate gene expression of transforming growth factor-beta(1) (TGF-beta(1)) and its two upstream signalling factors (smad2 and smad3) in fetal skin at different gestational ages and postnatal skin and its potential biological significance. METHODS: Fetal skin samples of human embryo were obtained from spontaneous abortions at different gestational ages ranging from 13 to 32 weeks, and also skin collected from patients undergoing plastic surgery. After morphological characteristics of skin at different developmental stages were examined histologically, gene expressions of TGF-beta(1), smad2 and smad3 in skin specimens at different developmental stages were examined with reverse transcription-polymerase chain reaction analysis (RT-PCR). RESULTS: Gene expression of TGF-beta(1), smad2 and smad3 could all be detected in fetal skin and skin after birth. In skin from early gestational fetus, gene expressions of TGF-beta(1) and smad2 were weak. Along with advance in gestational age, gene expression of these two genes in skin became progressively stronger. In skin from late gestational fetus and skin after birth, the transcription contents of these two genes were significantly increased compared with early gestation fetus (P<0.05). On the contrary, gene expression of smad3 was apparently higher in younger fetal skin versus elder compared with that of late fetal skin (P<0.05). In skins after birth, the levels of smad3 gene expression were elevated to the level similar to that in early gestational fetal skin. CONCLUSION: The signal pathway mediated by TGF-beta(1) might be involved in regulating development of the skin at embryonic stage and in designating cetaceous structure and function, and also in wound healing after birth. The relative lack in expression of TGF-beta(1) and smad2 genes in skins from younger fetuses might contribute to fetal scar-less healing, in which the role of smad3 needs to be further investigated.

[Gene expression of angiogenesis-related factors in fetal skin at different developmental stages and childhood skin].

OBJECTIVE: To explore the change in gene expression of angiogenesis-related factors vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1), acid fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF), in fetal skin at different developmental stages and children skin and their potential biological significances. METHODS: Fetal skin samples of human embryo were obtained from spontaneous abortion at different gestational ages ranging from 13 to 32 weeks, and children skin specimens were collected from child patients (4-12 years) undergoing plastic surgery. After morphological characteristics of skin at different developmental stages were defined histologically gene expressions of VEGF, Ang-1, aFGF and bFGF were examined with reverse transcription-polymerase chain reaction analysis (RT-PCR). RESULTS: The trend of changes in gene expression of VEGF, Ang-1, aFGF and bFGF was not same for different skin specimens at various developmental stages. In early gestational fetal skin, genes of VEGF and Ang-1 were strongly expressed, while in late gestational and childhood skins, gene expressions of VEGF and Ang-1 were apparently decreased. In skin of middle gestational stage, the level of aFGF gene expression was highest, and then it was progressively reduced. In childhood skin, this gene was weakly expressed. In marked contrast, the contents of transcripts of bFGF showed no substantial change in fetal skin at different developmental stages, whilst the mRNA content of bFGF was significantly decreased in childhood skin. CONCLUSION: VEGF, Ang-1, aFGF and bFGF might be involved in regulating angiogenesis in skin from fetuses of different gestational stages and children. The relative increase in gene transcription of VEGF and Ang-1 in younger fetal skin might be one of the reasons why cutaneous cells proliferate rapidly and the wounds heal without scar.

[Effects of acute hypoxia and intermittent hypoxic acclimatization on vascular endothelial growth factor gene expression in HepG2 cells].

AIM: To explore the influence of acute hypoxia and intermittent hypoxic acclimatization on vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1alpha (HIF-1alpha) gene expression in HepG2 cells underlying their possible biological significance. METHODS: HepG2 was cultured in 1% O2 for 24 hours, then in 21% O2 for another 24 hours, which composed a hypoxic exposure cycle. After 6 cycles, HepG2 cells reached the status of hypoxic acclimatization. Gene transcription and translation of VEGF and HIF-1alpha were detected with Northern blot and Western blot methods. RESULTS: Acute hypoxia could induce gene transcription and translation of VEGF and HIF-1alpha. After intermittent hypoxia acclimatization, the contents of VEGF and HIF-1alpha mRNA were 108.6% +/- 17.7% and 116.7% +/- 19.8% of those in normoxic control cells, while the protein contents were significantly increased to 1.4 and 2.7 times of those in control cells, respectively (P < 0.05). The protein expression levels of VEGF and HIF-1alpha were decreased in cells subjected to hypoxia acclimatization compared to cells treated with acute hypoxia. CONCLUSION: When HepG2 cells reached the status of hypoxic acclimatization, the acute hypoxia-induced increment of VEGF gene transcription and translation in cells were inhibited, in which HIF-1alpha might play an important role.

[Gene expression of extracellular-signal regulated protein kinase 5 and their MAPKK in fetal skin hypertrophic scars].

OBJECTIVE: To explore the change of gene expression of extracellular-signal regulated protein kinase 5 (ERK5) and its upstream signaling molecule (MEK5) in fetal skin of differentially developmental stages and hypertrophic scars. METHODS: After morphological characteristics of skin of different developmental stages and hypertrophic scars were detected with pathological methods, gene expression of ERK5 and MEK5 was examined with reverse transcription-polymerase chain reaction analysis (RT-PCR). RESULTS: In early gestational fetal skin, genes of ERK5 and MEK5 were strongly expressed, while in late gestational skin and children skin, the expression of ERK5 and MEK5 was apparently decreased (P < 0.05). In normal skin, the level of gene expression of ERK5 was lower. In proliferative hypertrophic scars, mRNA content of this gene was apparently increased. In mature scars, the content of this gene transcript was 3.2 times the normal skin. In contrast, the levels of MEK5 transcript in normal skin and hypertrophic scars of various phases showed no substantial changes (P > 0.05). CONCLUSION: ERKS medicating signaling pathway might be involved in regulating cutaneous development at the embryonic stage and determining cutaneous structure ad function. The increase of gene transcription of ERK5 and MEK5 in younger fetal skin might be a reason for rapid proliferation of the skin cells and scraless healing of skin. The activation of ERK5 gene expression in hypertrophic scars versus normal skin might be one of the mechanisms controlling the formation of hypertrophic scars, in which the role of MEK5 needed to be further studied.