ABSTRACT
Objective To investigate the function of Kiss1 gene and estrogen receptor α gene (ERα gene) in puberty of rats,by detecting the expressions of Kiss1 mRNA and ERα mRNA in the hypothalamus and the serum luteinizing hormone (LH) and estradiol (E2) level of female Sprague-Dawley (SD) rats at various stage of development with Real-time PCR and enzyme-linked immunosorbent assay(ELISA).Methods Thirty-five female SD rats of 3 days were weaned on postnatal(PND)22 and then the vaginal opening condition was observed daily.The rats were sacrificed at PND 15(juvenile group,n =19) and PND 35 (pubertal group,n =16).The hypothalamus were segregated and the serum were extracted from heart blood.All of the samples were stored at-80 ℃ prepared.Then the mRNA were extracted from the hypothalamus and the cDNA obtained by reverse transcription were tested with real-time PCR.The relative mRNA expression level of Kiss1 gene and ERα gene were calculated.Results 1.Entire level:it was found that the pubertal group vaginal opening time was (32.1 ± 1.0) days,while the juvenile group was not found with vaginal opening until sacrificed.2.Real-time PCR:the expressions of Kiss1 and ERα gene were significantly increased in pubertal group (Kissl gene:5.39-± 2.52,ERα gene:1.57 ±1.87) compared with juvenile group(Kiss1 gene:1.06 ± 1.09,ERα gene:0.59-± 0.68),and the differences were statistically significant (all P ＜ 0.001).3.ELISA:the serum LH and E2 in pubertal group [LH (11.61 ± 0.95) IU/L,E2 (167.53 ± 31.09) ng/L] were significantly higher than LH [(5.46-± 1.89)IU/L] and E2 [(58.59 ± 29.96) ng/L] in juvenile group,and the differences were statistically significant (all P ＜0.001).Conclusion Kiss1 gene and ERα gene are involved in the start of the sexual development of female SD rat.
ABSTRACT
<p><b>OBJECTIVE</b>Fluctuations in arterial oxygen are associated with development of severe retinopathy of prematurity (ROP) in humans. However, the causal relationship between oxygen variability and ROP remains unknown. The authors developed a rat model of retinal neovascularization by repeated fluctuations of inhaled oxygen between hypoxia and hyperoxia to investigate the mechanism of the development of retinal neovascularization, the regulation of vascular endothelial growth factor (VEGF) and KDR/Flk-1 (VEGFR-2) expression.</p><p><b>METHODS</b>Two hundred and eight newborn Sprague Dawley rats were randomly divided into oxygen and air groups. The oxygen concentration in the oxygen group was alternated between 50% and 10% every 24 hours for 14 days. The control group were kept in room air environment. VEGF and Flk-1 expression was observed at 14, 18 and 25 days after birth in both exposed group and control group by immunohistochemical staining and semiquantitative RT-PCR. The status of retinal vasculature on day 4 after oxygen exposure was also observed. The retinas were dissected and stained by using a histochemical method for detecting adenosine diphosphatase (ADPase) activity, digital images of the retinas were captured and the peripheral avascular retina were measured. HE staining on methacrylate sections of eyes was used for counting the number of nuclei extending from retinal area into vitreous to identify extraretinal neovascularization. Numeric data were expressed as the mean +/- standard deviation (SD). Statistical calculations were performed using the SAS 8.1 statistical package. Differences in measured variables between experimental and control groups were determined using comparison of the means using two-way analysis of variance (ANOVA) statistical calculations and T-test. AP value less than 0.05 was regarded as significant.</p><p><b>RESULTS</b>(1) The animal model was successfully established: the avascular areas of retina of 18-day-old rats were larger than those of the control group and the numbers of nuclei extending from retinal area into vitreous in exposed group were significantly higher compared to the control (P < 0.05). (2) The expression of VEGF and Flk-1 on the 14(th) day in the oxygen group was significantly stronger than that of the control group (P < 0.05). In the oxygen group, VEGF and Flk-1 expression was the strongest in the retina on the 18(th) day, the result had significant difference as compared with the 14(th) and 25(th) day (P < 0.05), and they were also stronger than that of the control group (P < 0.05). The expression of VEGF and Flk-1 decreased on the 25(th) day and had no significant difference as compared with the control group (P > 0.05). (3) Both VEGF-mRNA and Flk-1-mRNA significantly increased on the 14(th) day and the 18th day (P < 0.05). On the 25(th) day, the amounts of VEGF-mRNA and Flk-1-mRNA were similar between the control and oxygen group (P > 0.05).</p><p><b>CONCLUSION</b>Fluctuation in oxygen is associated with the development of retinal neovascularization in the retinopathy. Increased expressions of VEGF and Flk-1 in the oxgen fluctuations-induced neovascularized retina suggested that VEGF and Flk-1 might play a critical role in the pathogenesis of ROP. The results also indicated the positive feedback in the pathogenesis of ROP that the synergistic interaction of VEGF and Flk-1 in the retinal vascular proliferation. These findings provide insight into the effect of repeated oxygen fluctuation on the development of severe ROP in preterm infants.</p>