Small hairpin RNA (shRNA) was used to silence the HIF1alpha gene in human retinal pigment epithelial cells (RPE) under hypoxia in order to observe the effect of gene silencing on the expression of matrix metalloproteinase tissue inhibitor 1 (TIMP1). By using chemical hypoxic inducer CoCl2, to mimic RPE hypoxic environment, shRNA against the targeting region of HIF1alpha mRNA sequence was synthesized by a method of in vitro transcription, and the HIF1alpha was interfered in RPE cultured under hypoxia (induced by 150 micromol/L CoCl2). RT-PCR was employed to detect the expression of HIF1alpha and TIMP1. The expression levels of HIF1alpha and TIMP1 were measured by using Western blotting. The results showed that after the RPE were transfected with specific shRNA against HIF1alpha mRNA, RT-PCR revealed that under hypoxia, the efficacy of HIF1alpha gene silencing in RPE was 83.4%. Western blotting revealed that the expression levels of HIF1alpha protein was dramatically dropped. In addition. RT-PCR results demonstrated that the expression of TIMP1 mRNA was decreased by 28.9%, and the expression levels of TIMP1 protein were also significantly reduced by Western blotting. It was suggested that shRNA targeted against HIF1alpha mRNA could effectively silence the HIF1alpha gene, subsequently effectively inhibit the hypoxia-induced up-regulation of TIMP1.
Cell Hypoxia ; Cells, Cultured ; Gene Silencing ; Hypoxia-Inducible Factor 1, alpha Subunit/*genetics ; Pigment Epithelium of Eye/cytology ; Pigment Epithelium of Eye/*metabolism ; RNA, Messenger/biosynthesis ; RNA, Messenger/genetics ; RNA, Small Interfering/*pharmacology ; Retina/cytology ; Retina/*metabolism ; Tissue Inhibitor of Metalloproteinase-1/*biosynthesis ; Tissue Inhibitor of Metalloproteinase-1/genetics

Background Hypoxia is an important cause resulting in many retinal diseases,such as retinal edema,diabetic retinopathy,proliferative retinopathy and so on.ObjectiveThis study is to investigate the effects of hypoxia on the expression of AQP-4 in Müller cells in vitro.MethodsMüller cells were isolated from New Zealand white rabbits and primarily cultured in DMEM containing 20% fetal bovine serum by the explant culture method.The cells were identified by immunostaining for the glial fibrillary acidic protein(GFAP).Generation 2 of cells was cultivated with the chemical hypoxia inducer,CoCl_2,for 24 hours in the hypoxic group and only with DMEM in the control group.The expression of the AQP-4 protein in Müller cells was detected by immunocytochemistry.The expression of AQP-4 mRNA was determined by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR).ResultsAbout 90% of Müller cells(generation 2) presented a positive immunoreactivity for GFAP,showing a brown staining in the cytoplasm.Cultured cells displayed the presence of intermediate filaments,microvillus and various cellular organs.The Integralabsorbance of the AQP-4 protein in Müller cells was markedly increased 24 hours after incubation with CoCl_2 in comparison with the control group (t=6.74,P＜0.05).The expression level of AQP-4 mRNA in Müller cells was significantly enhanced 24 hours after incubation with CoCl_2 in comparison with the control group (t=21.79,P＜0.05). ConclusionHypoxia enchances the expression of AQP-4 in Müller cells and further increases fluid accumulation in the retina.These results suggest that Müller cells play an important role in the formation of retinal edema in diabetic retinopathy or proliferative retinopathy.

In order to investigate the application of par plana vitrectomy in the treatment of combined rhegmatogenous retinal detachment (RRD) and choroidal detachment (CD), 12 eyes of 12 cases of combined RRD and CD were retrospectively analyzed. Twelve eyes of 12 cases were subjected to par plana vitrectomy. Six mm infusion channels were used. Pars plana vitrectomy, membrane peeling and internal fluid－gas exchange with encircling scleral buckle placement were performed in a standard fashion. One patient received injection of silicone oil. Hormones were routinely administered pre- and post-operation. The results showed that the intraocular pressure was rapidly reconstructed in the 12 eyes of 12 cases, the fluid in the subchoroidal cavity was drained via the three sclera incisions. The detached choroidea replaced. No other sclera incision was needed to drain the fluid in the subchoroidal cavivity. The follow－up after operation lasted 2 to 16 months. The 12eyes were replaced anatomically. No postoperative proliferation of vitreous body and retina was induced. It was suggested that par plana vitrectomy was the first choice in the treatment of CD combined with RCD.

In order to investigate the effects of TGF-beta1 on the expression of MMP-2, -9 and TIMP-1 in human retinal pigment epithelial (RPE) cells, the third-sixth passage cultured RPE cells were treated with TGF-beta1 at different concentrations (0.01, 0.1, 1.0, 10 ng/mL), the expression of MMP-2, -9 and TIMP-1 mRNA was detected by semi-quantitative RT-PCR assays. MMP-2, -9 and TIMP-1 mRNA were expressed in the cultured RPE cells. The values of MMP-2/beta-actin in the cells treated with 0.1, 1.0, 10 ng/mL TGF-beta1 were 1.04 +/- 0.04, 1.07 +/- 0.02 and 1.11 +/- 0.03, respectively, significantly higher than in the control group (0.96 +/- 0.03, P < 0.05-0.01). The expression of MMP-2 mRNA could be up-regulated by TGF-beta1, in a dose-dependent manner. The expression of MMP-9 mRNA in the cultured RPE cells was slightly up-regulated by various TGF-beta1 concentrations treatment. The values of TIMP-1/beta-actin in the cells treated with 0.01 and 0.1 ng/ mL TGF-beta1 were 0.85 +/- 0.01 and 0.97 +/- 0.02 respectively, significantly lower than in the control group (1.07 +/- 0.04, P < 0.01), indicating that the expression of TIMP-1 mRNA was down-regulated by TGF-beta1 at low concentrations. But along with the increase of TGF-beta1 concentrations (1.0 and 10 ng/mL), the expression of TIMP-1 mRNA was slightly up-regulated, not significantly different from that in the control group (P > 0.05). It was concluded that TGF-beta1 might play an important role in the up-regulation of the expression of MMP-2 in RPE cells and result in a directional shift in the balance between MMP and TIMP. This may be facilitated for RPE cells to migrate in the pathogenesis of vitreoretinopathy.

BACKGROUND:Researches on attentional blink (AB) emphasized the importance of the distractors directly following targets and that of the leading distractors in rapid serial visual presentation (RSVP) streams in the production of the AB,but little has been mentioned about the subsequent distractors after the second target (except the one in direct succession to it).OBJECTIVE: To investigate the effect of subsequent distractors in RSVP stream on AB after the second target (T2).DESIGN: A randomized and controlled study.SETTING: Laboratory of Cognitive Science, South-central University for Nationalities.PARTICIPANTS: Twenty-five undergraduates aging 18-21 years with the mean age of 19 years were selected from South-central University for Nationalities. All undergraduates participated in the experiment did not have nervous mental diseases but had normal sight or corrected visual acuity; meanwhile, all of them provided the confirmed consent. The subjects were divided into experimental group (n =14) and control group (n =11). There were no significant differences between the two groups in age and sex (P ＞ 0.05).METHODS: The experiment was carried out in the Laboratory of Cognitive Science, South-central University for Nationalities from January to April 2007. ① Experimental intervention: The stimuli were RSVP streams consisted of digit distractors and two letter targets (T1 and T2). ② Experimental grouping: The experimental group participated in the omitted condition and the control group participated in the preserved condition. ③ Experimental analysis: One-way ANOVA was used to evaluate the data for statistically significant difference in the same group, and t test was used to compare data between two groups.MAIN OUTCOME MEASURES: Report accuracy of T1 and T2.RESULTS: A total of 25 subjects were involved in the final analysis. ① Report accuracy in the preserved condition:Correct identification of the first target, averaged across all lags, was 94.6%. The percentages of correct identification of the second target as a function of lag was high at Lag1 (92.7%), dropped dramatically at Lag2 (79.8%), then kept improving with increase of lag, revealing a significant effect of lag [F(4,40) = 10.98, P＜ 0.01]. ② Report accuracy in the omitted condition: Correct identification of the first target T1, averaged across all lags, was 96.2%. The percentages of correct identification of the second target as a function of lag, T2 report was high at Lag1 (94.4%), decreased at Lag2(84.4%), then improved at Lag3 (91.1%), but dropped remarkably again at Lag5 and Lag7 (44.9 vs. 43.9%), revealing a significant effect of lag [F (4,52) = 224.0, P ＜ 0.01]. ③ Comparison of the results in two conditions: T2 accuracy in the omitted condition was significantly lower than that in the preserved condition at Lag5 [t (23)=34.44, P ＜ 0.01], and Lag7 [t (23)=42.56, P ＜ 0.01], but did not differ from each other at Lag1 [t (23)=0.65, P ＞ 0.05], at Lag2 [t (23)=1.04, P ＞0.05], and at Lag3 [t (23)=0.64, P ＞ 0.05].CONCLUSION: The absence of subsequent distractors after T2 can introduce a bias on the attentional status of subjects to bring the T2 accuracy down to unusual low level at long lags.

Objective To assess the application value of NCE-MRA using iMSDE prepared bSSFP sequence in lower limb in patients with diabetes. Methods This prospective study included a total of 35 patients with type II diabetes who underwent CE-MRA on the 1.5T MR scanner after the NCE-MRA. The obtained MIP images were independently rated by two radiologist with a four score table and using CE-MRA as a reference standard to evaluate the diagnostic accuracy of NCE-MRA for the narrowed arteries. The difference of the percent age of diagnostic arterial segments between NCE-MRA and CE-MRA on diabetic patients was evaluated by the χ2 test. Results Compared with CE-MRA, the diagnostic-value arterial segment ratio of pelvic arteries on NCE-MRA is decreased significantly. The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of two readers in NCE-MRA were 95%/87%, 96%/95%, 57%/72%, 99%/99%, and 96%/95%(k=0.76), respectively. Conclusions The NCE-MRA using iMSDE prepared bSSFP sequence is capable of depicting vascu-lar lesions for the lower extremities in diabetic patients with the advantages of contrast agent free, short scan times and good diagnostic value in the thigh and calves.

Small hairpin RNA (shRNA) was used to silence the HIF1α gene in human retinal pigment epithelial cells (RPE) under hypoxia in order to observe the effect of gene silencing on the expression of matrix metalloproteinase tissue inhibitor 1 (TIMP1). By using chemical hypoxic inducer CoCl2 to mimic RPE hypoxic environment, shRNA against the targeting region of HIF1α mRNA sequence was synthesized by a method of in vitro transcription, and the HIF1α was interfered in RPE cultured under hypoxia (induced by 150 μmol/L CoCl2 ). RT-PCR was employed to detect the expression of HIF1α and TIMP1. The expression levels of HIF1α and TIMP1 were measured by using Western blotting. The results showed that after the RPE were transfected with specific shRNA against HIF1α mRNA, RT-PCR revealed that under hypoxia, the efficacy of HIF1α gene silencing in RPE was 83. 4 %. Western blotting revealed that the expression levels of HIF1α protein was dramatically dropped. In addition, RT-PCR results demonstrated that the expression of TIMP1 mRNA was decreased by 28.9 %, and the expression levels of TIMP1 protein were also significantly reduced by Western blotting. It was suggested that shRNA targeted against HIF1α mRNA could effectively silence the HIF1α gene, subsequently effectively inhibit the hypoxia-induced up-regulation of TIMP1.

In order to investigate the effects of TGF-β1 on the expression of MMP-2, -9 and TIMP1 in human retinal pigment epithelial (RPE) cells, the third-sixth passage cultured RPE cells were treated with TGF-β~ at different concentrations (0.01, 0. 1, 1.0, 10 ng/mL), the expression of MMP-2, -9 and TIMP-1 mRNA was detected by semi-quantitative RT-PCR assays. MMP-2, -9 and TIMP-1 mRNA were expressed in the cultured RPE cells. The values of MMP-2/β-actin in the cells treated with 0.1, 1.0, 10 ng/mL TGF-β1 were 1.04±0.04, 1.07±0.02 and 1.11±0.03, respectively, significantly higher than in the control group (0. 96±0.03, P＜0. 05-0. 01). The expression of MMP-2 mRNA could be up-regulated by TGF-β1, in a dose-dependent manner. The expression of MMP-9 mRNA in the cultured RPE cells was slightly up-regulated by various TGF-β1 concentrations treatment. The values of TIMP-1/β-actin in the cells treated with 0.01 and 0. 1 ng/mL TGF-β1 were 0. 85±0.01 and 0.97 ± 0.02 respectively, significantly lower than in the control group (1.07±0.04, P＜0.01), indicating that the expression of TIMP-1 mRNA was down-regulated by TGF-β1 at low concentrations. But along with the increase of TGF-β1 concentrations (1.0 and 10 ng/mL), the expression of TIMP-1 mRNA was slightly up-regulated, not significantly different from that in the control group (P＞0.05). It was concluded that TGF-β1 might play an important role in the up-regulation of the expression of MMP-2 in RPE cells and result in a directional shift in the balance between MMP and TIMP. This may be facilitated for RPE cells to migrate in the pathogenesis of vitreoretinopathy.

Objective:To explore the chain mediating effect of future orientation and academic engagement between perceived teacher support and academic procrastination in high school students.Methods:From December 2021 to February 2022, a survey was conducted on 550 high school students by the perceived teacher support questionnaire, the adolescent future orientation scale, the academic engagement scale, and the general procrastination scale-for student populations (GPS). Data were entered by EpiData 3.1 software, and SPSS 26.0 software was used to process and analyze the data by one-way ANOVA, Pearson correlation analysis and Bootstrap method test.Results:The scores of perceived teacher support, future orientation, academic engagement and academic procrastination were (3.77±0.80), (3.22±0.48), (68.11±18.08) and (52.32±11.78) respectively.The results of correlation analysis showed that academic procrastination was negatively correlated with perceived teacher support, future orientation and academic engagement ( r=-0.32, -0.38, -0.49, all P<0.01), while perceived teacher support was positively correlated with future orientation and academic engagement ( r=0.40, 0.43, both P<0.01). Future orientation was positively correlated with academic engagement ( r=0.56, P<0.01). The mediating effect analysis showed that perceived teacher support had a significant direct effect on academic procrastination (effect value: -0.10, 95% CI =-0.19--0.02), accounting for 32.26% of the total effect.The mediating effect between perceived teacher support and academic procrastination was found between future orientation and academic engagement (effect value: -0.05, 95% CI =-0.09- -0.02; effect values: -0.09, 95% CI=-0.15--0.05), accounting for 16.13% and 29.03% of the total effect respectively.Future orientation and academic engagement had a chain mediating effect between perceived teacher support and academic procrastination (effect value: -0.07, 95% CI=-0.10--0.04), accounting for 22.58% of the total effect. Conclusion:Perceived teacher support can influence academic procrastination, not only through the direct path, but also through the indirect path of future orientation and academic engagement, as well as chain mediating path between these two variables.

Objective:To evaluate the surgery combined chemotherapy and radiation in locally advanced neuroendocrine carcinoma of the cervix (NECC) .Methods:This is a single-center retrospective cohort study. Locally advanced NECC patients admitted to Peking Union Medical College Hospital, Chinese Acadmy of Medical Sciences from January 2011 to April 2022 were enrolled. They were divided into concurrent chemoradiotherapy group, and surgery combined with chemotherapy and radiation group. The Kaplan-Meier method was used to analyze the progression free survival (PFS), overall survival (OS), recurrence rate, and mortality rate.Results:(1) Forty-six cases were included, 22 in concurrent chemoradiotherapy group, 24 in surgery combined chemotherapy and radiation group. With 16 patients (35%, 16/46) received neoadjuvant chemotherapy (NACT), the NACT effective rate was 15/16. (2) The median follow-up time was 27.5 months (range: 10-106 months), with 26 (57%, 26/46) experienced recurrences. There were 4 (9%, 4/46) pelvic recurrences and 25 (54%, 25/46) distant recurrences, and 3 (7%, 3/46) both pelvic and distant recurrences. Compared with concurrent chemoradiotherapy group, surgery combined chemotherapy and radiation group had lower pelvic recurrence rate [14% (3/22) vs 4% (1/24); χ2=1.296, P=0.255] but without statistic difference. Both groups had similar distant recurrence rate [55% (12/22) vs 54% (13/24); χ2=0.001, P=0.979] and overall recurrence rate [59% (13/22) vs 54% (13/24); χ2=0.113, P=0.736]. (3) During the follow-up period, 22 cases (48%, 22/46) died, with 11 cases (50%, 11/22) in concurrent chemoradiotherapy group and 11 cases (46%, 11/24) in surgery combined chemotherapy and radiation group, without significant difference ( χ2=0.080, P=0.777). The postoperative 3-year and 5-year OS rates were 62.3% and 36.9%. Compared with concurrent chemoradiotherapy group, the patients in surgery combined chemotherapy and radiation group showed an extended trend in PFS (17.0 vs 32.0 months) and OS (37.0 vs 50.0 months) but without statistic differences ( P=0.287, P=0.125). Both groups had similar 3-year OS rate (54.2% vs 69.9%; P=0.138) and 5-year OS rate (36.1% vs 38.8%; P=0.217). Conclusions:Our study supports the multi-modality treatment strategy (including surgery, chemotherapy and radiation) as an important component in the treatment of locally advanced NECC. The combination of surgery, chemotherapy and radiation seems to have advantages in the treatment of locally advanced NECC, but needs to be confirmed by further multicenter studies.