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Objective:To explore the changes in morphology and function of meibomian gland and the expressions of inflammatory factors and lipid metabolic factors in meibomian gland of diabetic mice.Methods:Fifty 8-week-old male C57BL/6 mice of clean degree were divided into normal control group ( n=20) and diabetes model group ( n=30) according to a random table.Diabetes model was established by the intraperitoneal injection of streptozotocin (60 mg/kg, 10 mg/ml). Mouse tail vein blood glucose ≥16.7 mmol/L was considered as successful modeling.Blood glucose was measured weekly, and body weight was compared between the two groups.Ten mice were randomly selected for fluorescein sodium staining of the cornea to evaluate the integrity of the corneal epithelium from both groups at an interval of 4 weeks.Five mice were randomly selected from the two groups and were sacrificed via anesthesia to collect meibomian gland tissue for hematoxylin and eosin staining in order to observe morphological changes at 8 and 16 weeks after modeling, respectively.At 16 weeks following modeling, mebomian gland of 5 mice randomly selected from both groups was stained with oil red O staining to observe the distribution of lipid.Real-time fluorescence quantitative-PCR was performed to detect the relative expressions of tumor necrosis factor (TNF)-α, pigment epithelium derived factor (PEDF), peroxisome proliferators-activated receptor γ (PPARγ), and adipose differentiation-related protein (ADFP) mRNA in meibomian gland.The use and care of animals complied with the ARVO statement.This study protocol was approved by the Institutional Animal Care and Use Committee of Tianjin Medical University Eye Hospital (No.TJYY20190630009). Results:The successful modeling rate of diabetes in mice was 100%, and the survival rate was 83.3% (25/30). The weight was significantly lower and the blood glucose level was higher in diabetes model group at 8 and 16 weeks after modeling in comparison with normal control group (all at P<0.05). There were significant differences in corneal fluorescein staining score among different time points in diabetes model group ( F=27.155, P<0.05). In diabetes model group, thinner wall of meibomian gland duct, enlarged lumen of the duct, dilated acini and oil red-stained lipid deposition in most acini were observed.At 16 weeks after modeling, the expressions of TNF-α, and PPARγ mRNA in meibomian gland of diabetes model group were 3.33±0.91 and 1.55±0.25, which were significantly higher than 1.00±0.16 and 1.00±0.27 of normal control group (both at P<0.05). The expression of PEDF mRNA in diabetes model group was 0.42±0.08, which was significantly lower than 1.00±0.34 in normal control group ( P<0.05). There was no significant difference in the ADFP mRNA expression between the two groups ( t=0.943, P=0.38). Conclusions:Inflammatory factors and lipid metabolic factors such as TNF-α, PEDF, and PPARγ may be involved in the pathogenesis of meibomian gland dysfunction induced by diabetes.
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Objective To study the regulation of microRNA - 22(miR - 22)on glycometabolism of hemato-poietic stem cell TF - 1 and its molecular mechanism. Methods TF - 1 cells were cultured for 2 h under hypoxic con-ditions. The expression levels of Glut4 and miR - 22 was detected by quantitative real-time PCR(qRT - PCR). The sgRNA vector of the miR - 22 gene was constructed and miR - 22 gene of TK - 1 cells was knocked out by the CRISPR/ Cas9 technique. Overexpression vectors were constructed,and miR - 22 knocked - out cells were introduced to overexpress miR - 22,the expression of miR - 22 was detected by qRT - PCR and the expression levels of Glut4 and PPAR - γ were detected by qRT - PCR and Western blot. Results Compared with the control group,the expression of miR - 22 in TF - 1 cells decreased (0. 015 ± 0. 000 vs. 0. 056 ± 0. 001)and the expression of Glut4 (0. 351 ± 0. 038 vs. 0. 152 ± 0. 005)and PPAR - γ (0. 421 ± 0. 017 vs. 0. 248 ± 0. 008)increased,when TF - 1 cells were cultured for 2 h under hypoxic conditions,and the differences were statistically significant (all P < 0. 05). Compared with the control group,the expression levels of Glut4 (0. 019 ± 0. 00 vs. 0. 008 ± 0. 000)and PPAR - γ (0. 038 ± 0. 001 vs. 0. 019 ± 0. 000)were significantly increased after miR - 22 gene silencing,and they were significantly decreased (Glut4:0. 005 ± 0. 000 vs. 0. 008 ± 0. 000;PPAR - γ:0. 137 ± 0. 000 vs. 0. 019 ± 0. 000)after overexpression of miR - 22,and the differences were statistically significant (all P < 0. 05). Conclusions It suggests that miR - 22 ex-erts a negative regulation on glycometabolism of hematopoietic stem cell TF - 1 by downregulating the expression of PPAR - γ. A new regulatory factor of TF - 1 glycometabolism and the mechanisms are identified,which has provided new ideas for the targeted medication of diseases induced by hematopoietic stem cell dysfunction.