The occurrence and development of myopia is closely related to scleral remodeling. Therefore, in order to effectively prevent and cure myopia, it is very important to clarify the mechanism of scleral remodeling. In recent years, Chinese scholars have found that endoplasmic reticulum stress can regulate the expression of apoptotic proteins through the inositol demand protein-1/X box binding protein-1 pathway in the unfolded protein response, thus it is involved in regulating the state of scleral fibroblasts under hypoxia and regulating the occurrence and development of scleral remodeling. At the same time, some studies have found that inhibiting and knocking out protein kinase RNA-like endoplasmic reticulum kinase and activated transcription factor 6 in endoplasmic reticulum stress can effectively inhibit the growth of ocular axis. This proves that endoplasmic reticulum stress plays an important role in the occurrence and development of scleral remodeling. However, the comprehensive analysis of endoplasmic reticulum stress and scleral remodeling has not been reported at home and abroad. In-depth analysis of the relationship between endoplasmic reticulum and scleral remodeling is of great significance for the follow-up analysis and study of the mechanism of scleral remodeling.
Objective To observe the effect of “Luo’s Roujin Technique” on the inflammatory response and joint capsule fibrosis in white rabbits with scapulohumeral periarthritis model. Methods Thirty healthy male New Zealand white rabbits were randomly divided into a control group, a model group, and a treatment group, with 10 rabbits in each group. Scapulohumeral periarthritis models were established in the model group and the treatment group, while the control group received identical restraint procedures at the same timepoints. Six rabbits in the model group and seven in the treatment group were successfully modeled. The subsequent experiment included all six successfully modeled rabbits from the model group, along with six rabbits randomly selected from each of the control and treatment groups. On the second day after successful modeling, blood samples were collected from the auricular marginal vein in all three groups. After blood collection, the treatment group began massage therapy for 21 consecutive days, while the other two groups underwent the same restraint procedure simultaneously. On Day 22, all the three groups were euthanized after blood collection from the auricular marginal vein, and the synovial tissue of the affected shoulder joint was completely collected. Hematoxylin-eosin staining was used to examine the histopathological features of the synovial tissue. Enzyme-linked immunosorbent assay was employed to measure the concentrations of interleukin (IL)-1β, IL-6, IL-17, and tumor necrosis factor-α (TNF-α). Western blot and reverse transcription polymerase chain reaction were used to assess the protein and mRNA expression levels of vascular endothelial growth factor (VEGF), connective tissue growth factor (CTGF), transforming growth factor-β1 (TGF-β1), and Smad3. Results After treatment, the control group showed no significant inflammatory cell infiltration or fibrous tissue proliferation in the synovial tissue. The model group exhibited synovial cell hyperplasia in the lining layer and inflammatory cell infiltration in the sublining layer. The treatment group displayed mild inflammatory cell infiltration in the sublining layer. Compared with the control group, the model group showed significantly increased concentrations of IL-1β, IL-6, IL-17, and TNF-α in both serum and synovial homogenate, as well as elevated protein and mRNA expression of VEGF, CTGF, TGF-β1, and Smad3 in synovial tissue (P<0.05). Compared with the model group, the treatment group exhibited significantly lower serum levels of IL-1β, IL-6, and TNF-α, as well as reduced synovial homogenate levels of IL-1β, IL-6, IL-17, and TNF-α (P<0.05); furthermore, protein expression of VEGF, CTGF, TGF-β1, and Smad3 and mRNA expression of VEGF and CTGF in synovial tissue were significantly decreased in the treatment group (P<0.05). Conclusions “Luo’s Roujin Technique” can significantly alleviate local inflammatory infiltration in the synovial tissue of rabbits with scapulohumeral periarthritis, and reduce the levels of IL-1β, IL-6, IL-17, and TNF-α in both serum and synovial tissue. The underlying mechanism may involve suppression of VEGF, CTGF, TGF-β1, and Smad3 expression, leading to attenuated inflammatory responses and inhibition of fibroblast-to-myofibroblast transition. Thereby, it mitigates fibrotic changes in the shoulder joint capsule, exerting anti-inflammatory and analgesic effects and improving joint mobility.
Objective To microencapsulate a genetically engineered cell line which stably secrete human endostatin (hES).Methods Endostatin gene fragment was amplified from plasmid pcDNA3-Endo by polymerase chain reaction, and inserted into mammalian eukaryotic expression vector pEGFPN1, resulting into recombinant plasmid pEGFP-N1-ES.Hek-293 cells were transfected with pEGFP-N1-ES via cationic liposome and selected by G418, and were measured by Western blot for endostatin protein expression.The hES/293 cells were further entrapped by alginate-chitosan-alginate (ACA) microcapsules, and the expression of endostatin in the supernatant of cultured hES/293 cell microcapsules was examined by western blot at different time points.Results Recombinant plasmid pEGFP-N1 endostatin was digested by HindIII and BamHI, and resulted into 2 DNA fragments of 7 kb and 600 bp. The sequence of the 600 bp fragment was identical to human endostatin. Western blot of the supernatant of cultured hES/293 cells or hES/293 cell microcapsules detected a positive band with the relative molecular mass of 20times;103.Conclusion The hES protein was expressed in HeK-293 transfected with pEGFP-N1-endostatin, and secreted to the culture medium,and can freely diffused outside the micro-capsule.
Objective To investigate the effect of blue light on mRNA expression of L-type calcium channel subtypes of human retinal pigment epithelial (RPE) cells in vitro. Methods The fourth-generation of human RPE cells were randomly divided into four groups including control group (no light group), light group, light + nifedipine group, and light + (-) BayK8644 group. The cells were exposed to blue light (2000plusmn;500) lux for 6 hours, and then cultured for another 24 hours. Reverse transcription polymerase chain reaction real time (RT-PCR) and fluorescence quantitative PCR technologies were used to analyze mRNA expression of L-type calcium channel subunit of cardiac subtype ( 1C or CaV1.2), neuroendocrine subtype ( 1D or CaV1.3) and retinal subtypes ( 1F or CaV1.4) in each group. Results The length of PCR product of 1C, 1D, 1F subunit and actin was 68, 157, 125 and 186 base pairs respectively. (1) 1C mRNA expression in light, light + nifedipine and light + (-) BayK8644 group was higher than that in control group, the difference was statistically significant (P＜0.05). 1C mRNA expression in light +nifedipine group and light + (-) BayK8644 group was higher than in light group (P＜0.05). 1C mRNA expression in light + (-) BayK8644 group was higher than that in light + nifedipine group (P＜0.05). (2) Comparing with control group, 1D mRNA expression was higher in light, light +nifedipine and light + (-) BayK8644 group, the difference was statistically significant (P＜0.05). Light + (-) BayK8644 group was higher than light group and light + nifedipine group (P＜0.05), light group and the light + nifedipine group was not statistically significant (P＞0.05). (3) 1F mRNA expression in light, light + nifedipine and light + (-) BayK8644 group was higher than those in control group, there was statistically significant (P＜0.05), light +nifedipine group and light + (-) BayK8644 group was higher than light group (P＜0.05), light + nifedipine group and the light + (-) BayK8644 group was not statistically significant (P＞0.05). Conclusions The human RPE cells mRNA expression of L-type calcium channel 1C, 1D and 1F subunit was increased after exposing to blue light. Application of the 1times;10-5 mmol/L (-) BayK8644 can increase mRNA expression of 1C, 1D and 1F subunit.
ObjectiveTo observe the transthyretin (TTR) gene mutation, protein and mRNA expression in patients with familial vitreous amyloidosis. MethodsSubjects were divided into three groups: (1) illness group: seven patients with familial vitreous amyloidosis. (2) No-illness group: 9 unaffected family members. (3) Control group: 9 healthy individuals in same area. Subjects' peripheral venous blood were collected and DNA were extracted, 4 exons of TTR gene were amplified by reverse transcription polymerase chain reaction(RT-PCR), the gene fragments were sequencing by the fluorescence labelling method. Serum TTR protein expression was detected by Western blot, and TTR mRNA in leukocyte was assayed by RT-PCR. Results4 exons of TTR gene of all samples were amplified, and DNA sequencing data showed that 7 patients and 3 subjects DNA from unaffected family members had mutated in the 3rd exon of 107th base, changing from G to C. Heterozygous mutation occurred in codon of the 83th amino acid in exon 3, namely, Gly83Arg, resulted in the change of GGC to CGC. The protein and mRNA expression of TTR was lower in illness group than no-illness group and control groups(P < 0.05). Compared with control group, TTR mRNA expression in unaffected family members groups was significant decreased(P < 0.05). ConclusionHeterozygous mutation occurred in codon of the 83th amino acid in exon 3, namely Gly83Arg, and suggested that Gly83Arg is connected with the change of TTR mRNA and protein expression.
Objective To observe biological characteristics of microencapsulated human endostatin/293 (hES/293) cells at different density and their inhibitory effects on the proliferation of human umbilical vein endothelial cells (HUVEC). Methods The microencapsulated hES/293 cells at different cellular density of 1×104 (group A), 1×106 (group B) and 1×108 (group C) cells/ml were made by polyelectrolyte complexometry technology. The empty microcapsules were set as control group (group D). Each group has 6 samples. After 1, 3, 7, 14 and 35 days in culture, the number of total cells, viable cells was counted by trypan blue staining, and the survival fraction was measured. The grow status of hES/293 cells was measured by MTT assay, and the concentration of endostatin protein in supernatant was measured by enzyme linked immunosorbent assay (ELISA). HUVECs were cocultured with hES/293 cells of group A, B and C. The proliferation of HUVEC at the 24, 72 and 120 hours after coculture was measured by MTT assay. Results The number of total cells and viable cells were increasing and the survival fraction reached its peak after 3 days in culture in group A, B and C. The growth rate in group A was higher than that in group B and C after 3 days in culture (P＜0.05), but the growth rate in group B was higher than that in group A and C after 7, 14 and 35 days in culture (P＜0.05). The concentration of endostatin protein in the supernatant was the same in group A, B and C after 1 and 14 days in culture (P＞0.05). However, group A had higher endostatin than group B and C after 3 days in culture, group B had higher endostatin higher than group A and C after 7 and 35 days in culture (P＜0.05). The hES/293 cells of group A, B and C had no effects on the proliferation of HUVEC(P＞0.05) after 24 hours coculture, but can inhibit the proliferation of HUVEC after 72 or 120 hours co-culture (P＜0.05). Conclusions The microencapsulated hES/293 cells at a density of 1×106 cells/ml can grow and survive, and release endostatin protein stably. The microencapsulated hES/293 cells at different density all can inhibit the proliferation of HUVEC.