Characterization of Glucocorticoid Receptor on Lymphocytes in Chinese Patients with Glucocorticoid-induced Glaucoma

Purpose : We studied the pathogenesis of glucocorticoid-induced glucoma (GIG) through characterization of glucocorticoid receptor (GR) on lymphocytes in Chinese patients with GIG.Methods:By radioligand receptor binding followed by Scatchard analysis, the specific binding sites were characterized and quantitated for glucocorticoid receptors on peripheral blood lymphocytes obtained from patients with GIG and the control group.Results:The binding sites we detected were as follows; 12.7 ± 1.47 × 103 receptors per cell with a KD of 3.02 ± 0.62nmol/L in patients with GIG, 7.26 ± 0.45 × 103 receptors per cell with a KD of 3.03 ± 0.56nmol/L in the control group. The statistical difference of receptors per cell is significant between two groups (p < 0.05), patients with GIG having more GR binding sites, while the difference of Kd is not significant ( p > 0.05 ) . Conclusion: The preliminary findings suggest that patients with GIG are more sensitive to glucocorticoid and the increase of binding sites of GR     

The Study on Culture and Ultrastructure of Glaucomatous Trabecular Cells in Vitro

Purpose : To establish the culture system of human glaucomatous trabecular cells in vitro and study their ultrastructures.Methods : The trabecular specimens from trabeculectomy were cultured in vitro and passaged 3 times, then identified. Moreover, the glaucomatous cells were observed with electron microscope while compared with the normal ones.Result: Cultured human glaucomatous trabecular cells were obtained. The ultrastructure of the cells showed the decrease in vilious project, coated vesicle and lysosomal inclusion. Conclusion : The establishment of human glaucomatous trabecular cells culture in vitro made the culture system more perfect. The morphologic changes might be related to the abnormal functions of human trabecular meshwork cells. Eye Science 1998; 14 : 134 - 737.     

利用皮肤成纤维细胞重建兔角膜基质组织的初步研究

Objective To explore whether skin fibroblasts could be used as a cell source for reconstruction of the corneal stroma. Methods It was an experimental study. Skin fibroblast cells were isolated from newborn rabbits, cultured and expanded in vitro. Cells were labeled with green fluorescence protein (GFP) gene by retro-viral infection. Fibroblasts at passage 3 were seeded on polyglicolic acid (PGA) non-woven fibers to form a cell-scaffold construct. Constructs were then implanted into the adult rabbit corneal strema layer after being cultured in vitro for 1 week. Engineered stroma were observed continuously and harvested after 8 weeks of transplantation for gross, histological evaluation and Keratocan examination. PGA alone was used as control. Results The engineered tissue in the cornea became transparent gradually over a period of 8 weeks. Histological analysis showed that engineered stromal lamellar was relatively regular and the orientation of fibers was parallel to the surface of cornea, which is similar to normal cornea. The implanted cells were confirmed by GFP expression under fluorescent microscope, which also express Keratocan. By transmission electron microscopy examination, no significant difference in the diameter of collagen fiber was observed between engineered stroma (33.08 ± 2.47 ) nm and normal stroma ( t = 1.80, P = 0.0771 ). Conclusion Skin fibroblast cells could be used as seed cells for reconstruction of the corneal stroma.     

A New Arg54Gly Transthyretin Gene Mutation Associated with Vitreous Amyloidosis in Chinese

 Purpose:To analyse the hereditary features of a Chinese pedigree with familial vitreous amyloidosis in Liaoning Province, China, and to investigate the correlation between the clinical appearance of the disease and transthyretin（TTR）gene mutation, including the locus and type of TTR gene mutation． Methods:Five patients (10 eyes) from one Chinese family were diagnosed with vitreous amyloidosis between July 1996 and April 2009. Family members were followed up subsequently, and peripheral venous blood was obtained from 13 subjects (including 2 patients, and 11 controls without clinical signs of disease). DNA samples were extracted and 4 exons of the TTR gene were amplified by polymerase chain reaction (PCR). The gene fragments were subjected to sequencing analysis. The results were analyzed with DNAMAN Windows 5.2.2.0 and Chromas sequence chart analysis software, TTR gene exons were compared between affected patients and normal controls． Results:Family pedigree analysis revealed that patients were distributed in three generations. Male and female subjects had equal prevalence, and only one parent of affected patients had signs of disease. TTR gene exon sequencing showed that the sequence of patients was identical to that of normal individuals. No TTR gene mutations were noted in 10 un-affected family members. However, a TTR Gly-54 point mutation in the 2nd exon was detected in two patients and 1 unaffected family member (one of the patients' daughters)．Vitreous samples in 4 cases (7 eyes) showed positive Congo red staining, suggesting that these family members suffered from familial vitreous amyloidosis. Conclusion:This pedigree affected with familial vitreous amyloidosis was characterized by autosomal dominant inheritance; a TTR Gly-54 point mutation in the 2nd exon is presumed to be the cause. This Gly-54 point mutation of the TTR gene is a novel mutation in vitreous amyloidosis.     

Acellular porcine corneal matrix as a carrier scaffold for cultivating human corneal epithelial cells and fibroblasts in vitro

AIM: To investigate the feasibility of corneal anterior lamellar reconstruction with human corneal epithelial cells and fibroblasts, and an acellular porcine cornea matrix (APCM) in vitro. METHODS: The scaffold was prepared from fresh porcine corneas which were treated with 0.5% sodium dodecyl sulfate (SDS) solution and the complete removal of corneal cells was confirmed by hematoxylin-eosin (HE) staining and 4’, 6-diamidino-2-phenylindole (DAPI) staining. Human corneal fibroblasts and epithelial cells were cultured with leaching liquid extracted from APCM, and then cell proliferative ability was evaluated by MTT assay. To construct a human corneal anterior lamellar replacement, corneal fibroblasts were injected into the APCM and cultured for 3d, followed by culturing corneal epithelial cells on the stroma construction surface for another 10d. The corneal replacement was analyzed by HE staining, and immunofluorescence staining. RESULTS: Histological examination indicated that there were no cells in the APCM by HE staining, and DAPI staining did not detect any residual DNA. The leaching liquid from APCM had little influence on the proliferation ability of human corneal fibroblasts and epithelial cells. At 10d, a continuous 3 to 5 layers of human corneal epithelial cells covering the surface of the APCM was observed, and the injected corneal fibroblasts distributed within the scaffold. The phenotype of the construction was similar to normal human corneas, with high expression of cytokeratin 12 in the epithelial cell layer and high expression of vimentin in the stroma. CONCLUSION: Corneal anterior lamellar replacement can be reconstructed in vitro by cultivating human corneal epithelial cells and fibroblasts with an acellular porcine cornea matrix. This laid the foundation for the further transplantation in vivo.     

人角膜内皮细胞压力调节培养的初步研究

Objective To investigate the effect of pressure bionic culture on the morphology and function of rabbit corneal endothelial cells. Methods Corneal endothelial cells were separated and purified by tearing apart the descemet and digesting with trypsin and EDTA, then cultured in the plate. The cells were divided into two groups: group A were cultured under atmosphere; cells exposed to 2 kPa( 14. 66 mm Hg) pressure in vitro was group B; the morphology and growth pattern of cells were observed by inverted microscope; cells origin were identified by neuron-specific enolase immunoassay. Cellular changes in the structure were observed by HE staining and scanning and transmission electron microscopy (SEM and TEM) analysis. Cells activity was detected by flow cytometry. Results NSE antibody of the primary corneal endothelial cells was positive without corneal epithelial cells and corneal stroma cells. Two groups of cells were cultured for 120-144 h respectively, the morphology was flat, polygon, most of cells were hexagon and abundant cytoplasms in group B (pressure bionic culture), but in group A, the cells size was not uniform and there were much granules in the cytoplasm. There was no difference in the time of formation of monolayer in two groups. SEM showed that cells exposed to pressure connected tightly and the surface was rich in microvilli, extended foot processes and attached to the substrate tightly, while cells cultured under atmosphere with more off-chip. In group B, Annexiv-FITC/PI detection of apoptosis showed cell survival rate was 98.2%, early apoptosis rate was 0.7%, late apoptosis rate was 1.0%, death rate was 0. 1%; the corresponding data were 92.2%, 5.2%, 2.3%, and 0.3% in group A, respectively; There was statistically significant difference between the two groups (x2 =594. 0,P ＜0. 01 ). After cultured for 96 h,the expression of ZO-1 protein in cells exposed to pressure was higher than those in control. Conclusions The biological activity of endothelial cells is regulated positively by bionic pressure. The establishment of a new biomimetic pressure model will help to investigate the physiological function and injury repair of corneal endothelial cells in vitro.     

人角膜内皮细胞压力调节培养的初步研究

Objective To investigate the effect of pressure bionic culture on the morphology and function of rabbit corneal endothelial cells. Methods Corneal endothelial cells were separated and purified by tearing apart the descemet and digesting with trypsin and EDTA, then cultured in the plate. The cells were divided into two groups: group A were cultured under atmosphere; cells exposed to 2 kPa( 14. 66 mm Hg) pressure in vitro was group B; the morphology and growth pattern of cells were observed by inverted microscope; cells origin were identified by neuron-specific enolase immunoassay. Cellular changes in the structure were observed by HE staining and scanning and transmission electron microscopy (SEM and TEM) analysis. Cells activity was detected by flow cytometry. Results NSE antibody of the primary corneal endothelial cells was positive without corneal epithelial cells and corneal stroma cells. Two groups of cells were cultured for 120-144 h respectively, the morphology was flat, polygon, most of cells were hexagon and abundant cytoplasms in group B (pressure bionic culture), but in group A, the cells size was not uniform and there were much granules in the cytoplasm. There was no difference in the time of formation of monolayer in two groups. SEM showed that cells exposed to pressure connected tightly and the surface was rich in microvilli, extended foot processes and attached to the substrate tightly, while cells cultured under atmosphere with more off-chip. In group B, Annexiv-FITC/PI detection of apoptosis showed cell survival rate was 98.2%, early apoptosis rate was 0.7%, late apoptosis rate was 1.0%, death rate was 0. 1%; the corresponding data were 92.2%, 5.2%, 2.3%, and 0.3% in group A, respectively; There was statistically significant difference between the two groups (x2 =594. 0,P ＜0. 01 ). After cultured for 96 h,the expression of ZO-1 protein in cells exposed to pressure was higher than those in control. Conclusions The biological activity of endothelial cells is regulated positively by bionic pressure. The establishment of a new biomimetic pressure model will help to investigate the physiological function and injury repair of corneal endothelial cells in vitro.     

常染色体显性遗传核性白内障合并小角膜的CRYAA基因突变研究

Objective To identify the gene mutation in a four-generation Chinese family with autosomal dominant congenital cataract associated with microcornea. Methods Experimental research.Twelve members in this family (including six affected and six unaffected individuals) were enrolled into this study. They underwent full ophthalmological and clinical examinations to rule out any concomitant disorders.Blood samples were collected and genomic DNA was extracted. Microsatellite markers near the reported loci,which are associated with congenital cataract and microcornea were selected and amplified from DNA samples using polymerase chain reaction. Linkage analysis was performed. The exons and exon/intron junction of candidate gene in the related chromosome were sequenced. The product of the first exon was digested by ApaL Ⅰ restriction enzyme to certify the mutation. Results The phenotype studied in this family was nuclear cataract accompanied with microcornea. At markers D21S1885 and D21S1890 near the locus 21q22. 3, the affected members had the same allele, but the unaffected did not. The Lod scores were 2. 11in both markers, indicating that this locus were linked to the congenital cataract in this family. DNA sequencing of candidate gene CRYAA showed a heterozygous mutation c. 34C ＞ T in exon 1, which led to condon 12 in peptide chain encoding arginine substituted by cysteine. ApaL Ⅰ enzyme digestion certified that all of the affected members had the same mutation c. 34C ＞T, but the unaffected and normal individuals did not. Conclusion Mutation (p. R12C) of CRYAA is the genetic change that causes the occurrence of congenital cataract with microcornea in this family.     

常染色体显性遗传核性白内障合并小角膜的CRYAA基因突变研究

Objective To identify the gene mutation in a four-generation Chinese family with autosomal dominant congenital cataract associated with microcornea. Methods Experimental research.Twelve members in this family (including six affected and six unaffected individuals) were enrolled into this study. They underwent full ophthalmological and clinical examinations to rule out any concomitant disorders.Blood samples were collected and genomic DNA was extracted. Microsatellite markers near the reported loci,which are associated with congenital cataract and microcornea were selected and amplified from DNA samples using polymerase chain reaction. Linkage analysis was performed. The exons and exon/intron junction of candidate gene in the related chromosome were sequenced. The product of the first exon was digested by ApaL Ⅰ restriction enzyme to certify the mutation. Results The phenotype studied in this family was nuclear cataract accompanied with microcornea. At markers D21S1885 and D21S1890 near the locus 21q22. 3, the affected members had the same allele, but the unaffected did not. The Lod scores were 2. 11in both markers, indicating that this locus were linked to the congenital cataract in this family. DNA sequencing of candidate gene CRYAA showed a heterozygous mutation c. 34C ＞ T in exon 1, which led to condon 12 in peptide chain encoding arginine substituted by cysteine. ApaL Ⅰ enzyme digestion certified that all of the affected members had the same mutation c. 34C ＞T, but the unaffected and normal individuals did not. Conclusion Mutation (p. R12C) of CRYAA is the genetic change that causes the occurrence of congenital cataract with microcornea in this family.     

糖皮质激素性青光眼患者小梁细胞体外培养和超微结构研究

目的 探索糖皮质激素性青光眼患者小梁细胞体外培养方法及超微结构的特点。方法 从小梁切除术中取得的巩膜内板层,应用组织学培养方法进行患者小梁细胞体外培养及鉴定,并将糖皮质激素性青光眼患者小梁细胞与正常小梁细胞的超微结构进行分析和比较。结果 糖皮质激素性青光眼患者小梁组织培养的细胞经鉴定确为小梁细胞,但与正常小梁细胞相比,其细胞的微绒毛、吞饮小泡及胞浆的溶酶体含量较少。结论 糖皮质激素性青光眼患者小梁     

Clinical relevance of the glucocorticoid receptor gene polymorphisms in glucocorticoid–induced ocular hypertension and primary open angle glaucoma

AIM: To avoid the side effects of ocular hypertension of glucocorticoid (GC) usage in eye, we must identify susceptible individuals, which exists in about one-third of all population. Further, the majority of all primary open angle glaucoma (POAG) patients show this phenotype. Glucocorticoid receptor (GR) regulates C responsiveness in trabecular meshwork (TM) cells. In this study, single nucleotide polymorphism (SNP) genotyping was used to determine whether there are differences in the BclI (rs41423247) and N363S (rs6195) polymorphisms of the GR gene in healthy and POAG patients, and glucocorticoid-induced ocular hypertension (GIOH) populations. METHODS: Three hundred and twenty-seven unrelated Chinese adults, including 111 normal controls, 117 GIOH subjects and 99 POAG patients, were recruited. DNA samples were prepared and the BclI and N363S polymorphisms were screened using real-time polymerase chain reaction (RT-PCR)-restriction fragment length polymorphism (RFLP) analysis. Frequencies of the BclI and N363S polymorphisms were determined and compared using Fisher's exact test and the Chi-squared test. RESULTS: Only the BclI polymorphism was identified in the Chinese Han population. The frequency of the G allele was 21.6 % in normal controls, 18.3% in GIOH patients, and 13.64% in the POAG patients. There was no significant difference in polymorphism or allele frequency in the 3 groups. Furthermore, no N363S polymorphism was found in the study subjects. CONCLUSION: The BclI polymorphisms in GR gene had no association with GIOH and POAG patients, and N363S polymorphism might not exist in the Chinese Han population. Therefore, the BclI polymorphism might not be responsible for the development of GC-induced ocular hypertension or POAG.     

IL-6对体外培养的牛眼小梁细胞中纤维连接蛋白的影响

目的：探讨不同浓度白细胞介素-6(IL-6)刺激下体外培养的牛眼小梁细胞中纤维连接蛋白的表达变化。

方法：采用组织块培养法取新鲜牛眼的小梁网组织,提取并培养第3代牛眼小梁细胞,采用细胞形态学对细胞进行鉴定。经终浓度为0、0.1、0.5、1ng/mL的IL-6药物刺激24h后,采用荧光定量PCR和蛋白质免疫印迹法检测各浓度IL-6刺激下牛眼小梁细胞中FN mRNA和蛋白的表达。

结果：培养出的牛眼小梁细胞符合第3代牛眼小梁细胞形态特征。实时荧光定量PCR和蛋白质免疫印迹法显示,不同浓度IL-6刺激下的牛眼小梁细胞所产生的FN mRNA量分别为1.000±0.000、0.213±0.004、0.056±0.001、0.019±0.002,FN蛋白表达量分别为1.167±0.012、0.662±0.009、0.238±0.011、0.061±0.011,均呈下调趋势(r_s=-0.713、-0.901,均P<0.05),4组间FN mRNA和蛋白表达均有差异(P<0.05)。

结论：体外培养的牛眼小梁细胞在外源性IL-6刺激下影响FN mRNA和蛋白的表达,且IL-6浓度与蛋白表达呈负相关性,推测IL-6可能通过影响FN基因与蛋白的表达,进而改变小梁网组织结构。     

原发性开角型青光眼小梁细胞的体外培养及其生物学特性

背景原发性开角型青光眼（POAG）是一种常见致盲性眼病,其特点是房水外流阻力增加导致眼压增高。位于房水外流通道的小梁网调节房水的外流,因此研究小梁网细胞的生物学特性有着重要的意义。目的探讨POAG小梁细胞体外培养的方法及其生物学特性。方法经小梁切除术收集8例开角型青光眼患者患眼的带小梁网的深层巩膜组织块进行体外原代和传代培养,用鼠抗人层黏连蛋白（LM）单克隆抗体、兔抗人纤维连结蛋白（FN）单克隆抗体、鼠抗人神经元特异性烯醇化酶（NSE）单克隆抗体进行免疫组织化学检测以对传代细胞进行鉴定,在透射电子显微镜下对传代细胞的超微结构进行观察,并将传代小梁细胞的生物学特性与本研究组前期培养的正常小梁细胞进行比较。结果组织块培养10d左右,可见细胞从其边缘向外生长。传代细胞在4d内处于对数生长期,其后进入平台期,第7天细胞基本融合。第3代POAG小梁细胞及正常人眼小梁细胞中可见FN、LM和NSE均呈阳性表达,证实传代细胞为小梁细胞,而空白对照组细胞未见FN、LM和NSE表达。第3代POAG小梁细胞和正常小梁细胞中FN的A450值分别为0．354±0．06和0．26±0．01,LM的A450值分别为0．34±0．03和0．25±0．02,差异均有统计学意义（FN：t=14．446,P=0．001;LM：t=9．346,P=0．001）。与正常小梁细胞比较,第3代POAG小梁细胞表面的微绒毛、细胞质的溶酶体及吞噬小泡含量减少。结论采用组织块培养法可成功在体外培养POAG小梁细胞,该研究结果为研究青光眼的发病机制提供了细胞学基础。     

Presence of an established calcification marker in trabecular meshwork tissue of glaucoma donors

PURPOSE: To determine the presence of calcification markers in the trabecular meshwork tissue from glaucoma donors and in trabecular meshwork cells insulted by dexamethasone (DEX) and transforming growth factor beta2 (TGFbeta2), factors associated with glaucoma. To investigate as well the effect of silencing the inhibitor of calcification matrix Gla (MGP) in the trabecular meshwork cells. METHODS: Trabecular meshwork tissue was obtained from perfused postmortem anterior segments of glaucomatous and normal eyes. Primary trabecular meshwork cells were obtained from residual corneal rims after surgical corneal transplantation. Calcification marker alkaline phosphatase (ALP) enzyme activity was assayed by fluorescence produced after substrate cleavage. DNA quantification was evaluated by fluorescence produced after binding to the Hoechst dye. Transfection of siRNA to primary cells was accomplished by nucleofector electroporation with trabecular meshwork-optimized conditions. cDNA quantification was performed with the use of TaqMan real-time PCR. RESULTS: Human trabecular meshworks from glaucoma donors exhibited significantly higher levels of ALP activity than their matched counterparts with normal eyes. The normalized ALP of the control specimens was 7.3 +/- 1.6 ng ALP/microg DNA (n = 4), whereas that of the glaucomatous tissue was 37.0 +/- 10.7 ng ALP/microg genomic DNA (n = 5; P 相似文献   

表皮生长因子对牛眼小梁细胞c—fos基因表达的诱导

目的研究表皮生长因子（ｅｐｉｄｅｒｍａｌｇｒｏｗｔｈｆａｃｔｏｒ，ＥＧＦ）对小梁细胞的作用。方法应用分子杂交技术研究ＥＧＦ对体外培养的牛眼小梁细胞ｃ－ｆｏｓ基因表达的诱导和３Ｈ胸腺核苷酸（３Ｈ－ｔｈｙｍｉｄｉｎｅｉｎｃｏｒｐａｒａｔｉｏｎ，３Ｈ－ＴｄＲ）掺入法观察细胞ＤＮＡ的合成。结果小梁细胞的３Ｈ－ＴｄＲ掺入率随着ＥＧＦ浓度不同而变化，浓度为２０～１５０ｎｇ／ｍｌ时，细胞掺入率随浓度增加升高（Ｐ＜０．０１）。与对照组相比，ＥＧＦ刺激停止生长的小梁细胞０．５小时后，ｃ－ｆｏｓ基因开始表达，１小时后达高峰，至２小时后消失。不同浓度ＥＧＦ刺激小梁细胞１小时后，ｃ－ｆｏｓ基因表达呈浓度依赖性。结论实验结果表明ＥＧＦ刺激小梁细胞增殖或进入生长状态，可能与ｃ－ｆｏｓ基因产物的信号传递作用有关。     

地塞米松诱导人小梁细胞bcl—2基因的表达

目的:探讨地塞米松对小梁细胞bcl-2基因表达的影响。方法:应用组织块培养的方法建立人小梁细胞培养。取第三代小梁细胞培养于载玻片上,含10~(-7)mol/L地塞米松的培养液作用6小时、12小时、24小时后,应用LSAB观察bcl-2基因表达的情况。结果:地塞米松作用6小时后可见bcl-2蛋白阳性细胞,且随时间的延长,阳性细胞有所增多。结论:地塞米松可诱导小梁细胞bcl-2基因表达,可能在激素性青光眼发病中起着一定的作用。眼科学报1998;14:187～189。