CD147对高糖培养下RPE细胞生物学行为影响及衣霉素对CD147抑制作用研究
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摘要
糖尿病视网膜病变是糖尿病发展过程中常见的严重并发症,是目前世界范围内致盲和视力受损的主要原因之一,目前对于其发病机制仍不十分清楚,治疗效果亦不佳。CD147是一种分子量为50-60kD高度糖基化的单次跨膜糖蛋白,属于免疫球蛋白超家族成员,在眼组织中尤其是视网膜色素上皮细胞中高表达。目前的研究证实,CD147在多种疾病中发挥重要的作用,和MMPs、VEGF、MCT等多种重要的分子关系密切,因此推测CD147在糖尿病视网膜病变中可能发挥了重要的作用。RNA干扰是指由内源性或外源性短双链RNA诱导的同源mRNA的降解过程,可使基因的表达沉默。通过构建针对目标基因的siRNA或shRNA,导入细胞内使目标基因沉默,是研究基因和蛋白功能的重要工具。衣霉素作为N-糖链抑制剂多用于研究蛋白的糖基化。
本研究首先通过免疫组织化学的方法检测了CD147在正常人眼球各组织中的表达情况。选取视网膜色素上皮细胞进行体外培养,观察在低糖及高糖培养条件下RPE细胞中CD147的表达、细胞增殖和迁移能力变化以及VEGF、MMP-2、MMP-9变化。构建CD147表达载体pBS/U6/CD147-shRNA,转染到RPE中,检测其对细胞CD147内源性表达的抑制作用,筛选出抑制作用最强的重组质粒,并观察其对细胞增殖、运动性以及对MMP-2、MMP-9和VEGF表达的影响,证实CD147作为VEGF及MMP-9的上游因子在高糖条件下视网膜色素上皮细胞的生物学特性的变化中起到了重要的作用。在培养基中加入衣霉素,使CD147蛋白去糖基化,观察CD147的表达变化、RPE细胞增殖和迁移能力变化以及VEGF、MMP-2、MMP-9变化,提示CD147主要是通过自身的糖基化程度不同来发挥作用,使蛋白去糖基化后可有效抑制其功能。为研究糖尿病视网膜病变的发病机制及治疗提供新的思路。
The influence of CD 147 on retinal pigment epithelium cells under high glucose culture and the inhibition effect of tunicamycin on CD 147
Background Diabetic retinopathy(DR) is a severe complication of diabetes mellitus, and is the main cause of blindness of diabetes mellitus patients.The research on mechanism of DR and searching effective treatment is hot point.It is not entirely clear about the mechanism of DR. Many studies indicate that VEGF,MMPs and other cytokines play a important role during the DR.CD147 is a member of immunoglobulin superfamily,and it works by protein glycosylation.The expression of CD147 is more higher in malignant tumor and corneal ulcer etc..CD147 can stimulate fibroblast and endothelial cells to secrete matrix metalloproteinases(MMPs), then destroy basement membrane and cause series of pathological reaction. RNA interference(RNAi) means degradation of homologous mRNA induced by endogenous or exogenous short double-stranded RNA(dsRNA) molecule,its effector molecule is 21-23 nucleotide(nt) dsRNA called small interfering RNA(siRNA).The gene knockout effect caused by RNAi technology can inhibit the expression of oncogene,tumor suppression gene and gene mutatin et al.The short hairpin RNA(shRNA) expressed by vector transfected into target cells can be recognized and splitted into siRNA.Use the plasmid vector system which can steadily express shRNA can inhibit gene expression permanently. Tunicamycin is a natural nucleoside antibiotic, can inhibit the production of dolichol of the protein glycosylation pathway, hamper the generation of sugar chain, produce desugar protein, is a effective tool in sugar protein research. In this study, the expression and distribution of CD147 on each tissue of normal eyeball was detected by immunohistochemistry; detect the changes of CD 147 expression of RPE cells cultured by different concentration glucose in vitro; then construct the CD147-shRNA vector and transfect RPE cells and detect the inhibition effect of CD147-shRNA on the expression of CD147,and observe the migration ability of RPE cell and the effect on the secretion of MMP-2,MMP-9 and VEGF.Add tunicamycin on RPE cells,detect the deglycosylation effect on effect, and the changeof proliferation,migration and secretion ability of RPE cells.
Methods
1 Detect the expression and distribution of CD147 on each tissue of normal human eyeball by immunohistochemistry.
2 Detect the changes of CD147 expression of RPE cells cultured by different concentration glucose in vitro and the influence of CD 147 on biological behavior of RPE cells.
Detect the changes of expression of CD147 under different concentration glucose culture by immunofluorescent staining, study the migration ability by wound-healing assays,detect the expression of CD147,MMP-2,MMP-9 and VEGF by Western blot.
3 Construction and identification of CD147-shRNA and the inhibition effect of CD147-shRNA on the expression of CD 147 of RPE cells.
According to the CD147 mRNA sequence in Genebank,use the siRNA design software of Promega to find the right target sequence,BLAST to confirm no homologous with known human gene.Three pairs of oligonucleotide strand were synthesized.Cloned the sequence to pBS/U6 vector.Rename the recombinant plasmid pBS/U6/CD147-shRNA1、pBS/U6/CD147-shRN2、pBS/U6/CD147-shRNA3,and restriction enzyme digestion and sequencing to confirm the correctness.Transfect the plasmid into RPE cells to detect the expression of CD147 by Western blot, and the expression of MMP-2,MMP-9 and VEGF.Also the migration ability was detected by wound-healing.
4 The inhibition effect of tunicamycin on CD 147 of RPE cells
Add lOug/ml tunicamycin to RPE cells cultured by high and low glucose,detect the expression of CD147 by immunofluorescent staining;detect the degree of CD147 protein glycosylation and the secretion of MMP-2,MMP-9 and VEGF.Study the migration ability by wound-healing.Detect the influence of tunicamycin on cell cycle of RPE cell by flow cytometry.
Results
1 CD147 was expressed on human cornea epithelial cell, iris epithelial cell, lens epithelial cell and fibre suface, sclera vascular endothelial cell, choroid membranes pigment epithelial cell and small vascular endothelial cell, optic nerve fibre and all layers of retina.
2 The expression of CD 147 of RPE cell was increased under the high concentration glucose culture,and the migration ability was strengthened, more MMP-2,MMP-9 and VEGF was secreted.
3 CD147-shRNAs recombinant plasmid is completely correct comfirmed by restriction enzyme digestion and sequencing.All the plasmids have gene silencing effect and the pBS/U6/CD147-shRNA3 is most significant.The result of Westernblot indicates that CD 147 expression was significantly decreased after RPE cells was transfected by pBS/U6/CD147-shRNA3, and the migration ability was declined, and the secretion of MMP-2,MMP-9 and VEGF was significantly decreased.
4 CD147 was deglycosylated by tunicamycin, CD147 on the cell membrane was decreased showed by immunofluorescence.And the results of Western blot showed glycosylated CD147 significantly declined, and also the MMP-2,MMP-9 and VEGF. RPE cell migrated slowly.
Conclusions
1 CD 147 was expressed on human cornea epithelial cell and endothelial cells, lens epithelial cell and suface fibre cell, iris surface pigment cell, choroid membranes pigment epithelial cell, optic nerve fibre and all layers of retina and vascular endothelial cell of all tissues, and located mainly in cell membrane and cytoplasm.So in eyeball CD147 located mainly in pigment epithelium,epithelial cell and vascular endothelial cell.
2 Under high glucose culture,migration ability of RPE cell was strengthened, express more CD147, and the expression of MMP-2,MMP-9 and VEGF was increased, there is a positive correlation between them. pBS/U6/CD147-shRNA3 can inhibit the migration of RPE cell and the secretion of MMP-2,MMP-9 and VEGF.It indicates that high glucose circumstance can upgrade the expression of MMP-2,MMP-9 and VEGF, and promote the migration of RPE cell.
3 Tunicamycin works on the N terminal of CD147 protein, makes CD147 deglycosylation, and them inhibit its biological activity, result in a weakness of proliferation,migration and secretion of RPE cells.It indicates that as a membrane protein CD147 works by the degree of glycosylation.
本研究首先通过免疫组织化学的方法检测了CD147在正常人眼球各组织中的表达情况。选取视网膜色素上皮细胞进行体外培养,观察在低糖及高糖培养条件下RPE细胞中CD147的表达、细胞增殖和迁移能力变化以及VEGF、MMP-2、MMP-9变化。构建CD147表达载体pBS/U6/CD147-shRNA,转染到RPE中,检测其对细胞CD147内源性表达的抑制作用,筛选出抑制作用最强的重组质粒,并观察其对细胞增殖、运动性以及对MMP-2、MMP-9和VEGF表达的影响,证实CD147作为VEGF及MMP-9的上游因子在高糖条件下视网膜色素上皮细胞的生物学特性的变化中起到了重要的作用。在培养基中加入衣霉素,使CD147蛋白去糖基化,观察CD147的表达变化、RPE细胞增殖和迁移能力变化以及VEGF、MMP-2、MMP-9变化,提示CD147主要是通过自身的糖基化程度不同来发挥作用,使蛋白去糖基化后可有效抑制其功能。为研究糖尿病视网膜病变的发病机制及治疗提供新的思路。
The influence of CD 147 on retinal pigment epithelium cells under high glucose culture and the inhibition effect of tunicamycin on CD 147
Background Diabetic retinopathy(DR) is a severe complication of diabetes mellitus, and is the main cause of blindness of diabetes mellitus patients.The research on mechanism of DR and searching effective treatment is hot point.It is not entirely clear about the mechanism of DR. Many studies indicate that VEGF,MMPs and other cytokines play a important role during the DR.CD147 is a member of immunoglobulin superfamily,and it works by protein glycosylation.The expression of CD147 is more higher in malignant tumor and corneal ulcer etc..CD147 can stimulate fibroblast and endothelial cells to secrete matrix metalloproteinases(MMPs), then destroy basement membrane and cause series of pathological reaction. RNA interference(RNAi) means degradation of homologous mRNA induced by endogenous or exogenous short double-stranded RNA(dsRNA) molecule,its effector molecule is 21-23 nucleotide(nt) dsRNA called small interfering RNA(siRNA).The gene knockout effect caused by RNAi technology can inhibit the expression of oncogene,tumor suppression gene and gene mutatin et al.The short hairpin RNA(shRNA) expressed by vector transfected into target cells can be recognized and splitted into siRNA.Use the plasmid vector system which can steadily express shRNA can inhibit gene expression permanently. Tunicamycin is a natural nucleoside antibiotic, can inhibit the production of dolichol of the protein glycosylation pathway, hamper the generation of sugar chain, produce desugar protein, is a effective tool in sugar protein research. In this study, the expression and distribution of CD147 on each tissue of normal eyeball was detected by immunohistochemistry; detect the changes of CD 147 expression of RPE cells cultured by different concentration glucose in vitro; then construct the CD147-shRNA vector and transfect RPE cells and detect the inhibition effect of CD147-shRNA on the expression of CD147,and observe the migration ability of RPE cell and the effect on the secretion of MMP-2,MMP-9 and VEGF.Add tunicamycin on RPE cells,detect the deglycosylation effect on effect, and the changeof proliferation,migration and secretion ability of RPE cells.
Methods
1 Detect the expression and distribution of CD147 on each tissue of normal human eyeball by immunohistochemistry.
2 Detect the changes of CD147 expression of RPE cells cultured by different concentration glucose in vitro and the influence of CD 147 on biological behavior of RPE cells.
Detect the changes of expression of CD147 under different concentration glucose culture by immunofluorescent staining, study the migration ability by wound-healing assays,detect the expression of CD147,MMP-2,MMP-9 and VEGF by Western blot.
3 Construction and identification of CD147-shRNA and the inhibition effect of CD147-shRNA on the expression of CD 147 of RPE cells.
According to the CD147 mRNA sequence in Genebank,use the siRNA design software of Promega to find the right target sequence,BLAST to confirm no homologous with known human gene.Three pairs of oligonucleotide strand were synthesized.Cloned the sequence to pBS/U6 vector.Rename the recombinant plasmid pBS/U6/CD147-shRNA1、pBS/U6/CD147-shRN2、pBS/U6/CD147-shRNA3,and restriction enzyme digestion and sequencing to confirm the correctness.Transfect the plasmid into RPE cells to detect the expression of CD147 by Western blot, and the expression of MMP-2,MMP-9 and VEGF.Also the migration ability was detected by wound-healing.
4 The inhibition effect of tunicamycin on CD 147 of RPE cells
Add lOug/ml tunicamycin to RPE cells cultured by high and low glucose,detect the expression of CD147 by immunofluorescent staining;detect the degree of CD147 protein glycosylation and the secretion of MMP-2,MMP-9 and VEGF.Study the migration ability by wound-healing.Detect the influence of tunicamycin on cell cycle of RPE cell by flow cytometry.
Results
1 CD147 was expressed on human cornea epithelial cell, iris epithelial cell, lens epithelial cell and fibre suface, sclera vascular endothelial cell, choroid membranes pigment epithelial cell and small vascular endothelial cell, optic nerve fibre and all layers of retina.
2 The expression of CD 147 of RPE cell was increased under the high concentration glucose culture,and the migration ability was strengthened, more MMP-2,MMP-9 and VEGF was secreted.
3 CD147-shRNAs recombinant plasmid is completely correct comfirmed by restriction enzyme digestion and sequencing.All the plasmids have gene silencing effect and the pBS/U6/CD147-shRNA3 is most significant.The result of Westernblot indicates that CD 147 expression was significantly decreased after RPE cells was transfected by pBS/U6/CD147-shRNA3, and the migration ability was declined, and the secretion of MMP-2,MMP-9 and VEGF was significantly decreased.
4 CD147 was deglycosylated by tunicamycin, CD147 on the cell membrane was decreased showed by immunofluorescence.And the results of Western blot showed glycosylated CD147 significantly declined, and also the MMP-2,MMP-9 and VEGF. RPE cell migrated slowly.
Conclusions
1 CD 147 was expressed on human cornea epithelial cell and endothelial cells, lens epithelial cell and suface fibre cell, iris surface pigment cell, choroid membranes pigment epithelial cell, optic nerve fibre and all layers of retina and vascular endothelial cell of all tissues, and located mainly in cell membrane and cytoplasm.So in eyeball CD147 located mainly in pigment epithelium,epithelial cell and vascular endothelial cell.
2 Under high glucose culture,migration ability of RPE cell was strengthened, express more CD147, and the expression of MMP-2,MMP-9 and VEGF was increased, there is a positive correlation between them. pBS/U6/CD147-shRNA3 can inhibit the migration of RPE cell and the secretion of MMP-2,MMP-9 and VEGF.It indicates that high glucose circumstance can upgrade the expression of MMP-2,MMP-9 and VEGF, and promote the migration of RPE cell.
3 Tunicamycin works on the N terminal of CD147 protein, makes CD147 deglycosylation, and them inhibit its biological activity, result in a weakness of proliferation,migration and secretion of RPE cells.It indicates that as a membrane protein CD147 works by the degree of glycosylation.
引文
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