重组人骨形态发生蛋白7对原发性开角型青光眼小梁网细胞增殖的影响
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摘要
目的建立原发性开角型青光眼(POAG)小梁网细胞体外原代培养体系;分析不同终浓度重组人骨形态发生蛋白7(rhBMP7)对POAG小梁网细胞增殖的影响;探讨rhBMP7与POAG发生、进展的相关性。方法取术中切除的带小梁网组织块(未使用丝裂霉素C),进行体外原代及传代培养,取3代小梁网细胞,在CFDA SE标记后,分别加入终浓度为0(对照组),20,50,80,100,200ng/mL的rhBMP7无血清培养基,采用CCK8、荧光显微镜、流式细胞仪等方法检测POAG小梁网细胞增殖情况。结果细胞经传代培养,经鉴定为POAG小梁网细胞;采用CCK8法检测发现:经终浓度为0(对照组),20,50,80,100,200ng/mL的rhBMP7处理后,POAG小梁网细胞吸光度(OD值)分别为:(0.561 2±0.026 9),(0.724 2±0.039 3),(1.416 0±0.016 2),(1.740 4±0.039 2),(1.853 8±0.014 5),(1.936 4±0.054 6);实验组细胞增殖率分别为1.37%,2.96%,3.70%,3.96%,4.15%;实验组与对照组及各实验组间增殖率比较,差别具有统计学意义(P<0.05);荧光显微镜示:随着rhBMP7终浓度的增加,经CFDA SE标记的POAG小梁网细胞荧光染色变浅、细胞量及密度增加;采用流式细胞仪检测经20,50,80,100,200ng/mL终浓度rhBMP7干预的POAG患者小梁网细胞,分裂、增殖细胞所占的比例分别为17.85%,18.63%,20.10%,27.45%,72.41%。结论运用组织块培养法,可体外原代培养出POAG患者的小梁网细胞;rhBMP7在一定程度上可促进POAG小梁网细胞的增殖,且在一定范围内呈剂量依赖性。
Objective To establish a primary culture system for primary open-angle glaucoma trabecular meshwork cells in vitro. To analyze the effects of different final concentrations of recombination human bone morphogenetic protein 7(rhBMP7)on proliferation on the basis of culturing the trabecular meshwork cells from primary open-angle glaucoma eye. To discuss the relationship between rhBMP7 and beginning and progression of POAG. Methods The trabecular tissues from trabeculectomy without mitomycin were cultured in vitro and passaged 3 times. The culture cells were proven as trabecular meshwork cell. The third-fifth passage cells were incubated with different final concentrations of rhBMP7[0(control group),20,50,80,100,and 200 ng/mL]for 24 h,48 hrespectively,and the proliferation effect of rhBMP7 on trace meshwork cells from POAG was detected by Cell Counting Kit-8 assay(CCK-8),fluorescence microscopy,and flow cytometry. Results The primarily trabecular meshwork cells from POAG were identified as purified trabecular meshwork cells through continuous cell culture,inverted microscope and immunocytochemical method. The OD value of trabecular meshwork cells from POAG which were treated with different final concentrations of rhBMP7 [0(control group),20,50,80,100,200 ng/mL]for 24 h were [0.561 2±0.026 9],[0.724 2±0.039 3],[1.416 0±0.016 2],[1.740 4±0.039 2],[1.853 8±0.014 5],[1.936 4±0.054 6]by CCK-8 assay. The difference between experimental groups and control group was statistically significant(P<0.05). The difference among experimental groups was statistically significant(P<0.05). Observed by fluorescence microscopy,as the rhBMP7 final concentration increased,the counts of the signed POAG trabecular meshwork cells by CFDA SE were enhanced. After the treatment of 20,50,80,100,200 ng/mL rhBMP7 for 48 h,the proliferation rate of POAG trabecular meshwork cells were 17.85%,18.63%,20.10%,27.45%,72.41%.Conclusion The POAG trabecular meshwork cells can be successfully cultured and identified in vitro.The proliferation ability of POAG trabecular meshwork cells can be accelerated by rhBMP7 in a certain range and the change is rhBMP7 concentration dependent in a certain range.
Objective To establish a primary culture system for primary open-angle glaucoma trabecular meshwork cells in vitro. To analyze the effects of different final concentrations of recombination human bone morphogenetic protein 7(rhBMP7)on proliferation on the basis of culturing the trabecular meshwork cells from primary open-angle glaucoma eye. To discuss the relationship between rhBMP7 and beginning and progression of POAG. Methods The trabecular tissues from trabeculectomy without mitomycin were cultured in vitro and passaged 3 times. The culture cells were proven as trabecular meshwork cell. The third-fifth passage cells were incubated with different final concentrations of rhBMP7[0(control group),20,50,80,100,and 200 ng/mL]for 24 h,48 hrespectively,and the proliferation effect of rhBMP7 on trace meshwork cells from POAG was detected by Cell Counting Kit-8 assay(CCK-8),fluorescence microscopy,and flow cytometry. Results The primarily trabecular meshwork cells from POAG were identified as purified trabecular meshwork cells through continuous cell culture,inverted microscope and immunocytochemical method. The OD value of trabecular meshwork cells from POAG which were treated with different final concentrations of rhBMP7 [0(control group),20,50,80,100,200 ng/mL]for 24 h were [0.561 2±0.026 9],[0.724 2±0.039 3],[1.416 0±0.016 2],[1.740 4±0.039 2],[1.853 8±0.014 5],[1.936 4±0.054 6]by CCK-8 assay. The difference between experimental groups and control group was statistically significant(P<0.05). The difference among experimental groups was statistically significant(P<0.05). Observed by fluorescence microscopy,as the rhBMP7 final concentration increased,the counts of the signed POAG trabecular meshwork cells by CFDA SE were enhanced. After the treatment of 20,50,80,100,200 ng/mL rhBMP7 for 48 h,the proliferation rate of POAG trabecular meshwork cells were 17.85%,18.63%,20.10%,27.45%,72.41%.Conclusion The POAG trabecular meshwork cells can be successfully cultured and identified in vitro.The proliferation ability of POAG trabecular meshwork cells can be accelerated by rhBMP7 in a certain range and the change is rhBMP7 concentration dependent in a certain range.
引文
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[6]Wordinger R J,Aqarwal R,Talati M,et al.Expression of bone morphogenetic proteins(BMP),BMP receptors,and BMP associated proteins in human trabecular meshwork and optic nerve head cells and tissues[J].Mol Vis,2002,8:241-250.
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[8]贺翔鸽,李美玉.人眼小梁细胞培养及免疫组化特性研究[J].中华眼科杂志,1998,34(4):280-282.
[9]Yu A L,Birke K,Moriniere J,et al.TGF-{beta}2induces senescence-associated changes in human trabecular meshwork cells[J].Invest Ophthalmol Vis Sci,2010,51(11):5718-5723.
[10]赵太宏,王林农,方芳,等.原发性开角型青光眼小梁网内皮细胞凋亡的探讨[J].中国煤炭工业医学杂志,2009,12(11):1665-1666.
[11]Fuchshofer R,Tamm E R.Modulation of extracellular matrix turnover in the trabecular meshwork[J].Exp Eye Res,2009,88(4):683-688.
[2]Sebald W,Mueller T D.The interaction of BMP-7and ActRII implicates a new mode of receptor assembly[J].Trend Biochem Sci,2003,10:518-521.
[3]Inatani M,Tanihara H,Katsuta H,et al.Transforming growth factor-β2levels in aqueous humor of glaucomatous eyes[J].Clin Exp Ophthalmol,2001,239(2):109-113.
[4]Shepard A R,Fleenor D L,Hellberg P E,et al.TGF2-induced changes in human trabecular meshwork:implications for intraocular pressure[J].Invest Ophthalmol Vis Sci,2006,47(1):226-234.
[5]Fuchshofer R,Yu A H,Tamm E R,et al.Bone morphogenetic protein-7is an antagonist of transforming growth factor-beta2in human trabecular meshwork cells[J].Invest Ophthalmol Vis Sci,2007,48(2):715-726.
[6]Wordinger R J,Aqarwal R,Talati M,et al.Expression of bone morphogenetic proteins(BMP),BMP receptors,and BMP associated proteins in human trabecular meshwork and optic nerve head cells and tissues[J].Mol Vis,2002,8:241-250.
[7]Zhao S,Chen Q,Hung F C,et al.BMP signaling is required for development of the ciliary body[J].Development,2002,129(19):4435-4442.
[8]贺翔鸽,李美玉.人眼小梁细胞培养及免疫组化特性研究[J].中华眼科杂志,1998,34(4):280-282.
[9]Yu A L,Birke K,Moriniere J,et al.TGF-{beta}2induces senescence-associated changes in human trabecular meshwork cells[J].Invest Ophthalmol Vis Sci,2010,51(11):5718-5723.
[10]赵太宏,王林农,方芳,等.原发性开角型青光眼小梁网内皮细胞凋亡的探讨[J].中国煤炭工业医学杂志,2009,12(11):1665-1666.
[11]Fuchshofer R,Tamm E R.Modulation of extracellular matrix turnover in the trabecular meshwork[J].Exp Eye Res,2009,88(4):683-688.