大麻素WIN55，212-2对体外培养牛眼小梁细胞MMP-3，MMP-9及TIMP-1表达的影响
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摘要
目的：研究大麻素WIN55,212-2对体外培养的牛眼小梁细胞MMP-3、MMP-9及TIMP-1表达的影响，从而探讨其降眼压的机制。
方法：1.牛眼小梁细胞的培养和鉴定：应用组织块培养法对牛眼小梁细胞进行原代培养，传代培养，采用免疫组织化学方法对第3代细胞（波形蛋白，NSE，VIII因子相关抗原染色）进行鉴定，应用透射电镜对细胞进行生长特性及形态学的观察,确定所得细胞大部分为小梁细胞组织来源；2.免疫组化SP染色法测定MMP-3,MMP-9的量：将经过消化离心后的传3代的小梁细胞接种于孔底铺有盖玻片的6孔培养板（密度：5×104个/mL），当细胞铺满大部分孔底时，改换培养液（不含FBS）进行饥饿培养（48h），后随机分为6个组（1孔/组）。1~5组施加含终浓度0（对照组）、1、10、20、40μmol/L大麻素WIN55,212-2的无血清培养液，第6组为阴性对照组（以PBS液为一抗）。继续培养48h后对各组细胞爬片进行免疫细胞化学染色（MMP-3及MMP-9），重复该实验共4次。结果进行计算机图像分析并进行统计学检验。3.提取细胞上清液用ELASA法检测随浓度的不同TIMP-1量的变化：将经消化离心后的传3代小梁细胞接种于96孔板（密度：5×104个/mL），当细胞铺满大部分孔底时，改换培养液（不含FBS）进行饥饿培养（48h），以使所有细胞同步。随后将其随机分为5组（15孔/组），施加含终浓度0（对照组）、1、10、20、40μmol/L大麻素WIN55,212-2的无血清培养液，继续培养48h。后分组吸出各孔上清液置于EP管中，-20℃冰箱保存，应用ELASA法检测随浓度的不同TIMP-1量的变化情况。
结果：体外培养的牛眼小梁细胞表达MMP-3及MMP-9；含大麻素WIN55,212-2终浓度为1、10、20、40μmol/L的无血清培养液可促进牛眼小梁细胞MMP-3及MMP-9的表达，且与浓度的呈正相关性，并抑制TIMP-1的表达，与浓度呈负相关性。
结论：一定剂量的大麻素可以促进牛眼小梁细胞MMP-3及MMP-9的表达（P<0.05），并抑制TIMP-1的表达（P<0.01），从而达到降低眼压的目的。
Aim: To study the effect of WIN55,212-2 on the expression of MMP-3,MMP-9and TIMP-1 incultured bovine trabecular meshwork cells (TMCs) and discuss its mechanism of reducing theintraocular pressure.
Methods: 1.Culture and identify TMC:TMCs were obtained through the cultured tissue, bovineTMCs were prmiarily cultured and subcultured.We apply immunohistochemistry (NSE,VIIIfactor related antigen staining) to identify the cell and apply transmission electron microscopy toobserve morphology and growth characteristics of cells，To determine the received cells in themajority of the trabecular tissue sources.2. Immunohistochemical SP staining the determineamount of MMP-3, MMP-9:Pass three generations from the trabecular meshwork cells afterdigestion and centrifugal inoculation density of 5×104 cells/mL in the preset coverslip 6-wellplates until the cells close to the fusion, the replacement of serum-free culture starvation for 48hafter cultured.The wells were randomly divided into 6 groups. 1-5 groups were imposedserum-free medium containing the cannabinoid WIN55,212-2 with final concentration of 0(control group) and 1,10,20,40μmol/L,and the 6 group as a negative control group (an anti-PBS).Remove the cover slip after 48h for MMP-3 and MMP-9 immunocytochemistry.Eachconcentration was repeated four times. The results of the computer image analysis and statisticaltests.3. Levels of TIMP-1 in cell media were quantified by ELASA:Take to pass on behalf of thetrabecular meshwork cells after digestion and centrifugal inoculation density of 5×104cells/mL in96-well plates until the cell fusion, the culture medium with serum-free culture hunger 48h cellstate as far as possible to achieve synchronizationof. Cells were randomly divided into 5 groupswith 10 wells in each group,separately applied with cannabinoid WIN55,212-2 finalconcentration of 0,1,10,20,40μmol/L,serum-free medium.Cultured for 48h suction correspondinghole in the supernatant were put in the EP tube ELASA assay with the concentration of TIMP-1in the amount of change.
Result：The cultured bovine TMCs expressed MMP-3 and MMP-9. Containing marijuanaprime WIN55,212-2 final concentration 1,10,20,40μmol / L of serum-free culture fluid with increasing concentration of bovine trabecular meshwork cells can promote MMP-3 and MMP-9expression and suppression of TIMP-1 expression.
Conclusion：Certain concentration of WIN55,212-2 may promote the expression of MMP-3and MMP-9 of TMCs(P<0.05)and decrease TIMP-1 expression(P<0.01),so as to reduce theintraocular pressure.
方法：1.牛眼小梁细胞的培养和鉴定：应用组织块培养法对牛眼小梁细胞进行原代培养，传代培养，采用免疫组织化学方法对第3代细胞（波形蛋白，NSE，VIII因子相关抗原染色）进行鉴定，应用透射电镜对细胞进行生长特性及形态学的观察,确定所得细胞大部分为小梁细胞组织来源；2.免疫组化SP染色法测定MMP-3,MMP-9的量：将经过消化离心后的传3代的小梁细胞接种于孔底铺有盖玻片的6孔培养板（密度：5×104个/mL），当细胞铺满大部分孔底时，改换培养液（不含FBS）进行饥饿培养（48h），后随机分为6个组（1孔/组）。1~5组施加含终浓度0（对照组）、1、10、20、40μmol/L大麻素WIN55,212-2的无血清培养液，第6组为阴性对照组（以PBS液为一抗）。继续培养48h后对各组细胞爬片进行免疫细胞化学染色（MMP-3及MMP-9），重复该实验共4次。结果进行计算机图像分析并进行统计学检验。3.提取细胞上清液用ELASA法检测随浓度的不同TIMP-1量的变化：将经消化离心后的传3代小梁细胞接种于96孔板（密度：5×104个/mL），当细胞铺满大部分孔底时，改换培养液（不含FBS）进行饥饿培养（48h），以使所有细胞同步。随后将其随机分为5组（15孔/组），施加含终浓度0（对照组）、1、10、20、40μmol/L大麻素WIN55,212-2的无血清培养液，继续培养48h。后分组吸出各孔上清液置于EP管中，-20℃冰箱保存，应用ELASA法检测随浓度的不同TIMP-1量的变化情况。
结果：体外培养的牛眼小梁细胞表达MMP-3及MMP-9；含大麻素WIN55,212-2终浓度为1、10、20、40μmol/L的无血清培养液可促进牛眼小梁细胞MMP-3及MMP-9的表达，且与浓度的呈正相关性，并抑制TIMP-1的表达，与浓度呈负相关性。
结论：一定剂量的大麻素可以促进牛眼小梁细胞MMP-3及MMP-9的表达（P<0.05），并抑制TIMP-1的表达（P<0.01），从而达到降低眼压的目的。
Aim: To study the effect of WIN55,212-2 on the expression of MMP-3,MMP-9and TIMP-1 incultured bovine trabecular meshwork cells (TMCs) and discuss its mechanism of reducing theintraocular pressure.
Methods: 1.Culture and identify TMC:TMCs were obtained through the cultured tissue, bovineTMCs were prmiarily cultured and subcultured.We apply immunohistochemistry (NSE,VIIIfactor related antigen staining) to identify the cell and apply transmission electron microscopy toobserve morphology and growth characteristics of cells，To determine the received cells in themajority of the trabecular tissue sources.2. Immunohistochemical SP staining the determineamount of MMP-3, MMP-9:Pass three generations from the trabecular meshwork cells afterdigestion and centrifugal inoculation density of 5×104 cells/mL in the preset coverslip 6-wellplates until the cells close to the fusion, the replacement of serum-free culture starvation for 48hafter cultured.The wells were randomly divided into 6 groups. 1-5 groups were imposedserum-free medium containing the cannabinoid WIN55,212-2 with final concentration of 0(control group) and 1,10,20,40μmol/L,and the 6 group as a negative control group (an anti-PBS).Remove the cover slip after 48h for MMP-3 and MMP-9 immunocytochemistry.Eachconcentration was repeated four times. The results of the computer image analysis and statisticaltests.3. Levels of TIMP-1 in cell media were quantified by ELASA:Take to pass on behalf of thetrabecular meshwork cells after digestion and centrifugal inoculation density of 5×104cells/mL in96-well plates until the cell fusion, the culture medium with serum-free culture hunger 48h cellstate as far as possible to achieve synchronizationof. Cells were randomly divided into 5 groupswith 10 wells in each group,separately applied with cannabinoid WIN55,212-2 finalconcentration of 0,1,10,20,40μmol/L,serum-free medium.Cultured for 48h suction correspondinghole in the supernatant were put in the EP tube ELASA assay with the concentration of TIMP-1in the amount of change.
Result：The cultured bovine TMCs expressed MMP-3 and MMP-9. Containing marijuanaprime WIN55,212-2 final concentration 1,10,20,40μmol / L of serum-free culture fluid with increasing concentration of bovine trabecular meshwork cells can promote MMP-3 and MMP-9expression and suppression of TIMP-1 expression.
Conclusion：Certain concentration of WIN55,212-2 may promote the expression of MMP-3and MMP-9 of TMCs(P<0.05)and decrease TIMP-1 expression(P<0.01),so as to reduce theintraocular pressure.
引文
[1].刘慧峰，王万辉，赵俊华。大麻素（WIN55,212-2）对高眼压家兔模型降眼压作用的研究[J]。眼科研究，2008,26（12）:929-931.
[2]杨晓岗，张德秀。大麻素对牛眼小梁细胞增殖、粘附和迁移的影响。国际眼科杂志，2008,8(7)1343-1345.
[3] Mahmut Yilmaz, Gerhard Christofori, Francois Lehembre.Distinct mechanisms of tumorinvasion and metastasis[J].Trends in Molecular Medicine,2007,13(12):535－540.
1杨新光，李美玉.牛眼小梁细胞体外培养及吞噬功能的研究.中华眼科杂志,1996;32(2):136- 139。
2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.
3 Acott TS,Wirtz MK.Biochemistry of aqueous humor outflow[M].The Glaucomas,1996;281-305.
4宋哲,周喜岩,张德秀.拉坦前列素对青光眼治疗作用.国际眼科杂志,2002;2(3):16-17.
5 Oltmanns MH,Samudre SS,Caztillo IG.Topical WIN55212-2 alleviates intraocularhypertension in rats through a CB1 receptor mediated mechanism of action[J]. Ocul PhannacolTher.2008;24:104-115.
6 Rosch S, Ramer R, Brune K, et al. R(+)-methanandamide and other cannabinoids induce theexpression of cyclooxygenase-2 and matrix metalloproteinases in human nonpigmented ciliaryepithelial cells[J].Pharmacol Exp Ther,2006;316:1219-1228.
7 Chew PT, AungT, AquinoMV, et al. Intraocular pressure-reducing effects and safety oflatanoprostversustmi olol in patientswith chronic angle-closure glaucoma. Ophthalmology,2004;111:427-434
8 KooK MS,Cho HS,Yang SJ,et al.Efficacy of latanoprost in patients with chronic angle-closure glaucoma and no visible ciliary-body face:aprelmi inary study[J].Ocul PharmacolTher,2005;21:75-84.
9 Parshiey DE et al.Curr Eye Res，1995:14:537
10 Alexander JP, Sample JR, Van Buskirk EM. Expression of matrix metalloproteinases andinhibitor by human trabecular meshwork[J]. Invest Ophthalmol Vis Sci ,1991;32(4):172-195
11张静，王万辉.大麻素WIN55 212-2对牛眼小梁细胞内钙离子及组织型转谷氨酰胺酶表达的影响。中国药物与临床，2009，10（2）162-163.
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[2]杨晓岗，张德秀。大麻素对牛眼小梁细胞增殖、粘附和迁移的影响。国际眼科杂志，2008,8(7)1343-1345.
[3] Mahmut Yilmaz, Gerhard Christofori, Francois Lehembre.Distinct mechanisms of tumorinvasion and metastasis[J].Trends in Molecular Medicine,2007,13(12):535－540.
1杨新光，李美玉.牛眼小梁细胞体外培养及吞噬功能的研究.中华眼科杂志,1996;32(2):136- 139。
2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.
3 Acott TS,Wirtz MK.Biochemistry of aqueous humor outflow[M].The Glaucomas,1996;281-305.
4宋哲,周喜岩,张德秀.拉坦前列素对青光眼治疗作用.国际眼科杂志,2002;2(3):16-17.
5 Oltmanns MH,Samudre SS,Caztillo IG.Topical WIN55212-2 alleviates intraocularhypertension in rats through a CB1 receptor mediated mechanism of action[J]. Ocul PhannacolTher.2008;24:104-115.
6 Rosch S, Ramer R, Brune K, et al. R(+)-methanandamide and other cannabinoids induce theexpression of cyclooxygenase-2 and matrix metalloproteinases in human nonpigmented ciliaryepithelial cells[J].Pharmacol Exp Ther,2006;316:1219-1228.
7 Chew PT, AungT, AquinoMV, et al. Intraocular pressure-reducing effects and safety oflatanoprostversustmi olol in patientswith chronic angle-closure glaucoma. Ophthalmology,2004;111:427-434
8 KooK MS,Cho HS,Yang SJ,et al.Efficacy of latanoprost in patients with chronic angle-closure glaucoma and no visible ciliary-body face:aprelmi inary study[J].Ocul PharmacolTher,2005;21:75-84.
9 Parshiey DE et al.Curr Eye Res，1995:14:537
10 Alexander JP, Sample JR, Van Buskirk EM. Expression of matrix metalloproteinases andinhibitor by human trabecular meshwork[J]. Invest Ophthalmol Vis Sci ,1991;32(4):172-195
11张静，王万辉.大麻素WIN55 212-2对牛眼小梁细胞内钙离子及组织型转谷氨酰胺酶表达的影响。中国药物与临床，2009，10（2）162-163.
1 Elsohly MA, Slade D. Chemical constituents of marijuana: the complex mixture of naturalcannabinoids. Life Sci,2005,78:539-548
2 Kearse EC, Green K. Effect of vehicle upon in vitro transcorneal permeability andintracorneal content of Delta9-tetrahydrocan-nabinol. Curr Eye Res,2000,20: 496-501
3 Jarvinen T, Pate DW, Laine K. Cannabinoids in the treatment of glaucoma. PharmacolTher,2002,95:203-22。
4 Matsuda LA,Lolait SJ,BrownsteinMJ,et al.Structure of a cannabinoid reactor and afunctionalexpression of the cloned cDNA[J].Nature,990,346(6284):561-564.
5 Hertzog DL.Recent advances in the cannabinoids.Expert opin T-her Pat,2004,14:1435-1452.
6 Thakur GA,Nikas SP,Makriyannis A.CBl cannabinoid receptarligands.Mini Rev MedChem,2005,5:631-640.
7 Straiker AJ,Maguire G,Mackie K,et a1.Localization of cannabi—noid CBI receptors in thehuman anterior eye and retina.Invest Ophthalmol Vis Sci,1999,40:2442-2448.
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