水蛭提取液对体外培养牛晶状体上皮细胞影响的实验研究
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摘要
目的 观察水蛭提取液对体外培养的牛晶状体上皮细胞(LEC)生长、贴壁及在人工晶状体(IOL)表面黏附生长的影响，探讨中药水蛭用于防治后发性白内障的可能性。
方法 1、采用植块培养法，对牛晶状体前囊膜进行培养，并利用形态学观察和体外培养LEC特异性的“晶状体小体”进行细胞鉴定。
2、利用五种常用水蛭提取方法(水醇双提法、乙醇回流法、水提法、水提醇沉法、渗滤法)提取液。观察体外培养的LEC，加入不同浓度的各种提取液培养24h、72h后的生长情况，以MTT法测定OD值，并求出半效抑制量(ID_(50))。
3、传代的LEC加入各种提取液高剂量(ID_(50))及低剂量(1／10ID_(50))培养24h，观察贴壁抑制情况。
4、接种LEC到PMMA材质的IOL表面，并加入各种高剂量和低剂量提取液。培养24h后，观察IOL表面LEC的黏附生长抑制情况。
结果 五种水蛭提取液抑制LEC生长的24h、72hID_(50)介于20.56mg／ml至68.78mg/ml之间，其中水提醇沉液、渗滤液具有更明显生长抑制作用(P＜0.05)；与空白组比较，水蛭水醇双提液、水提醇沉液、渗滤液对LEC贴壁和在IOL表面黏附生长有明显抑制作用(P＜0.01或P＜0.05)。
结论 五种水蛭提取液均能抑制体外培养的LEC生长，其中水提醇沉液、渗滤液具有更明显生长抑制作用；水蛭水醇双提液、水提醇沉液、渗滤液对LEC贴壁和在IOL表面黏附生长有明显抑制作用；水蛭水提
成都中医药大学硕士研究生学位论文
醇沉液、渗滤液能同时明显抑制体外培养的LEC生长、贴壁及在IOL
表面猫附生长;实验为水蛙提取液用于防治后发性白内障提供了实验
依据。
Objective To examine the effects of extracting solution of Hirudo on the proliferation, adhesion and adherence to intraocular lens(IOL) of bovine lens epithelial cells(LEC) cultivated in vitro, for evaluating the possibility of Hirudo on the prevention of after cataract.
Methods 1. Bovine lens anterior capsule was cultured in vitro with explant culture method, and the cultured cells were identified by morphological examination and the characteristic lens-like spherules of LEC cultured in vitro.
2. Five kinds of conventional extracting solution of Hirudo (alcohol and water extracting, alcohol refluxing, water extracting, water extracting-alcohol precipitating, percolation filtration) were used in this study. The passage LEC was incubated for 48h, then exposed the cells to different extracting solution in different concentrations for 24h and 72h. MTT method was used to detect the OD values and ID50 was calculated.
3. The passage LEC and different extracting solution in high dosage(ID50) and low dosage(l/10ID50) were placed and cultured for 24h to investigate the inhibitive rate of cell adherence.
4. LEC was incubated with IOL in high and low dosage of different extracting solution for 24h to investigate the inhibitive rate of cell adherence on the surface of PMMA IOL.
Results The ID50 of these extracting solution exposed to LEC for 24h and 72h were between 20.56mg/ml to 68.78mg/ml, and inhibition of water extracting-alcohol precipitating solution and percolation filtration solution was more strongly(P<0.05); Compared with the control group, there was
significant inhibition to LEC on cell adhesion and adherence on IOL of alcohol and water extracting solution, water extracting-alcohol precipitating solution and percolation filtration solution ( P<0.01 or P<0.05).
Conclusion Five kinds of conventional extracting solution of Hirudo all can inhibit the proliferation of LEC cultivated in vitro, and inhibition of water extracting-alcohol precipitating solution and percolation filtration solution was more strongly among them. There was significant inhibition to LEC on cell adhesion and adherence on IOL of alcohol and water extracting solution, water extracting-alcohol precipitating solution and percolation filtration solution. Water extracting-alcohol precipitating solution and percolation filtration solution of Hirudo can significantly inhibit the proliferation, adhesion and adherence to IOL of LEC cultivated in vitro at the same time. This study gives some experiment foundation for extracting solution of Hirudo to prevent after cataract.
方法 1、采用植块培养法，对牛晶状体前囊膜进行培养，并利用形态学观察和体外培养LEC特异性的“晶状体小体”进行细胞鉴定。
2、利用五种常用水蛭提取方法(水醇双提法、乙醇回流法、水提法、水提醇沉法、渗滤法)提取液。观察体外培养的LEC，加入不同浓度的各种提取液培养24h、72h后的生长情况，以MTT法测定OD值，并求出半效抑制量(ID_(50))。
3、传代的LEC加入各种提取液高剂量(ID_(50))及低剂量(1／10ID_(50))培养24h，观察贴壁抑制情况。
4、接种LEC到PMMA材质的IOL表面，并加入各种高剂量和低剂量提取液。培养24h后，观察IOL表面LEC的黏附生长抑制情况。
结果 五种水蛭提取液抑制LEC生长的24h、72hID_(50)介于20.56mg／ml至68.78mg/ml之间，其中水提醇沉液、渗滤液具有更明显生长抑制作用(P＜0.05)；与空白组比较，水蛭水醇双提液、水提醇沉液、渗滤液对LEC贴壁和在IOL表面黏附生长有明显抑制作用(P＜0.01或P＜0.05)。
结论 五种水蛭提取液均能抑制体外培养的LEC生长，其中水提醇沉液、渗滤液具有更明显生长抑制作用；水蛭水醇双提液、水提醇沉液、渗滤液对LEC贴壁和在IOL表面黏附生长有明显抑制作用；水蛭水提
成都中医药大学硕士研究生学位论文
醇沉液、渗滤液能同时明显抑制体外培养的LEC生长、贴壁及在IOL
表面猫附生长;实验为水蛙提取液用于防治后发性白内障提供了实验
依据。
Objective To examine the effects of extracting solution of Hirudo on the proliferation, adhesion and adherence to intraocular lens(IOL) of bovine lens epithelial cells(LEC) cultivated in vitro, for evaluating the possibility of Hirudo on the prevention of after cataract.
Methods 1. Bovine lens anterior capsule was cultured in vitro with explant culture method, and the cultured cells were identified by morphological examination and the characteristic lens-like spherules of LEC cultured in vitro.
2. Five kinds of conventional extracting solution of Hirudo (alcohol and water extracting, alcohol refluxing, water extracting, water extracting-alcohol precipitating, percolation filtration) were used in this study. The passage LEC was incubated for 48h, then exposed the cells to different extracting solution in different concentrations for 24h and 72h. MTT method was used to detect the OD values and ID50 was calculated.
3. The passage LEC and different extracting solution in high dosage(ID50) and low dosage(l/10ID50) were placed and cultured for 24h to investigate the inhibitive rate of cell adherence.
4. LEC was incubated with IOL in high and low dosage of different extracting solution for 24h to investigate the inhibitive rate of cell adherence on the surface of PMMA IOL.
Results The ID50 of these extracting solution exposed to LEC for 24h and 72h were between 20.56mg/ml to 68.78mg/ml, and inhibition of water extracting-alcohol precipitating solution and percolation filtration solution was more strongly(P<0.05); Compared with the control group, there was
significant inhibition to LEC on cell adhesion and adherence on IOL of alcohol and water extracting solution, water extracting-alcohol precipitating solution and percolation filtration solution ( P<0.01 or P<0.05).
Conclusion Five kinds of conventional extracting solution of Hirudo all can inhibit the proliferation of LEC cultivated in vitro, and inhibition of water extracting-alcohol precipitating solution and percolation filtration solution was more strongly among them. There was significant inhibition to LEC on cell adhesion and adherence on IOL of alcohol and water extracting solution, water extracting-alcohol precipitating solution and percolation filtration solution. Water extracting-alcohol precipitating solution and percolation filtration solution of Hirudo can significantly inhibit the proliferation, adhesion and adherence to IOL of LEC cultivated in vitro at the same time. This study gives some experiment foundation for extracting solution of Hirudo to prevent after cataract.
引文
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