D,L-聚乳酸降解产物对内皮细胞生长影响的体外实验

In vitro effects of D, L-lactic acid degradation products on the growth of endothelial cells

目的:观察D,L-聚乳酸体外降解规律和降解产物对内皮细胞生长的影响。方法:实验于2006-06/12在四川大学细胞相容性研究室完成。将D,L-聚乳酸膜在生理盐水中体外无菌降解120d。①计算失质量率:失质量率(%)(降解前本体的质量-降解后本体的质量)/降解前本体的质量×100%。②测定黏均相对分子质量:用乌=氏黏度计外推法测降解后本体的特性黏度,特性黏度与黏均相对分子质量关系式为:η=2.21×104M0.77。③测定降解液的pH值。④乳酸标准曲线的制备:将乳酸稀释为0.16,0.08,0.048,0.032,0.016,0.008g/mL,于262nm的波长处测定吸光度值,对乳酸质量浓度做标准曲线。⑤将不同降解时间的降解液与内皮细胞共培养,分为3组,实验组分别加入不同降解时间的未经稀释的降解液100μL和新鲜培养基100μL,阴性对照组加入生理盐水100μL和培养基100μL,空白对照组加入培养基100μL,各组分别培养1,2,3,4,5,6d。采用MTT法测定内皮细胞的生长。结果:①失质量率:在最初2周内,失质量率增大较快。在20～70d失质量率变化不大。在80～120d失质量率快速增加。②黏均相对分子质量:从开始降解到70d,黏均相对分子质量呈直线下降趋势,之后随时间延长而趋于平缓,且相对分子质量很小。③pH值:前30dpH值下降很快,从6.32降到4.56;在30～70dpH值反而有所上升,上升到5.39;之后急剧下降,在120d时达到2.29。④乳酸的质量浓度:随降解时间延长而增大,前20d达到5.8g/L,70d为16.7g/L,120d时达到24.4g/L。⑤不同降解时间的降解液作用下内皮细胞的生长情况:前70d的降解液对内皮细胞生长有促进作用,70d后的降解液明显抑制内皮细胞生长,到120d时降解液使内皮细胞表现显著的细胞毒性,细胞出现凋亡。结论:D,L-聚乳酸膜降解后,70d前降解液促进内皮细胞生长,70d后至120d降解液对内皮细胞生长的影响从抑制作用到细胞毒性逐渐增强。本实验表明D,L-聚乳酸的降解产物在局部积累达一定浓度后对内皮细胞生长的微生态环境有极大的改变,将导致细胞毒性增强,甚至细胞凋亡。

AIM： To investigate the rule of poly （D,L-lactic acid） （PDLLA） degradation in vitro and the growth of endothelial cells cultured with PDLLA degradation products. METHODS： The experiment was performed at the Biocompatibility Laboratory of Sichuan University from June to December in 2006. The degradation of PDLLA film was carded out during 120 days in sterilized physiological saline solution. ①Weight loss rate （%）=（noumenal weight before degradation-noumenal weight after degradation）/noumenal weight before degradation×100%. ②By using extrapolation method of Ubbelohde viscometer, the noumenal intrinsic viscosity after degradation was measured, then viscosity average relative molecular weight was calculated according to the formula of η=2.21×10^4 M^0.77.③The pH value of degradation fluid was detected.④The absorbance at the wavelength of 262 nm was determined when the concentration of lactic acid was 0.16, 0.08, 0.048, 0.032, 0.016, and 0.008 g/mL. Then the concentration curve was prepared.⑤Endothelial cells were cultured with degradation fluid of PDLLA and divided into three groups. Experimental group was added with 100 μL undiluted degradation fluid and 100 μL fresh medium; negative control group was added with 100 μL saline and 100 μL medium; blank control group was added with 100 μL medium. Three groups were cultured for 6 days. The growth of endothelial cells was studied by MTT method. RESULTS： ①Weight loss rate was increased in the initial two weeks, stable during 20-70 days, and quickly increased during 80-120 days. ②The viscosity average relative molecular weight was lineady degressive in the first 70 days, then gradually came to an equilibrium value and the relative molecular weight was very small. ③The pH value rapidly decreased from 6.32 to 4.56 in the first 30 days of degradation time, and increased to 5.39 during 30-70 days, then sharply decreased to 2.29 after 120 days. ④The concentration of lactic acid increased with the increase of degradation time, and the value reached 5.8 g/L in the first 20 days, 16.7 g/L after 70 days, and 24.4 g/L after 120 days. ⑤The endothelial cells grew well when cultured with degradation fluid in the first 70 days, then the cell growth was obviously inhibited, and after 120 days the cell apoptosis appeared, showing significant cytotoxicity. CONCLUSION： The degradation fluid of PDLLA at first 70 days accelerates the growth of endothelial cells, and inhibits the growth from 70 days to 120 days, appearing cytotoxicity. The degradation products of PDLLA will greatly change the ecological micro-environment of endothelial cells and result in the enhancement of cytotoxicity even cell apoptosis.