微囊化人内皮抑素／293细胞生物学性状及其对人脐静脉内皮细胞增生的抑制作用

The biological characteristics of microencapsulated human endostatin/293 cells and their inhibitory effects on the proliferation of human umbilical vein endothelial cells

目的观察不同细胞密度的微囊化人内皮抑素／293（hES／293）细胞生物学性状及其对人脐静脉内皮细胞（HUVEC）增生的抑制作用。方法采用聚电解质络合法制作不同密度微囊化hES／293细胞，分为1×10^4个／ml组（A组）、1×10^6个／ml组（B组）、1×10^8个／ml组（C组）；同时将微囊内不包裹hES／293细胞者设为对照组（D组）；每组6个样本。培养后1、3、7、14、35d，锥虫蓝染色计数微囊囊内细胞总数、活细胞数和存活率；噻唑蓝（MTT）比色法检测各组微囊化hES／293细胞的生长情况；酶联免疫吸附试验（ELISA）法检测微囊化hES／293细胞囊外内皮抑素（ES）蛋白释放量。取生长状态良好的HUVEC分别与A、B、C、D组微囊化hES／293细胞共同培养。于共同培养后24、72、120h，MTT比色法检测微囊化hES／293细胞对HUVEC增生的抑制作用。结果A、B、C组微囊囊内hES／293细胞总数和活细胞数均随时间延长而增多，囊内细胞存活率于培养后3d最高。A、B、C组微囊化hES／293细胞生长速率比较，培养后1d时组间差异无统计学意义（P〉0．05）；培养后3d，A组较B、C组高，差异有统计学意义（P〈0．05）；培养后7、14、35d，B组较A、C组高，差异有统计学意义（P〈0．05）。A、B、C组微囊化hES／293细胞囊外ES蛋白释放量比较，培养后1、14d时组间差异无统计学意义（P〉0．05）；培养后3d，A组较B、C组高，差异有统计学意义（P〈0．05）；培养后7、35d，B组较A组高，差异有统计学意义（P〈0．05）。共同培养后24h，A、B、C组对HUVEC均未表现出抑制作用（P〉0．05）；共同培养后72、120h，A、B、C组均明显抑制HUVEC增生，差异有统计学意义（P〈0．05）。结论细胞密度为1×10^6个／ml的微囊化hES／293细胞生长稳定、存活率较高，可持续稳定的释放ES蛋白。不同密度的微囊化hES／293细胞均可抑制HUVEC增生。

Objective To observe biological characteristics of microencapsulated human endostatin/ 293 （hES/293） cells at different density and their inhibitory effects on the proliferation of human umbilical vein endothelial cells （HUVEC）. Methods The microencapsulated bES/293 cells at different cellular density of 1 × 10^4 （group A）, 1× 10^6 （group B） and 1 ×10^8 （group C） cells/ml were made by polyelectrolyte complexometry technology. The empty microcapsules were set as control group （group D）. Each group has 6 samples. After 1, 3, 7, 14 and 35 days in culture, the number of total cells, viable cells was counted by trypan blue staining, and the survival fraction was measured. The grow status of hES/293 cells was measured by MTT assay, and the concentration of endostatin protein in supernatant was measured by enzyme linked immunosorbent assay （ELISA）. HUVECs were co cultured with hES/293 cells of group A, B and C. The proliferation of HUVEC at the 24, 72 and 120 hours after co-culture was measured by MTT assay. Results The number of total ceils and viable cells were increasing and the survival fraction reached its peak after 3 days in culture in group A, B and C. The growth rate in group A was higher than that in group B and C after 3 days in culture （P〈0.05）, but the growth rate in group B was higher than that in group A and C after 7, 14 and 35 days in culture （P〈0.05）. The concentration of endostatin protein in the supernatant was the same in group A, B and C after 1 and 14 days in culture （P〈0.05）. However, group A had higher endostatin than group B and C after 3 days in culture, group B had higher endostatin higher than group A and C after 7 and 35 days in culture （P〈0.05）. The hES/293 cells of group A, B and C hadno effects on the proliferation of HUVEC （P〈0.05） after 24 hours co-culture, but can inhibit the proliferation of HUVEC after 72 or 120 hours co-culture （P〈0.05）. Conclusions The microencapsulated hES/293 cells at a density of 1 × 10^6 cells/ml can grow and survive, and release endostatin protein stably. The microencapsulated hES/293 cells at different density all can inhibit the proliferation of HUVEC.