透明质酸支架复合小鼠3T3-L1细胞及VEGF注射充填的实验研究

The study of injection of 3T3-L1 preadipocyte and hyaluronic acid scaffold combined with VEGF

目的观察透明质酸（HA）支架复合小鼠3T3- L1细胞及血管内皮细胞生长因子（VEGF）注射充填后移植细胞的存活情况。方法体外培养细胞并诱导后分组，PCR法检测分组后的脂肪分化相关基因[氧化物酶体增殖物激活受体γ（PPARγ）、转录因子CCAAT增强子结合蛋白α（C- EBPα）]的表达。根据移植物成分分为4组：实验组（3×106诱导后脂肪细胞＋0.6mlPBS缓冲液＋0.6mlHA＋20ng/mlVEGF），HA对照组（3＆#215;106诱导后脂肪细胞＋0.6mlPBS缓冲液＋0.6mlHA），VEGF对照组（3＆#215;106诱导后脂肪细胞＋1.2mlPBS缓冲液＋20ng/mlVEGF），空白对照组（3＆#215;106诱导后脂肪细胞＋1.2mlPBS缓冲液）。术后2、4、8周处死小鼠后取材，标本行形态学及病理学检查，并测定移植物质量及微血管计数。结果体外培养后各组小鼠PPARγ及C/EBPα条带密度相近。实验组较其他各组存活的细胞多，形态均一，坏死较少。2、4、8周实验组小鼠移植物质量均明显高于其他组，HA对照组均明显高于VEGF对照组及空白对照组，差异均有统计学意义（均P＜0.05）；2、4、8周实验组小鼠微血管计数均明显高于其他组，VEGF对照组均明显高于空白对照组，HA对照组均明显低于VEGF对照组，差异均有统计学意义（均P＜0.05）。结论 HA支架复合小鼠3T3- L1细胞及VEGF体外培养后移植入小鼠体内可以提高移植细胞的存活水平。

Objective To evaluate the effect of increasing the survival rate of mouse 3T3- L1 after transplantation with hyaluronic acid （HA） gel scaffold and vascular endothelial growth factor （VEGF）. Methods After cellculture and differentiation induction in vitro, gene expression of peroxisome proliferator activated receptor γ （PPARγ） and CCAAT/enhancer- binding proteinα （C/EBPα） was tested. HA- VEGF group （3＆#215;106 induced 3T3- L1＋0.6ml PBS＋0.6ml HA）, HA group （3＆#215;106 induced 3T3＋0.6mlPBS ＋0.6mlHA）, VEGF group （3 ＆#215;106 induced 3T3＋1.2mlPBS ＋20ng/mlVEGF） and control group （3 ＆#215;106 induced 3T3＋1.2mlPBS） were set up to transplant the components. The mice were executed after 2, 4, and 8 weeks. Morphology and pathology evaluation were performed to exam the tissue specimen. The weight measurement of the tissue specimen and the microvessel counting were also performed. Results After culture and differentiation induction in vitro, the band density of PPARγand C/EBP α were similar between groups by PCR test. The HE and Sultan III staining result indicated that VEGF combined with hyaluronic acid transplantation group had regular cellsize, more cellsurvival, less necrosis than other groups. After 2, 4 and 8 weeks, Graft weight of HA- VEGF group was significant higher than other groups. Graft weight of HA groups was significant higher than VEGF group and control group. The results had statistical difference （P〈0.05）. After 2, 4 and 8 weeks, the microvessel density of HA- VEGF group was significant higher than other groups. Microvessel density of VEGF groups was significant higher than HA group and control group. The results had statistical difference （P〈0.05）. Conclusion Intradermal injection of 3T3- L1 preadipocyte by hyaluronic acid scaffold combined with VEGF increases the rate of survival of transplanted tissue.