载肝细胞生长因子的纳米粒子对大鼠移植肝细胞生存的影响

Influence of hepatocyte growth factor loaded nanoparticles on survival of rat transplanted hepatocytes

目的 探讨载肝细胞生长因子（HGF）的聚乳酸-氧-羧甲基壳聚糖（PLA-O-CMC）纳米粒子对大鼠肝细胞移植后移植肝细胞生存的影响.方法 Seglen原位两步灌流法分离大鼠肝细胞,培养24 h后分别将5ml常规胶原大鼠肝细胞悬液（5.0＆#215;107个细胞,下同）（A组）、5 ml PLA-O-CMC纳米粒子大鼠肝细胞（B组）、5 ml载HGF的PLA-O-CMC纳米粒子大鼠肝细胞（C组）移植到大鼠腹腔内,5 ml PLA-O-CMC纳米粒子大鼠肝细胞腹腔移植加每日静脉注射HGF 10 μg/kg ＆#215;7 d（D组）.观察各组培养1d和腹腔肝细胞移植后移植肝细胞的超微结构变化.观察移植前培养液和受体腹腔渗液丙氨酸转移酶（ALT）和门冬氨基转移酶（AST）水平变化.流式细胞仪检测培养和移植肝细胞凋亡率、坏死率及存活率.结果 移植肝细胞的存活时间以C组最长,达7d,术后3～7 d移植肝细胞的电镜变化A组核质浓缩最为明显,C组内质网最为丰富.移植后1d时腹腔渗液ALT A组＞C组（1 002.68 ±229.37） U/L比（770.62 ±120.77） U/L,P＜0.05].移植后1、3、5d,活率B、C、D组＞A组（P＜0.05）,其中5d时C组[（59.51±1.59）％]＞D组[（55.98±2.27）％,P＜0.05];移植后3、5d凋亡率A组＞B、C、D组（P＜0.05）;移植后1、5d,坏死率A组＞B、C、D组.结论 载HGF的PLA-O-CMC纳米粒子培养的大鼠肝细胞腹腔内移植后有提高移植肝细胞活率的作用,延长其生存时间并促进其功能表达.

Objective To evaluate the hepatocyte growth factor （HGF） loaded polylactic acid-Ocarboxymethylated chitosan （PLA-O-CMC） nanoparticles on the survival of the transplanted hepatocytes after hepatocyte transplantation.Methods Two-steps in situ collagenase perfusion method described by Seglen was used to isolate rat hepatocytes.Respectively 5ml rat free hepatocyte suspension （5.0 ＆#215; 107 cells） （Group A）,5 ml rat hepatocytes co-cultured with PLA-O-CMC nanoparticles （5.0 ＆#215; 107 cells） （Group B） and 5ml rat hepatocytes co-cultured with HGF loaded PLA-O-CMC nanoparticles （5.0 ＆#215; 107 cells） （Group C） were transplanted into the abdominal cavity of SD rats at 48 h after D-Gal injection.As Group B,plus intravenous injection HGF 10 μg/kg everyday as comparison （Group D）.The pathological changes and ultramicrostructure of hepatocyte culured 1d or transplanted hepatocyte of each group were observed.The alanine transaminase （ALT） and aspartate aminotransferase （AST） of culture solution and effusion of abdominal cavity was detected.The rate of apoptosis,viability and necrosis of the transplanted hepatocytes were observed.Results The survival time of the transplantated hepatocytes in group C achieved 7 d was the longest in all groups.Transplanted hepatocytes nuclear chromatin condensation phenomenon is most obvious in group A while Group C is the opposite.Peritoneal exudate ALT level of Group A [（1 002.68 ± 229.37） U/L] 〉 Group C [（770.62 ± 120.77） U/L] after transplantation 1d.After transplantation 1,3,5 d,motility of Group B,C,D 〉 A （P〈0.05）,and Group C [（59.51 ±1.59）%] 〉 Group D [（55.98 ± 2.27） %] 〉 D after 5d.After transplant 3d,5d apoptosis rate in group A 〉 B,C,D.After 1,5 d,necrosis rate in group A 〉 B,C,D.Conclusion HGF loaded PLA-O-CMC nanoparticles may increase the viability of the transplantated hepatocytes after abdominal cavity transplantation,prolong the survival time and promote their functional expression.