微囊小肝细胞样前体细胞移植治疗大鼠急性肝衰竭

目的探索大鼠海藻酸钠-聚赖氨酸-海藻酸钠(APA)微囊小肝细胞样前体细胞(SHPC)移植治疗急性肝衰竭(AHF)大鼠模型的可行性和有效性。方法选择体质量150~180 g健康雄性Wistar大鼠10只。采用Retrosine(30 mg/kg)腹腔内注射联合2/3肝切除诱导雄性Wistar大鼠SHPC增殖模型,改进Seglen胶原酶灌注联合Percoll密度梯度离心分离SHPC,倒置显微镜、电子显微镜下观察,并进行免疫组织化学染色;静电液滴法APA微囊包埋。选择体质量180~220 g健康雌性Wistar大鼠20只,随机分为A组(微囊化SHPC腹腔内移植)、B组(空囊对照组);采用D-氨基半乳糖腹腔注射制备大鼠AHF模型;观察模型一般状况及生存时间,术后不同时间点检测血清丙氨酸转氨酶(ALT)、天门冬氨酸氨基转移酶(AST)、总胆红素(TBiL)及血氨水平,检测肝脏病理改变。结果Retrosine腹腔内注射联合2/3肝切除成功建立SHPC增殖模型,分离获得细胞符合大鼠SHPC;APA微囊呈光滑规则球形,直径为(300±40)μm,强度、韧性良好,囊内细胞形态完整,存活率为(90.0±1.3)%。微囊SHPC移植后,A组较B组中位生存时间延长(119.00 h vs 70.33 h),差异有统计学意义(P<0.05);随时间延长,A组大鼠血清ALT、AST、TBiL及血氨水平明显下降,均低于移植前水平,各时间点A组明显低于B组,差异有统计学意义(P<0.05);肝组织病理显示A组肝细胞损伤较B组明显改善;存活48 h大鼠腹腔内微囊保持完整光滑球形,囊内SHPC形态完整,1周微囊有散在破损皱缩,囊内细胞有增多聚集趋势,细胞存活率85%以上。结论APA微囊化大鼠SHPC腹腔内移植能改善大鼠AHF模型生存时间、生物化学指标(ALT、TBiL等)及病理损伤。

Microencapsulated rat small hepatocyte-like progenitor cells transplantation in treatment of acute hepatic failure

Objective To study aginate-polylysine-alginate(APA)microencapsulated small hepatocyte-like progenitor cells(SHPC)transplantation in treatment of acute hepatic failure(AHF)rats.Methods The SHPC proliferation model were induced in 10 male Wistar rats(body weight 150-180 g)with administration of Retrosine(30 mg/kg)combined with 2/3 partial hepatectomy,then SHPC were isolated by modified Seglen collagenase perfusion method and purified by Percoll gradient centrifugation protocol,the obtained cells were observed under inverted phase-contrast microscope and electron microscope,identified by immunohistochemical staining,and APA microencapsulated by electrostatic liquid droplet technique.Twenty healthy female Wistar rats(body weight 180-220 g)were randomly divided into group A(microencapsulated SHPC intraperitoneal transplantation)and group B(blank control group).The rat AHF model was prepared by D-galactosamine.General condition and survival time of the models,serum alanine transarninase(ALT),aspartate aminotransferase(AST),total bilirubin(TBiL)and ammonia levels after operation at different time points were observed,and liver histopathology was examined.Results Satisfactory rat model of SHPC proliferation were established and obtained cells met characteristics of SHPC under phase-contrast microscope,electron microscopic examination and phenotypic makers.APA microcapsule was smooth regular sphere,and the size was(300±40)μm with good intensity and tenacity,SHPC viability in microcapsule was(90.0±1.3)%.After SHPC transplantation,the survival time of group A was longer than that of group B(119.00-hour vs 70.33-hour),and the difference was statistically significant(P<0.05).Biochemical parameters(serum ALT,AST,TBiL and ammonia)at each time points in group A were declined after microencapsulated SHPC transplantation as time increased,which lower than that of group B,and the difference was statistically significant(P<0.05).The liver histopathology observation results showed that hepatocyte injury in group A was significantly improved compared with that of group B.The microencapsulated SHPC obtained from 48-hour survived rat remained sooth round shape and shrinked or damaged after one week,the cells were spherical aggregated and cell viability was higher than 85%.Conclusion It is demonstrated that APA microencapsulated SHPC transplantation could prolong survival time,improve biochemical indexes(ALT,TBiL)and liver pathological injury of AHF rats.