睾丸支持细胞与肝细胞混合共微囊化移植治疗大鼠急性肝功能衰竭

Treatment of acute liver failure by xeno-transplantation of co-microencapsulated Sertoli cells and hepatocytes

目的观察大鼠睾丸支持细胞（Sertoli细胞）对微囊化肝细胞腹腔移植治疗大鼠急性肝功能衰竭疗效的影响。方法气流法制备含单独肝细胞或Sertoli细胞与肝细胞混合的微囊。D-氨基半乳糖诱导大鼠急性肝功能衰竭模型，设模型组、裸肝细胞移植组、微囊化肝细胞移植组和Sertoli细胞与肝细胞混合微囊化移植组，每组24只，检测ALT、AST、TBil、Alb水平，RT—PCR法测定肝脏凋亡指标Smac／Diablo、caspase一3表达情况，同时各组另取15只大鼠进行生存率分析。观察腹腔内微囊的变化以及腹水中淋巴细胞数的改变。样本比较采用重复测量的多元方差分析或单因素力差分析，组间比较采用，检验。结果Sertoli细胞与肝细胞混合微囊化治疗组ALT、AST在48h时即分别降至（533．7±76．5）U／L、（381．2±46．7）U／L，TBil在72h降至（7．36±2．18）gmol／L，血清Alb水平在48h已升至（28．4±2．5）g／L，与其他各组相比，差异有统计学意义（F值分别为10．7、6．5、12．2和8．4，均P〈0．05）；且Smac／Diablo、caspase-3mRNA在48、72h的表达水平也较其他组低（F笸分别为3．7、4．8和3．6、4．2，均P〈0．05）。微囊化肝细胞移植组和Sertoli细胞与肝细胞混合微囊化移植组大鼠生存率无明显差别，但明显高于其他两组。不同组的微囊均未与腹腔粘连。混合微囊组腹水中的淋巴细胞数也较肝细胞微囊组少（t=4．21，P〈0．05）。结论Sertoli细胞与肝细胞混合微囊化移植治疗肝功能衰竭的效果优于单纯微囊化肝细胞移植。纯肝细胞微囊在腹腔内仍可引起一定量淋巴细胞聚集，而Sertoli细胞的存在有助于减少这一现象。

Objective To evaluate the treatment effect of acute liver failure（ALF） by xenotransplantation of co-microencapsulated Sertoli cells and hepatocytes and the intraperitoneal immune privilege effects of Sertoli cells on hepatocytes. Methods ALF rats were induced by intraperitoneal injection of D-galactosamine and, thereafter, were treated with physical saline, free hepatocytes, microencapsulated hepatocytes, or co-microencapsulated Sertoli cells and hepatocytes （CMSH）, respectively. Alanine aminotransferase （ALT）, aspartate aminotransferase fAST） and total bilirubin （TBil） were detected in rats＇ blood samples from various groups. Expression of Smac/Diablo and caspase-3 were determined by reverse transcription-polymerase chain reaction （RT-PCR）. Fifteen rats in each group were used for survival rate analysis. The intraperitoneal microeneapsules were observed and lymphocytes in ascites were counted. The data were analyzed by multi-factor or single factor analysis of variance and the comparison between groups was done by t test. Results In CMSH treatment group, ALT level decreased to （533. 7±76. 5） U/L, AST level decreased to （381. 2±4 46.7） U/L after 48 h. TBil level reduced to （7. 36±2. 18） U/mol/L after 72 h. Albumin level increased to （28.4±2.5） g/L after 48 h. All these values were significantly different from those in other groups （F= 10.7,6.5,12.2,8.4;P〈0.05）. The expression levels of Smac/Diablo and caspase- 3 mRNA at 48 h and 72 h were lower in CMSH group than in other groups （F=3. 7,4. 8,3. 6,4. 23 P〈0.05）. Survival rates in microencapsulated hepatocytes group and CMSH group were similar while both of them were higher than other groups. Microencapsules neither in microencapsulated hepatocytes group nor in CMSH group were adhered to intraperitoneal mucosa. Lymphocyte counts in ascites of CMSH group were lower than those in microencapsulated hepatoeytes group （t= 4.21, P〈0.05）. Conclusions Intraperitoneal transplantation with CMSH is a promising approach for ALF treatment. Furthermore, Sertoli cells can help reduce lymphocytes＇ aggregation caused by encapsulated hepatocytes in ascites.