造血干细胞移植抑制肝癌术后复发转移的实验研究

Effect of allogenic hematopoietic stem cell transplantation on tumor recurrence and metastasis of hepatocellular carcinoma after radical hepatectomy

目的证实异基因造血干细胞移植（allo hematopoietic stem cell transplantion，allo-HST）对严重联合免疫缺陷（severe combined irnmunodeficiency，SCID）小鼠肝癌根治性切除术后的复发转移有一定抑制作用。方法采集足月健康胎儿新鲜脐血，以6％羟乙基淀粉（hydroxyethyl starch，HKS）＋NH4Cl破红细胞法分离人脐带血有核细胞（nucleated cells，NC）。SCID小鼠22只随机分为A组：程序移植组（n=8）；B组：单次移植组（n=8）；C组：生理盐水对照组（n=6）。两于细胞移植组内每只小鼠经尾静脉注入人脐带血NC共5×10^7个，三组小鼠移植前都给予低剂量的环磷酰胺预处理，并连续使用甲泼泥龙1周。6周后观测小鼠存活情况，流式细胞仪检测两干细胞移植组人源细胞嵌合情况。同时将HCCLM6肿瘤组织块原位接种于SCID小鼠肝脏，10d后对荷瘤肝叶行根治性切除，4周后处死小鼠，观察肝内复发和肝外转移的情况。结果通过化疗预处理及使用免疫抑制剂能够成功建立了人一鼠造血干细胞移植模型，程序移植组及单次移植组外周血CD3^＋细胞嵌合率分别为（1．66±0．47）％（0．68±0．56）％，差别有统计学意义（P〈0．01）。移植瘤切除后A、B、C各组肝内复发率均为100％，但复发瘤体积大小差异显著，分别为（367．18±31．86）mm^3、（648．26±155．22）mm^3、（811．38±127．36）mm^3，两两比较差别均有统计学意义（P〈0．01）。A、B两组的抑瘤率分别为54．7％和20．1％。各组间肺转移率分别为14．3％（1／7）、66．7％（4／6）、100％（5／5），差别有统计学意义（P〈0．01）。淋巴结转移率分别为14．3％（1／7）、33．4％（2／6）、40％（2／5），差别无统计学意义（P=0．58）。结论在小鼠实验中，造血干细胞移植对肝癌根治性切除术后的复发和转移显示出一定的抑制作用，并且随着人源细胞嵌合率的增加，抗肿瘤效应也相应得到提高。

Objective To evaluate the effect of alloHCT on recurrence and metastasis of HCC after hepatic radical resection. Methods Umbilical cord blood were collected after labor. The efficacy of separation by 6% hydroxyethyl starch （6% HES） and NH4CL lysing solution were examined. Twenty-two SCID mice were randomized into the scheduled transplantation group （n=8）, the single transplantation group （n= 8） and the normal saline group （n= 6）. Human nucleated cells （NC）at a amount of 5 × 10^7 were transfused through the tail vein into the 2 groups of transplantation. All the mice in the 3 groups received cyclophosphamide （CTX） as conditioning regimen before tansplantation and Methylprednisolone （MP） for 1 week continuously after trasplantation. Hematopoietic and immune recovery, graft versus host disease （GVHD）, engraftment and survival rate were observed after transplantation. Six weeks after alloHST, the orthotopic tumor model in SCID mice was established by implanting histologically intact tissue under the embrane of liver. Ten days later, the mice received radical resection of lobe bearing tumor. The condition of recurrence and metastasis was observed 4 weeks after operation. Results A murine model of umbilical cord blood transplantation using CTX and MP as conditioning regimen could be successfully established in SCID mice. The percentage CD34^＋ cells of peripheral blood NC in scheduled transplantation group and single transplantation group was 1.66% ± 0. 47 % and 0. 68%±0. 56 %, respectively. There was significant statistical significance （ P〈0.01）. The intrahepatic recurrence rate after operation was 100% in all the 3 groups. However, the recurrent tumor volume was （367.18±31.86） mm^3 , （648.26±155.22） mm^3 , （811.38±127. 36） mm^3 , respectively in the 3 groups. There was marked difference among the 3 groups （P〈0. 01）. The inhibitory rate of group A and B was 54.7% and 20.1%. The incidence of lung metastasis was 14.3 （1/7）, 6.7% （4/6） and 100% （5/5）, respectively in the 3 groups and there was remarkable difference （P〈0.01）. The incidence of celiac lymph node metastasis was 14.3 （1/7）, 33.4% （2/6） and 40% （2/5）, respectively in the 3 groups and there was no significant difference （P=0.58）. Conclusion AlloHSCT is a useful method for decreasing metastasis and recurrence of liver cancer after radical resection in early stage.