外源性mFasL-cDNA基因干预后骨髓造血细胞功能的实验研究

目的:探讨FasL基因转移后造血细胞活性是否到影响,骨髓移植(BMT)后受者骨髓造血功能是否受改变。方法:FasL-cDNA经鉴定后,常规收集BalB/c小鼠和BAC小鼠股骨和胫骨骨髓单个核细胞(BMMNC),采用脂质体转移法将FasL基因转入BalB/c小鼠BMMNC,然后按1∶0.625体外与BAC鼠BMMNC混合培养,6d后实验组(第3组)尾静脉输注给经致死量照射的BalB/c小鼠,同时设立:第1组(空白对照组即未移植细胞);第2组(未转染基因BalB/c鼠BMMNC+BAC鼠BMMNC);第4组(同基因BMT组)。观察移植后受者鼠造血功能及细胞来源,移植物抗宿主病(GVHD)及生存率。结果:BMT后10,20d,第4组白细胞及血小板计数明显高于第2、3组(P<0.01);第2、3组间差异不显著(P>0.05);30d,各组间均无显著性差异(P>0.05);第1组小鼠相继于1周内死亡,死亡后取脾观察未见脾结节形成。第2组、第3组存活的11只雌性BalB/c小鼠BMMNC中,Y染色体出现率为(79.35±6.77)%。第2组、第3组、第4组2月生存率分别为30%、80%、100%。第3组生存率明显高于第2组(P<0.01),与第4组无显著差异(P>0.05)。第3组少数及第2组中大部分鼠移植30d出现GVHD表现,两组死亡鼠及第2组存活鼠组织因子改变符合Ⅱ-Ⅲ度GVHD病理变化,第3组存活7只鼠中有Ⅰ度GVHD病理变化。结论:转基因自体造血细胞与异基因供体BMMNC混合培养后移植,能使受者获给供体来源的造血重建,并明显降低GVHD发生。

Experimental Study on Hemotopoietic Function of Recipient Mice after mFasL-cDNA Transfected Hematopoietic Cell Transplantation

Objective: To explore the influence on the hematopoietic cells activity and hematopoietic function of the recipient mice after transfected BMMNC transplantation. Methods: The exogenous mFasL-cDNA gene was transferred to BalB/c mouse BMMNCs by liposomes. Then the transferred BMMNCs were co-cultured with BMMNC from BAC mouse(H-2 d b, male) at the ratio of 0.625 to 1 for 6 days. In the experimental group(the 3 rd group), 1×10~7(0.5 ml) mixed viable cells were injected into whole bodily irradiated (~(60)Co-γ, via the tail vein) mice (6 to 8 weeks old female BalB/c). The following three groups of grafted mice were simultaneously transplanted as: the 1st group with 0.5 ml culture medium; the 2nd group with the mixture of untransferred BalB/c mouse BMMNC and BAC mouse BMMNC; the 4th group with BalB/c mouse BMMNC. The hematopoietic reconstitution, the origin of bone marrow cells responsible for the reconstitution, GVHD and the survival rate for the recipient mice were observed after bone marrow transplantation (BMT). Results: The counts of leukocytes and platelets in recipient mice's blood of the 4th group on the 10th day and 20th day after BMT were higher than in the 3rd and 2nd group (P<(0.01), but on the 30th day after BMT the counts for all groups were at their normal levels. The Y Chrosome from the donor mice was discovered in (79.35±6.77)% BMMNCs of recipient mice which had survived over two months after BMT in the 2nd and 3rd group. The survival rates of the recipient mice two mouths after BMT in all groups were 0% for the 1st group, 30% for the 2nd group, 80% for the 3rd group, 100% for the 4th group, respectively, and the total survival rate of recipient mice in the 3rd group was obviously higher than that of the 2nd group (P<0.01). Grade Ⅱ to Ⅲ GVHD signs were found on the histology from death mice after BMT in the 3rd group and 2nd group, and the mice had survived over two months in the group two. Grade Ⅰor no GVHD signs were found on histology from the mice survived over two months after BMT in the experimental group. Conclusion: The transplantation of mixed cells into recipient mice made recipient mice to get hematopoietic reconstitution from donor BMMNC.