脐血源性T淋巴细胞移植在BALB/c小鼠红白血病治疗中的作用

Role of cord blood-derived T lymphocytes transplanting for BALB/c mice with erythroleukemia

目的研究脐血源性T淋巴细胞在小鼠体内的存活、浸润情况，以及对红白血病（EL）小鼠的治疗作用，为探讨脐血源性T淋巴细胞的生物学特性及其治疗自血病提供实验和理论依据。方法48只BALB／c小鼠随机分为对照组、实验1组和实验2组。分离制备脐血源性T淋巴细胞，对其标记后经尾静脉或腹腔移植入红白血病BALB／c小鼠体内，采用免疫荧光、免疫组织化学、透射电镜等方法观察脐血源性T淋巴细胞在小鼠体内的存活时间、浸润状况及其对白血病小鼠脾脏结构的影响。结果脐血单个核细胞（CBMNCs）经植物血凝集素（PHA）诱导后，绝大多数细胞为CD3＋T淋巴细胞，阳性率可达（83．42±1．26）％；移植后第3、7天均可在小鼠外周血和肝、脾组织中检测到人CD3＋细胞和Brdu＋细胞；两实验组（EG）小鼠肝、脾组织内均见CD25＋细胞散在浸润；两实验组EL小鼠平均存活时间分别为（27．25±7．06）d和（24．74±2．93）d，与对照组比较，均有统计学意义（P〈0．05）；Hoechsd3258染色法检测显示，在荧光显微镜下见对照组细胞核荧光均匀，凋亡细胞较少；实验组部分白血病细胞呈现凋亡的形态学改变：染色质高度凝聚，致密浓染，或裂解成碎块状，边缘光滑清晰；透射电镜下见对照组小鼠脾内白血病细胞广泛浸润，实验组小鼠部分白血病细胞呈现典型凋亡形态学改变：核染色质浓缩、边集；核碎裂，可见凋亡小体，粗面内质网（RER）扩张，线粒体（Mi）呈现空泡样变及髓样变。结论脐血源性T淋巴细胞能在白血病小鼠体内存活并趋化至白血病细胞浸润部位，发挥其相应的功能，对白血病有一定的治疗作用。

Objective To study the survival and infiltration condition of cord blood derived-T lymphocytes in mice and its therapeutic action for erythroleukemic （EL） mice; and to provide the experimental and theoretical basis for studying the biological characteristics and anti-leukemia effects of cord blood-derived T cells. Methods Fourty-eight BALB/c mice were randomly divided into three groups, the control group （CG）, the experimental group 1 （EG 1 ）and the experimental group 2 （EG 2）. Cord blood derived-T lymphocytes were separated and prepared, and transplanted it into EL mice via the tail vein or peritoneal injection after being signed to their group. This was done in order to observe the survival time and infiltrate condition of the cord blood- derived T cells in mice and its effect on the morphosis of EL mice spleen by immunofluorescence （IMF）, immunohistoehemistry （IMC） and transmission electron microscope （TEM）. Results Most activated cells were CD3＋ T lymphoeytes by PHA, and the positive rate was （83.42 ± 1.26） % ; human CD3 ＋ ceils and Brdu＋ cells were detected in peripheral blood, spleens and liver of all mice at the 3rd and 7th day after cord blood derived-T lymphoeytes were transplanting; CD25 ＋ cells could be detected in the liver and spleen of EL mice in both experimental groups ; and the average survival time of EL mice in the two experimental groups was （27.25 ± 7.06） days and （24.74 ± 2.93 ） days respectively （ this was statistical significance compared to control group, P 〈 0.05 ）. The results of Hoechst 33258 staining showed ： 1. Nuclear fluorescence was uniform in the control group and there were few apoptotic cells ; 2. In the experimental group, some of the leukemia cells appeared to have morphological changes consistent with apoptosis; chromatin had a high degree of cohesion; dense concentration, or splited into massives, edge was smooth and clear; Under TEM, leukemic cells infiltrate was generally observed in the spleens of the control group mice. Parts of the leukemic ceils showed typical apoptosis morphological changes in the experimental groups： nuclear chromatin concentration and edge accumulation was seen ; nuclei fragmentation and formation of apoptotic bodies ; the expansion of RER, and the vacuolar or medullar degeneration of mitochondria could be found. Conclusion The cord blood-derived T lymphocytes could be activated in vivo, and transferred to the site of leukemia, and educed relevant function, thus having some therapeutic action for leukemia.