Objective:To investigate the effects of estradiol on ~(60)Coγ-ray induced apoptosis of bone marrow hematopoietic cells of mice,and to discuss the related anti-irradiation mechanism.Methods:KM mice were randomly divided into 3 groups(15 mice/each group):control group(without radiation),pure radiation group and estradiol+radiation group(ER group).The pure radiation group was irradiated by 4.0 Gyγ-ray at a dose rate of 1.15Gy/min;the ER group was administered with 0.1 mg estradiol(IM)at 10 days before 4.0 Gyγ-ray radiation;and the control group received no special treatment.The apoptotic DNA segments of bone marrow hematopoietic cells were analyzed by DNA agarose gel electrophoresis;flow cytometry was used to examine the apoptosis rate of cells and expression of Fas and Bcl-2 at 4 h,8 h,and 12 h after irradiation.Results:Eight hours after radiation,the apoptotic DNA segments were obviously increased and apoptotic DNA ladder appeared,which was not seen in the other 2 groups.The apoptosis rate of bone marrow hematopoietic cells in ER group was significantly lower than that in the pure radiation group at 4,8,and 12 h after irradiation(P0.01).Fas expression was only slightly elevated in the ER group than in the control group,but was markedly lower than that in pure radiation group(P0.01),and showed no typical crest-time; Bcl-2 expression was significantly higher in the ER group than in the pure radiation group(P0.01).Conclusion:Estradiol can decrease the apoptosis of mice bone marrow hematopoietic cells induced byγ-ray,probably through down-regulation of Fas and up-regulation of Bcl-2 expression.

BACKGROUND & OBJECTIVE:STAT3 protein has been found constitutively activated in a wide variety of human tumor tissues and cell lines. It may be involved in tumorigenesis and development. This study was to observe the activation of STAT3 protein in mouse melanoma cell line B16, human liver cancer cell lines SMMC-7721 and HepG-2, human lung cancer cell line A549, and human cervical cancer cell line HeLa, and to investigate the effects of STAT3 antisense oligodeoxynucleotides (ASODN) on proliferation and apoptosis of B16 cells. METHODS:B16 cells were transfected with STAT3 ASODN or STAT3 sense oligodeoxynucleotides (STAT3 SODN), respectively. The expression and phosphorylation levels of STAT3 protein in all the tumor cells were measured by Western blot. The proliferation of B16 cells was detected by MTT assay. Cell apoptosis was determined by Hoechst33258 staining and Annexin V/PI using flow cytometry. RESULTS:STAT3 was highly expressed and phosphorylated in all the tumor cells. Transfection of STAT3 ASODN suppressed the expression and phosphorylation levels of STAT3 protein in B16 cells. Forty-eight hours after transfection, the proliferation of B16 cells was inhibited the higher the concentration (0-200 nmol/L) of STAT3 ASODN was, the heavier the inhibition was (P<0.01); when transfected with STAT3 SODN over 250 nmol/L, the proliferation of B16 cells was also inhibited (P<0.05). Inhibitory effects appeared 24 h later after transfection of STAT3 ASODN, and became more obvious 48 h later (P<0.01). The early apoptosis rate was significantly higher in 400, 800, 2,000 nmol/L STAT3 ASODN groups and 2,000 nmol/L STAT3 SODN group than in blank control group (8.22%, 9.99%, 16.97%, and 13.31% vs. 5.52%, P<0.01); no significant difference was found among 400 and 800 nmol/L STAT3 SODN groups and blank control group (5.87%, 5.36% and 5.52%, P>0.05). CONCLUSIONS:STAT3 is highly expressed and activated in all the tumor cells detected. STAT3 ASODN can abrogate the activity of STAT3 protein, inhibit proliferation and induce apoptosis of B16 cells, which support that STAT3 may be a potential molecular target for tumor therapy.

ObjectiveTo investigate the influence of STAT3 antisense oligodeoxynucleotides (ASO-STAT3) on radiosensitivity of B16 cells. Methods Oligodeoxynucleotides were tansfected into B16 cells with oligofectamine reagent. Cell survival was measured using CCK-8 assay. Apoptotic assays were performed using Hoechst33258 staining and Annexin V/PI with FACS analysis. Results Cell survival decreased significantly in those groups tansfected with ASO-STAT3 and irradiated with different dose of γ-rays. Compared with those cells treated with irradiation alone or AO-STAT3 transfection with subsequent exposure to radiation, fraction of early apoptosis increased in those cells treated with ASO-STAT3 transfection combined with irradiation. Conclusion These results showed that targeting ASO-STAT3 can enhance the therapeutic effect of γ-ray irradiation on B16 cells, which implied that STAT3 can act as a potential molecular target for tumor therapies, and also a potential molecular target for enhancement of radiosensitivity of radioresistant tumors.

STATs(Signal Transducers and Activators of Transcription)家族是一种存在于细胞浆并在激活后能够转入核内与DNA结合的独特的蛋白家族,它与酪氨酸磷酸化信号通道耦联,发挥信号转导和转录调控作用.目前在哺乳动物中已发现STATs家族有7个结构和功能相似的成员(STAT1-4、STAT5a、STAT5b、STAT6),分别由不同的基因编码.STATs激活后形成二聚体,转入到核内,结合特定的靶DNA启动子序列,促进相应的基因转录,进而调节细胞的生长、分化、生存和凋亡;STATs异常激活将导致细胞的异常增殖和凋亡障碍,促进肿瘤的形成、发展;针对STATs家族成员的各种阻断治疗,已经取得一定的进展,本文试对此类研究进展作一综述.

Objective To investigate the effects of transplantation times and number of transplanted cells on homing of these cells after scheduled bone marrow transplantation. Methods An allogeneic mouse model and flow cytometric detection were used to observe homing efficacy of transplantation times and number of cells transplanted. Results After different times of scheduled transplantation,the highest number of homing cells and homing efficiency were both observed after the third time of transplantation in every group,and detecting at same time point (the fourth day) showed that the highest results was obtained in the transplanted group ( P 0.05),suggesting that there might be different homing patterns after different times of transplantation.After the scheduled transplantation of different number of cells,the number of homing cells of low cell number group was not diffent from that of high number group,but the homing efficiency was higher than the latter ( P 0.01),suggesting that the homing efficacy of multi-transplantation with low cell number may be equal or superior to that of a single injection of a large number of cells and the accumulated number of cells may be reduced to 10 +5. Conclusion The routine method of transplanting a large number of cells by a single injection may be waste of the stem cells.In a certain range of cell number,transplantation efficiency may be enhanced by changes of transplantation schedule. ;

目的:探讨移植细胞数对小鼠骨髓移植回巢及生存率的影响.方法:采用不同细胞数移植、不同细胞数加不同时间间隔程序移植及流式细胞仪检测等方法,观察不同细胞数骨髓移植后小鼠骨髓内回巢效果和生存情况.结果:不同细胞数骨髓移植中5×105组的回巢细胞数与高细胞数组无差异,回巢率远远高于后者(P<0.01);生存率1×105组为95%,与高细胞数组无差异.不同细胞数加不同时间间隔程序移植以细胞总数在1×105、间隔24 h移植组存活率高,组间差异非常显著(P<0.01).结论:在一定细胞数量范围内,分次移植可能提高移植干细胞的回巢效率.

造血干细胞移植,是将具有造血潜能的健康造血干细胞,通过静脉输入受者体内并植入骨髓,帮助患者重建造血功能,达到治疗或辅助治疗相关疾病的目的.

Objective To identify homing of bone marrow cells after every fractionation during scheduled transplantation. Methods The recipient mice were transplanted with homologous (H 2K d) and allogenic(H 2K b) mouse bone marrow cells after lethal irradiation,and the homing status of allogenic bone marrow cells in host bone marrow and spleen was observed. Results A quantity of allogenic homed cells were observed in host bone marrow,and the percentage of homing cells in second fraction was the highest in all groups ( P 0 01).The allogenic homed cells in spleen declined along with increase of the number of fraction,suggesting that regulation of homing to spleen was different from that to bone marrow. Conclusion In scheduled bone marrow transplantation niche may be more effectively utilized and thus transplation efficiency be enhanced.\;

我们曾观察过少量多次的程序移植对移植效率的影响及不同移植细胞数与回巢效果的关系[1],认为一次性大剂量移植可能造成造血干细胞的浪费,而少量多次的程序移植则可能是解决这个问题的方法.在此基础上我们进一步观察了移植细胞数与回巢效果的关系、不同移植细胞数、移植时间和移植次数对生存率的影响.

Objective To investigate the role of chemoattractant SDF 1 in of homing hematopoietic cells. Methods A mouse allogeneic bone marrow transplantation model and double staining of immunohistochemistry were used in this study.Relationship between expression of chemoattractant SDF 1 and of homing hematopoietic cells was observed. Results In bone marrow,SDF\|1 was mainly expressed at sites near endosteum,in microvessel endothelium,osteocyte and around donor cells.Contrast to non\|irradiation group,SDF\|1 was significantly expressed by osteocytes,and at sites near endosteum in the irradiation group, suggesting that pretreatment with irradiation might enhance secretion of SDF\|1 by bone marrow stromal cells and SDF\|1 could have a close relation with homing cells. Conclusion Pretreatment with irradiation might be one of the stimulating factors significantly increasing the level of SDF\|1 produced by bone marrow stromal cells and the chemoattractant SDF\|1 could play an important role in homing of hematopoietic cells.;

To assess the mechanisms of modest hypothermia (MH) and its effects on cellular radiation response, a model of anesthesia-induced modest hypothermia (AIMH) in the adult mice and a model of pure MH in the newborn mice were established. The survival rate of lethally irradiated mice was increased to 72% through AIMH before irradiation. Both apoptosis and necrosis of human fetal bone marrow CD34+ hematopoietic stem cells cultured under MH were significantly decreased as detected by MTT and flow cytometry, with three-color labeled by PE-CD34+/ FITC-AnnexinV /7AAD. The survival and proliferation of mouse bone marrow MNC treated with MH after irradiation were also increased. The MH exerted similar protective effects on the leukemia cell lines A20, HL60, K562 to the normal bone marrow cells, but it enhanced the radiation sensitivity of leukemia cell line FBL3 and mouse melanoma B16F10. No effects have been found on the radiation sensitivity of those cells treated with MH before irradiation. The results also showed that MH mediated the effects on radiation sensitivity, in addition to increasing the oxygen tension. These results show different effects of MH on different cells: (i) AIMH exerts a protective effect on the normal hematopoietic stem cells, some leukemia cell lines A20, HL60, K562, and some neoplasma 3LL, LOVO. And MH exhibits a synthetic effect with anesthetic. (ii) MH enhances the radiation sensitivity of another leukemia and neoplasma cell lines FBL3, B16F10 and CT26. Therefore, AIMH has a potential to enhance the effects of radiation-therapy and decrease side effects on some tumors.

为探讨中度低温对辐射敏感性的影响及其机制, 分别建立了正常成年小鼠和新生小鼠30℃中度低温模型, 表明照射前麻醉诱导中度低温能提高小鼠存活率达72%(P＜0.001 ; 流式细胞术三色荧光法和MTT法检测结果表明, 正常骨髓单个核细胞和造血干(祖)细胞在受照射后经中度低温处理后, 凋亡和死亡细胞数量减少; 对受照射后的白血病细胞系A20, HL60, K562和实体瘤3LL, LOVO中度低温也有相似的保护作用, 但是对小鼠白血病FBL3细胞系、小鼠实体瘤B16, F10和CT26却提高其辐射敏感性. 结果提示, 中度低温对不同组织细胞的辐射敏感性的作用是不同的; 低温和麻醉有协同作用; 中度低温对正常骨髓造血干(祖)细胞能减少辐射所致的细胞凋亡和死亡, 但是对某些白血病细胞和实体瘤细胞却增加了辐射敏感性, 这就有可能通过麻醉诱导中度低温以增强放疗效果并减轻毒副作用, 有临床运用潜力, 同时也提示除低温所致低氧效应外, 存在低温诱导性因子的可能性.

Objective To investigate the characterization of death of the human hematopoietic stem cells after irradiation. Methods Human fetal bone marrow mononuclear cells were irradiated with different doses of 60Co γ-rays at different high dose rates.Apoptosis and necrosis of CD34+ cells were analyzed by flow cytometry,following three-color labeling with PE-CD34/FITC-Annexin V/7AAD at different times after irradiation. Results The death of CD34+ cells after 5 Gy and 8 Gy irradiation showed a continuous process of reproductive death during the first week,and the main death type was apoptosis.A majority of CD34+ cells died of necrosis during the first day after 10 Gy and 12 Gy irradiation,and all of them died within a week. Conclusion Niches are continuously vacated every day within a week following irradiation and reproductive death of hematopoietic stem cells occurred.

Objective To explore the homing regularity of different doses of bone marrow cell transplantation.Method An allogeneic mouse model was used in this study.The homing status of different dose groups from the first day to the forth day after transplantation were observed.Results The rate of positive cells in bone marrow and spleen:differences among four groups was not significant.The rate of positive cells of third day was highest among four days (P<0.01).A phenomenon that homing-mobilization-rehoming could be observed.The homing efficiency:low dose groups were higher than that high dose groups (P<0.01).Conclusion The homing efficiency of low dose groups is higher than that of the high dose groups in certain range,the routine method of transplanting a large quantities cells by a single injection may be an waste.

硫酸葡聚糖(DS)作为一种有效的造血干细胞动员剂已有很多研究报道,并可适于体内研究[1].人脐血移植SCID小鼠是造血干细胞体内研究的较好模型,对受体的预处理可促进外源干细胞的植入[2].本实验在该动物模型及作者等先前分次移植[3]的研究基础上,观察了DS作为预处理剂的作用.

归巢是造血干细胞移植中的关键一步［1］，建立一种较为精确的检测方法是进行造血干细胞归巢研究的一项重要课题。目前检测归巢的各种方法皆有不足之处：流式细胞仪精确度低，价格昂贵；放射性核素标记干/祖细胞则会对细胞造成伤害，影响其归巢能力。本实验建立了一种新式的微量荧光细胞计数板来检测造血干/祖细胞归巢的情况。

Objective Using a new method to expand the number of umbilical cord blood stem cells culture in vitro, at the same time to keep their originality. Methods under the same condition with hematopoietic factors in the liquid culture, compared the sequential expansion group and once for all expansion group with or without stroma celllayer. Results When cultured for 28 days, the absolute number of MNC cells in the sequential expansion group was lessthan that in the once for all expansion group,but the number of CD34 + cell in the former was more than that in thelatter. Furthermore, the total number of MNC and progcnitor were greater in that with presence of stromal cell layerthan that with sbsence of stromal cell layer. Conclusion Sequential expansion of umbilical blood stem cells is an effective method for expansion of srem cells which enables more extensive use of umbilical blood. (Shanghai Med J, 2001,24: 95-98)

为进一步提高骨髓移植效果.以昆明种小鼠急性放射病为模型,进行了胎肝-骨髓程序移植(FL-BMST)后小鼠活存及造血重建、急性移植物抗宿主病(GVHD)的研究.胎肝-骨髓程序移植1 106个骨髓细胞时,于照射后第17d外周血白细胞、骨髓有核细胞计数接近正常,CFU-E、CFU-GM、CFU-F、CFU-S已达正常;GVHD较一次骨髓移植组轻;活存率达60%,显著高于一次骨髓移植组.胎肝-骨髓程序移植既充分利用了每次腾出的龛位''(Niche),又通过移植胎肝基质细胞改善了龛位'',增加了龛位'',从而增加了干、祖细胞的植入并加快增殖分化,减少了移植细胞数,提高了移植效果,是一种较好的移植新方法.

目的:进一步了解脐血造血细胞回巢潜能及硫酸葡聚糖(DS)的动员作用机制.方法:重症联合免疫缺陷小鼠经亚致死剂量γ射线照射或DS预处理后,进行经冻存后的人脐血移植,利用人粒-单系祖细胞培养及流式细胞术,检测植入小鼠骨髓、脾脏等器官中的人源性细胞.结果:与未预处理组相比,DS或照射预处理组移植受体鼠内人源CD45+,CD34+及CD19+细胞植入水平明显提高;DS预处理组植入水平虽较照射预处理组差,但毒性较低,小鼠存活率高达100%.结论:DS是一种安全、有效的预处理方案;DS对造血干细胞既有动员作用又有促进龛位腾出以供移植干细胞回巢的作用.

目的:观察造血生长因子对受照射小鼠辐射损伤的防治作用.方法: 用大鼠浆细胞株培养上清(RSP-CM)和小鼠成纤维细胞株培养上清(LP3-CM)来源的集落刺激因子,治疗受不同剂量60Co γ射线照射的ICR小鼠.观察小鼠30 d存活率、死亡动物平均存活日、保护系数及部分动物外周血白细胞计数.研究GM-CSF对受照射小鼠重建造血功能的疗效及量效、时效关系.结果:RSP-CM能显著提高受7.5 Gy照射小鼠的存活率,而LP3-CM则无效.照射剂量为8.0 Gy时,只有RSP-CM治疗组有效,预防组和防治组无效.照射剂量为8.5 Gy时,小鼠30 d内全部死亡,表明GM-CSF对大剂量照射小鼠疗效差,但从保护系数看,各实验组均有效,且存在量效、时效关系.结论:RSP-CM源性GM-CSF对受照小鼠的骨髓损伤有促进造血恢复的作用,且在一定程度上存在量效、时效关系,而LP3-CM源性M-CSF则无效.