Conversion of mononuclear cells from human umbilical cord blood into hepatocyte-like cells

Objective: To evaluate the differentiation of human umbilical cord blood cells into hepatocyte-like cells. Methods: Mononuclear cells (MNCs) derived from human umbilical cord blood were isolated using Ficoll. The experiment was derived into 3 categories:(1) MNCs co-cultured with 50 mg minced liver tissue separated by a trans-well membrane and then collected at 0 h,24 h,48 h and 72 h; (2) MNCs cultured along supplemented with 100 ml/L FBS. 100μ/ml penicillin, 100μg/ml streptomycin. 4. 7μg/ml linoleic acid, 1×ITS, 10-4 mol/L L-Ascorbic acid 2-P and a combination of FGF4 (100 ng/ml) and HGF (20 ng/mL). Cells were then collected at 0 d and 16 d to examine the expression profile of hepatocyte correlating markers;(3) 0. 2-0. 3 ml of MNCs with a cell density of 2×107/ml were transplanted into prepared recipient mice [n = 12, injected with 0. 4 ml/kg (20%) CCl4 and 150 ng/kg 5-fluorouracil (5-Fu) prior the transplant 24 h and 48 h, respectively] via injection through tail vein. Mice were sacrificed 4 weeks after transplantation. The hepatocyte correlating mRNAs and proteins were determined by RT-PCR, immunohistochemical analysis and immunoflurence technique. Results: (1) After 72 h. a number of glycogen positive stained cells were observed with MNCs co-cultured with damaged mouse liver tissues. The expression of hepatocyte markers, human albumin (ALB),α-fetal protein (AFP) and human GATA4 mRNA and proteins were detected by RT-PCR and immunohistochemistry as well. For the confirmation, the DNA sequencing of PCR products was performed. In control groups, MNCs co-cultured with normal mouse hepatocytes or MNCs cultured alone, all markers remained negative. (2) In growth factor supplemented culture system, MNCs developed into larger volume with richer cytoplasm and binucleation after 16 d. Positive expression of ALB, AFP, CK18 and CK19 mRNA were detected with RT-PCR, and ALB positive staining was observed by immunocytochemistry as well. In contrast. MNCs cultured without exogenous growth factors scarcely attached to the culture dish and ALB mRNA was not detected. (3) In transplantation experiment, both of ALB and AFP mRNA were detected by RT-PCR and HSA, PCNA and ALB positive staining were observed in the livers of recipient mice by immunocytochemistry. Conclusion: MNCs from human umbilical cord blood could convert into hepatocyte-like cells in 3 different ways, indicating their potential use in the clinic applications for the treatment of human liver diseases.     

非亲缘性双份脐血移植治疗成人慢性髓系白血病

目的 :探索非亲缘性双份脐血移植 (CBT)治疗成人恶性血液病的可行性及并发症的防治。方法 :1例 2 7岁体重为 6 0 kg的慢性髓系白血病急变期 (CML- BP)患者 ,由北京大学人民医院脐血库提供 HL A配型部分相合的双份脐血。予改良的环磷酰胺 +6 0 Co全身照射 (TBI)方案进行预处理 ,经股动脉同时回输双份脐血 ,有核细胞数 (MNC)分别为 1.5 4× 10 7/ kg和 1.2 5× 10 7/ kg。移植物抗宿主病 (GVHD)的预防用环胞菌素 A(Cs A)、甲氨蝶呤 (MTX)及霉酚酸酯 (MMF)、白细胞介素 2(IL - 2 )及甲泼尼龙 ,肝静脉闭塞病 (VOD)的预防用前列腺素 E1 (PGE1 ) ,以更昔洛韦预防巨细胞病毒(CMV)感染。结果 :移植后第 2 2天 (+2 2天 )中性粒细胞计数 >0 .5× 10 9/ L ,+2 8天血小板计数 >2 .0× 10 9/ L。移植 +40天骨髓血 DNA位点检测与其中一份编码为 980 2 180 2的脐血完全相符。移植过程仅出现 度急性移植物抗宿主病 (a GVHD) ,+30天出现出血性膀胱炎。+44天出现低热 ,血尿 CMV检查 (+) ,加用更昔洛韦 2周 ,CMV病毒转阴。移植过程共输血小板 7次。随访 2 5个月 ,患者一般情况好 ,骨髓细胞 Ph染色体和 bcr/ abl融合基因阴性。结论 :同一受者接受 HL A配型部分相合的两份脐血移植在临床上是可行的 ,其造血功能获得及时恢复     

两种类型内皮祖细胞的体外分化及非对称性二甲基精氨酸对其增殖的抑制作用

目的:研究人脐带血中两种类型内皮祖细胞(endothelial progenitor cells,EPC)的体外分化特征及非对称性二甲基精氨酸(ADMA)对其增殖的影响.方法:采用6%羟乙基淀粉沉降法和密度梯度离心法联合分离脐带血中单个核细胞,在含有血管内皮生长因子和碱性纤维细胞生长因子的培养基中培养.通过形态学、免疫荧光、逆转录多聚酶链式反应(RT-PCR)和流式细胞分析细胞的生长特点和生物学特征.在贴壁细胞中加入不同浓度的ADMA(1,5和10 μmol/L)作用不同时间(24,48和72 h),用四甲基偶氮唑蓝(MTT)比色法和细胞克隆计数的方法评价ADMA对EPC增殖的影响.结果:贴壁细胞分为早期内皮祖细胞和晚期内皮祖细胞两种类型细胞.早期内皮祖细胞的形态从小的圆形细胞变成以集落为中心,周围呈发芽式向外生长的细胞群,晚期内皮祖细胞形成典型的"铺路石样".DiL标记的乙酰化低密度脂蛋白(Dil-acLDL)和FITC标记荆豆凝集素I(FITC-UEA-I)双染色阳性细胞初步鉴定为内皮祖细胞.RT-PCR提示随着培养时间延长,内皮特异性基因表达变化逐渐增强,流式细胞分析提示早期EPC有AC133,CD34和KDR表达;晚期EPC AC133不表达,CD34表达弱,KDR表达增强.MTY法提示早期EPC增殖能力弱,而晚期EPC增殖能力强;同时ADMA呈量效和时效减少EPC的数目和抑制它的增殖能力.结论:在人的脐带血中获得两种类型EPC,加入ADMA能抑制EPC的增殖,这为进一步研究ADMA对EPC的作用提供了实验基础.     

短期体外扩增对脐带血干／祖细胞归巢相关功能影响的研究

目的 探讨体外扩增对脐血(UCB)于/祖细胞(HSPC)的归巢相关功能的影响.方法 将从新鲜UCB中纯化CD34 细胞接种于无血清无基质悬浮扩增体系,分别于培养4、7、10和14d检测扩增细胞归巢相关黏附分子的表达、黏附功能和趋化功能变化.结果 [1]表达各种黏附分子的CD34 细胞亚群的数量在扩增过程中明显增加(7～32倍),而且扩增后CD34 细胞表面黏附分子CD11a、CD49d的表达与原代CD34 细胞比较升高,CD62L、CD54和CD49e的表达则有不同程度的下凋;[2]在前7d的扩增中,CD34 细胞与FN间的自发黏附率和SDF-1诱导黏附率均呈上升趋势,扩增10d后呈下降趋势.[3]在前4d的扩增中,CD34 细胞的趋化因子受体CXCR-4的表达及体外迁移能力呈上升趋势,扩增7d后呈下降趋势.结论 所建立的培养体系1周内可以提高UCB HSPC归巢相关功能,而继续扩增则不利于细胞这些能力的保持.     

婴幼儿过敏症的家庭预防

大约有40%的过敏性疾病始发于10岁以内的儿童,其中约有半数在婴儿时期已开始发病了。还有一些过敏性疾病是专门发生在婴儿期的,如婴儿过敏性湿疹、尿布皮炎等。婴儿患过敏性疾病虽然算不上什么大病,可是,却容易影响宝宝的身心发育,因此,年轻父母非常着急。为什么婴幼儿特别容易发生过敏性疾病呢?我们该如何预防呢?     

孕母维生素C、钙缺乏对胎儿骨发育的影响

[目的]探讨孕期VitC、钙缺乏对胎儿骨发育的影响.[方法]常规生化方法测定68对母婴血清VitC、ALp和Ca 含量,采用SAS6.12软件包对检测数据进行分析处理.[结果]脐血VitC浓度为:(16.49±5.99)μmol/L.其中38.24%的样本<17μmol/L.ALP活性为:(141.72±41.63)U/L.其中35.29%的样本活性145U/L.Ca 浓度为:(2.43±0.48)mmol/L,其中8.82%样本含量<2.23mmol/L.脐血低VitC的样品的ALP活性比正常样本略有增高,但差异无统计学意义(P>0.05),而低VitC组的Ca 浓度明显低于正常组(P<0.05).低Ca 样本的ALP活性明显高于正常样本(P<0.05).母血VitC浓度为:(9.06±3.68)μmol/L,低于正常者占80.88%.ALP活性为:(117.26±61.43)U/L,其中高于正常者占17.65%.Ca 浓度为:(2.44±0.55)mmol/L,其中低于正常者占20.59%.[结论]母婴普遍存在VitC水平低,提示母孕期应适当增加VitC的摄入量.低血钙与低VitC有关,孕母补充维生素D的同时应考虑VitC的补充.低维生素C组胎儿骨代谢较为活跃.     

109对母婴叶酸营养状况观察

配对观察１０９对母婴间叶酸的营养状况，发现孕产妇叶酸低于正常组，有显著性差异（Ｐ＜０．０００１）。其中孕产妇叶酸降低者有９７例（８８．９９％）。母婴叶酸同时降低者有８９对（８１．６５％）。经多元回归分析，婴儿叶酸与孕产妇叶酸呈极显著正相关。但脐血叶酸水平仍高于孕产妇血叶酸水平，两者有显著性差异（Ｐ＜０．００１）。     

脐带血中衍生树突状细胞及其生物学特性的研究

目的利用脐带血衍生培养树突状细胞(DC),以获得可用于临床治疗的一种新的血细胞制品.方法从脐带血中分离单个核细胞,联合使用细胞因子GM-CSF、IL-4和TNF-α在体外诱导和扩增DC;采用扫描电镜、流式细胞仪(FACS)和混合淋巴细胞反应(MLR)试验对脐带血来源的DC进行生物学特性分析.结果从30ml脐血诱导培养12d后可获得6.6×106的DC.扫描电镜观察具有典型的树突状突起;FACS分析显示,获得的DC细胞群高表达CD1a(90.6%)、CD80(96.8%)、CD86(84.8%)和HLA-DR(91.0%);MLR显示,脐带血诱导培养的DC对同种异体的脐带血幼稚型T细胞均具有强烈的激发和促增殖作用.结论从脐带血中成功诱导出大量高纯度成熟DC,为依赖DC免疫治疗的临床应用打下基础.     

Successful transplantation of double unit umbilical-cord blood from unrelated donors in high risk leukemia with a long follow-up

Umbilical-cord blood (UCB) is a source of hematopoietic stem cells that has been successfully used for transplantation, primarily in children. But the low cell dose severely limits the application of unrelated umbilical-cord blood transplantation (UCBT) in adult patients,