VIP对人脐血CD34^+细胞向肝系分化的影响初探  

Initial Research for Effects of Vasoactive Intestinal Polypeptide(VIP) on Differentiation of Human Umbilical Cord Blood Derived CD34^+ Cells into Hepatic Related Cells

作　　者：王利[1] 唐承薇[1] 王春晖[1] 李献[1] 强欧[1] 黄明慧[1] 

机构地区：[1]四川大学华西医院人类疾病生物治疗教育部重点实验室人类疾病相关多肽研究室

出　　处：《四川大学学报（医学版）》2006年第4期578-582,共5页Journal of Sichuan University(Medical Sciences)

基　　金：国家杰出青年科学基金(39725012)资助

摘　　要：目的探讨血管活性肠肽(V IP)对人脐血造血干细胞(HSC)向肝系分化的影响。方法采用免疫磁分选技术纯化人脐血CD 34+细胞,流式细胞术鉴定纯度;V IP和混合因子(包括V IP、EGF、HGF)作用CD 34+细胞后,酶联免疫化学法测定CD 34+细胞及上清AFP水平,免疫细胞化学法检测CD 34+细胞上肝系标志AFP、白蛋白(ALB)、细胞角质素19(CK-19)的表达,W estern b lot方法检测CD 34+细胞上ALB表达;巢式RT-PCR方法检测CD 34+细胞上AFP、ALB的mRNA表达,随机选送ALB产物测序。结果免疫组化结果显示CD 34+细胞上不表达CK-19蛋白,但有AFP和ALB蛋白表达;V IP作用CD 34+细胞14 d后,细胞内的AFP质量浓度(165.00±8.51 pg/mL)较对照组(270.00±11.37 pg/mL)显著下降(P<0.05)。W estern b lot显示V IP作用后CD 34+细胞内ALB蛋白表达有减弱趋势。人脐血CD 34+细胞表达了AFP mRNA和ALB mRNA,随机选取ALB产物测序与G eneB ank中ALB基因序列完全相同。结论人脐血CD 34+细胞表达肝系标志AFP、ALB,虽未见CK-19表达,但仍提示造血干细胞有向肝细胞横向分化的可能。V IP降低了人脐血CD 34+细胞AFP及ALB表达。这种对造血干细胞内胚层细胞标志物表达的抑制,提示V IP对HSC向肝细胞横向分化具有抑制作用。Objective To investigate the effect of vasoaetive intestinal polypeptide （VIP） on differentiation of hematopoietic stem cells （HSC） into hepatic related cells and probe into the possibility that VIP affects HSC transdifferentiation. Methods Mini MACS assay was used to purify human CD34^＋ cells from mononuelear cell （MNC）, the purity of the CD34^＋ cells was evaluated by flow eytometry. Alpha fetal proteins（AFP） in cultured CD34^＋ cells and their supernatant were measured with ELISA assay. Liver tissue markers on CD34^＋ cells, AFP, albumin （ALB） and CK-19, were measured by immunohistochemistry. Western blot assay was used to detect the expression of ALB on CD34^＋ cells. Nest RT-PCR was used to measure the expression of AFP mRNA and ALB mRNA on CD34^＋ cells, the product of ALB was chosen to measure the sequence. Results Immunohistochemistry showed that human CD34^＋ cell contained AFP and albumin but no CK-19 protein. When human CD34^＋ cells were cultured with VIP for 14 days, the concentration of AFP within CD34^＋ cells was decreased from （270.00±11.37） pg/mL to （165.00±8.51） pg/mL （P〈0.05）. Western blot revealed that albumin in CD34^＋ cells treated with VIP faded, compared to that of control. Both human cord blood MNC and CD34^＋ cells expressed AFP mRNA and albumin mRNA; the sequence of the product of nest RT-PCR for albumin matched completely with the sequence of albumin in GenBank. Conclusion Human cord blood CD34^＋ cells have been note to express hepatoeyte related markers such as AFP and albumin, although there is no evidence of CK-19 expression. These findings suggests the possibility that human HSC could be transdifferentiated into hepatocyte. The expressions of AFP and albumin on human CD34^＋ cells depressed by VIP indicate that VIP might inhibit the transdifferentiation of HSC.

关 键 词：血管活性肠肽(VIP) 造血干细胞(HSC) 肝系细胞 分化 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]