VIP对人脐血CD34+细胞向肝系分化的影响初探

目的探讨血管活性肠肽(VIP)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CD34+细胞,流式细胞术鉴定纯度;VIP和混合因子(包括VIP、EGF、HGF)作用CD34+细胞后,酶联免疫化学法测定CD34+细胞及上清AFP水平,免疫细胞化学法检测CD34+细胞上肝系标志AFP、白蛋白(ALB)、细胞角质素19(CK-19)的表达,Western blot方法检测CD34+细胞上ALB表达;巢式RT-PCR方法检测CD34+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CD34+细胞上不表达CK-19蛋白,但有AFP和ALB蛋白表达;VIP作用CD34+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).Western blot显示VIP作用后CD34+细胞内ALB蛋白表达有减弱趋势.人脐血CD34+细胞表达了AFP mRNA和ALB mRNA,随机选取ALB产物测序与GeneBank中ALB基因序列完全相同.结论人脐血CD34+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.VIP降低了人脐血CD34+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VIP对HSC向肝细胞横向分化具有抑制作用.

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

OBJECTIVE: To investigate the effect of vasoactive 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 mononuclear cell (MNC), the purity of the CD34+ cells was evaluated by flow cytometry. 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 hepatocyte 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.