脐血CD34^+细胞向肝细胞分化的体内外实验

Differentiation of cord blood origin CD34^+ cells into hepatocytes: An in vivo and ex vivo trial

目的:以脐血CD34+细胞为起始细胞,分别在人体外和肝受损的重度联合免疫缺陷小鼠体内诱导CD34+细胞向肝细胞转化。方法:实验于2004-09/2005-06在反应器工程国家重点实验室进行。取健康足月产妇的新鲜脐血,产妇知情同意。采用密度梯度离心法分离脐血单个核细胞。将单个核细胞悬浮于免疫磁珠法缓冲液中,获取CD34+细胞。将CD34+细胞在干细胞因子+白细胞介素3+白细胞介素6细胞因子组合中培养1周,然后在肿瘤抑制素+成纤维细胞因子1+成纤维细胞因子2+白血病抑制因子+肝细胞生长因子+表皮生长因子组合中诱导其向肝细胞分化。将2-乙酰氟氨以0.4mg/只的剂量通过腹腔注射输入到重度联合免疫缺陷小鼠体内。7d后再腹腔注射入0.4mL/kg的CCl4,同时向实验组小鼠尾部静脉注射CD34+细胞,对照组小鼠则仅输注2-乙酰氨芴和CCl4。分别于4,6周后采用流式细胞术检测小鼠肝脏中的人源细胞,并用RT-PCR和免疫组化方法检测人源血清白蛋白基因和抗原的表达。结果:①CD34+细胞体外培养过程中,细胞总数扩增了近30倍,并且在培养21d收获的细胞中可检测到人血清白蛋白基因和抗原的表达,CD34+在体外被诱导成肝样细胞。②采用流式细胞术检测重度联合免疫缺陷小鼠肝脏中的人源细胞,并用RT-PCR和免疫组化的方法检测人源血清白蛋白基因和抗原的表达,4周时发现小鼠肝脏中含有7.66%的人源细胞,但人源细胞不表达血清白蛋白基因和抗原。6周时重度联合免疫缺陷小鼠肝脏中的人源细胞的比例增加至31.10%,并且人源细胞开始表达血清白蛋白基因和抗原。结论:CD34+细胞无论在体外培养还是在肝脏受损的重度联合免疫缺陷小鼠体内均能成功转化为肝实质细胞。

AIM： Using the CD34^＋ cells as the primary cells, induced the cells to hepatic cells both in ex vivo cultures and severe combined-immunodeficiency disease （SCID） mice models. METHODS： From September 2004 to June 2005, the experiment was performed at the State Key Laboratory of Bioreactor Engineering. The fresh umbilical cord blood was obtained from healthy full term puerpera after getting the puerpera＇s agreement. Cord blood mononuclear cells （MNCs） were firstly separated by density gradient centrifugation, and then were suspended in buffer by immunomagnetic beads method. CD34^＋ cells were extracted from the suspension. CD34^＋ cells were firstly cultured with stem cell factor plus interleukin 3 plus interleukin 6 （SCF＋IL-3＋IL-6） for a week, and then calls were cultured with OSM＋ fibroblast growth factor （FGF）-I＋FGF-2＋ leukaemia inhibitory factor （LIF）＋ hepatocyte growth factor （HGF）＋ epidermal growth factor （EGF） for differentiation into liver cells. SCID mice were firstly injected with 0.4 mg/per mice 2-AAF by intraperitoneal injection. 7 days later, mice were intraperitoneally injected with 0.4 mL/kg CCl4 plus CD34^＋cells into tail vein in the experimental group. The mice of control group were only subjected with 2-AAF and CCl4. In situ hybridization （ISH） was used to detect human origin cells of mice livers after 4 weeks and 6 weeks, respectively. At the same time, RT-PCR and immunohistochemical method were used to detect human albumin gene and human hepatic cells antigen, respectively. RESULTS： ①In ex vivo cultures, the total number of cells expanded nearly 30 folds, and albumin gene expression and antigen could be detected in 21-day cultured cells. As a result, CD34^＋ cells could differentiate into hepatocytes in ex vivo cultures.② In situ hybridization （ISH） was used to detect human origin cells of mice livers. RT-PCR and immunohistochemical method were used to detect human albumin gene and human hepatic cells antigen. At week 4, 7.66% of human CD45^＋ cells were found, but no human albumin gene expression and human hepatocytes, but 2 weeks later, the percentage of human CD45^＋ cells increased to 31.10%, and human ALB gene expression and human hepatocytes were found. CONCLUSION： Human CD34^＋ cells had successfully differentiated into hepatocytes both in ex vivo cultures and SCID mice liver.