GFP转基因小鼠胎肝干细胞体外分离培养及脾内移植

Embryonic hepatic stem cells from GFP transgenic mice:in vitro separation,cultivation and intra-spleen transplantation

背景:近年研究证实,干细胞移植的成功与否、移植后细胞功能的发挥均与体内微环境密切相关。正常生理情况下,肝脏不易接受外源性细胞,因此,在目前的细胞移植研究中,通常会先建立一个移植动物模型,通过造成宿主肝脏的损伤或降低宿主肝脏的免疫反应等方式,为外源细胞的植入和增殖创造一个良好的移植条件。目的:观察GFP转基因小鼠胎肝干细胞在脂质体包裹的二氯亚甲基二磷酸盐/倒千里光碱联合1/3肝切除(L-CL2MDP/RS/PH)模型大鼠肝脏内的定植和分布情况。方法:(1)以孕15 d GFP转基因小鼠为供体,分离培养胎肝干细胞,荧光显微镜观察GFP表达,并行免疫细胞化学鉴定;(2)将30只SD大鼠随机分为实验组和对照组,实验组腹腔注射倒千里光碱溶液(30 mg/kg,共2次,每次间隔2周),术前1 d每只大鼠静脉注射1 m L脂质体包裹的二氯亚甲基二磷酸盐(L-CL2MDP)溶液,行1/3部分肝脏切除后经脾内移植GFP转基因小鼠胎肝干细胞,对照组用生理盐水分别代替L-CL2MDP和倒千里光碱溶液,不进行1/3部分肝脏切除;(3)移植后3,15,30 d取出两组大鼠肝脏行冰冻切片,荧光显微镜下观察GFP阳性肝干细胞在大鼠肝脏的分布,并通过免疫组化染色方法检测抗小鼠C-Kit抗体在移植大鼠肝内的表达。结果与结论:(1)镜下观察分离的肝干细胞大小均匀,呈鹅卵石形态。培养5 d后细胞体积逐渐增大,呈类圆形或梭形,荧光显微镜下可见绿色荧光,免疫细胞化学检测第3代肝干细胞表达CD34、AFP;(2)胎肝干细胞移植后3 d在荧光显微镜下发现实验组大鼠肝内散在分布GFP阳性细胞,移植后30 d大鼠肝脏内仍可见GFP阳性表达;(3)移植后3,15,30 d在实验组大鼠肝脏组织可见C-Kit阳性细胞表达;(4)结果表明,GFP转基因小鼠胎肝干细胞成功移植于L-CL2MDP/RS/PH模型大鼠脾内,植入的细胞能够在大鼠肝脏中存活,并出现不同程度增殖。

BACKGROUND:Recent studies have confirmed that the success of stem cell transplantation and the function of transplanted cells are closely related to the in vivo microenvironment.Under normal physiological conditions,the liver is not susceptible to exogenous cells.Therefore,a transplant animal model is usually established to damage the host liver or reduce the immune response of the host liver,creating a good condition for the transplantation and proliferation of exogenous cells.OBJECTIVE:To observe the colonization and distribution of embryonic hepatic stem cells from GFP transgenic mice in a rat model of liposome-coated dichloromethylene diphosphate/retrorsine and 1/3 liver resection(L-CL2MDP/RS/PH).METHODS:GFP transgenic mice at gestational 15 days were taken as donors to separate and cultivate embryonic hepatic stem cells.Fluorescence microscope was used to observe the GFP expression followed by immunocytochemical identification.Thirty Sprague-Dawley rats were randomized into experimental group and control group.Rats in the experimental group were given intraperitoneal injection of retrorsine(30 mg/kg,twice,with an interval of 2 weeks).At 1 preoperative day,these rats were subjected to intravenous infusion of L-CL2MDP(1 mL)and 1/3 liver resection followed by intra-spleen transplantation of embryonic hepatic stem cells from GFP transgenic mice.Rats in the control group were only given injection of normal saline instead of L-CL2MDP and retrorsine,but no liver resection.Frozen liver sections were made in the two groups to observe the distribution of GFP-positive hepatic stem cells under the fluorescence microscope at 3,15,30 days after transplantation.Moreover,immunohistochemical staining was used to detect the expression of anti-mouse C-Kit antibody in the rat liver.RESULTS AND CONCLUSION:(1)The hepatic stem cells under microscope were uniform with a cobblestone shape.After 5 days of culture,the cells with increased size presented with the shape of fusiform or polygon,and the green fluorescence was observed in cells under the fluorescence microscope.Passage 3 hepatic stem cells expressed CD34 and AFP.(2)At 3 days after spleen transplantation of embryonic hepatic stem cells,GFP positive cells were scattered in the experimental group under the fluorescence microscope,and the GFP positive expression was still visible in the liver at 30 days after transplantation.(3)At 3,15,and 30 days after transplantation,C-Kit positive cells were visible in the liver of rats in the experimental group.All these findings reveal that embryonic hepatic stem cells from GFP transgenic mice have been successfully transplanted into the spleen of the L-CL2MDP/RS/PH rat model,and the transplanted cells can survive and proliferate in the rat liver.