单克隆人胰腺干细胞分离培养体系的建立

ESTABLISHMENT OF THE ISOLATION AND CULTURE SYSTEM FOR MONOCLONING HUMAN PANCREATIC STEM CELL

本研究探讨了建立单克隆人胰腺干细胞分离培养体系及单克隆人胰腺干细胞系,对一些影响干细胞增殖的因素进行了分析。无菌取人流产胎儿胰腺组织。切碎至1 mm^3,0.1%Ⅳ型胶原酶消化。低糖DMEM、10%FBS、3.7g/L NaHCO_3、0.08 g/L青霉素及0.1 g/L链霉素培养液贴壁培养细胞。2.5 g/L胰蛋白酶+0.4g/L EDTA消化传代。克隆环筛选单克隆干细胞,培养液中添加10 ng/mL EGF,扩增单克隆干细胞。采用核型分析法检测干细胞染色体,MTT法测定干细胞生长曲线。胶原酶消化胰腺组织.获得单个细胞和细胞团。贴壁培养,原代上皮样胰腺干细胞克隆性生长。胰蛋白酶消化传代,上皮样胰腺干细胞逐渐被纯化。克隆环筛选,获得单克隆人胰腺干细胞。扩增培养。1例来源于4月龄男性流产胎儿胰腺组织干细胞建系,传50代。染色体核型分析,该干细胞系为正常的二倍体细胞。细胞生长曲线显示培养1-4 d,干细胞生长缓慢,5-6 d,进入倍增期。培养液中添加15%FBS,干细胞增殖较快,再添加15 ng/mL EGF或10 ng/mL IGF-Ⅱ,干细胞增殖更快。研究结果表明应用本实验建立的细胞分离培养体系获得了单克隆人胰腺干细胞系。

The present study aimed to establish an isolation and culture system for the monoclonal human pancreatic stern cells and monoclonal human pancreatic stem cell line. Some factors which would influence the proliferation of the monoclonal human pancreatic stem cells were assessed. Pancreatic tissues, taken from abortive fetuses by sterile procedures, were dissected into 1 mm3 segments and digested with 0.1% type Ⅳ collagenase. The isolated cells were grown in media containing low glucose Dulbecco＇s Modified Eagles＇s Medium supplemented with 10% fetal bovine serum （FBS）, 3.7 g/L sodium pyruvate, 0.08 g/L penicillin and 0.1 g/L streptomycin. These cells were further digested with 0.25 g/L trypsin and 0.4 g/L EDTA for propagation. The monoclonal human pancreatic stem cells were selected by clone-ring, and further proliferated after addition of 10 ng/mL epidermal growth factor （EGF） in culture media. The cell chromosome set was determined by karyotype analysis. The growth curve was made by the 3-（4, 5）-dimethyhhiahiazo （-z-y1）- 3, 5-di-phenytetrazoliumromide （MTF） method. The results showed that pancreatic tissues were digested to many single cells and cell clusters with collagenase. Adherently cultured, primary epidermal-like pancreatic stem cells grew clonally. After several times of dissociation and propagation, pancreatic stem cells were gradually purified during generations. Using clone-ring selection, the monoclonal human pancreatic stem cells were obtained. Continuously propagated, a monoclonal human pancreatic stem cell line which was derived from a male abortive fetus of 4 month-old had been passed through 50 generations. Karyotype analysis demonstrated that the chromosome set of the monoclonal human pancreatic stem cell line was normal diploid. Growth curve revealed that monoclonal human pancreatic stem cells grew slowly in initial 1-4 days. Then, they entered the logarithmic growth period in next 5-6 days. Their proliferation was speeded up by supplementation with 15% FBS in culture media, which was even more quickly after addition of the 15 ng/mL EGF or 10 ng/mL insulin growth factor Ⅱ （IGF-Ⅱ）. The result identified that the monoclonal human pancreatic stem cell line could be obtained by the cell isolation and culture system.