大鼠胰腺间质细胞体外培养过程及其形态学特征

Process of in vitro culturing rat pancreatic mesenchymal cells and their morphological characteristics

目的:对胰腺间质细胞进行原代及传代培养,观察其形态学的变化。以探索一种简单稳定的获取及培养胰腺间质细胞的方法。方法:用机械剪切加胰蛋白酶消化的方法制备SD大鼠胰腺单细胞悬液,取新生SD大鼠1只麻醉断髓处死,体积分数0.75的乙醇浸泡消毒,无菌条件下取出胰腺组织,去除红细胞,剪切,加入胰蛋白酶消化,消化终止后细胞筛过滤,收集滤过的细胞悬液离心,弃上清液,重复1次。收集获得的单个胰腺间质细胞。接种于含体积分数0.1的胎牛血清、0.05g/L的亚胺培南的DMEM/F-12(1∶1)培养基,培养48h后换液,弃上清及悬浮细胞,继续培养48h后,换用含1×10-5mol/L阿糖胞苷的完全培养基培养24h,再换正常培养基继续培养,在培养细胞达到80%～90%的细胞汇合时,按1∶3进行传代培养。测定细胞生长曲线及贴壁率,原代细胞及传代细胞用双硫腙染色,鉴定有无胰腺β细胞。结果:①原代细胞及传代细胞形态学观察:原代培养换液前,显微镜下可见细胞大小不等,悬浮,有较多细胞碎片。48h首次全量换液后,悬浮细胞减少,经3次换液可基本去除。加用阿糖胞苷培养24h,大量细胞相继死亡。至第10-14天,培养细胞明显增加,基本铺满瓶底,以星形、梭形为主,细胞呈放射状或漩涡状生长,部分区域呈灶状。传至第5代细胞出现老化。②细胞生长曲线:细胞从传代后第2天,数量即开始增加,第4天增加幅度最大,至第6天细胞数量最大,以后数量略有下降。传代培养对数增殖期为2-4d,对数增殖期结束后,第6天细胞生长缓慢,进入平台期。③细胞的贴壁率:传代细胞培养2h已有部分细胞贴壁;培养4h贴壁细胞接近50%;培养10h贴壁达79%;培养12h细胞全部贴壁,而且部分细胞开始分裂增殖,细胞形态与原代细胞基本相似。④胰腺β细胞的化学鉴定:对胰腺组织分离所得单个细胞、原代培养细胞及传代培养细胞分别用双硫腙染色。培养前细胞染色,可见少量腥红色细胞,随着培养时间延长腥红色细胞逐渐减少,在原代培养10～14d,腥红色细胞消失,传代培养细胞染色未见腥红色细胞。结论:细胞培养12h全部贴壁,传代培养对数增殖期为2-4d,第6天细胞进入平台期。原代培养胰腺间质细胞形态多样,主要呈梭形,少数呈三角形或星形,贴壁爬行生长,第1周增殖较慢,随后增殖加速,细胞团块多见,偶可见大的圆形细胞生长,核大,胞浆内颗粒多。传代培养,细胞能迅速贴壁生长,细胞形态逐渐均一,主要为梭形、三角形细胞,呈集落样生长,双硫腙染色阴性;但细胞培养至第5代,胞浆颗粒增多,细胞增殖减慢,大部分细胞逐渐老化死亡。

AIM： To primarily culture and subculture the pancreatic mesenchymal cells（PMCs） of rats, and observe the morphological changes of the cultured PMCs so as to explore a simple and stable method for isolating and culturing PMCs in vitro. METHODS： The single cell suspension of Sprague-Dawley （SD） rats pancreas was gotten by mechanical shearing and trypsin digestion. A newborn SD rat was put to death by breaking the cervical cord and sterilized with alcohol 75%. The pancreas of the rat was cut off under germ-free condition, and the erythrocytes were washed out, followed by shearing and digestion with trypsin. After the cells were filtered and washed, the cell suspension was collected to centrifuge, and supernatant was discarded; The above procedure was repeated once. The collected single mesenchymal cells were maintained in DMEM/F-12 medium （1：1） consisting of 10% fetal bovine serum（FBS） and 0.05 g/L imipenem. Then 48 hours later, the supernatant and suspension cells were discarded, and culture medium were changed. After another culture of 48 hours, the cells were cultured in the medium consisting of 1 ×10^-5 mol/L cytarabine for 24 hours, and then cultured in the replaced normal medium. When the cultured cells reached 80% to 90% of confluence, they were subcultured in the proportion of 1 to 3. The growth curves of cells were drawn and the adhesion rate was measured. The primarily cultured and subcultured cells were stained with dithizone（DTZ） to identify the pancreatic β cells. RESULTS： ① Morphological observation of primarily cultured and subcultured cells： Before the medium was changed, the primarily cultured cells showed inequality of size in suspension and there were many cell debris. When the medium was changed completely for the first time 48 hours later,and the suspension cells were decreased which could be removed after the medium were changed three times. Most of the cells died one after another after cultured with cytarabine for 24 hours. The number of the cells increased quickly and the cells were nearly confluent in starlike and fusiform shape on 10^th to 14^th day of culture. They proliferated radially or circinately, among which some were focal. They appeared aging at the 5^th generation of subculture. ②Growth curve of PMCs： The cells proliferated on the 2^nh day after subculture, proliferated quickest on the 4^th day and the number of PMCs was maximum on the 6^th day. The logarithm multiplication period of subculture was from 2^nd to 4^th day. After the 6^th day, the PMCs grew slowly and entered platform period. ③Adhesion rate of subcultured cells： Partial cells adhered to the wall 2 hours after subculture, nearly 50% of PMCs adhered to the wall at 4 hours, 79% on the 10^th hour and all on the 12^th hour. Some of the cells began to proliferate on the 12^th hour. The above results were similar to the observation results of primarily cultured cells in shape. ④Cytochemical identification of PMCs： The single cells isolated from pancreas, the primarily cultured and subcultured cells were stained with DTZ successfully.The cells were stained scarlet in a small quantity before culture, however they were decreased as the culture time went on and few were seen on the 10^th to 14^th day of primary culture. No scarlet cells were identified in the subcuhured cells. CONCLUSION： Almost all of the subcuhured PMCs adhered to the wall at 12 hours after culture. The logarithm multiplication period was from the 2^nd to 4^th day, and the platform period from the 6^th day. The primarily cultured PMCs were diverse, in which most were fusiform, and a few triangle or star-shaped, growing creepingly. The PMCs proliferated slowly on the first week, whereafter speeded; Mass of PMCs was observed, and big and round-shaped cells were found occasionally with large nuclei and many granules in cytoplasm. After subculture, PMCs adhered to the wall rapidly, whose form were uniform gradually, mainly fusiform and triangular, forming a colony; they were stained negative by DTZ. When the PMCs were cultured to the fifth generation, granules in cytoplasm were increased, the proliferation speed was decelerated, and most of the PMCs were aging gradually.