Isolation Of Hepatic Oval Cell From Different Rat Models

Background and AimDiabetes mellitus is one of the most common diseases in China with an increasing incidence rate every year and threatens peoples' health. Patients have to receive therapy in all their lives, which is really a great burden to home and national economy. Reconstruction of internal insulin secretary system may cure type 1 diabetes and part of advanced type 2 diabetes. With the induction of specific environmental factors, adult stem cells could transdifferentiate into other certain types of cells, so transdifferentiation of adult stem cell could solve immunological rejection completely which is the cutting-edge research of regeneration medicine. Liver and ventral pancreas are specified at the same time and in the same general domain of endodermal cells, so same progenitor cells may be shared and adult hepatic stem cells are possible to transdifferentiate into insulin-producing cells. This study aims to establish a simple and effective method to acquire abundant highly purified stem cells from adult rat liver.Materials and MethodsSprague-Dawley (SD) rats were divided into 4 groups including control, 2-AAF, 2-AAF/CC14 and 2-AAF/PH. Control and 2-AAF groups were fed corn oil and 20 mg/kg body mass 2-AAF (dissolved in corn oil) by oral gavage throughout this experiment respectively. 2-AAF/PH group was fed 20 mg/kg body mass 2-AAF (dissolved in corn oil) daily by oral gavage for 5 d. On the 6th d, rats were partially hepatectomized under anesthesia by injection with sodium pentobarbital (0.1 ml/100 gm body weight) and were not fed 2-AAF on the same day. 2-AAF/CCl₄ group was fed 20 mg/kg body mass 2-AAF daily for 7d and on the 8th d a single CCl₄ dose of 1.9 ml/kg of body weight in a 1:1 (vol/vol) dilution in corn oil was administered by intraperitoneal injection. The time points of this study were counted when partial hepatectomy and CC14 injection were performed.Diabetic rat models were induced by intraperitoneal injection of a single STZ dose of 60mg/kg of body mass diluted in pH 4.5 sodium citrate buffer. After 3days, if blood glucose from tail vein was larger than 16.6mmol/L, the construction would be successful.On the 13th d after PH and CCl₄ injection, rats were anesthetized by injection with sodium pentobarbital (0.1 ml/100 gm body weight). The digestion was done by using the two-step collagenase perfusion protocol of Seglen and oval cells were isolated by Percoll density gradient centrifugation and flow cytometry sorting for Thy1.1 positive cells. Oval cells were cultured in Dulbecco's minimum Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 10ng/ml leukemia inhibitory factor (LIF), 10ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF), 10ng/ml stem cell factor (SCF), 10ng/ml IL-3 and 10ng/ml IL-6.ResultsAfter cell sorting by flow cytometry, we found there were different Thy1.1 positive rates in different rat models including diabetic models. Sequence from largest to smallest rate was 2-AAF (0.5%), 2-AAF/PH (0.3%), 2-AAF/CCl₄ (0.3%), diabeticmodel (0.1%) and control (0.0%). The newly isolated cells presented dissociative with each other. After cultured for 12h, some cells started to adhere to the plate. 48-72 hours later most of the cells adhered to the plates. The adherent cells were fusiform or polygon which were in phenotypes of epithelium cells. 1 week later cells began to form cell clusters.Conclusion2-AAF, 2-AAF/CCl₄ and 2-AAF/PH groups seem to be the ideal model of hepatic oval cell activation. Two-step collagenase perfusion protocol of Seglen, Percoll density gradient centrifugation and flow cytometry sorting for Thy1.1 positive cells were effective ways to isolate hepatic oval cells. We suspect islet damage may stimulate the activation of stem cells such as pancreatic ductal cells, bone marrow derived stem cells and hepatic oval cells in some tissues. This discovery could have important implication of future application of transdifferentiation into insulin producing cells.