| Objective1. In the first part, islets of langerhans were obtained from Wistar rats by injecting collagenase-Ⅴinto common bile duct to digest the pancrease of the laboratory animals, and then gradient centrifugation were made to purify the islets. The objective of this part is to find out reliable and feasible way of islet isolation and purification.2. We isolated pancreatic stem cells (PSCs) by using collagenase to digest rat embryonic pancreas, then purified and cultured the cells we got. After that, marks of PCSs were detected to decide the cells'property and to prove reliability of the method.3. The PSCs were co-cultured with islets on basis of traditional inducing culture medium, and then several factors were detected, the purpose of which is to evaluate the differentiating effect on PSCs that and study its mechanism. All in all, we want to prove that, PSCs transplantation is a feasible and promising treatment of diabetes mellitus, although there will be lots of problem to be solved.MethodsPart one:Isolation and purification of islet.We chose Wistar rat as donor of islet by injecting collagenase-Ⅴinto common bile duct to digest the pancreas, and then discontinued density gradient centrifugation were made to purify the islets. In order to decide the procedure of islet isolation and purification is feasible or not, the production, purity, livability and basal insulin level of islets were detected, insulin release test and insulin stimulation index were done also.Part two:Isolation, culture and identification of PSCs.We used pancreata of fetal wistar rats for cultivation of PSCs by collagenase digestion. The PSCs can be separated and purified through differential adhesion method. To determine cells we obtained have the same characteristics of PSCs and the method is faithful, several factor were detected by immunohistochemical stains and RT-PCR simultaneously, Which is CK-19, Nestin, Glucogon and Insulin.Part three:Make PSCs differentiate into islet-producing cells.There were three groups:PSCs culture group, islet culture group and islet-PSCs co-culture group. Every group were given the same inductive medium, and 50 islets (diameter>100μm) were added into the inductive media in co-culture group. In the process of differentiation, basal insulin level, insulin release test, insulin stimulation index, CK-19, Nestin, Glucogon and Insulin were detected again, the purpose of which is to evaluate the differentiating effect on PSCs and study its mechanism.Data are expressed as mean±SD and analysised by SPSS 13.0. Statistical analysis was performed using repeated-measures ANOVA and the Student's t-test as appropriate. P values<0.05 were considered to be statistically significant.ResultsPart oneThere were (345±38) islets been obtained from every single pancreas of Wastar rat, and (254±42) were left after purification, whose average purity and recovery rate was (87±13)%and 74.3%. The AO/PI straining showed that livability of the islets were better than 95%. The average basal insulin level and insulin stimulation index was 4.21mIU/L and 2.31.Part twoThe cultured cells had the ability of well growth and self-replication, which showed the characteristics of undifferentiated cells, expressing the markers of PSCs: CK-19, Nestin and Glucogon, but Insulin was negative by methods of immunostaining and RT-PCR.Part three After induction, some of these cells started expressing insulin, besides CK-19, Nestin and Glucogon. The further comparison showed that, positive expression rate in co-culture group is higher than in PSCs culture group, and the difference has statistical significance (P<0.05).Conclusions(1) The islet has satisfactory production, purity, livability and biological activity, by injecting collagenase-V into common bile duct to digest the pancreas, and discontinued density gradient centrifugation were made as purification.(2) The procedure that digest pancreata of fetal wistar rats by collagenase and purify cells through differential adhesion, is a reliable method for cultivation of PSCs.(3) The cells obtained from showed the characteristics of pancreas of fetal wistar rats showed the characteristics of PSCs.(4) Small amount of PSCs can differentiated to be induce to insulin-secreting cells by using traditional inductive medium.(5) Islet is maturation promoting factor in the process of PSCs'differentiation, it can secrete multiple of cytokine to establish mostly like internal environment of pancreas.(6). One of the most important factors contributing to the maturation of PSCs is a significant increased expression of CK-19 in PSCs after co-cultured with islets. Keywords:Diabetes; Islets of langerhans transplantation; Stem cell;Induction; Differentiation.
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