Experimental Studies On The Xenotransplantation Of Microencapsulated Bovine Retinal Pigment Epithelial Cells Into Parkinsonian Rats

To find the most suitable and effective donor cells that will allow widespread application is a focus in the field of neural transplantation of parkinson's disease (PD). Human retinal pigment epithelial (hRPE) cells are support cells in the inner layer of the retina. They have been reported to contain tyrosinase and tyrosine hydroxylase (TH)-like activity, and shown to secrete dopamine (DA) and many kinds of neurotrophic factors. Furthermore, hRPE are easily harvested and expanded in culture. These properties of hRPE cells make it a promising donor cell of PD. Studies in rats, primates and parkinsonian patients have demonstrated that striatally implanted hRPE cells attached to gelatin microcarriers are able to improve parkinsonian symptoms and are well tolerated for extended periods. However, hRPE cells bring out severe ethical concerns which limit its clinical application. Using xenogenic RPE cells to replace hRPE is a new approach. Bovine RPE cell line is easy to get and purify, and it has no ethical problems. So it was used as an alternative source in rat PD model in the present study.Alginate-polylysine-alginate (APA) is a kind of semipermeable membrane material which has fine biocompatibility. The encapsulation of living cells made with APA can relief the immune rejection even in xenotransplantation. The present study was focused on: 1. To detect the biological characteristics of cultured bovine RPE cells. 2. Examine the host immune response and therapeutical effect of transplantation of bovine RPE cells encapsulated with APA into the striatum of unilateral PD rats. 3. Apply the encapsulated bovine RPE cells to not only 6-OHDA PD rat model but also another PD rat model made with lactacystin which containing Lewy bodies in its nigral cells.Part I The culture and identification of bovine RPE cells in vitroAim: To get a large number of high purity bovine RPE cells for later transplantation. Methods: Harvest cells using enzyme digestion. Then cells were identified by their morphological features and were further confirmed with the RPE cell markers, S-100and cytokeratin, through immunocytochemistry. The growth curve of the cells was also observed. Results: We harvest enough purified bovine RPE cells for our study. The growth curve showed that exponential growth occurred at the first 1-4 days.Part II Study on the biological characteristics of cultured bovine RPE cellsAim: To study the biological characteristics of cultured bovine RPE cells. To detect whether they can express TH on mRNA and protein levels with the intention to expand the possibility of the RPE cells transplantation. Methods: The secretion of DA was determined by high performance liquid chromatography electrochemical (HPLC) assay. Using ELISA technique to detect the secretion of BDNF and GDNF under basal condition. Using RT-PCR and immunocytochemistry methods to test the expression of TH on mRNA and protein levels with PC 12 cells as positive control. Results: Dopamine content was first detected at the time point of 2 hour. The level of DA> BDNF and GDNF all arrived at the peak at about 48 hour after inoculation. The secretion of these substances was not different between passages. Small quantity of TH mRNA and TH protein were detected in bovine RPE cells.Part III The making of APA microcapsules and the influence on thebioactivity of donor cellAim: To improve our microcapsule preparation conditions fit for the bovine RPE cells. The effect of microencapsulation on growth and secretion of cells were investigated. Methods: APA microencapsulated cells were made with a high voltage electostatic system. We search for the other optimal parameter at the fixed temperature in making the microcapsule such as the concentration of the alginate, the distance from liquid surface and the microencapsulated voltage. The shape, diameter, strength of the microcapsule were investigated. The growth of the cell were observed at 3rd , 7th,14th,21th days by microscope. The activity of the cells at above time in the microcapsule was detected by trypan blue staining. The secretion of DA, BDNF, GDNF was determined before and after microencapsulation. Results: The optimal concentration of alginate was 1.5%, the corresponding distance from liquid surface was 22-25mm and the voltage is 3.28kv. The microcapsules were near round with smooth surface and the size is uniform. The average diameter is 348±39p.m. The shape of the microcapsules remained well after aspiration with syringe. The sum ofthe cells in microcapsules was gradually increased from the 7th day to 21th day, P<0.05. The survival rate of the cells in microcapsules was decreased gradually at the same time, P<0.05. But the sum of the living cell still keep raising at the period of observational time, P<0.05. DA, BDNF, GDNF secretion was dramatically decreased after microencapsulation in the first 4 or 5 days (p<0.01), but showed no difference at the time point of 5-8 days between the two groups.Part IVEstablishing two kinds of rat PD model and the transplantation ofbovine RPE cellsAim: To set up the stable 6-OHDA and the lactacystin unilateral PD rat model. To examine the host immune response and therapeutical effect after transplantation of the cell into the striatum of the PD rats. Methods: 45 SD rats were randomly divided into three groups (n=15): NS group, 6-OHDA group and lactacystin group, which were respectively injected with NS, 16|ig 6-OHDA and 8(Xg lactacystin by stereotaxic unilateral injection into the substantia nigral pars compacta. The spontaneous and apomorphine-induced rotational behaviors of rats were observed; nigral degeneration and Lewy body were viewed by HE staining; expressions of tyrosine hydroxylase in nigral cells were investigated by immunohistochemistry; contents of dopamine (DA) and homovanillic acid (HVA) were determined by HPLC. Then transplantation was carried out in the two kinds of PD models. Each group was divided into 4 subgroups: NS control, RPE group, APA control group and RPE-APA group. 3*104 RPE cells or about 50 microcapsules with RPE cells were transplanted. The behavior changes of the rats were observed at the 1st, 2nd, 4th, 8th, 14th week posttransplantation. 14 days after transplantation the contents of DA, HVA; the expression of TH, GFAP, MHC-II in the striatum and the activity of cells in microcapsules were detected. Results: The two models were identified through ethology, neurobiochemistry and pathological feurure. In 6-OHDA model, RPE-APA group showed an obvious behavior improving 2 weeks after transplantation (compared to APA group, P<0.05). The improving was more significant at the 4th week and last to the 14' week. Although the RPE group also displayed an obvious behavioral amelioration 2 weeks after transplantation (compared to NS group, P<0.05), the rotational behavior reversed to the beginning after the 4th week. The changing of DA and HVA contents in each group after transplantation were consistent with their changing of behavior. On histological...