Objective To introduce the basic research and cl inical appl ication of stem cells transplantation for treating diabetic foot. Methods The recent original articles about the stem cells transplantation for treating diabetic foot were extensively reviewed. Results Transplanted different stem cells in diabetic foot could enhanced ulceration heal ing in certain conditions, increase neovascularization and avoid amputation. Conclusion Stem cells transplantation for treating diabeticfoot may be a future approach．
Purpose To investigate the development of embryonic stem cells (ESC)in the subretinal space． Methods ESC were cultivated in suspension for 4 days till they developed into cell aggregates,i.e.embryonic body(EB).ESC as well as EB combined with or without RA were respectively transplanted into vitreous cavity and subretina1 space in SD rats,and the subretinal transplanted eyes,transient ischemia-reperfusion injuries were made by ligating the ophthalmic artery for 40 seconds before the transplantation .The experimental eyes were enucleated for histological and immunohistochemical assays after 14～28 d. Results The EB was found to develope into photoreceptors induced by RA in the subretinal space under an ischemia-reperfusion condition，and EB transplantation without RA induction induced multiple differentiations in the subretinal space.The single injection of RA without EB induced hyperplasia of the neural retinal cells.ESC transplanted into vitreous cavity rapidly proliferated and developed into atypical hyperplastic mass. Conclusion EB derived from ESC can differentiate into photoreceptors induced by RA in the host subretinal space under an ischemia-reperfusion condition. (Chin J Ocul Fundus Dis，2000，16：213-284)
Objective To establish a culture method for human fetal retinal progenitor cells (RPC) in vitro. Methods Retinal neuroepithelium of 8-12-week human fetal were isolated and cultured in suspension and adherent methods. The passage cells were cultured and differentiated for 14 days with 5% fetal bovine serum without basic fibroblast growth factor (bFGF). The expressions of RPC and retinal final cells markers before and after the differentiation were detected by immunohistochemical analysis. Results The isolated cells cultured in suspension method congregated as the neurospheres and expressed the neuroectodermal marker nestin, but failed in passage and expansion; while the expression of nestin and serial passage were found in the cells cultured in adherent way. The differentiated passage cells expressed retinal final cells markers including glial fibrillary acid protein, beta;-tubulin and recoverin. Conclusions RPC derived from human fetal neural retina at the 8th12th week of gestation are capable of expansion and multipotentiality. (Chin J Ocul Fundus Dis, 2007, 23: 98-100)
Objective To summarize the research progress of CD90 protein. Methods The demestic and international published literatures related to CD90 protein in recent years were collected and reviewed. Results CD90 protein was involved in the cell-cell and cell-cytoplasm function. CD90 protein could promote axons growth and neural regeneration， and could induce apoptosis of thymus gland cells and stromal cells. CD90 protein participated in cell adhesion， extravasation and transfer， and the regulation of fibrosis. CD90 protein was a potential marker for cancer stem cells. Conclusion CD90 protein is very important in development of many diseases， and can provide a new molecular target to diagnose and treat neoplasms.
ObjectiveTo observe the effect of interleukin (IL) 10 modified endothelial progenitor cells (EPC) in diabetic retinopathy (DR). MethodsEPC cells were collected and cultivated from the bone marrow of rats and identified by immuno-fluorescence staining. EPC cells were infected with lentivirus (LV) of EPC-LV-IL10-GFP (EPC-LV-IL10-GFP group) or EPC-LV-NC-GFP (GFP group). EPC cells without lentivirus infection was the EPC group. Enzyme-linked immuno sorbent assay (ELISA) was used to measure the concentrations of tumor necrosis factor (TNF)-α, IL10, IL8 and vascular endothelial growth factor (VEGF) in the supernatant of these three groups. 168 male Wistar rats were divided into normal control group (28 rats), diabetes mellitus (DM) group (28 rats), DM-blank control group (56 rats) and DM-intervention group (56 rats). DM was introduced in the latter 3 groups by streptozotocin intravenous injection. Three months later, the rats in the DM-blank control group and DM-intervention group were injected with EPC-LV-NC-GFP or EPC-LV-IL10-GFP by tail vein, respectively. Immunohistochemistry was used to observe the GFP expression in rat retinas. The blood-retinal barrier breakdown was detected by Evans blue (EB) dye. The retinal histopathologic changes were observed by transmission electron microscope. The mRNA level of VEGF, matrix metallproteinases-9 (MMP-9), angiopoietin-1 (Ang-1), inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) in retina were measured by reverse transcription-polymerase chain reaction (RT-PCR). ResultsELISA showed that the levels of TNF-αand IL8 in the supernatant significantly decreased, while the levels of IL10 and VEGF increased (P < 0.05) in EPC-LV-IL10-GFP group. GFP expressed in the retina of blank control group and intervention group, mainly in the ganglion cell layer, inner nuclear layer and outer plexiform layer. The retinal blood vessel pathological change and EB permeability significantly decreased in intervention group compared with DM group (P < 0.05), and blank control group (P < 0.05). RT-PCR revealed that the mRNA level of VEGF, MMP-9 and Ang-1 significantly increased, and eNOS decreased in DM group compared to the normal control group (P < 0.05). The mRNA level of VEGF and iNOS decreased, eNOS increased while Ang-1 and MMP-9 had not changed in DM-blank control group and DM-intervention group compared with DM group (P < 0.05). ConclusionsIL10 modified EPC can improve the inflammative microenvironment and suppressed the pathogenesis of DR. Furthermore, EPC transplantation can increase the number of EPC and exerted their effect.
OBJECTIVE To explore the possible mechanisms of skin regeneration through the epidermal stem cells stimulated by epidermal growth factor (EGF). METHODS At 8 and 14 days after treatment with EGF, the tissue specimens from 8 skin ulcered patients who were treated with EGF were used to evaluate the distribution and differentiation of epidermal stem cells. The expression of beta 1 integrin, keratin 19 (K19), keratin 14(K14) and keratin 10 (K10) in skin was detected with SP immunohistochemical methods. Hematoxylin and eosin staining method were used to observe the tissue structure. Another 7 biopsies from ulcered patients without EGF management were used as the control. RESULTS The results from the hematoxylin and eosin staining showed that the epidermis in EGF treated wounds was thick and the epidermal ridges were enlarged both in 8 and 14 days compared with those in control skin. Immunohistochemical staining from beta 1 integrin and K 19 showed that all tissues treated with EGF were rich in epidermal stem cells both in 8 and 14 days. These stem cells were bigger in size and larger in number and localized at the base of the epidermis. In contrast, the positive expression cells of beta 1 integrin and K 19 in control group in the same time were scanty. It was found that there were some stem cell islands in epidermis treated with EGF in day 14 and absent from the control group. The expression of K14 and K10 could be observed in those terminally differentiating epidermal cells in both groups. CONCLUSION The results indicate that the possible mechanisms of skin regeneration stimulated by EGF comes from the mitogenic effects and differentiation of skin stem cells.
Objective To investigate the experimental condition and mechanism of differentiation of human umbilical cord blood derived mesenchymal stem cells（hUCB-MSC）into neuron-like cells induced by recombined human epidermal growth factor （rhEGF） and taurine in vitro.Methods hUCB-MSC were primary cultured in Dulbeccoprime;s modified Eagle's medium/F12 （DMEM/F-12）which supplemented with 105U/L penicillin G, 100 mg/L streptomycin sulfate, 10% fetal bovine serum (FBS),5% autologous plasma,4 mmol Lglutamine, 30 ng/ml rhEGF.The DMEM/F-12 medium was replaced by taurine medium after 3 passages.The expression of surface antigen CD90,CD29,CD34,CD44 and CD45 were detected by flow cytometry；the expression of neuron specific enolase,rhodopsin and nestin were investigated by immunocytochemistry. The statistical method was chi square test.Results Morphologically similar to bonemarrow MSC,hUCB-MSC became attached cells after the first 5 to 7 days in culture，and reached 80% to 90% confluent after 3 to 4 weeks. Growth accelerated after passage. hUCB-MSC were positive for CD29,CD44 and CD90 but negative for CD34 and CD45. After taurine induction, 2515/3120 cells expressed NSE, 1168/3175 cells expressed rhodopsin and 903/3050 cells expressed nestin while only 234/2965 cells expressed NSE in the control group(P＜0.01).Conclusion rhEGF and taurine can induce hUCB-MSC differentiating into neuronlike or rhodopsin positive cells.
Objective To review the latest research progress of full-thickness tissue engineered skin (FTTES), to thoroughly understand its current state of research and appl ication so as to lay a sol id foundation for developing new type FTTES and improving the qual ity of skin substitutes. Methods Domestical and international l iterature concerning FTTES in recent years was extensively reviewed and comprehensively analyzed. Results Some progress of FTTES had made in seedcells, scaffold materials and construction, and some therapeutic efficacy had also been achieved in cl inical appl ication. ButFTTES grafting successful rate was lower, and it had no complete skin structure and had not reached the requirements of cl inicalappl ication. Conclusion FTTES is an ideal skin substitute and has great development prospects. However, in seed cells, scaffold materials, construction and appl ications of FTTES, further studied is still needed.
Objective To investigate the viability and the characters of proliferation and differentiation of retinal stem cells (RSCs) after cryopreservation and anabiosis. Methods The RSCs of a Long Evans rat with the embryonic age of 17 days were separated and cultured in vitro. The third-passage RSCs in the cryopreservation liquid consisted of 80% Dulbecco modified Eagle medium (DMEM)/F12，10% bovine serum albumin (BSA)，10% dimethylsulfoxide (DMSO)，and basic fibroblast growth factor (bFGF) (20 ng/ml), were stored in liquid nitrogen. After 1, 2, 4, 8, 12, and 16 weeks of freezing period, these cells were thawed. The livability of the cells was counted and the differentiation was induced while the proliferation and characters of differentiation were detected by immunofluorescence. Results The effects of different durations of cryopreservation on the livability of RSCs did not differs much (Pgt;0.05). These cells were reculturd well and presented specific marker of RSCs. In addition, they also could be induced and differentiated into several types of retinal cells. Conclusion Cryopreservation and anabiosis of RSCs does not affect the cellular intrinsic characters of proliferation and differentiation. (Chin J Ocul Fundus Dis, 2007, 23: 94-97)
Objective To isolate and purify the melanoma stem cells (MSC) in choroidal melanoma OCM-1 cells. Methods OCM-1 cells were resuscitated, and after cultured in standard Dubecco's modifided Eagle's medium (DMEM)/F12, they were cultured in serum-free medium (SFM). The cultured MSC were isolated and purified, and the positive rate of CD133, the specific markers of neurostem cells, was observed by flow cytometry (FCM). The 6th generation of the cells were stained by musashi-1 immunocytochemistry, and the rate of the positive cells was observed under the microscope. Results After the Adherent OCM-1 cells cultured in SFM, the number of the adherent number decreased obviously. The cells at the 6th generation grew as the suspended gobbets, which represented the typical grow manner of the stem cells. Positive CD133 could be found in the cells of different generations, which was 2.5％, 21.7％, and 57.8％ in the non-isolated OCM-1 cells, the 1st generation of isolated cells, and the 2nd generation cells, respectively. The positive rate of CD133 in the cells at the sixth generation was 79.8% with b positive expression of musashi-1. Conclusion MSC is in the human choroidal melanoma OCM-1 cells. The suspended stem cells may be purified by limited differentiation and serial passage in SFM. (Chin J Ocul Fundus Dis, 2007, 23: 87-90)