Objective To investigate the effect of inducible nitric oxide synthase (iNOS) inhibitor aminoguanidine on pancreas islets cultured with cytokines TNF-α and IL-1β in rats. Methods Islets isolated from Wistar rats were purified and cultured. According to whether cytokines TNF-α, IL-1β and aminoguanidine were added into the medium respectively or not, islets were divided into 4 groups: cultured with islet only was taken as blank control group, cultured with TNF-α+IL-1β as cytokine group, cultured with aminoguanidine as aminoguanidine group, and cultured with TNF-α+IL-1β and aminoguanidine as aminoguanidine+cytokine group. NO level in culture medium and iNOS activity in islets tissue (Test Kit), apoptosis (TUNEL method) and viability of islets cell (acridine orange/ethidium bromide stain), and the function of islets (insulin release test) were measured. Results Compared with blank control group, the activity of iNOS in islet tissue and level of NO in culture medium increased, and the mass mortality and apoptosis appeared in islet cells, while insulin secretion decreased in cytokine group (P＜0.01). Compared with cytokine group, the activity of iNOS 〔（3.17±0.51） U/ml vs. （38.93±4.72） U/ml〕 and level of NO 〔（50.5±10.4） μmol/L vs. （313.0±35.4） μmol/L〕 decreased, the survival 〔（72.73±3.14）% vs. （57.07±5.07）%〕 increased and the apoptosis rate 〔（20.11±8.48）% vs. （41.17±6.87）%〕 decreased, the insulin secretion (secretion index: 3.50±0.27 vs. 1.96±0.19) improved； There were all significant differences in 2 groups (P＜0.01). Conclusion The iNOS inhibitor aminoguanidine could prevent the islet from the damage of iNOS/NO, alleviate the impairment of cytokines to islets, and ameliorate the survival and function of islets.
ObjectiveTo establish an efficient method of isolating and culturing high activity and high purity of Schwann cells, and to identify the cells at the levels of transcription and translation. MethodsThe sciatic nerves harvested from a 4-week-old Sprague Dawley rat were digested in the collagenase I for 15 minutes after dissecting, and then the explants were planted in culture flask directly. The cells were cultured and passaged in vitro, the growth state and morphological changes of the cells were observed under inverted phase contrast microscope. MTT assay was used to test the proliferation of cells and the cells growth curve was drawn. RT-PCR and immunohistochemistry staining were used to detect S100 and glial fibrillary acidic protein (GFAP) at the levels of transcription and translation, respectively. The purity of cells was caculated under microscope. ResultsAfter the digestion of collagenase I, fibroblast-like cells appeared around explants within 24 hours, with slender cell body and weak refraction. After tissues were transferred to another culture flask, a large number of dipolar or tripolar cells were seen after 48 hours, with slender ecphyma, plump cell body, and b refraction, and the cells formed colonies within 72 hours. The cells were covered with the bottom of culture flask within 48-72 hours after passaging at a ratio of 1∶2, and spiral colonies appeared. Cells showed vigorous growth and full cytoplasm after many passages. MTT assay results showed that the cells at passage 3 entered the logarithmic growth phase on the 3rd day, reached the plateau phase on the 7th day with cell proliferation, and the growth curve was “S” shape. RT-PCR results showed that the cells expressed S100 gene and GFAP gene, and immunohistochemistry staining showed that most of the cells were positively stained, indicating that the majority of cells expressing S100 protein and GFAP protein. The purity of Schwann cells was 98.37% ± 0.30%. ConclusionHigh activity and high purity of Schwann cells can be acquired rapidly by single-enzyme digestion and explant-culture method.
Objective To establish a model of the human marrow mesenchymal stem cells (hMSCs) cultured under the hypoxic condition in adults and to investigate the biological features of MSCs under hypoxia.Methods The bone marrow was obtained by aspiration at the posterior superior iliac spine in 3 healthy adult subjects. hMSCs were isolated by the gradient centrifugation and were cultured in the DMEM-LG that contained 20％ fetal bovine serum. The serial subcultivation was performed 10-14 days later. The second passage of the hMSCs were taken, and they were divided into the following 4 groups according to the oxygen concentrations and the medium types: the normoxic group（20％O2, DMEM-LG, Group A）, the hypoxic group（1％O2, DMEM-LG,Group B）, the normoxic osteoblast induction group（20％O2, conditioned medium, Group C）, and the hypoxic osteoblast induction group（1％O2, conditioned medium, Group D）. The biological features of the cultured hMSCs under hypoxia were assessed bythe cell count, the MTT method, the colony forming unit-fibroblast, the real-time RT-PCR, and the alkaline phosphatase (ALP) activity, and the alizarinred staining. Results The hMSCs cultured in the Group B and Group D had a significantly higher proliferation rate than those in the Group A (Plt;0.01), and the culture effect was not influenced by the medium type. The hMSCs in the Group B had a significantly higher level of the colony-forming unit capability than the hMSCs cultured in the Group A(Plt;0.01). After the induction, hMSCs in the Group B had a decreasednumber of the osteoblasts than hMSCs in the Group C. The hMSCs in the Group D had a gradually-increasedactivity of ALP, which was significantly lower than that in the Group C(Plt;0.01). The RT-PCR examination revealed that ALP,osteocalcin, and mRNA expressions of collagen type Ⅰ and osteonectin in the Group Csignificantly increased （P＜0.01). By comparisonamong the 3 groups, after the 4-week culture the obvious calcium salt deposit and the red-stained calcium nodus could be observed.ConclusionHypoxia can promote the proliferation rate of hMSCs, enhance the colonyforming ability and inhibit the differentiation of the osteoblasts.
Objective To isolate neural stem cells (NSCs) from rabbit retina and brain, and induce differentiation of those NSCs using different culture media. Methods Single-cell suspensions of retina and cerebral cortex were prepared from rabbit embryo, cultured in 5 types of different media to isolate the NSCs by continual passages. After 3 passages, NSCs were induced to differentiation in 2 types of different media for 8 to 10 days. NSCs and inducedretinal cells were examined by immunofluorescence and flow cytometry for the expression pattern of some specific antigens.Results Immunofluorescence showed that NSCs from retina and brain, cultured in serumfree media, both expressed Nestin partially. Flow cytometry showed that Nestin positive cells were significantly decreased while the Rhodopsin and Thy1.1 positive cells were increased after induction. Compared with the combined induction of alltrans retinoid acid (ATRA) and serum, 5%FBS (fetal bovine serum) led to higher expression of Rhodopsin（P＜0.01），but lower expression of Thy1.1（P=0.01）.Conclusion Serumfree media with N2, EGF, bFGF, LIF is the best for NSCs purification. Both induciton media can induce NSCs to differentiate.Retina NSCs have higher potentials to differentiate into retinal neuroepithelial cells than brain NSCs.
Purpose
To investigate the effects of human vitreous fluid on proliferation of cultured human retinal pigment epithelial (RPE) cells and vascular endothelial cell lines(VEC304).
Methods
Human RPE cells and VEC304 were cultured and treated in different human vitreous-conditioned medium (VCM) with or without serum, vitreous volume concentrations of VCM were 1∶8, 1∶4 and 1∶2. Cells proliferation was assayed by tetrazolium (MTT) colorimetry at the 2nd, 4th and 6th day respectively.
Results
In the presence of serum, 1∶4, 1∶2 VCM had a significantly stimulative effect on RPE cells proliferation compared with control group at the 2nd, 4th, and 6th day retrospectively (P＜0.01), but exerted a bly inhibitory effect on VEC304 proliferation compared with control group at the 2nd, 4th, and 6th day retrospectively (Plt;0.05). In the absence of serum, only 1∶2 VCM had a stimulative effect on RPE cells growth compared with control group at the 2nd day (P＜0.05) and obviously at the 4th and 6th day respectively (P＜0.01).
Conclusion
Human vitreous fluid influences human RPE cells and VEC304 growth in vitro. This result suggests that vitreous may play different role in proliferative vitreoretinopathy and intraocular neovascular disease.
(Chin J Ocul Fundus Dis, 2002, 18: 140-142)
Objective To isolate and culture the chondroid cells and notochord cells from New Zealand rabbit immature nucleus pulposus (NP) in monolayer, and to valuate the responsiveness of rabbit disc-derived chondroid cells to notochord cells with respect to cell prol iferation and phenotype. Methods The NP cells were released from the minced immature NP of 6 New Zealand rabbits (4-week-old) by 0.2% collagenase II digestion. The chondroid cells and notochord cells were purified by discontinuous gradient density centrifugation. The chondroid cells were cultured alone (group A) andco-cultured with notochord cells (group B) (1 ∶ 1), and cell prol iferation and phenotype including proteoglycan and collagen II were evaluated. The cells in both groups were observed by the inverted microscope, and the survival rates of the primary and passage cells were detected by toluidine blue staining. The growth curves of the second passage cells in both groups were determined by MTT. Besides, the expressions of proteoglycan and collagen II of the primary and passage cells were examined by toluidine blue and immunocytochemistry staining. Results The notochord cells and chondroid cells were isolated and purified. With the diameter of 10-15 μm, the notochord cell had abundant intracytoplasmic vesicles, while the chondroid cell, with the diameter of 4-6 μm, had no intracytoplasmic vesicle. The cell survival rate was 89.0%-95.3% in group A and 91.3%-96.3% in group B. There was no significant difference between the same passages in both groups (P gt; 0.05). The co-cultured cells (group B) increased in cell prol iferation compared with the chondroid cells alone (group A) in repeated experiments. The cells in group A reached their logarithmic growth phase after 3-4 days of culture, while the cells in group B did after 2 days of culture. The cell prol iferation in group B was more than that in group A after 4-day culture (P lt; 0.05). The cocultured cells retained their phenotype for 5 passages, while parallel-cultured chondroid cells lost the expression of proteoglycan and collagen II after the third passage. Conclusion The notochord cells are conducive for the prol iferation and phenotypekeeping of the chondroid cells and may play a key role in preventing degeneration of the disc.
Objective Dexamethasone is one of the basic agents which could induce osteogenic differentiation of mesenchymal stem cells. To investigate the optimal concentration of dexamethasone in osteogenic differentiation of adiposederivedstem cells (ADSCs) so as to provide the theoretical basis for further bone tissue engineering researches. Methods FiveNew Zealand rabbits (2-3 kg) of clean grade, aged 3 months and male or female, were obtained. ADSCs were isolated from the subcutaneous adipose tissue of inguinal region, and cultured with collagenase digestion, then were detected and identified by CD44, CD106 immunofluorescence staining and adi pogenic differentiation. ADSCs at passage 3 were used and the cell density was adjusted to 1 × 105 cells/mL, then the cells were treated with common cultural medium (group A) and osteogenic induced medium containing 0 (group B), 1 × 10-9 (group C), 1 × 10-8 (group D), 1 × 10-7 (group E), 1 × 10-6 (group F), and 1 × 10-5 mol/ L (group G) dexamethasone, respectively. The cell prol iferation and the mRNA expressions of osteocalcin (OC) and core binding factor α1 (Cbfα1) were detected by MTT and RT-PCR, respectively. The activity of alkal ine phosphatase (ALP) was measured, and the percentage of mineral area was calculated. The mineral nodules were also detected by al izarin red staining. Results ADSCs mostly presented fusiform and polygon shape with positive expression of CD44 and negative expression of CD106. The result of oil red O staining was positive after ADSCs treated with adipogenic induced medium. The result of MTT revealed that the absorbance (A) value decl ined with the ascending of the concentration of dexamethasone, and there was significant difference in A value between groups D and E at 5 and 7 days after osteogenic induction (P lt; 0.05). The mRNA expressions of OC and Cbfα1 reached the peak in groups E and D at 7 days after osteogenic induction, respectively. The activity of ALP and the percentage of mineral area had the maximum value in group D at 14 days, then decl ined gradually. There was no significant difference in the mRNA expressions of OC and Cbfα1, the activity of ALP, and the percentage ofmineral area between groups D and E (P gt; 0.05), but significant differences were found between groups D and E and other groups (P lt; 0.05). After 14 days, the cells of group G died, and the result of al izarin red staining was positive in groups B, C, D, E, and F. Conclusion When the concentration of dexamethasone in osteogenic medium is 1 × 10-8 mol/L, it could not only reduce the inhibitive effect on cells prol iferation, but also induce osteogenic differentiation of ADSCs more efficiently.
ObjectiveTo explore the distribution and features of the optic cup stem cells in embryonic rat at tailbud stage.MethodsThe distribution of optic cup stem cells in optic cup tissue in 12.5-embryonic-day-old rats was observed by immunohistochemistry. The separated cells from optic cup were cultured with serum-free media, and immunofluorescence technique was used to detect the ability of hyperplasia of stem cells and expression of CHX10 antigen and specific antigens of mature retinal cells before and after differentiation.ResultsThe optic cup stem cells in embryonic rat at tailbud stage were mainly located at inner, outer, and marginal layer of optic cup. No expression of specifically marked protein of mature retinal cells was detected. The cells separated from optic cup had the ability of single-cell clone, positive expression of CHX10 and expression of several specific antigens of mature retinal cells after the inducement, including Thy1.1, glial fibrillary acidic protein (GFAP), protein kinase C (PKC) α, and rhodopsin.ConclusionOptic cup of 12.5-embryonic-day-old rats composes of undifferentiated cells, and the stem cells are mainly located in optic cup inner and marginal. High ability of hyperplasia of the optic cup stem cells cultured in vitro is found. The cells, which are retinal stem cells, can express several specifically marked proteins of mature retinal cells after inducement and differentiation.(Chin J Ocul Fundus Dis, 2005,21:159-162)
Objective To study the effects of platelet-rich plasma(PRP) on the proliferation and osteogenetic activity of the marrow mesenchymal stem cells(MSCs) cultured in vitro to elucidate the cellular and molecularmechanism by which PRP accelerates bone repair.Methods The human MSCs were cultured in vitro and randomly divided into the experimental group(n=9) and control group(n=9). In the experimental group, the MSCs were interfused with PRP(10 μl/ml culture media). The proliferation ability of the cells was tested by flow cytometry and MTT, the osteogenetic activity by alkaline phosphatase(ALP) measuring and tetracycline fluorometry, and cbfal mRNA expression by reverse transcriptPCR.Results PRP could stimulate the MSCs proliferation. The flow cytometry assay showed that the MSCs proportion of S period of the experimental group significantly increased 14.5±0.4 in comparison with that of the control group 7.2±0.5 (P＜0.01) after 24 hours. MTT value showed that MSCs proliferatedto platform period earlier in the experimental group than in the control group. There was a significant increase in ALP activity of the experimental group 7.79±1.98,9.51±2.31and 14.03±3.02 when compared with that of the control group 2.06±0.77,2.84±0.82 and 2.58±0.84 after 3, 6 and 9 days(P＜0.05). The number of mineral nodes increased. Reverse transcript-PCR showed that the expression of cbfal mRNA were elevated gradually at 2,4 and 8 hours after interfused with PRP.Conclusion The effect of PRP on accelerating bone repair is related to its effects on stimulating the proliferation of MSCs, increasing the cbfal expression and promoting the osteogenetic activity.
Objective To investigate the feasibility oftissue engineered intervertebral disc for regeneration of discs. Methods A three-dimensional porous poly(L-lactic-co-glycolic acid) (PLGA) scaffold was fabricated by temperature induced phase separation method. Human fetal disc cells were isolated and cultured in vitro. The disc cells labeledwith a PKH-26 fluorescent dye were seeded into a threedimensional porous scaffold. The proliferation of disc cells with PKH-26 fluorescent labels was assessed by using MTT uptake, laser fluorescence microscopy and SEM. Results Human fetal disc cells displayed a polygonal shape in primary monolayer culture. A regular arrangement and microtubules orientationstructure scaffold with 50-300 μm in diameter was fabricated by thermal-induced phase separation technique. MTT uptake and fluorescent microscopy examination indicated that the seeded disc cells were viable and showed proliferation activity within a porous scaffold. Conclusion The above findings support potential applications of tissue engineered disc in treatment of disc degenerative diseases.
