Objective To establish a better method of isolating andculturing ofneural stem cells(NSCs) in neonatal rat brain. Methods Tissue of brain was isolated from neonatal rats. Different medium and culture concentration were used toculture NSCs of neonatal rat. The culture concentration used were 1×10 4, 1×105, 1×106and 1×107/ml respectively. Ingredient of medium was classified into group 1 to 8 respectively according to whether to add 2% B27, epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) as well as the difference in culture concentration. The cells were induced to differentiate asto be confirmed as NSCs, and then were checked by phase contrast microscopy and identified by immunocytochemistry. Results The cells isolated and cultured gathered into neurospheres. The cells were capable of proliferating and maintaining longterm survival in vitro. The cells could be differentiated into neurons and glia.It was to the benefit of the survival of NSCs to add 5% fetal bovine serum(FBS)into the medium at the beginning of the culturing. When 10% FBS was added intothe medium, the neurospheres differentiated quickly. When concentration 1×106/ ml was used, the growth rate of the cells was the highest of all the concentrations. Reasonably higher cell concentration promoted the proliferation of NSCs. It was necessary to add 2% B27, EGF, and bFGF into the medium. The cells had the best growth when 2% B27, 20 ng/ml bFGF and 20 ng/ml EGF were added into the culture medium. EGF and bFGF had cooperative effect. Conclusion A better method of isolating and culturing of NSCs in neonatal rat brain is established and the foundation for future research is laid.
OBJECTIVE: From the point of view of material science, the methods of tissue repair and defect reconstruct were discussed, including mesenchymal stem cells (MSCs), growth factors, gene therapy and tissue engineered tissue. METHODS: The advances in tissue engineering technologies were introduced based on the recent literature. RESULTS: Tissue engineering should solve the design and preparation of molecular scaffold, tissue vascularization and dynamic culture of cell on the scaffolds in vitro. CONCLUSION: Biomaterials play an important role in the tissue engineering. They can be used as the matrices of MSCs, the delivery carrier of growth factor, the culture scaffold of cell in bioreactors and delivery carrier of gene encoding growth factors.
Objective To investigate the expression and significance of growth-associated protein 43 (GAP-43) in the dorsal root ganglion (DRG) and intervertebral disc in the rat model of intervertebral disc inflammation. Methods A total of 103 adult male Sprague Dawley rats (weighing, 200-250 g) were randomly divided into the experimental group (n=48), the control group (n=48), and the blank control group (n=7). Fluoro-gold (F-G) as tracer was injected into the L5, 6 intervertebral disc of 3 groups; after 7 days of F-G injection, complete Freund’s adjuvant (50 µL) and the same volume of saline were injected in the experimental group (to prepare the model of intervertebral disc inflammation) and the control group, respectively, and the blank control group had no further treatment. After 1, 3, 7, and 14 days, T13-L6 DRG and L5, 6 intervertebral disc of experimental group and control group were harvested to detect the GAP-43 by using fluorescent immunohistochemistry, in situ hybridization, and RT-PCR. The DRG and intervertebral disc of blank control group were also harvested after 8 days of F-G injection. Results Fluorescent immunohistochemistry results showed that the number of F-G-labeled GAP-43 immunoreaction (GAP-43-IR) cells of the DRGs in the experimental group was significantly higher than that in the control group (P lt; 0.05) at 3 days, and no significant difference was found at the other time points (P gt; 0.05). There was no significant difference in the cross-sectional area of F-G-labeled GAP-43-IR cells between the experimental group and the control group at each time point (P gt; 0.05). The co-expression of GAP-43 with calcitonin gene-related peptide (CGRP) and isolectin B4 (IB4)-binding glycoprotein exhibited that the expression of CGRP was 91.4% ± 7.4% in the control group and was 87.6% ± 7.8% in the experimental group, showing no significant difference between 2 groups (P gt; 0.05). There was no IB4-binding glycoprotein expression in GAP-43-IR cells of the DRGs in 2 groups. The expressions of GAP-43, CGRP, and IB4-positive nerve fibers in the intervertebral disc exhibited that the GAP-43-IR nerve fibers in the experimental group were significantly more than that in the control group (P lt; 0.05), but no significant difference was found in the expression of CGRP between 2 groups (P gt; 0.05); and there was no IB4-binding glycoprotein expression in GAP-43-IR nerve fibers of the intervertebral disc in 2 group. In situ hybridization and RT-PCR detection showed that the positive expression cells ratio of GAP-43 mRNA and the level of GAP- 43 mRNA were significantly higher in the experimental group than in the control group at 1 day (P lt; 0.05), and no significant difference was found at the other time points (P gt; 0.05). Conclusion Intradiscal inflammatory environment may induce the expression of GAP-43, and potentially promote the nerve fiber ingrowth of rat.
Objective To review the recent progress of the researches in the field of cartilage tissue engineering, and to discuss the challenges in construction of tissue engineered cartilage. Methods Literature related with cartilage tissue engineering was reviewed and analyzed. Results Some techniques have been appl ied in cl inical. As far as the seeding cells, induced pluripotent stem cells have attracted much more attention. Current strategies of scaffold designing are trying to imitate both component and structure of natural extracellular matrix. Cartilage regeneration through the autologous cell homing technique el iminate the transplantation of exotic cells and has become the hot topic. Conclusion Successful treatment of the damaged cartilage using tissue engineering method will depend on the advances of stem cell technology development, biomimetic scaffolds fabrication and proper appl ication of growth factors.
Objective To investigate the effect of acid, basic fibroblast growth factor (aFGF, bFGF) and epidermal growth factor (EGF), andtheir combination on the proliferation of rabbit anterior cruciate ligament (ACL) and medial collateral ligament (MCL) in vitro. Methods Thecells of ACL and MCL were isolated and subcultured from the knee joints of tenweek-old New Zealand white rabbits. The cells were seeded into 96-well corning cluster plates. Three growth factors of different concentration alone or in combination were added into the culture medium respectively, which were 0, 1, 5, 10, 50 and 100 ng/ml for aFGF, bFGF and 0, 1.56, 3.13, 6.25, 12.5, 25 and 50 ng/ml for EGF. The proliferation of the fibroblasts was measured for 48 h with XTT method. Results All of the three growth factors alone promoted the cell proliferation of ACL and MCL fibroblasts. The concentration of aFGF hada significant effect on the proliferation of both ACL and MCL fibroblasts. The concentration of 1 ng/ml bFGF and 5 ng/ml EGF was most effective in promoting the proliferation of ACL, and both bFGF and EGF had a significant effect on MCL. 5ng/ml aFGF with 50 ng/ml EGF had effect on ACL. 1 ng/ml aFGF with 3.13 ng/ml EGF had effect on MCL. Conclusion The three growth factors may promote the cell proliferation of ACL and MCL. These findings suggest that topical application of aFGF, either alone or in combination with EGF may have the potential to promote the proliferation of rabbit ACL and MCL,and aFGF of low concentration in combination with EGF is more effective than single growth factor.
Objective To explore the effects of recombinant human growth hormone （rhGH） on senile patients after pancreaticoduodenectomy. MethodsFortysix patients were divided into the therapeutic group (rhGH, n=17) and control group (n=29). Both were treated with parenteral nutrition. In the therapeutic group, rhGH (8 u/d) was given hypodermically for 7 days. After operation the levels of albumin, prealbumin, transferrin, and immunoglobulin were measured. Postoperative fatigue syndrome and the average length of stay in hospital were observed too. ResultsAfter operation the levels of albumin, prealbumin, transferrin, and immunoglobulin in the therapeutic group were significantly higher than those of control group. The degree of postoperative fatigue syndrome in the therapeutic group was less than that of control group. The average length of stay in hospital was significantly shortened. Conclusion The early application of rhGH in senile patients after pancreaticoduodenectomy can enhance immune function, reduce the incidence of infection, promote the postoperative recovery, shorten the average length of stay in hospital,decrease the mortality, increase the safety of operation and improve the postoperative life quality of senile patients.
OBJECTIVE :To investigale effect of subretinal fluld(SRF)on proliferalion of the cellular elements of PVR. METHOD:The effect of SRF of 28 patients with rhegmatogenous retinal detachment proliferation of the cultured human retinal pigment epithelial cells(RPE),retinal glial cells (RG),and fibroblast (FB)was observed and detected by the methods of cell-counting and 3H-TdR in DNA synthesis. RESULTS:The range of proliferatinn-stimulating activity was 52.5%~233.3%, 36.4% ~ 177.8%，55.4% ~277.8% above the baseline in 1:10 dilution of these 3 kinds ,of cellular elements,and there was no significant difference among them. CONCLUSION ;The stimulating effect of SRF on the cellular proliferation was thougt to be due to the actions from certain growth factors. （Chin J Ocul Fundus Dis,1996,12: 233-235）
Objective To study the effect of transforming growth factor β1(TGF-β1) and insulin-like growth factor 1(IGF-1) during the induction course from marrow mesenchymal stem cells (MSCs) to chondrocytes and to observe the effect of cell density on cell induction. Methods Differential time adherent methods were used to purify MSCs obtained from the bone marrow of Kunming mice. MSCs were cultured under special conditionsto induce themto differentiate into chondrocytes. Toluidine blue staining and immunofluoresence were used to identify those induced chondrocytes.TGF-β1 and IGF-1 were used individually or in combination under two different culture patterns: pellet culture and monolayer culture. According to different growth factors, experiment included 3 experimental groups(TGF-β1+IGF-1 group,10 ng/mland 50 ng/ml respectively;TGF-β1 group, 10 ng/ml; and IGF-1 group, 50 ng/ml) and control group(without growth factor). In TGF-β1+IGF-1 group, toluidine blue staining and immunofluoresence staining were carried out at 14 days and 21 days. The effect ofTGF-β1 and IGF-1 on the expression of collagen Ⅱgene was detected by RT-PCR at 7, 14 and 21 days of induction; the expressionsof collagen Ⅱ were compared between two culture patterns. Results In TGF-β1+IGF-1 group, the histological examination and immunofluoresence showed that those inducted chondyocytes could express collagen Ⅱ at 14 days. The gel electrophoresis results showed that the fragment of collagen Ⅱ gene was seen in TGF-β1+IGF-1 group andTGF-β1 group and that no fragment ofcollagen Ⅱ gene was seen in IGF-1 group and control group. The expression of collagen Ⅱ gene was ber in TGF-β1+ IGF-1 group than inTGF-β1 group, showing significant difference(Plt;0.05). Cells expressed more collagen Ⅱ under pellet culture than under monolayer culture. Conclusion IGF-1 could enhance the effect ofTGF-β1 during the induction course from MSCs to chondrocytes. A certain extent of high cell density is more effective for MSCs to differentiate into chondrocytes.
Objective To investigate the effect of growth differentiation factor 7 (GDF-7) on the tenogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in vitro, to provide evidence for improving the efficacy of BMSCs on tendon repair. Methods BMSCs were isolated from bone marrow tissue of green fluorescent protein rats by density gradient centrifugation method. Chondrogenic, osteogenic, and adipogenic differentiation assays were used to demonstrate the multi-differentiation potential of the BMSCs. BMSCs at passage 3 were cultured and divided into 4 groups according to different concentrations of GDF-7 (0, 12.5, 25.0, and 50.0 ng/mL): group A, B, C, and D, respectively. After cultured for 2 weeks in vitro, the mRNA expressions of scleraxis, tenomodulin, tenascin C, and collagen type I were detected by real-time fluorescent quantitative PCR method, the protein expressions of tenomodulin, tenascin C, and collagen type I by immunocytochemistry staining in 4 groups, and the protein expressions of tenomodulin by Western blot in groups A and C. Results BMSCs had osteogenic, chondrogenic, and adipogenic differentiation potentials. The mRNA expressions of tenomodulin in groups B, C, and D were 2.85, 3.41, and 3.07 times higher than that in group A, respectively; the mRNA expressions of scleraxis in groups B, C, and D were 2.13, 1.50, and 2.56 times higher than that in group A, respectively; and the mRNA expressions of tenascin C in groups B, C, and D were 2.45, 2.86, and 1.88 times higher than that in group A, respectively. There were significant differences between groups B, C, D and group A (P lt; 0.05), while there was no significant difference among groups B, C, and D (P gt; 0.05). The mRNA expressions of collagen type I in groups B and C were 1.92 and 2.45 times higher than that in group A, showing significant differences between groups B, C and group A (P lt; 0.05), but no significant difference between groups A and D (P gt; 0.05). Immunocytochemistry staining showed that the protein expressions of tenomodulin, tenascin C, and collagen type I were detected in groups B, C, and D but not in group A. The results were further confirmed by Western blot results which showed higher protein expression of tenomodulin in group C than in group A. Conclusion GDF-7 can be used to promote tenogenic differentiation of rat BMSCs in vitro.
OBJECTIVE To prevent early closure of growth plate and developmental deformities of limbs by allografts of cultured cartilages into growth plate defects of rabbits. METHODS Chondrocytes isolated from articular cartilage of 1-month rabbits formed cartilage after cultivation in centrifuge tubes. The cartilages cultured for two weeks were implanted into growth plate defects of proximal tibiae of 6-weeks rabbits. At 4th and 16th weeks, X-ray, histologic and immunohistochemical examination were performed. RESULTS The tibiae had no marked deformities after 4 weeks of operation. Histologic examinations showed that the defects were filled with cartilage. Immunohistochemical results of type II collagen were positive. The tibiae with allografts of cultured cartilages had no evident deformities after 16 weeks of operation. Histologic examination showed nearly closure of growth plates. On the contrary, the tibiae on control side formed severe deformities and growth plate were closed. CONCLUSION Allograft of cultured cartilages into growth plate defects may replace lost growth plate tissues, maintain normal growth of limbs and prevent developmental deformity.