Abstract An experiment was carried out to investigate the possibility of the establishment of an osteoblasts bank which could supply osteoblasts in repairing bone defect. Osteoblasts were isolated from thetibial periosteum of eight New-Zealand rabbits and cultured in votro. A bone defect, 1.5cm in length was made in both radii of each of the 8 rabbits. The cultivated osteoblasts, gelfoam as a carrier were randomly implanted into the defects of the radii of rabbits. Accordingly, the contralateral radial defects wereimplanted with gelfoam absorbed with the Hanks solution as control. The healing of bone defects was evaluated by roentgenographic examination at 2, 4, 8 and 12 weeks after operation, respectively. It was shown that the implanted cells had osteogenetic capability and could be possible to promote healing of the bone defects. It was suggested that further study needed to be carried out in this field.
Objective To clarify the relationship between inhibition of proliferation and cxpression of Ki-67 in cultured human retinal pigment epithelial(RPE) cells. Methods The cultured human RPE cells were treated with daunoblastina at a dose of 180 mu;g/L for 12h.Twenty-four hours later,DNA inhibiting rate was studied by using tritium-labelled thymidine deoxyribose(3H-TdR)incorporation assay.The expression of Ki-67 was evaluated by immunocytochemical staining technique and image analysis system.Flow cytometry was used to analyse cell cycle. Results DNA inhibiting rate was directly proportional to the dosage of daunoblastina.The proportion of the cells positive staining to Ki-67 in the control and the daunoblastina-treated group were 89.3% and 45.6%(Plt;0. 01),and the integral optical density values for expression of Ki-67 in the two groups were 68.1plusmn;6.2 and 27.3plusmn;5.5(Plt;0.01),respectively.The percen tage of cells in G2 phase of cell cycle increased from 8.9% to 29.5%. Conclusion G2 block was induced and poliferation was inhibited by daunoblastina in cultured human RPE cells.There is a relatively good correlation between Ki-67 immunostaining and inhibition of RPE cell proliferation. (Chin J Ocul Fundus Dis，2000，16：1-70)
Objective To discuss the strategies for building the framework of team culture of multi-disciplinary team (MDT) for colorectal cancer. Methods By comprehending the traditional concept of volunteer and probing into the value of traditional team culture, combining the needs of MDT for colorectal cancer, build appropriate team culture and core idea of MDT for colorectal cancer. Results Confirm that building of volunteers groups and the volunteers culture is the core of the team culture of MDT for colorectal cancer. Analyze characters of volunteers groups and the operation strategies, and find the way of maintaining the volunteers culture. Conclusion With the development of volunteers groups and increased participants, the team culture of MDT for colorectal cancer will show more sociality and extent. And it is also the important idea and direction for development in future. As team culture, organization structure and personnel structure supplements each other, adjusting and perfecting the team culture in practice continually is a long-term work for MDT.
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 research the methods and techniques of SD rat bone marrow stromal cells （MSCs）culture in vitro and to provide a large number of MSCs for cell therapy. Methods Bone marrow from the femur and tibia of the early age SD rats was taken to culture to the passage 1-4 （P1-P4）, its growth was observed and P3 cells were evaluated by HE staining and immunohistochemisty. Results The growing speed of P1-P3 was faster than that of P0, cells fusion was 85%-90% after 3-4 d and the cells were arranged in groups liked whirlpool shape or parallel; （3.4-3.6）×104/cm2 cells were gained and the total cell number of P1-P3 was 4.08×106, 2.44×107 and 2.85×108 respectively, the rate of trypan blue rejecting stained was 95%-97%. P4’s growing speed was slower than before, 1.42×109 in 3.0×104/cm2 cells were gained, and the rate of rejecting stained was 95%. P4-cell output was amplified nearly 2 000-fold higher than P0-cell. P3 immunohistochemical analysis indicated CD105+ cells 61.9%， CD44+ 45.4%， CD29+ 16.8%， CD45+ 8.2%， CD31+ 13.7%， CD34+ 8.3% and CD11b+ 1.5%, respectively. Conclusion The culture of whole bone marrow is suitable for a large number of MSCs provision in vitro, and can meet the needs of the cell therapy research.
In thiis study,we show thai carbachol stimulates the accumulation of inositol phosphates(InsPs)in human rellnal pigment epithelium (RPE)cells and atropine blocks the carbachol-induced effect ,suggesting the existence of musearinie acelyleholine receptors in human RPE cells. In contrast,noradrenaline,serotonin, cpidermal growth factor (EGF),isoproterenol,and NECA (5'-[N-ethyl]-carboxamido-adenosine)do not influence the basal levels of InsPs.Moreover,isoprmerenol and NECA do not affect the carhaehol elevated levels of InsPs.EGF,howcvcr,does potentiate the carhaehol stimulated elevation of InsPs in a dose-dependent manner ,suggesting an interaction between EGF and musearinie receptors in cultured human RPE cells. （Chin J Ocul Fundus Dis,1994,10:220-222）
OBJECTIVE: To construct a tissue engineering skin containing melanocytes by employing tissue engineering method. METHODS: The keratinocytes, dermal fibroblasts and melanocytes were isolated and purified. Then the cells were used to construct a tissue engineering skin containing melanocytes. The location of melanocytes in the tissue engineering skin were detected by Dopa staining, transmission electron microscope (TEM) and S-100 immunohistochemical staining. RESULTS: Melanocytes can be detected in the basal layer of the constructed tissue engineering skin. The results of TEM showed that the melanocytes were in good conditions. CONCLUSION: The artificial skin containing melanocytes was successfully constructed in vitro and can be used to repair the full-thickness skin defects.
Objective To investigate the impact of photodynamic therapy (PDT) with verteporfin on the expression of pigment epithelial derivative factor (PEDF) mRNA and vascular endothelial growth factor (VEGF) mRNA in adult retinal pigment epithelial (RPE) cells in vitro. Methods The changes of cellular viability before and after PDT were assessed by methyl thiazolyl tetrazolum (MTT) colorimetric assay. Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) was conducted to detect the expression of PEDF and VEGF mRNA in RPE cells before and after PDT. Results PDT caused the death of RPE cells. The cellular mortality was positively correlated with the power of photocoagulation and the concentration of verteporfin. Conclusion PDT could downregulate the expression of PEDF and VEGF mRNA in adult RPE cells in vitro, which may relate to the cure or relapse of subfoveal choroidal neovascular membrane after PDT. (Chin J Ocul Fundus Dis, 2006, 22: 256-260)
Objective To investigate the expression of Human leucocyte antigen(HLA)-DP, -DQ, -DR and CD40 in human retinal pigment epithelial (RPE) cells, to determine their molecule expression in immune response process, and their abilities to stimulate T lymphocyte activation. Methods Human RPE cells were cultured with or without (IFN respectively. Expression of HLA-DP, -DQ, -DR and CD40 was measured by immunohistochemical staining. Meanwhile, peripheral blood mononuclear cells (PBMC) were cocultured with RPE cells in vitro, and then the expression of activated lymphocytes CD69 was measured by fluorescence activated cell sorter(FACS). Results Expression of HLA-DP, -DQ, -DR and CD40 antigen were enhanced by gamma;-interferon inducement. Increasing amount of CD69 positive lymphocytes were found in the co-culture system of RPE cells and PBMC. Conclusion T-lymphocytes in the peripheral blood were activated by human RPE cells which is antigen presenting cells with immunological characteristics potential.
Objective To observe the expression of integrin αVβ3 in vascular endothelium cultured in vitro at different time points under different level of shear stress. Methods（1）We established a vascular culture system in vitro which could provide steady flow with different level of shear stress, and tested the flow stability when loading different level of shear stress. （2） A total of 50 rabbits were randomly divided into low shear stress group （5 dyn/cm2, n=25）and normal shear stress group（20 dyn/cm2, n=25）. Rabbits in each group were further randomly divided into five different time points as 2 h, 4 h, 8 h, 16 h and 24 h（n=5 at each time point）. The descending aorta of rabbits were harvested and cultured in the vascular culture system in vitro under different level of shear stress. The expression sites and intensity of αVβ3-Integrin in vascular endothelium were examined at 5 different time points in both groups by immunohistochemical staining. Results The vascular culture system in vitro was stable in providing laminar flow with different level of shear stress required for the experiment. Vascular endothelium expressions of αVβ3-Integrin in the low shear stress group were in high level at all the 5 time points and reached its summit at 16 h, when the mean optical density（MOD）value was （1.995±0.194）×10-2. In the normal shear stress group, the MOD value decreased time-dependently at the 5 time points. The MOD values at 2 h （0.059±0.005）×10-2 and 4 h（0. 049±0.002）×10-2 were significantly higher than those at other time points （P＜ 0.05）. The αVβ3-Integrin MOD values of the low shear stress group were significantly higher than those of the normal shear stress group at all the 5 respective time points （P=0.000）. Conclusion Low shear stress can significantly promote the expression of αVβ3-Integrin while normal shear stress decreases the expression of αVβ3-Integrin in vascular endothelium cultured in vitro.