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BACKGROUND: The mitochondrial apoptotic pathway is an important pathway in cell apoptosis. Previous studies have found that Bushen Zhuangdu Fang can improve intervertebral disc degeneration by inhibiting the mitochondrial apoptotic pathway in animal experiments. However, its mechanism of action is to be clarified. OBJECTIVE: To observe the effect of serum containing Bushen Zhuangdu Fang on mitochondrial apoptotic pathway key proteins of human nucleus pulposus cells, and to explore the mechanism by which this drug-containing serum improves intervertebral disc degeneration. METHODS: Thirty-seven male Sprague-Dawley rats were randomly divided into blank group, low-dose Chinese medicine group (0.506 g/kg per day), medium-dose Chinese medicine group (1.012 g/kg per day) and high-dose Chinese medicine group (2.024 g/kg per day). After 2 weeks of continuous administration, drug-containing serum was prepared. Human nucleus pulposus cells were randomly divided into normal group, cell model group, low-dose drug-containing serum group, medium-dose drug-containing serum group, and high-dose drug-containing serum group. The cell model group was treated with 200 μmol/L H2O2 for 6 hours, and the normal group received no treatment. The three drug-containing serum groups were treated with corresponding treatments for 48 hours. The pathological changes of nucleus pulposus cells were observed by transmission electron microscopy. Apoptotic rate of nucleus pulposus cells was detected by flow cytometry and mitochondrial membrane potential was detected by flow cytometry. Apaf1, Bcl-2, Bax and Cytc expressions were detected by real-time quantitative PCR and western blot assay. RESULTS AND CONCLUSION: Compared with the normal group, the apoptotic rate of nucleus pulposus cells with obvious apoptotic morphology was significantly increased (P < 0.05), the expression of Apaf1, Cytc, and Bax were significantly increased at mRNA and protein levels (P < 0.05), and the mitochondrial membrane potential and expression of Bcl-2 mRNA and protein were significantly decreased in the cell model group (P < 0.05). After treatment with drug-containing serum, the apoptotic rate of nucleus pulposus cells decreased significantly (P < 0.05), the expression of Apaf1, Cytc, Bax and their proteins decreased significantly (P < 0.05), and the mitochondrial membrane potential, Bcl-2 and their proteins increased significantly (P < 0.05). Therefore, the serum containing Bushen Zhuangdu Fang can effectively inhibit apoptosis of nucleus pulposus cells in a dose-dependent manner. The drug-containing serum may alleviate intervertebral disc degeneration by reducing the expression of Apaf1, Cytc and Bax and increasing the expression of Bcl-2 at protein and gene levels, and inhibiting the mitochondrial apoptotic pathway.
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Objective To investigate the effect of direct intercell contact on the bone mesenchymal stem cells(MSCs)differentiate into nucleus pulposus cells(NPs)when cocultured with NPs in constant magnetic field.Methods The primary NPs labeled by DAP1 were cocultured with the 3 rd generation of MSCs through direct and indirect intercell contact in the presence or absence of constant magnetic field(0.05,0.10,0.50 and 1.00 mT,respectively). Observation of morphological changes was performed every 24 hours.The method of MTT was employed to evaluate the level of proliferation.The gene expression of collagen Ⅱ,Sox-9 and Aggrecan was measured by using RT-PCR. Results MSCs cocultured with direct intercell contact with the NPs rounded up and presented a round ring-like structure appearance.The expression of marker genes including Collagen type Ⅱ,Aggrecan and Sox-9 were significantly increased when cells cocultured in constant magnetic field of 0.05 mT compared with those without constant magnetic field(P＜0.05).There were no significantly changes with regard to the expression of the above genes in 0.10 mT field(P＞0.05).The growth of NP-like cells was suppressed when the intensity of magnetic field was higher than 0.10 mT(P＜0.05).Conclusion It is suggested that 0.05 mT constant magnetic field and direct intercell contact facilitate differentiation of MSCs into NPs.
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@#Objective To explore the changes of rabbit adipose stem cells(ASCs)and bone mesenchymal stem cells(BMSCs)cultured in vitro and the anabolism under basic fibroblast growth factor(bFGF).Methods BMSCs and ASCs were cultured with DMEM,DMEM/F12(2∶1)or α-MEM respectively.The 3rd generation ASCs and BMSCs were divided into 2 groups respectively:group A:ASCs cultured in chondrogenic medium(CM),group B:ASCs cultured in CM supplemented with bFGF 5 ng/ml,group C:BMSCs cultured in CM,group D:BMSCs cultured in CM supplemented with bFGF 5 ng/ml.Morphological changes were observed under inverted microscope.The 35SO42-incorporation and total hydroxyproline were measured.Results BMSCs and ASCs showed much higher growth rate when cultured in α-MEM medium comparison with that in DMEM or in DMEM/F12(2:1).Both stem cells attachment cultured in monolayer greatly increased and cell clones were abundant,while the cells attachment became rather difficult and cell clones were less after cutured in CM.All stem cells possessed a round-like morphology,and the cells in group B and D were more than that in the other 2 groups.The 35SO42-incorporation and total hydroxyproline synthesis of group B or D increased compared with that of group A or C,but there was no diference between group D and B.Conclusion The rabbit ASCs and BMSCs cultured in CM suppling with bFGF grow well and their metabolism increased.
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[Objective]To evaluate whether transplanted marrow mesenchymal stem cells interfered in TGF-?1 can differentiate to nucleus pulposus cells and increase the amount of proteoglycan and collagenase Ⅱ content in intervertebral discs.[Method]We used an in vivo model to investigate the feasibility of marrow mesenchymal stem cells that cultured in vitro and interfered in TGF-?1 delivery,retention,and survival in the degeneratived disc space.In 2,4,6,8 weeks we used immunohistochemical staining to determine the change of collagenase Ⅱ content;spectrophotometry to determine the change of amount of proteoglycan with Phlorglucinol;the experiment date were analyzed by SPSS 11.5 soft ware.[Result]We found MSCs could maintain viability and proliferate within the rabbit inter vertebral disc.The amount of proteoglycan and collagen Type Ⅱ content of the intervertebral in matrix synthesis in the experiment group was increased in 8 weeks.We found no changes in the modle group.[Conclusion]Our data suggest that transplanted marrow mesenchymal stem cells in vivo can survive and increase proteoglycan and collagen Type Ⅱ amount interfered in TGF-?1 in some periods,which support its potential use as a treatment of intervertebral disc degeneration.
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STUDY DESIGN: In-vitro experimental study. OBJECTIVES: To determine the proteoglycan synthesis of the rabbit nucleus pulposus cells in various concentration of extracellular collagen type I and II under the stimulation of TGF-beta1. SUMMARY OF LITERATURE REVIEW: Therapeutic effect of growth factor and gene therapy can be altered by composition of extracellular matrix. However, the effect of extracellular collagen types I and II on synthetic activity of intervertebral disc cells is not thoroughly studied before. MATERIALS AND METHODS: The nucleus pulposus cells were isolated and cultured from 10 skeletally mature rabbits. Cultures were trypsinized and incorporated into alginate beads with different concentration of extracellular collagen type I and II (0.5%, 1.0% and 1.5%). Those cultures with TGF-beta1 (10 ng/ml) served stimulated condition of matrix synthesis. Newly synthesized proteoglycans were assessed by 35 S-sulfate incorporation using chromatography on Sephadex G-25 in PD-10 columns. Scintillation count was normalized with DNA content by Hoechst dye method. RESULTS: In basal condition, difference in proteoglycan synthesis in given concentration of extracellular collagen type I and II were statistically insignificant. In stimulated condition with TGF-beta1, difference in proteoglycan synthesis in given concentration of extracellular collagen type I and II was also statistically insignificant. However, cultures in stimulated condition with TGF-beta1 showed increased amount of newly synthesized proteoglycans compared to those of basal condition regardless of the concentration of extracellular collagen type I and II (p < 0.05). CONCLUSION: Anabolic response of rabbit nucleus pulposus cells is relatively insensitive to extracellular matrix composition, which facilitates application of gene therapy in various conditions of disc degeneration.
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Conejos , Cromatografía , Colágeno Tipo I , Colágeno , ADN , Matriz Extracelular , Terapia Genética , Degeneración del Disco Intervertebral , Disco Intervertebral , Proteoglicanos , Factor de Crecimiento Transformador beta1 , TripsinaRESUMEN
Objective To compare the cellular characteristics of the nucleus pulposus(NP) cells of young and adult rabbits.Methods The structure and arrangement of the NP cells from young and adult rabbits were investigated on both tissue and ultrastructure level by confocal laser scanning microscope and transmission electron microscope. Results The NP cells from the young rabbit discs,which were round and contained numerous large inclusion bodies,often formed clusters with a diameter of(266?167)??m and a density of(8.0?2.4) per high power(?40);The NP cells from the adult rabbit discs were round or oval shape and had no large size inclusion bodies,formed less clusters or were singly diffusedly distributed,with the diameter of clusters to be(94?42)??m(single cell excluded) and density to be(14.9?4.3) per high power(?40).Conclusion There were remarkable morphological differences in the cell architecture of nuclei pulposi between the young and adult rebbits.These features of the aging and degenerating disc contribute to decreased biological function.
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Objective To explore morphologic characterizatics of cellular processes from adult human nucleus pulposus cells. Methods The nucleus pulposus of adult human intervertebral disc were obtained from 8 patients(Thompson's grade Ⅰ-Ⅱ) and then the tissues specimens were carried out by frozen section and electron microscopic section as well as cell isolation and cultured,processes of nucleus pulposus cells were examined using light microscopy,laser scanning confocal microscopy and transmission electron microscopy. Results When examined at both the confocal and electron microscope level,all the cells possessed the processes and adjacent nucleus pulposus cells processes possessed a gap junction.The elongated and round cells were examined when NP cells became monolayer in vitro.The rate of elongated cells to round cells was 2.3 to 1.The elongated cells protruded along with the long axis of cell body without second processes.Dendritic processes of round cells protruded to all directions from the cell body with multiple-level processes.Conclusion Processes are one of the morphologic characteristics of intervertebral disc cells.The research on processes functions could be helpful to understand pathomechanism of intervertebral disc degradation.