ABSTRACT
BACKGROUND: It has been widely accepted that both bone marrow-derived mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs) have the capacity to differentiate into neural lineages. Some scholars believe that in addition to HSCs and MSCs, bone marrow (BM) also harbors a highly mobile population of CXCR4+ tissue committed stem cells (TCSCs), including skeletal muscles, heart, liver, and neural tissue. OBJECTIVE: To make sure that neural tissue-committed stem cells (NTCSCs) reside in the bone marrow, and to establish a purification and culture method for bone marrow-derived NTCSCs.DESIGN: Opening animal study.SETTING: Department of Anatomy, the Second Military Medical University of Chinese PLA. MATERIALS: Adult Sprague-Dawley (SD) rats (pathogen-free) were provided by the Animal Center of the Second Military Medical University of Chinese PLA. Dulbecco's modified eagle's medium (DMEM)/F12, B27, N2 and epidermal growth factor (EGF, Invitrogen Company), basic fibroblast growth factor (bFGF, CytoLab Ltd), rabbit anti-rat Nestin,CXCR4, β-Tublin Ⅲ, glial fibrillary acidic protein (GFAP, Santa Cruz Company), mouse anti-rat microtubule associated protein 2ab (MAP2ab) (Clone11-5B), cyclic nucleotide 3'phosphohydrolase (CNPase, Clone AP20, NeoMarkers Company), fluorescent(fluorescein isothiocyanate, Cy3) marker reagents (Wuhan Boster Bioengineering Co., Ltd), nuclear fluorescent dyes 4,6-diamidino-2-phenylindole(DAPI)(Sigma), immunohistochemistry reagents (Vector Laboratories Company) , and NycoPrepTM separation liquid (1.077A, Axis-Shield Company) were used in this study.METHODS: This study was performed in the Department of Anatomy, the Second Military Medical University of Chinese PLA from January 2004 to December 2006. Bone marrow was harvested from bilateral femurs and tibias of 2-3 weeks SD rats. Mononuclear cell layer was isolated by NycoPrepTM separation liquid and suspended in DMEM/F12(1:1)serum-free medium supplemented with 2% B27,1% N2, 20 μg/L bFGF, 20μg/L EGF, 1×105 U/L penicillin and 100 mg/L streptomycin. NTCSCs were isolated and propagated by suspensive growing from adherent cells in bone marrow in DMEM/F12 free-serum medium. MAIN OUTCOME MEASURES: NTCSCs were identified by immunocytochemistry for CXCR4, a marker of TCSCs and nestin, a marker of neural stem cells, and neural lineages marker protein after differentiation of cellular spheres. RESULTS: The NTCSCs spheres expressed nestin, a neural stem cell marker as well as CXCR4, a marker of TCSCs. The NTCSCs' spheres were naturally differentiated in DMEM medium with 15% fetal bovine serum. The differentiated cells expressed β-Tublin Ⅲ, MAP2ab, CNPase and GFAP, markers of neural lineages. CONCLUSION: NTCSCs reside in bone marrow and naturally differentiate into neural lineages in vitro.
ABSTRACT
BACKGROUND: It is proved that acupuncture can remarkably promote recovery of nervous function after spinal cord injury (SCI). Previous studies in our groups have been proved that electro-acupuncture can inhibit apoptosis in early period of SCI, but the mechanism is unclear yet.OBJECTIVE: To study the effect of electro-acupuncture on expressions of apoptosis inhibitory gene Bcl-2 mRNA and protein with hybridization in situ and immunohistochemistry and discuss the possible mechanism of apoptosis inhibited by electro-acupuncture in early SCI.DESIGN:Opening animal study.SETTING: Department of Anatomy, the Second Military Medical University of Chinese PLA; Shanghai Acupuncture & Moxibustion and Meridian Research Center.MATERIALS:Adult male SD rats of pathogen-free aged 2-3 months were selected in this study. Bcl-2 hybridization in situ kit was provided by Wuhan Boshide Biotechnology Company Limited and Bcl-2 antibody (1:200) was provided by Santa Cruz Biotechnology Company.METHODS: The experiment was completed at the Laboratory of Anatomy of the Second Military Medical University of Chinese PLA from July 2004 to December 2005. All experimental rats were randomly divided into model group, electro-acupuncture group, methylprednisolone group and sham operation group. T10 spinal cord was injuried by the modified Allen's method and treated with electro-acupuncture immediately, and then the expressions of Bcl-2 mRNA and protein were evaluated with hybridization in situ and immunohistochemistry combined with image quantitative analysis.MAIN OUTCOME MEASURES: ① Expressions of Bcl-2 mRNA and protein in early SCI; ② effect of electro-acupuncture on expressions of Bcl-2 mRNA and protein.RESULTS: The rats were supplied when they died during the experiment,and all 42 rats were involved in the final analysis. ① Moderate expression of Bcl-2 mRNA and protein was observed in the sham operation group.Expression of Bcl-2 mRNA was increased in model group at 6 hours after SCI, but expression ofBcl-2 protein was not changed. At 24 hours after SCI, both expressions of Bcl-2 mRNA and Bcl-2 protein were increased.Expression of Bcl-2 mRNA and protein was higher in electro-acupuncture group than that in mo del group (P ＜ 0.05), and there was no significant difference from that in methylprednisolone group. ② Amount of positive Bcl-2 mRNA cells was increased in electro-acupuncture group and methylprednisolone group at 6 hours after treatment, and gray value was decreased.There was significant and remarkably significant difference from those in sham operation group and model group, respectively (P ＜ 0.05-0.01). After 24-hour treatment, amount of positive Bcl-2 mRNA cells was increased in electro-acupuncture group and gray value was decreased.There was significant and remarkably significant difference from those in sham operation group and model group, respectively (P ＜ 0.05-0.01). ③ After 24-hour treatment, amount of immunohistochenistry positive Bcl-2 cells was increased in electro-acupuncture group and gray value was decreased. There was significant and remarkably significant difference from those in sham operation group and model group, respectively (P ＜ 0.05-0.01).CONCLUSION: Electro-acupuncture can up-regulate the expressions of Bcl-2 mRNA and protein so as to inhibit apoptosis in early SCI.