| Objective: Through human osteoblasts and osteoclasts in vitro culture system, weexplored the possible mechanism of recombinant mycobacterium tuberculosis heat shockprotein10（r-Mtcpn10）affecting bone metabolism balance in osteoarticular tuberculosiswith the aim to give an insight in biological mechanism of targeted gene therapy forskeleton tuberculosis. Methods:1) Osteoblasts were isolated from iliac spongy boneusing enzyme digestion, and the characteristics of morphology and activity of thoseosteoblasts were evaluated according to the traditional criterias. Monocytes were isolatedby means of density gradient centrifugation from peripheral blood and planted on thecover glass or bone slices with culture medium. TRAP-positive cells containing morethan three nuclei were counted as osteoclast-1ike cells．The cells and resorption pits onthe bone slices were identified with toluidine blue stain and electrical microscope scan．2)Human Osteoblasts were cultured with various concentrations of r-Mt cpn10(0.1,1,10mg/L). Osteoblasts cultured without r-Mt cpn10were assigned as control group.Parameters including osteoblast proliferation, cell cycle, alkaline phosphatase activityand calcium nodule formation was assessed as usual. And the expression of OPG,RANKL, OPG／RANKL mRNA and their protein activity was detected usingRealtime-PCR and Western-blot, respectively;3)Human monocytes were cultured withvarious concentrations of r-Mt cpn10(0.01,0.1,1mg/L) with/without40ng/mL RANKLto observe osteocalst formation. Mononudeated cells cultured without r-Mt cpn10andRANKL were assigned as controls. The respectively osteoclast and lacunae formation,calcium concentration, tumor necrosis factor, the expression of NFATc1mRNA, c-FosmRNA and protein activity was detected by Realtime-PCR and Western-blot.4) Similaly, human monocytes and osteoblast were co-cultured with1mg/L concentrations of r-Mtcpn10to observe osteocalst formation. and the parameters including osteoclast andlacunae formation, calcium concentration, the expression of NFATc1mRNA, c-FosmRNA and OPG mRNA, RANKL mRNA, OPG／RANKL mRNA was evaluated as well.Results:1) Calcium nodules in osteoblast culture were observed after25days. And thelevels of ALP were gradually increased to peak at the10th days and proved by ALPstaining. At the21th days, the cells cultured with RANKL and M-CSF was observed withTRAP-positive expression．Bone absorb pits were found in the bone slice in theinvestigation of toluidine blue stain and electrical microscope scan, polynuclearmacrophage with pseudopodium were found in electric microscope scan．In control groupwe did not found the same result．2) MTT results showed, cell proliferation decreased45.3%(48h, P＜0.05),68.2%(72h, P＜0.05) in10μg/mL group,32.6%(48h, P＜0.05),56.7%(72h, P＜0.05) in1μg/mL group,25.3%(48h, P＜0.05),38.7%(72h, P＜0.05) in1μg/mL group. In cell cycle assay, G1-phase increased8.12%(24h, P＜0.05),22.77%(48h, P＜0.05), G2-phase increased5.58%(24h, P＜0.05),194%(48h, P＜0.05),S-phase decreased71.6%(24h, P＜0.05),89.01%(48h, P＜0.05), PI decreased29.8%(24h, P＜0.05),44.92%(48h, P＜0.05), S-phase decreased17.41%(24h,48h, P＜0.05),PI decreased15.15%(24h,48h, P＜0.05).The level of ALP assay, decreased6.33±1.67(7d, P＜0.05),6.97±1.40(10d, P＜0.05) in10μg/mL group, decreased4.95±1.57(7d,P＜0.05),5.68±1.23(10d, P＜0.05) in1μg/mL group. Color and area of calciumnodules obviously decreased in10μg/mL group. Realtime PCR results indicated, OPGmRNA decreased41.6%(48h, P＜0.05),52.6%(72h, P＜0.05) in10μg/mL group,(48h,P＜0.05),48.3%(72h, P＜0.05) in1μg/mL group,9.3%(48h, P＞0.05),26.7%(72h, P＜0.05) in0.1μg/mL group; RANKL mRNA increased98.2%(48h, P＜0.05),124%(72h, P＜0.05) in10μg/mL group,(48h, P＜0.05),67.3%(72h, P＜0.05) in1μg/mLgroup,(45.4%(72h, P＜0.05) in1μg/mL group; Western-blot result indicated, OPGprotein expression decreased and RANKL protein expression increased in10μg/mLgroup.3) Monocytes were cultured with various concentrations of r-Mt cpn10, ELISAdetection: r-Mtcpn10did not induced TNF-α production. TRAP-positive osteoclast andbone absorb pits were found in RANKL+r-Mtcpn10group and r-Mtcpn10group, Incontrol group we did not found the same result. Count result showed: the number ofTRAP-positive osteoclast, the number and area of bone absorb pits increased and calciumconcentration increased (P＜0.05); Realtime PCR results indicated NFATc1mRNA andc-Fos mRNA increased in various group with time and r-Mtcpn10concentration (P＜ 0.05). Western-blot result indicated, NFATc1protein expression increased and c-Fosprotein expression decreased with time in1μg/mL group.4) In osteoblast and monocytesco-cultured TRAP-positive osteoclast and bone absorb pits were found in1μg/mLr-Mtcpn10group and control group. Count result showed: the number of TRAP-positiveosteoclast, the number and area of bone absorb pits increased and calcium concentrationincreased (P＜0.05); Realtime PCR results indicated, NFATc1mRNA, c-Fos mRNA,RANKL mRNA, OPG mRNA expression respectively were3.878±0.5748,0.4192±0.1030,2.832±0.3075,1.135±0.0457in r-Mt cpn10group, repectively. Theresult of the control group were lower than cpn10group, which was statisticallysignificant (P＜0.05) in addition to the c-Fos mRNA expression. Conclusion:1)Recombinant mycobacterium tuberculosis heat shock protein10may suppress osteoblastproliferation by arrest cell cycle, and affect alkaline phosphatase activity, express ofRANKL, OPG mRNA, the ratio of OPG/RANKL mRNA, and result in the differentosteogenesis ability;2) r-Mtcpn10may induce osteoclastogenesis by up-regulating theexpress of NFATc1mRNA、c-Fos mRNA, and induce osteoclast formation via NF-κBsignal pathway;3) In osteoblast and mononuclear cell co-culture system, r-Mtcpn10mayinduce osteoclastogenesis by increasing osteoblast RANKL expression, which may leadto up-regulate the express of NFATc1mRNA、c-Fos mRNA of osteoclast precursor cells,caused osteoclast formation. Experimental result may provide new targeted gene fortreatment of skeleton tuberculosis. |
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