摘要
目的:探讨缝隙连接细胞间通讯在大鼠骨髓基质细胞成骨分化中的作用。
方法:采取全骨髓贴壁分离筛选法从雄性Sprague-Dawley大鼠中的股骨、胫骨中提取骨髓基质细胞并在L-DMEM中进行培养。细胞传代培养至第3代时,分成对照组、诱导组、实验组共3组,选用地塞米松+β-甘油磷酸钠+维生素C为成骨诱导剂、18α-甘草次酸为缝隙连接通讯阻滞剂,3组细胞分别加入培养液、培养液+成骨诱导剂、培养液+成骨诱导剂+18α-甘草次酸进行培养。培养过程中在不同时间点检测各组细胞的增殖、碱性磷酸酶相对活性,同时在第7天应用细胞免疫荧光技术定位缝隙连接蛋白43,RT-PCR法检测缝隙连接蛋白43、骨钙素、骨涎蛋白的mRNA水平。在第10天应用划痕标记染料示踪法评估缝隙连接细胞间通讯功能,在第21天茜素红S染色法检验基质中钙结节形成能力。
结果:在第7、9天的增殖能力,实验组<诱导组<对照组。第9、11天的碱性磷酸酶相对活性,对照组<实验组<诱导组。第7天,发现缝隙连接蛋白43定位于胞浆、胞膜,缝隙连接蛋白43的mRNA水平诱导组和实验组间无显著差别,但均高于对照组;骨钙素、骨涎蛋白的mRNA对照组<实验组<诱导组。第10天,显示缝隙连接通讯功能对照组<实验组<诱导组(均以P<0.05为有显著统计学意义)。第21天,钙结节形成能力方面,肉眼观察显示实验组<诱导组,对照组无钙结节形成。
结论:18α-甘草次酸能通过阻滞由缝隙连接蛋白43介导的缝隙连接细胞间通讯能力,从而减弱成骨诱导剂对大鼠基质细胞的成骨分化作用。
Objective To investigate the functions of connexin43(Cx43) on the osteogenic differentiation of rat bone marrow stromal cells(BMSCs) in vitro.
Methods The BMSCs were taken out from the femur and tibia of the male Sprague-Dawley rat by the whole bone marrow culture and purified by adhesive- screening method, and then were treated in the L-DMEM. When the passage culture reached the third generation we divided the BMSCs into three groups, ie,controled group(CG), induced group(IG) and experimental group(EG). At our experiment, dexamethasone,β-glycerophosphate and Vitamin C were combined used as the osteogenic inductor while 18α-glycyrrhetinic acid as the blocker of gap junctional intercellular communcation(GJIC). L-DMEM, L-DMEM+ osteogenic inductor, L-DMEM+ osteogenic inductor+AGA were added into CG, IG and EG, respectively. During the culture, the capability of proliferation and alkaline phosphatase(ALP) relative activity of different groups of cells were tested at various day. At 7th day, Cx43 was allocated by fluorescent technique. And semi-quantitative RT-PCR analysis were used for measuring the expression of Cx43, osteocalcin(OC), sialoprotein(SA) mRNA at the same day. At 10th day, we explored the function of GJIC by scrape-loading and dye transfer method. Then at 21st day, alizarin red S staining was used to observe the mineralization nodules among the matrix.
Results At day 7 and 9, the proliferated ability of 3 groups sequenced as: EG Conclusion The blocking of Cx43-mediated GJIC by 18α-glycyrrhetinic should weak the osteogenic differentiation of rat bone marrow stromal cells that caused by osteogenic indutors in vitro.
引文
[1] Mauney JR, Kirker-Head C, Abrahamson L, et al. Matrix-mediated retention of in vitro osteogenic differentiation potential and in vivo bone-forming capacity by human adult bone marrow-derived mesenchymal stem cells during ex vivo expansion.J Biomed Mater Res,2006,79A:464-475.
[2] Mese G, Richard G, White TW. Gap junctions: basic structure and function.J Invest Dermatol,2007,127:2516-2524.
[3] Kwak BR, Jongsma HJ. Regulation of cardiac gap junction channel permeability and conductance by several phosphrylating conditions. Moll Cell Biochem,1996,157:93-99.
[4] Saez JC, Nairn AC, Czernik AJ, et al. Phosphorilation of connexin43 and the regulation of neonatal rat cardiac myocyte gap junctions. J Mol Cell Cardiol,1997,29(8):2131-2134.
[5] Jiang JX, Siller-Jackson AJ, Burra S. Roles of gap junctions and hemichannels in bone cell functions and in signal transmission of mechanical stress. Front Biosci,2007,12:1450-1462.
[6] Schiller PC,D’Ippolito G,Brambilla R,et al. Inhibition of gap-junctional communication induces the trans-differentiation of osteoblasts to an adipo- cytic phenotype in vitro.J Biol Chem,2001,276:14133-14138.
[7] Davidson JS, Baumgarten IM. Related glycyrrhetinic acid derivatives: a novel class of inhibitors of gap-junctional intercellular communication structure-activity relationships.J Pharmacol Exp Ther,1988,246(3):1104 -1107.
[8] Opsahl H, Rivedal E. Quantitative determination of gap junction inter- cellular communication by scrape loading and image analysis. Cell Adhes Commun,2000,7(5):367-375.
[9] Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues.Science,1997,276(4):71-74.
[10] Van-Damme A,Vanden-Driessche T,Collen D,et al.Bone marrow stromal cells as targets for gene therapy.Curr Gene Ther,2002,2:195-209.
[11] Bruder SP, Jaiswal N, Haynesworth SE. Growth kinetic,self-renewal, and the osteogenic potential of purified human mesenchymal stem cells during extensive subcultivation and following cryopreservation.J Cell Biochem, 1997,64(2):278-294.
[12] Erices A, Conget P, Minguell JJ.Mesenchymal progenitor cells in human umbilical cord blood.Br J Haematol,2000,109(1):235-242.
[13] Zuk PA,Zhu M,Mizuno H,et al.Multilineage cells from human adipose tissue implications for cell-based therapies.Tissue Eng,2001,7(2):211-228.
[14]史春梦,程天民.大鼠真皮多能间充质干细胞的分离培养.第三军医大学学报,2001,23(9):1068-1070.
[15]闫西刚,贡志刚,兰青,等.大鼠骨髓基质干细胞体外分离、培养及分化的相关实验研究.中国微侵袭神经外科杂志,2007,12(4):175-178.
[16] Jose LR,Miguel H,Federieo G,et al. Value of deoxyribonucleic acid ploidy and nuclear morphometry for disease progression in renal cell carcinomal.The J Urological,1996,155:459-465.
[17]金丹,裴国敏,王前,等.骨髓基质细胞体外培养骨发生潜能及条件研究.中国矫形外科杂志,2000,7(6):565-568.
[18] Friedenstein AJ,Latzinik NV,Gorskaya YU,et al. Bone marrow srtomal colony formation requires stimulation by hameopoictic cells. Bone Mineral,1992, 36(3):725-736.
[19]罗飞,许建中.间充质干细胞在组织工程中的研究进展.第三军医大学学报,2003, 25(2):176-178.
[20]曲芸,刘惠民.骨髓基质细胞研究进展北华大学学报(自然科学版).2004,5(1): 42-44.
[21] Pittenger MF,Mackay AM,Beck SC,et al.Multilineage potential of adult human mesenchymal stem cells.Science,1999,284:143-147.
[22] Minguell JJ,Erices A, Conget P. Mesenchymal stem cells.Exp Biol Med,2001,226(6):507-520.
[23] Jiang Y,Jahagirdar BN,Reinhardt RL,et al. Pulripotency of mesenchymal stem cells derived from adult marrow. Nature,2002,418(6893):41-49.
[24] Reyes M,Verfaillie CM. Turning marrow into brain: Generation of glial and neuronal cells from adult bone marrow mesenehymal stem cells.Blood,1999,94: 377-380.
[25] Kopen GC,Prockop DJ,et al. Marrow stromal cells migrate throughout forebrain and cerebellum, and they differentiate into astrocytes after injection into neonatal mouse brains. Proc Natl Acad Sci USA,1999,96:10711-10716.
[26] Orlic D,Kajstura J, Chimenti Set al. Bone marrow cells regenerate infarcted myocardiumNature,2001,410(6829):701-705.
[27]胡军,邢达,张爱斌,等.冲击波诱导人骨髓基质细胞体内成骨研究.生物化学与生物物理进展,2006,33(5):452-457.
[28] Yoshikawa T, Peel SA, Gladstone JR, et al.Biochemical analysis of the response in rat bone marrow cell culture to mechanical stimu- lation.Biomed Mater Eng,1997,7(6):369-377.
[29] Kamalia N,MeCulloch CA,Tenebaum HC,et al. Dexamethasone recruitment of self-renewing osteoprogenitor cells in chick bone marrow stromal cell cultures.1992,79(2):320-326.
[30] Cui Q,Wang GJ,Balian G. Steroid-Induced adipogenesis in a pluri-potential cell line from bone marrow.Bone Joint Surg,1997,79:1054-1063.
[31] Otsuka E,Yamaguchi A,Shigehisa H,etal. Characterizations of osteoblastic differentiation of stromal cell line ST2 that is induced by ascorbic acid. Am J Physiol,1999,277(Cell Physiol 46):C132-C138.
[32] Mc Qillna DJ,Richardson MD,Bateman J F. Matrix deposition by a calcifying human osteogenic sarcoma cell line(SAOS22).Bone,1995,16(4):415-426.
[33] D'Andrea P, Vittur F. Gap junctions mediate intercellular calcium signalling in cultured articular chondrocytes.Cell Calcium,1996,20(5):389-97.
[34] Zhang W, Green C, Stott NS.Bone morphogenetic protein-2 modulation of chondrogenic differentiation in vitro involves gap junction-mediatedintercellular communication.J Cell Physiol,2002,193(2):233-243.
[35]鄂征,刘流.医学组织工程技术与临床应用.北京:北京出版社,2003:1-14.
[36] Lee DH,Suh H,Han DW,et al.The effects of recombinant human BMP-7,prepared from a COS-7 expression system,on the proliferation and differentiation of rat newborn calvarial osteoblasts.Yonsei Med J.2003,44(4):593-601.
[37] Dong J,Kojima H,Uemura T,et al.In vivo evaluation of a novel porous hydroxyapatile to sustain osteogenesis of transplanted bone marrow derived osteoblastic cells.J Biomed Mater Res,2001,57:208-216.
[38]郑创义,李学东,杜世新.缝隙连接蛋白43在骨骼发育和塑形中的作用.国际骨科学杂志.2009,30(2):134-136.
[39] Dorshkind K, Green L, Godwin A, et al.Connexin-43-type gap junctions mediate communication between bone marrow stromal cells. Blood,1993,82(1):38-45.
[40] Young MF. Bone matrix proteins:their function,regulation,and relationship to osteoporosis. Osteoporos Int,2003,14(Suppl3):S35-42.
[41] Wuttke M, Muller S, Nitsche DP, et al.Structural characterization of human recombinant and bone-derived bone sialoprotein.Functional implications for cell attachment and hydroxyapatite binding.J Biol Chem,2001,276(39): 36839 -36848.
[42] Plotkin LI, Manolagas SC, Bellido T. Transduction of cell survival signals by connexin-43 hemichannels.J Biol Chem,2002,277(10):8648-8657.
[43] Alford AI, Jacobs CR, Donahue HJ. Oscillating fluid flow regulates gap junction communication in osteocytic MLO-Y4 cells by an ERK1/2 MAP kinase- dependent mechanism small star, filled.Bone,2003,33(1):64-70.
[44] Stains JP,Civitelli R.Gap junctions regulate extracellular signalregulated kinase signaling to affect gene transcription. Mol Biol Cell,2005,16(1): 64-72.