| [Background]:Bone morphogenetic proteins(bone morphogenetic protein,BMP)is a multifunctional growth factor,belong to the growth factor TGF-? superfamily,is a highly conserved group of proteins having similar structural features,the BMPs have been found to have 15 kinds,depending on their amino acid sequence Similar divided into 3 groups:BMP-2,BMP-4,BMP-5,BMP-8,BMP-3 and GDF-101and among of them,BMP-2 is the most widely studied and one of the most active BMP to induce bone,BMP-2 was originally formed as a factor capable of inducing bone and cartilage for people to know,embryonic development and on bone plays an important role in regeneration.Recombinant human bone morphogenetic protein-2(rhBMP-2)by the US National Food and Drug Administration(FDA)approval to market in 2002,has become the most commonly used bone graft substitutes,the first case of domestic rhBMP-2 for the treatment of bone defects on July 6,2014 to succeed in Kunming General Hospital of Chengdu Military Region,marking the national multi-center clinical trial has begun.In addition to studies,BMP-2 suggesting to be involved in regulating the proliferation of many types of tumor cells,chemotaxis,anti-vascular,differentiation and apoptosis of biological processes,rhBMP-2 can reduce the proliferation of breast cancer cells,which can inhibit the highly metastatic cell lines and also can inhibit the proliferation of low metastatic cell lines,but it has a stronger ability to inhibit the activity of highly metastatic cells than that of the low-metastatic cells.BMP-2 in have significantly inhibited the growth of 6 colon cancer cell lines:HT29 CACO-2,DLD-1,SW480,HCT116 and LS174-T cells;however MAPK signaling through BMP-2 activation pathway stimulates ASPC-1 and CAPAN-1 growth of pancreatic cancer cell lines,and through SMAD-1/5 signaling pathway is activated,stimulate the growth of NSCLC lung cancer cell lines 9-10.So BMP-2 is a double-edged sword for different tumor have different effects,but there nearly had no study about BMP-2 on bone giant cell tumor in vitro studies to date and there was also no research on animal tumor models in vivo,assuming that BMP-2 can inhibit the growth of giant cell tumor with a adjuvant therapy in patients in inhibiting the growth of residual tumor cells,and simultaneously stimulate the surrounding normal osteoblasts,thereby reducing the recurrence rate of giant cell tumor of bone.[Objectives]1 To investigate the optimum concentration of rhBMP-2 that inhibited the growth of GCTS cells.2 To investigate the effect of rhBMP-2 on cell cycle and apoptosis of GCTS cells3 To investigate initially which pathway of cell apoptosis of GCTS cells does take effect by the treatment of rhBMP-24 To establish the subcutaneous xenograft of nude mice of GCTS cells,To investigate the inhibition of rhBMP-2 on GCTS cells in vivo.[Methods]1 GCTS cells were cultured in vitro.Experimental samples were taken from nine giant cell tumor patients from General Hospital of Guangzhou Military Command and Nanfang Hospital,Southern Medical University on February 2013 to December 2013.Three patients were male and the others were females.They aged from 16 to 35 years old.The tumors were graded by Campanacci grading standards.There were five cases of grade II and four cases of grade III.All patients had not received any adjuvant therapy before the operation.Postoperative pathological diagnosis was confirmed as giant cell tumor of bone.Primary cell cultures were got from resected fresh tissue of giant cell tumor.Purified giant cell tumor stromal cells were got after nine culture.The cultured giant cell tumor stromal cells were freezed in liquid nitrogen.2 By use of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT method)to observe the growth of GCTS cells by the addition of rhBMP2 of different concentration(0,10,100 test,300 ng/ml),and obtained the best inhibition concentration of rhBMP2.After Conventional resuscitation of giant cell tumor of bone stromal cells,the GCTS cells were cultured in DMEM medium.24 hours later,the digestive tumor cells were inoculated into 96 pore culture plates,and were divided into 4 groups:rhBMP2(0,10,100,300 ng/ml)groups,each group includes 3 wells,GCTSCs were plated and cultured in 96-well plates with rhBMP-2(R&D systems,USA)(0,10,100,300ng/ml)in 10%FBS DMEM medium for 1,3,5,7 days.MTT(sigma,USA)was added to the culture medium following the manufacturer's instructions,and the culture was continued for another 4 hours.Dimethylsulfoxide(10O?L/well)was added to each well to dissolve Fromazan crystals.The optical density of the resulting product was measured on an ELISA plate reader(Thermo,Multiskn Go)at 490nm.By use of SPSS 19 statistical software each photometric values were analyzed by one-way ANOVA(one-way ANOVA)analysis,multiple comparisons between groups using LSD test.According to the experimental results obtained by MTT,rhBMP2(10 ng/ml)on 5?7 days,significantly inhibited the growth of stroma cells in giant cell tumor of bone.so the subsequent study of flow cytometry cell cycle,apoptosis,and signal pathway of GCTS cells by Western method are all used to add rhBMP2(10 ng/ml)into the GCTS cells.control group of GCTS cells in DMEM without addition of rhBMP2.3 Flow cytometric quantitative analysis was used to detect the cell cycle change of GCTS cells in the experimental group with the addition of rhBMP2(10 ng/ml)and in the control group without the addition of rhBMP2(0 ng/ml)for 48 hours.The primary culture of giant cell tumor of bone stromal cell were digested,then were inoculated into 24 well plates,and divided into two groups:the experimental group with the addition of rhBMP2(10 ng/ml)in DMEM culture medium,the control group without addition of rhBMP2 in DMEM culture medium.Two groups were cultured for 48 hours in DMEM culture medium,flow cytometry PI staining was used to detect cell cycle of these two groups,the comparison of G0/G1,S and G2/M of GCTs cells between these two groups was used by SPSS 19 statistical software for independent samples t test.4 Annexin-V FIT Flow cytometry was used to detect the cell apoptosis of GCTS cells in the experimental group with the addition of rhBMP2(10 ng/ml)and in the control group without the addition of rhBMP2(0 ng/ml)for 48?72 hours.The primary culture of giant cell tumor of bone stromal cell were digested,then were inoculated into 24 well plates,and divided into two groups:the experimental group with the addition of rhBMP2(10 ng/ml)in DMEM culture medium,the control group without addition of rhBMP2 in DMEM culture medium.Two groups were cultured for 48?72 hours in DMEM culture medium,flow cytometry of double Annexin V-PE and 7-AAD staining was used to detect the apoptosis of these two groups.the comparison of cell apoptosis of GCTs cells between these two groups was used by SPSS 19 statistical software for independent samples t test.5 Western blot was used to detect the expression of Erk1/2?p38?JNK in non-Smad pathways mitogen-activated protein kinase(MAPK)pathways of GCTS cells in the experimental group with the addition of rhBMP2(10 ng/ml)and in the control group without the addition of rhBMP2(0 ng/ml)for 72 hours.The primary culture of giant cell tumor of bone stromal cell were digested,then were inoculated into 24 well plates,and divided into two groups:the experimental group with the addition of rhBMP2(10 ng/ml)in DMEM culture medium,the control group without addition of rhBMP2 in DMEM culture medium.Two groups were cultured for 72 hours in DMEM culture medium,then digested GCTS cells and by Western blot to detect the grey value of p-Erkl/2,Erkl/2,p-p38,p38,p-JNK,JNK,the comparison of gray value between the experimental group and the control group is applied by SPSS 19 statistical software for independent samples t test.6 Animal experiments in vivo,GCTS cells in the experimental group with the treatment of rhBMP2(10 ng/ml)and in the control group without the treatment of rhBMP2(0 ng/ml)were implanted in subcutaneous of BALB/C nude mice.To observe the growth of xenografts in nude mice.The primary culture of giant cell tumor of bone stromal cell were digested,then were inoculated into 25 cm3 culture flask,and divided into two groups:the experimental group with the addition of rhBMP2(10 ng/ml)in DMEM culture medium,the control group without addition of rhBMP2 in DMEM culture medium.Two groups were cultured for 72 hours in DMEM culture medium,then digested GCTS cells and collected,and calculated by the trypan blue exclusion assay.1×106/ml cell suspension were prepared by use of 10%PBS.Female BALB/C nude mice(6-8 week old,22-24g in weight)were raised in SPF environment.1×106/ml GCTS cell suspension were injected in four sites of subcutaneous of abdomen in 10 nude mice.continuous observation for 12 weeks,the grafted tumor were not observed and not touched.10 nude mice were sacrificed finally.By the same means rat osteosarcoma UMR106 cell line was recovered and cultured in DMEM medium for 72 hours,then were digested,and were calculated by the trypan blue exclusion assay.1×106/ml cell suspension were prepared by use of 10%PBS.1×106/ml UMR cell suspension were injected in four sites of rhBMP2(0 ng/ml)subcutaneous of dorsum in 10 nude mice.continuous observation for 1 week,9 nude mice were grown into tumors,7/9 nude mice,3 sites of the dorsum have loaded tumor.random 4 nude mice were executed for 1 week,the rest of nude mice were executed for 2 weeks.All of the tumor spicemens were tested by HE staining and PCNA immunohistochemical staining,to observe the proliferation of tumor.Because at osteosarcoma UMR106 cell line was not associated with this experiment in vivo,so the animal in vivo experiments ended.[Results]1 Giant cell tumor resected from bone tissue culture in the DMEM medium for nine times,we can see the tumor cells with a uniform,purified bone giant cell tumor of stromal cells under a microscope,then digest tumor cells,frozen in liquid nitrogen for this experiment.2 MTT assay at different concentrations rhBMP2(0,10,100,300 ng/ml),respectively,giant cell tumor of bone stromal cell cultured for 1,3,5,7 days to detect whether it has a growth inhibition effect.1)No significant difference(P>0.05)between days 1 and 3 in each group,wherein the first day rhBMP2(100,300 ng/ml)compared with the control group had a slight growth stimulation,but no significant difference(P>0.05);2)Five days rhBMP2(10,100 ng/ml)compared with the control group significantly inhibited tumor cell growth(P<0.001),rhBMP2(300 ng/ml)compared with the control group had no significantly difference of inhibiting tumor cells growth(P<0.05);3)After cultured for 7 days,rhBMP2(10,100,300ng/ml)group significantly inhibited tumor cell growth(P<0.001)compared to the control group.3 We used Flow Cytometry to detect the effect of rhBMP2(0,10ng/ml)on tumor cells of cell cycle after 48 hours,G0/G1,S and G2/M phase in both groups showed no significant difference(P>0.05).4 We used Flow Cytometry to detect the effect of rhBMP2(0,10ng/ml)on tumor cells of cell cycle after 72 hours.1)Treated with rhBMP-2(10ng/mL)for 48 hours,no significant difference in the number of apoptotic cells in the experimental group and the control group,but the number of dead cells in the experimental group was significantly more than the control group(P<0.05);2)After tumor cells were treated rhBMP-2(10ng/mL)72 hours,and the number of necrotic and apoptotic cells in the experimental group was significantly more than the control group(P<0.01);5 Western blotting analysis the expresssion level of the non-Smad pathways of mitogen-activated protein kinase(MAPK)signaling pathway p-Erk1/2,Erk1/2 and the expression of p-p38,p38,p-JNK,JNK after the rhBMP-2(0,10ng/ml)of giant cell tumor of bone stromal cells in the experimental group treated for 72 hours,1)the expression level of pErk1/2,p38,JNK rhBMP2(10ng/ml)t group and the control grou had no significant difference after 72 hours(P>0.05);2)the expression level of pErk1/2,p38,JNK rhBMP2(10ng/ml)t group and the control grou all showed a significant difference after 72 hours(P>0.05);6 In vivo animal experiments,because of no immature tumor formation for ontinuous observation of giant cell tumor of bone grown in nude mice after 12 weeks,declaring the end of the in vivo animal experiments.[Conclusions]1)rhBMP-2 for inhibition of giant cell tumor of bone stromal cell growth and induction of apoptosis.Its may be no highly relevant related to the drug concentration In vitro,after 10 ng/ml concentration rhBMP2 treatment of giant cell tumor of bone stromal cells for 72 hours,it can significantly induce apoptosis.Therefore,to maintain a low dose of rhBMP2 adjuvant treatment of giant cell tumor of bone residual tumor stromal cells can induce apoptosis of giant cell tumor of bone which can be avoided excess of normal bone resection in surgery,and can contribute to strengthen or rebuild the bone tissue surrounding the lesion,reducing fracture after surgery and other complications,and also can reduce the adverse reactions of high concentrations adjuvant systemic medicine.2)After using rhBMP2(10ng/ml)for 48 hours,the cell cycle of giant cell tumor of bone stromal cells(G0/G1,S and G2/M phase)was no significant difference(P<0.05),it was said that as rhBMP2 was not affect other tumors by G1 arrest cell growth like early study.3)The MAPK-associated protein ERK(1/2),p38 and JNK signaling pathway play an important role in a variety of physiological processes of osteoblast differentiation,proliferation,apoptosis.Treated with rhBMP2(10ng/ml)for 72 hours,the expression level of the MAPK-related protein pERK(1/2),p-p38 and p-JNK were significantly increased(P<0.05).suggesting that rhBMP2 induced apoptosis proteins through the MAPK signaling pathway by activating the ERK(1/2),p38 and JNK.Through in-depth study of cell apoptosis mechanism,it provide a new way for the selection and use of new chemotherapy drugs to treat the giant cell tumor.4)the in vitro experiments confirmed low concentrations rhBMP2(10ng/ml)induced apoptosis of the giant cell tumor cell,given that rhBMP2 has been used in clinical bone defect,nonunion,so used in vitro not only reduce residual tumor cells,act as a "safe handling wall" effect,for the tumor failling to be widely excised(such as the patients of limb salvage surgery for three degree giant cell tumor of bone)of the cases to avoid excessive resection and and prevention of giant cell tumor recurrence of bone and soft tissue,further optimization of surgical project,and may promote to strong bone lesions or rebuild the structure,with the dual role of reducing the incidence of fractures and other complications.5)The clinical application of any of anticancer drugs need to go through in vitro,in vivo animal experiments and this stage ?-? clinical trials before they can enter the clinic.According to planting technique in animal models of prostate cancer of the Master project,we failed to establish a giant cell tumor in nude mice in vivo subcutaneous model,while injecting the cultured osteosarcoma cells as the same way like giant cell tumor,we successfully established body giant cell tumor of bone grown in nude mice model.So infer that using giant cell tumor lines can not be successfully established subcutaneous tumor model in nude mice because of the mechanism of low degree of malignancy of the bone giant cell tumor,and so far no specific bone giant cell tumor cell lines,which need for further follow-up study. |
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