目的观察FTY720对聚乙烯颗粒诱导的破骨细胞前体细胞RAW264.7分化的影响,探讨其防治人工关节无菌性松动的可能性。方法构建破骨细胞-成骨细胞（RAW264.7-MC3T3）小室共培养体系及破骨细胞-骨片体系,用FTY720干预受聚乙烯磨损颗粒刺激的RAW264.7细胞的分化,倒置显微镜观察分化细胞的形态;抗酒石酸酸性磷酸酶（Tartrate-resistant acid phosphatas,TRAP）染色法对破骨细胞进行计数;扫描电镜观察破骨细胞的一般形态及骨吸收效应;ELISA法检测共培养体系中肿瘤坏死因子-α（Tumor necrosis factor-α,TNF-α）和白细胞介素-6（Lnterleuk-in-6,IL-6）的分泌水平;RT-PCR检测破骨细胞表面核因子κB受体活化子（Receptor activator of NFκB,RANK）和TRAP基因mRNA的转录水平。结果聚乙烯颗粒组RAW264.7细胞体积增大,胞质丰富,胞体边缘不齐呈云雾状,细胞核较多;FTY720组TRAP（+）细胞数明显低于聚乙烯颗粒组（P＜0.01）;破骨细胞呈圆形,细胞间可通过纤维样足突连接,骨片上有破骨细胞附着生长,FTY720组骨吸收陷窝和骨吸收面积均明显低于聚乙烯颗粒组（P＜0.01）;聚乙烯颗粒组TNF-α和IL-6的分泌水平均明显增加（P＜0.01）,FTY720组TNF-α和IL-6的分泌受到抑制;FTY720组破骨细胞表面TRAP和RANK基因mRNA的转录水平均明显低于颗粒组（P＜0.01）。结论 FTY720能有效抑制破骨细胞前体细胞RAW264.7分化成熟,减少破骨细胞的形成及对骨片的溶解吸收,减少TNF-α、IL-6等炎性因子的分泌,下调RANK、TRAP等破骨细胞特异细胞表型和功能基因mRNA的转录水平,有望成为防治人工关节无菌性松动的药物。

Objective To construct a recombinant adenovirus vector for expression of proline-arginine rich 39-amino acid peptide(PR-39).Methods PR-39 gene was amplified from the plasmid containing core encoding region of PR-39 and subcloned to shuttle plasmid pAdTrace-TO4.The constructed recombinant shuttle plasmid was linearized with PmeⅠ and transfected to competent AdEasier cells.The obtained recombinant adenovirus plasmid pAdPR-39 was transfected to HEK293 cells for packaging of recombinant adenovirus which was then propagated and determined for titer.Murine fetal mesenchymal stem cells C3H10T1/2 were infected with recombinant adenovirus AdPR-39,observed for expression of red fluorescent protein(RFP) by fluorescent microscopy,and determined for transcription level of PR-39 gene by RT-PCR. Results Restriction analysis with KpnⅠ and Hind Ⅲ and sequencing proved that recombinant adenovirus shuttle plasmid pAdTrace-TO4-PR-39 was constructed correctly.Digestion of pAdPR-39 with PacⅠ proved that the target gene was integrated into the genome of adenovirus.The titer of AdPR-39 of passage 4 was l0 10 IU/ml.RFP was expressed in C3H10T1/2 cells infected with AdPR-39.PR-39 gne was successfully transcribed in C3H10T1/2 cells.Conclusion Recombinant adenovirus vector for expression of PR-39,containing RFP reporter gene,was successfully constructed and highly expressed in murine fetal mesenchymal stem cells C3H10T1/2.

Objective： To investigate the effect of FTY720（Fingolimod） on osteolysis induced by ultra- high- molecular- weight polyethylene（UHMWPE） particles in mouse air pouch model. Methods： The air pouches model was established, mice were divided into four groups： ultra - high - molecular - weight polyethylene（UttMWPE） particles and FFY720 group （UHMWPE was introduced into the air pouches and mice were injected intraperitoneally with FTFY720）; UHMWPE group（ UHMWPE was introduced into the air pouches and mice were injected intraperituneally with DMSO） ; FTY720 goup （normal saline was introduced into the air pouches and mice were injected intraperitoneally with FFY720） ;blank group（ normal saline was intredueed into the air pouches and mice were injected intraporitoneally with DMSO）. Four weeks later, all the mice are sacrificed, pouch walls and implanted calvaria were collected. and imqanmmtory reaction and the number of exuded inflammatory cells of the pouch walls were detected through HE staining, some of inflanmuitory cytokines, IL - 6 and TNF - a, in tissue homogenate were detected with ELISA; the resorption of the bone slices were detected with scanning electron microscopy; the osteeclast associated gene TRAP in pouch tissues were detected with RT- PCR. Results： （1）HE staining results showed that the number of exuded inflammatfry ceils of the pouch walls in the UHMWPE and FFY720 group （4892 ± 457 ） had no difference compared with the UHMWPE group（ 4931 ± 572 ） （ P 〉 0.05 ）, there was no difference between the FTY720 group （ 2013 ± 375 ） and the blank group （2151 ± 310） （ P 〉 0.05 ） ; but inflammatory cell amounts in the UHMWPE groups were higher than the groups that without treatment UHMWPE （P 〈 0.05 }. （2）ELISA results showed that the quantity of inflannYuatory cytokines IL- 6 and TNF-α tissue hongenate in the UHMWPE and bq＇Y720 group[ （130.13± 3.22） , （ 129.22 ± 5.02） ]had no difference compared with the UHMWPE group[ （ 149.22 ± 6.09 }, （ 140.52 ± 2.68 } ] （P 〉 0.05） ,there was no difference between the FYY720 group[ （68.24 ± 2.83）, （82.88 ± 5.34） 3and the blank groul）[ （69.51 ± 2.31 ）, （74.15 ± 4.64） { P 〉 0. 05） ;but the cytokine（ IL- 6 and TNF- α） concentrations in the UHMWPE groups were higher than the groups that without treatment UHMWPE （P 〈 0.05）. （1）Scanning electron microscopy showed that the area of resorption of the bone slices in the UHMWPE and FRY720 group[ （ 10.21 ~ 1.78） % was lower than the UHMWPE group[ {17. 79 ± 2.56） %33,the FTY720 group[ （1.82 ± 1.37）% was lower thau the blank groups[ （6.27 ± 2.11 }% 3;and the area of resorption of the bone slices in the UHMWPE groups were higher than the groups that without treatment UHMWPE （P 〈 0. 05 ）. （（~）RT- PCR results showed that osteeclasts - associated TRAP mRNA in the UHMWPE and FI＇Y720 group （ 0.53 -± 0.04 ） was lower than the UHMWPE group （0.74 ±0. 05 ）, the FTY720 group （0.22 ± 0.03 ） was lower than the blank group （0.38 ± 0.04） ;and osteeclasts- associated TRAP mRNA in the UHMWPE groups were higher than the groups that without treatment UHMWPE（ P 〈 0.05 ）. Couclusion： FTY720 could inhibit the bone destruction that caused by the UHMWPE in mousc air pouch mode.

Objective： To study the clinical effect of large-diameter femoral head-cemented hip arthroplasty in the surgical treatment of 12 patients with Parkinsonism involving the femoral neck fracture.Methods： From January 2007 to October 2010,we retrospectively analyzed 12 cases,including 3 males and 9 females with an average age of 76.17 years.All patients were treated with artificial prosthesis replacement,including 2 cases received total hip arthroplasty and 10 underwent bipolar artificial femoral head replacement.The patients were followed up since 3 months and evaluated clinically and radiographically respectively for the safety of replacement,hip function by Harris score.Result： All patients were safe through the perioperative.One case had hip dislocation in 3 weeks after replacement.There were no infections or death during the perioperative period.The radiolucent line happened in 2,but there was no osteolysis,prosthesis,loosening and femoral stem subsidence.According to the Harris scoring system,the patients were rated as 90~100 in 3,80~89 in 5,70~79 in 3 and 70 in 1.Conclusion： Large-diameter femoral head-cemented hip arthroplasty in patients with femoral neck fracture in elderly patients with Parkinsonism may decrease complications and dislocation rate to improve joint functional recovery and quality of life.

Objective To observe whether RAW264.7 cells,a mouse monocytes cell line,can be induced to differentiate to mature osteoclasts with RANKL.Methods RAW264.7 cells were cultured and treated with RANKL for 7 to 9 days.Cell morphological changes were observed using light microscope,transmissional electron microscope(TEM),and scanning electron microscope(SEM).Multinucleated cells were stained with tartrate-resistant acid phosphatase(TRAP) and TRAP-positive cells were observed.Expressions of osteoclast phenotype and function genes were detected using Semi-quantitative RT-PCR.Osteoclastic resorption pits that formed on the bone slices were observed with SEM.Results Light microscopy and TEM showed that the size of the cells enlarged,and the cell shape was oval or irregular,and each cell contained 5 to 10 nucleus.SEM demonstrated that there were large amount of pseudopodia-like protrusions on the surface of the cells.Furthermore,RANKL induced RAW264.7 cells to differentiate to TRAP-positive multinucleated osteoclasts with more than 5 nucleus.The differentiated cells had bone resorption ability.Osteoclast phenotypic and function genes Cathepsin-K,TRAP,and RANK were up-regulated.Conclusion RAW264.7 cells could be used as a perfect model of osteoclast precursors.RANKL 50ng/mL treatment alone for over 7 days could induce the precursor cells to differentiate to mature osteoclasts in vitro.

Objective To observe whether osteoprotegerin(OPG) is effective in reducing bone destruction and treating aseptic loosening in a mouse air pouch model.Methods Air was injected into the mouse back to form an air pouch,and the skull from another homologous mouse was implanted into the air pouch to obtain an air pouch model.Mice were divided into a blank group,a particle group,and an OPG group.Normal saline was injected into the air pouches of mice in the blank group.Polyethylene particles and physiological saline were injected into the air pouches of mice in the particle group.Polyethylene particles and OPG were injected into the air pouches of mice in the OPG group.Three weeks later,pouch walls and implanted skull tissues were collected,and the inflammatory reaction and the quantity of exuded inflammatory cells were detected through HE staining.The concentrations of inflammatory cytokines IL-1 and TNF-α in tissue homogenate were measured with ELISA.The differentiation and maturation of osteoclast were observed through tartrate-resistant acidic phosphatase staining.The resorption of bone slices was detected with scanning electron microscopy.Results The quantities of exuded inflammatory cells in the particle group[(3 812±628)/eyeshot] and the OPG group[(3 665±297)/eyeshot] were significantly higher than that in the blank group[(1 820±598)/eyeshot,P<0.05].The IL-1 and TNF-α concentrations in the particle group[(210.57±4.26),(188.36±7.33)pg/ml] and the OPG group [(198.59±6.90),(179.28±2.11)pg/ml] were significantly higher than those in the blank group [(138.34±2.32),(156.14±4.17)pg/ml,P<0.05].The percentage of tartrate-resistant acidic phosphatase staining positive area to visual field area in the particle group(4.34±1.53)% was significantly higher than that in the blank group [(0.89±0.12)%,P<0.05] and that in the OPG group [(0.96±0.55)%,P<0.05].The percentage of bone slice resorption area to visual field area in the particle group(14.58±2.68)% was significantly higher than that in the blank group[(3.09±0.62)%,P<0.05]and that in the OPG group [(3.25±0.78)%,P<0.05].Conclusion Mouse air pouch model is established.OPG has no significant inhibitory effect on the inflammatory reaction induced by polyethylene wear particles,but can inhibit the differentiation and maturation of osteoclast and reduce the bone resorption induced by polyethylene wear particles.

Artificial joint replacement has been one of the revolutionary leaps in orthopaedics in the 20th century. An ideal artificial joint replacement can relieve pain of patients and reconstruct the move-ment funtion,which are the foundamental goals of the the artificial joint replacement. But there is a severe problem which is said to be the prosthesis lossening in the late-stage of operation. According to relative statistics,during 15-20 years after the replacement,the prosthesis lossening would happen to about 10-15% artificial joints,and thus leads to a failure of the artificial joint funtion. Wear particles produced after long-term wear of the artificial joint induce the large-scale generation of around osteo-clasts,which causes the bone disolusion and osteoporosis. That is one of the main reasons of prosthesisloosening happening in the late-stage of the replacement. However,the current review highlights the importance of the kinase activation in RANK/RANKL signaling pathway on the differentiation and activation of the osteoclastogenesis.