重组人白细胞介素1受体拮抗蛋白荧光质粒在软骨细胞的表达**☆

背景：白细胞介素1受体拮抗蛋白能延缓骨性关节炎进程,通过转基因方法可以使白细胞介素1受体拮抗蛋白表达的增加。 目的：观察重组人重组人白细胞介素1受体拮抗蛋白荧光质粒的构建及经脂质体转染软骨细胞的表达情况。 方法：双酶切法切取重组人白细胞介素1受体拮抗蛋白的c-DNA片段,通过T4DNA连接酶连接到pEGFP-C1载体上。体外分离培养兔关节软骨细胞,然后用构建的重组人白细胞介素1受体拮抗蛋白质粒经脂质体转染软骨细胞,通过荧光显微镜观察转基因的表达和荧光定量PCR检测其表达。 结果与结论：获得重组人pEGFP-C1-IL-1Ra真核表达载体质粒,酶切及测序结果证明表达质粒的DNA序列完全正确。荧光显微镜观察有绿色荧光蛋白表达,荧光定量PCR鉴定证实转染的软骨细胞基因得到表达。     

rAAV1-EGFP和rAAV2-EGFP转染犬骨髓间充质 干细胞的比较

 【目的】 研究不同血清型腺相关病毒(rAAV)载体介导的增强绿色荧光蛋白基因(EGFP)对体外培养犬骨髓间充质干细胞(MSC)的转染效率及其毒性作用&#65377;【方法】 应用密度梯度离心法及贴壁筛选法分离培养犬MSC,倒置显微镜及Giemsa染色观察细胞形态,透射电镜观察其超微结构,流式细胞仪鉴定其表面标记物&#65377;取第3代MSC,以MOI为104 ~ 105 v.g./cell 的rAAV1-EGFP 和rAAV2-EGFP进行转染,3 d&#65380;7 d&#65380;21 d后荧光显微镜观察检测转染效率;MTT法检测rAAV1-EGFP 和rAAV2-EGFP对MSC的毒性作用&#65377; 【结果】 原代及传代培养的MSC呈梭形外观,具有较强的增殖能力&#65377;MSC超微结构显示其胞体较小,细胞核/浆比例大,胞浆少,染色质较疏松&#65377;细胞表面抗原CD29&#65380;CD44表达阳性,CD34表达阴性&#65377;随MOI的增加及时间的延长,rAAV1-EGFP&#65380;rAAV2-EGFP对犬MSC的转染效率逐渐增加,rAAV2-EGFP转染效率明显高于rAAV1-EGFP(P < 0.01)&#65377;转染MSC细胞后,细胞生长正常,MTT法显示各转染组与未转染组的OD值无统计学差异(P > 0.05)&#65377; 【结论】 相对犬MSC,rAAV2是一种比rAAV1更优越的转基因载体,而且对细胞生长无明显抑制,做为组织工程种子细胞的载体是安全可行的&#65377;     

犬骨髓基质干细胞体外定向分化为软骨细胞

背景：骨髓基质干细胞的多向分化性不利于向软骨细胞单一方向分化,植入体内后成骨系细胞分泌的骨形态发生蛋白作用于非定向分化的前体细胞,使其向成骨细胞分化,而已定向分化的细胞则不受骨形态发生蛋白的影响,形成相应的组织。 目的：体外诱导犬骨髓基质干细胞定向分化为软骨细胞,探讨体外诱导成软骨的方法和条件。 设计、时间及地点：单一样本观察,于2005-03/2006-01在中山大学组织工程实验室完成。 材料：选取4个月龄雄性家犬1只,骨髓基质干细胞取自犬肋骨。 方法：自犬肋骨取骨髓2.0~3.0 mL行体外原代骨髓基质干细胞分离培养。8~11 d细胞接近融合时,加入胰酶消化,用含体积分数10%胎牛血清的L-DMEM合成培养液终止消化,收集细胞悬液,离心后,用培养液悬浮细胞,按1∶3接种传代。取第3代细胞培养扩增,换液时加入10 μg/L碱性成纤维细胞生长因子2 mL,换液2次后,再加入 1 mg/L转化生长因ß1 2 mL诱导骨髓基质干细胞向软骨细胞分化。 主要观察指标：行甲苯胺蓝、阿新蓝染色检测软骨基质的分泌,免疫组织化学染色检测软骨特异性Ⅱ型胶原表达。 结果：骨髓基质干细胞体外行原代和传代培养至P4代生长良好,经碱性成纤维细胞生长因子和转化生长因ß1扩增诱导的细胞甲苯氨蓝异染性、阿新蓝染色阳性;II型胶原免疫组织化学检测阳性,显示被诱导的骨髓基质干细胞具备软骨细胞特性。 结论：应用碱性成纤维细胞生长因子和转化生长因ß1体外可以诱导犬骨髓基质干细胞分化为软骨细胞,诱导的软骨细胞可作为软骨组织工程较理想的种子细胞。     

全髋关节置换术治疗强直性脊柱炎髋关节骨性强直的临床研究

目的探讨强直性脊柱炎(AS)髋关节骨性强直行全髋关节置换术(THA)的特点,寻找提高手术疗效,减少并发症的方法。方法对本组18例AS患者29个骨性强直的髋关节行THA手术,术前髋关节强直在屈曲0～65°(平均23.6°),术后平均随访4.2年,Harris评分进行术后临床疗效评定,X线检查观察假体有无松动、脱位及异位骨化。结果所有患者治疗经过顺利,无关节松动、脱位、骨折、感染等严重并发症发生,髋关节的屈曲和内收畸形得到纠正。平均髋关节屈伸活动度81°,屈伸、内收外展、内外旋总活动度142°,Harris评分平均81.6分,X线照片见假体位置良好,未见松动和下沉。2例2髋长期行走时轻度疼痛,异位骨化2例:BrookerⅠ级1例,BrookerⅡ级1例。结论THA是AS髋关节骨性强直重建关节功能的有效方法,个体化手术方案的设计包括手术入路的选择、假体位置的正确安放、术中软组织松解与平衡、髋臼周围骨赘的清除等是影响手术疗效的关键。     

关节镜下肘关节滑膜软骨瘤病的诊断和治疗

目的探讨肘关节滑膜软骨瘤病的临床特点及关节镜术在该病诊断和治疗中的应用价值、操作要点及临床疗效。方法 1997年1月～2007年1月15例肘关节滑膜软骨瘤病患者行关节镜下清理术,术前所有患者均有不同程度的疼痛及活动受限,10例患者有关节绞锁病史。术中采用多入路结合清除关节内游离体及病变滑膜。采用改良HSS肘关节评分作为疗效评定标准。结果 15例患者随访2.5～12年(平均5.6年)。肘关节平均伸屈活动范围从术前的(85.6±11.3)°提高到术后的(121.2±10.1)°。肘关节疼痛、肿胀症状减轻,关节绞锁消失。改良HSS评分由术前的(48.2±13.6)分提高到术后的(79.8±12.5)分。6例患者非常满意,7例满意,2例不满意,满意率为86.7%。结论关节镜下关节清理术治疗肘关节滑膜软骨瘤病可取得良好效果,适当的入路选择、规范的镜下操作和病变滑膜的彻底清除是影响疗效和预防复发的关键。     

Change of inflammatory cytokines after co-culture of inflamed cartilage blocks and chondrocytes transfected by interleukin-β1 receptor antagonist gene and transforming growth factor-β1 gene in vitro

Objective To evaluate the in vitro effects of recombinant human interluekin-β1 receptor antagonist(IL-1Ra) gene and transforming growth factor-β1 (TGF-β1) gene on rabbit osteoarthritis (OA).Methods Articular cartilages were extracted from mature New Zealand rabbits and by enzyme digestion,isolated for chondrocytes which were then identified with specific extracellular matrix collagen type Ⅱ stained immunocytochemistry.The chondrocytes were divided into IL-1Ra-transfected group (group A), TGF-β1?transfected group (group B) , combined IL-1Ra- and TGF-β1-transfected group (group C) , untransfected group (group D) and the blank control group (group E).LipofectamineTM 2000 Reagent was used as the vehicle for transfection among groups A, B and C.All the groups of chondrocytes were co-cultured with fragmented articular cartilages and added with 20 ng IL-β 1?expect for group E.The transgenic expression of chondrocytes was detected under fluorescence microscope at 12h,24h,2d,4d and 6 d after transfection and co-culture.In addition, radioimmunoassay (RIA) was used to determine the levels of IL-1βand TNF-α in each group at 2 d, 4 d and 6 d after transfection and co-culture.Results The chondrocytes were successfully isolated and cultured.Collagen type Ⅱ stained immunocytochemistry showed the brownish - yellow cytoplasm and unstained chromophobic nuclei.Under fluorescence microscope, the expression of enhanced green fluorescent protein was observed in groups A, BandC, which peaked at 24 hours after transfection (16.16±2.71)% vs (16.54±2.91)% vs (17.20±2.39)% and gradually declined 2 d later.At any time spots, the IL-1βevel was highest in group D, followed by group B, group A, group C, and group E.The level of TNF-a in each group was ordered by group D＞group A＞group B＞group C＞group E on days 2 and 6, and by group E＞group A＞group B＞group C＞group D on day 4.The level of TNF-α in group A was slightly higher than that of group B, but the difference was not statistical significance.There were statistical difference among the other groups.The expressions of IL-1βand TNF-α in groups A, B and C were significantly lower on day 6 than those on days 2 and 4.The level of IL-1βin groups D and E did not change with time, while the level of TNF -α was the lowest in group D and highest in group E on day 4.ConclusionsTransfection with IL-1Ra or TGF-β1 can reduce the level of inflammatory cytokines.Combined use of IL-1Ra and TGF-β1 genes may show control of inflammatory response and provide evidences for gene therapy of osteoarthritis.     

高能量Pilon骨折手术时机及方法选择探讨

目的 探讨高能量Pilon骨折手术时机及治疗方法的选择.方法 25例高能量Pilon骨折患者采用解剖型钢板进行急诊或延期手术治疗,骨折类型:Rüedi-Allg(o)wer骨折分型Ⅱ型4例、Ⅲ型21例;开放性骨折8例,闭合性骨折17例.8例开放性骨折和6例闭合骨折采用急诊清创复位、解剖型钢板内固定术,肿胀严重的11例闭合型骨折延期内固定术,合并腓骨骨折进行相应处理,术后平均随访32个月,根据Mazur等制定的踝关节症状与功能评分系统对手术关节进行功能评估.结果 手术关节功能评估:优10例、良11例、可3例、差1例,优良率84%;术后并发症:感染2例,皮肤坏死1例,创伤性关节炎2例.结论 高能量Pilon骨折根据不同的骨折类型、软组织损伤程度选择正确的手术方式和手术时机,可取得良好的治疗效果.     

股骨近端髓内钉在股骨转子周围骨折中的应用

目的探讨应用股骨近端髓内钉(PFN)治疗股骨转子周围骨折的疗效。方法应用PFN治疗股骨转子周围骨折48例。结果44例经4～18个月随访，平均11个月，所有患者均获得骨性愈合。术后骨折延迟愈合伴股骨颈螺钉退出1例，出现股骨颈螺钉退出、同时髋螺钉向内侧切出1例。根据Harris髋关节评分，优良率达90．9％。结论PFN治疗股骨转子周围骨折具有手术创伤小、固定可靠，应力分布分散、防旋转功能，可早期功能锻炼，其并发症可通过提高手术技术避免，是固定股骨转子周围骨折较理想的内固定物。     

Study on chondrogenic differentiation of canine mesenchymal stem cells induced by Type 2 recombinant adeno-associated viral mediated transfer of hTGF-β1

Objective To investigate the potential application of human transforming growth factor-beta-1 (hTGF-β1) gene mediated by type 2 recombinant adeno-associated virus (rAAV2) vector inducing chondrogenic differentiation of canine mesenchymal stem cells (MSCs) in vitro. Methods Canine MSCs from bone marrow were isolated and cultured in vitro by density gradient centrifngation and adherence screening methods. The morphology of MSCs was observed by inverted phase contrast microscope and Giemsa stain. Flow eytometry was used to detect surface antigens of MSCs, The third generation of MSCs were transfected by rAAV2-hTGF-β1 with or without MOI of 1 ×105 v.g./cell or 5×105 v.g./cell. The expression of hTGF-β1 was detected by Western blot after 10 days, and TGF-β1 synthesis was determined by ELISA at 3, 6 and 9 day, respectively. After 2 weeks of culturing, mRNA expressions of type Ⅱ collagen and aggrecan were determined by RT-PCR and the collagen Ⅱ protein was detected by immunocytochemistry. Results The MSCs appeared to be morphologically spindle-shaped and showed active capability of proliferation both in primary and passage generations. Flow cytometry analysis indicated that MSCs were universally positive for CD29, CD44 and CD105, but negative for CD34 and CD45. TGF-β1 expression can be observed by Western blot after 10 days in two transfection groups, MOI of 5 × 105 group and MOI of 1× 105 group. With the extension of time, the contents of hTGF-β1 increased in the two groups detected by ELISA, while there was a significant difference between them two (P < 0.01). After 2 weeks of transfection of MSCs by rAAV2-hTGF-β1, the expression of collagen Ⅱ and Aggreacan mRNAs were positive. It also showed positive of collagen Ⅱ detected by immunocytochemistry. Conclusion Canine MSCs show chondrogenesis differentiation after induction by Type 2 rAAV mediated transfer of TGF-β1 gene. The process is a potential application for cartilage tissue engineering.     

对桡骨远端粉碎性骨折及关节内骨折患者手术结合中药治疗的疗效评价

目的：评价手术结合中药治疗桡骨远端粉碎性骨折及关节内骨折的方法及疗效。方法：综合分析2000年4月-2003年4月采用不同方法治疗桡骨远端粉碎性骨折及关节内骨折患者的治疗效果。32例患者根据AO原则进行分类，按不同类型骨析相应采用钛板螺钉内固定、简单内固定加外固定架固定及单纯外固定架固定手术治疗，其中7例患者术后加中药外敷。结果：随访了全部患者，功能满意者26例，占81．25％；功能尚可者5例，占15．62％；功能不满意者1例，占3．13％。加敷中药者x线片显示骨折愈合时间及住院时间均显著缩短。结论：经合理的手术方法治疗桡骨远端粉碎性骨折及关节内骨折，能最大限度地恢复桡(尺)骨的相对长度、关节面平整、掌倾角及尺偏角，术后配合合理的锻炼，可以使腕关节功能得到最大恢复，加敷中药有助于骨折的愈合。