Experimental Study on Allogenic Decalcified Bone Matrix as Carrier for Bone Tissue Engineering

The biocompatibility and osteogenic activity of allogenic decalcified bone matrix (DBM) used as a carrier for bone tissue engineering were studied. Following the method described by Urist, allogenic DBM was made. In vitro, DBM and bone marrow stromal cell (BMSC) from rab-bits were co-cultured for 3-7 days and subjected to HE staining, and a series of histomorphological observations were performed under phase-contrast microscopy and scanning electron microscopy (SEM). In vivo the mixture of DBM/BMSC co-cultured for 3 days was planted into one side of muscules sacrospinalis of rabbits, and the DBM without BMSC was planted into other side as con-trol. Specimens were collected at postoperative week 1, 2 and 4, and subjected to HE staining, and observed under SEM. The results showed during culture in vitro, the BMSCs adherent to the wall of DBM grew, proliferated and had secretive activity. The in vivo experiment revealed that BMSCs and undifferentiated mesenchymal cells in the perivascular region invaded gradually and proliferated together in DBM/BMSC group, and colony-forming units of chondrocytes were found. Osteoblasts,trabecular bone and medullary cavity appeared. The inflammatory reaction around muscles almost disappeared at the second weeks. In pure DBM group, the similar changes appeared from the sur-face of the DBM to center, and the volume of total regenerate bones was less than the DBM/BMSC group at the same time. The results indicated that the mixture of DBM and BMSC had good bio-compatibility and ectopic induced osteogentic activty.     

大鼠骨髓间充质干细胞转染人TH基因的实验研究

目的评价酪氨酸羟化酶(tyrosine hydroxylase,TH)基因修饰骨髓间充质干细胞(mesenchymalstem cells,MSCs)后TH的表达情况及转染TH基因对MSCs的影响.方法构建含人TH基因的pCMV/hTH质粒,用脂质体转染原代培养的大鼠MSCs;Western blot及免疫细胞化学染色鉴定TH基因的表达及MSCs的分化情况;MTT比色法测定转染后的MSCs细胞活性,并与未转染的MSCs进行细胞活性比较.结果构建的pCMV/hTH质粒经ECoRI酶切后产生1.9okb和5.3kb的片段,与回收的目的基因及载体基因片段大小相符;转基因后的MSCs Western blot及免疫细胞化学染色显示TH染色阳性;转染后MSCs未见有NeuN,GFAP的表达;MTT比色法测定细胞活性,未转染与转染者差异无显著性.结论构建的TH基因能在体外培养的鼠MSCs中较好的表达,转染不会诱导MSCs向神经样细胞分化,对MSCs细胞活性无明显影响.     

Effect of Ligustrazine on Bone Marrow Microenvironment and Signal Transducti on Path in Irradiation Injured Mice

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Bone marrow punctures and pain

We prospectively analysed pain in 263 patients induced by a frequent diagnostic procedure for oncologists, specifically the bone marrow puncture. Substantial pain (5 and more out of 10 on a numerical rating scale) was reported by 30.4% of patients, but physicians did not realize this procedure-related pain of patients in more than 50% of such punctures. The necessity for improved analgesia is emphasized by the fact, that at least 50% of patients experiencing substantial pain wished to receive concomitant medication in future punctures. Duration of the procedure was identified as sole independent predictive factor for patients’ pain intensity, while patients’ characteristics like gender, age and body-mass index (BMI) played only a minor role. As premedication with analgesics or anxiolytics may be associated with significant side-effects and an early identification of patients prone to experience severe pain is therefore difficult, further studies are warranted to establish an adequate approach in terms of pain control and feasibility in an ambulatory setting. In the meantime, daily physicians’ practice should be changed, as a pain-focused patient interview and presented indicators can be used in order to increase physicians’ awareness to procedure-related pain and augment their application of analgesics.     

快速富集骨髓干细胞复合羟基磷灰石三钙在胸腰椎骨折中的应用

胸腰椎骨折在临床上是脊柱骨折的好发部位,后路手术多采用椎弓根钉系统复位固定加椎板间或横突间植骨融合。后外侧融合是降低内固定失败、减少纠正丢失等并发症的有效措施。传统植骨采用自体髂骨,也有运用同种异体骨,但有一定并发症发生率。近年来有单纯运用骨髓移植治疗骨缺损     

骨髓间充质干细胞输对移植肾的保护作用

目的体外培养大鼠骨髓间充质干细胞（MSCs），探讨其对移植肾的保护作用。方法无菌条件下取大鼠股骨、胫骨，用直接贴壁法分离纯化MSCs，体外扩增，在注射MSCs的基础上，行同种异体肾移植，实验大鼠分为注射大鼠骨髓MSCs肾移植组（Ⅰ组）和单纯肾移植组（Ⅱ组），环孢素A治疗肾移植组（Ⅲ组）术后5d结扎受体右肾动脉（左侧为移植肾）；观察右肾动脉结扎术后受体存活时间，检测右肾术后移植肾的形态学变化，肌酐水平。结果Ⅰ组、Ⅲ组大鼠生存期（15．50±6．35）d和（18．50±5．97）d较Ⅱ组（6．50±3．11）d明显延长，且前者移植肾形态学观察和功能状态均明显优于后者。结论骨髓间充质干细胞在体外很容易分离培养和扩增，骨髓间充质干细胞对早期移植肾具有保护作用。     

生长板软骨细胞促进骨髓基质干细胞血管内皮生长因子的表达

目的观察骨髓基质干细胞(BMSCs)在生长板软骨细胞旁分泌作用下血管内皮生长因子(VEGF)的表达规律及其与成骨分化的相关性。方法大鼠BMSCs与生长板软骨细胞进行间接共培养,培养终末期做细胞化学染色,定量测定碱性磷酸酶(ALP)活性,用RT-PCR方法半定量检测VEGFmRNA的表达。结果生长板软骨细胞持续高表达VEGF。BMSCs随共培养时间的延长,ALP活性升高,BMSCs的VEGF的表达也逐渐增强。培养液加入两种分泌型VEGF中和抗体后,VEGF表达趋势不变,ALP活性仍为升高趋势,也不影响培养终末期钙化结节的形成。培养终末期BMSCs的CD31和CD34均阴性。结论BMSCs成骨分化过程中VEGF的表达符合成骨细胞分化基因的表达规律,与成骨细胞特征性基因的表达趋势一致,体外条件共培养条件下,中和VEGF后并不能阻碍BMSCs的成骨分化。     

骨髓输液在PICU的应用探讨

目的　探讨骨髓输液在PICU的适应证、方法及临床效果。方法　选择PICU危重症建立静脉通道困难患儿 30例 ,采用 7号骨穿针或 7～ 9号头皮针于胫骨粗隆下 1～ 2cm穿刺、固定 ,接入医嘱液体 ,记录穿刺所需时间、入液速度及生命体征变化、并发症等。结果　 2 8例 1次成功 ,2例用头皮针者有堵塞 ,换针后重新穿刺成功 ,穿刺、固定到接入液体平均时间 (30± 10 )s。速率 :一般压力 (8± 3)ml (kg体重·h) ,加压下 (17± 6 )ml (kg体重·h) ,所有病例均达到了医嘱要求。骨髓输液持续时间 3～ 2 2h ,无 1例出现并发症。结论　骨髓输液在PICU危重症抢救中可迅速建立液体通道 ,争取抢救时间。头皮针比骨髓穿刺针易于固定 ,使用更方便     

骨髓间充质细胞与速固化磷酸钙支架共培养的实验研究

[目的]观察人骨髓间充质干细胞在速固化磷酸钙骨水泥支架中的增殖和生长情况。[方法]将速固化壳聚糖-磷酸钙骨水泥支架材料预先成形。采用全骨髓法分离人骨髓间充质细胞并在体外对细胞进行培养和扩增。72h首次换液,细胞融合80%传代,取第3代细胞用于实验。将细胞与磷酸钙骨水泥支架复合培养,分别利用四甲基偶氮唑蓝MTT法及扫描电镜等检测细胞在材料中的增殖和生长情况。[结果]用酶标仪检测OD值提示共培养后,支架内的细胞数量逐渐增加。扫描电镜结果显示支架材料内部为多孔结构,孔内见有细胞生长,细胞表面可见分泌颗粒。[结论]速固化壳聚糖-磷酸钙骨水泥对人骨髓间充质干细胞有较好的细胞相容性,细胞可在材料内黏附和增殖,是理想的骨组织工程支架材料。     

菲立磁标记兔BMSCs自体移植脊髓后的核磁共振活体示踪及形态学研究

目的通过观察菲立磁标记兔骨髓源性神经干细胞(BMSCs)自体移植脊髓后的核磁共振活体示踪及形态,期待找到一种应用非侵袭性方法来识别、跟踪BMSCs的存活状态及与宿主组织整合情况的方法。方法无菌条件下股骨取骨髓,梯度密度离心法分离获取兔骨髓基质细胞;使用“Feridex-多聚赖氨酸复合物(FE-PLL)”标记骨髓基质细胞,采用普鲁士兰染色和台盼蓝排除实验等方法鉴定FE-PLL标记兔骨髓基质细胞的效率和细胞的活力;体外标记的细胞自体脊髓移植,磁共振、免疫组织化学染色和透射电镜检查。结果普鲁士蓝染色显示FE-PLL标记骨髓基质细胞胞质内出现细小的蓝色铁颗粒;与正常未标记的细胞相比较,FE-PLL标记对骨髓基质细胞的活力、增殖和分化等能力没有明显的影响;经菲立磁标记的兔BMSCs自体脊髓移植后,可在核磁共振上活体示踪。结论菲立磁与核磁共振联合可无创性活体标记检测移植的神经干细胞基本的存在部位、存在方式及其一些生物学特性,可以用来活体示踪移植的BMSCs。