西司他丁钠+亚胺培南消除细胞培养中细菌污染的研究

目的 :研究西司他丁钠 +亚胺培南 (泰能 )清除细胞培养中细菌污染的可行性。 方法 :将 1 5批细胞培养中已发生细菌污染的细胞 ,用含泰能 1 0～ 1 0 0 μg/ ml的 PBS洗涤 3次 (PBS用量逐次递增 ) ,第 3次洗涤之前将细胞悬浮于含 1 0 0 μg/ m l泰能的完全培养液 1 .5 ml中 37℃孵育 30 min;然后细胞移入含 1 0 0 μg/ m l泰能的完全培养液中培养 ,更换含 1 0 0 μg/ ml泰能的完全培养液 1次 / d,共 3d。结果 :成功消除 1 4批细菌感染 ,另 1批加用万古霉素后亦获成功。结论 :泰能可以有效消除细胞培养中的细菌污染 ,其剂型和包装等尚可改进     

融合蛋白诱导机体免疫的现状与思考

融合蛋白是利用基因工程技术构建的人工蛋白,可以优化蛋白性能,甚至产生新功能。在抗肿瘤和抗寄生虫病免疫等方面具有巨大的应用前景。面对众多研究策略,本文试图从如何有效激发机体免疫,尤其是从细胞免疫角度,探讨融合蛋白的研究现状、作用机制、存在问题和可能的发展方向。     

硅胶膜管联合组织工程骨修复兔桡骨缺损实验研究

目的:探讨硅胶膜复合组织工程骨修复兔桡骨缺损的效果.方法:在新西兰大白兔左、右侧桡骨中段制作长1.5 cm的骨与骨膜缺损,左侧采用硅胶膜包裹组织工程骨修复缺损为实验组,右侧单纯用组织工程骨修复缺损为对照组;术后3、6、9个月通过肉眼观察,组织学观察和生物力学检测,评价硅胶膜复合组织工程骨修复兔桡骨缺损的效果.结果:术后3个月实验组可见局部成熟的成骨区域,优于对照组;实验组6个月成熟成骨区域增大,可见明显血管;实验组9个月成骨区域基本上都是成熟胶原纤维,且结构排列规则,血管明显;生物力学检测发现实验组的压缩刚度、抗扭转刚度、三点弯曲最大载荷、抗折弯刚度都明显优于对照组.结论:硅胶膜包裹组织工程骨修复骨缺损效果良好,不但能促进骨组织的生长和成熟,并且能明显提高骨组织的力学性能;硅胶膜除了可以限制纤维组织的长入,还可以引导血管的长入和其他营养物质聚集在组织工程骨内,很好地发挥屏障和引导作用.     

大鼠雪旺细胞对两种来源的成骨细胞增殖分化影响的研究

目的 研究应用雪旺细胞(SCs)对两种来源的成骨细胞(OB)的增殖与分化的影响,为构建神经化组织工程骨提供体外实验理论依据.方法 通过采用股骨、胫骨髓腔冲洗法获得SD大鼠骨髓基质干细胞(BMSCs),采用胰蛋白酶消化新生鼠颅骨获得OB及采用消化后组织块法获得SCs.将BMSCs在成骨诱导液作用3周,鉴定BMSCs向OB分化.采用96孔共培养板将第2代SCs种植于上室,颅骨来源OB种植下室,共培养为实验组,无SCs干预共培养为对照组.将诱导分化的OB种植于下室,第2代SCs种植于上室,共培养为实验组,无SCs干预共培养为对照组,分别于共培养后1、3、5、7、9d采用甲基噻唑基四唑(MTT)进行增殖比较.采用6孔共培养板,实验组上室种植SCs,下室分别种植颅骨来源及BMSCs来源的OB,不加SCs的两种来源的OB作为对照组,分别共培养后于3、7 d检测两种来源OB的碱性磷酸酶、骨钙素、骨桥蛋白、骨形态发生蛋白-2 mRNA表达.结果 SCs对颅骨来源的OB在共培养3、5、7、9 d 4个时间点均有明显促增殖作用,在碱性磷酸酶、骨桥蛋白、骨形态发生蛋白-2mRNA在各个时间段均起到抑制作用.SCs对大鼠BMSCs来源的OB在共培养1、3 d无明显促增殖作用,在5、7、9 d有明显促增殖作用,在成骨培养基的环境里,SCs可促进BMSCs诱导的OB分化.结论 选择BMSCs来源的OB 与 SCs共培养更适合用于构建神经化组织工程骨.     

血管束植入组织工程骨修复兔股骨缺损对血管内皮生长因子表达的影响

目的 探讨血管束植入组织工程骨修复兔股骨缺损对局部血管内皮生长因了(VEGF)表达的影响.方法 将兔自体骨髓基质下细胞经诱导后与β-磷酸三钙材料复合,植入制备的兔股骨缺损处,其中实验组在材料侧槽中植入股骨的动静脉血管束,对照组则单纯植入组织工稗骨,分别于术后2、4、8、12周通过形态学检测新生骨量,免疫组织化学方法检测新生骨中VEGF的表达量.结果 随着时间进展,各组成骨量逐渐增加,差异有统计学意义(P<0.05),并且从第4周开始实验组成骨量高于对照组,差异有统计学意义(P<0.05);符时问点实验组新生骨中VEGF的表达最均高于对照组,差异有统汁学意义(P<0.05),且4周时达到最人值.结论 血管束植入组织工程骨可明显增加新生骨中VEGF的表达并能促进骨缺损的修复.     

恒河猴体内组织工程骨血管化监测的实验研究

目的探讨不同方法监测组织工程骨血管化的优缺点,寻找较好的监测方法。方法在13只成年恒河猴的25侧胫骨上制备2cm的骨膜与骨缺损模型,根据骨缺损充填材料不同随机分为五组(每组5侧),A组：β-磷酸三钙(β-TCP) 骨髓基质下细胞(BMSCs) 血管束；B组：β-TCP 血管束；C组：β-TCP BMSCs；D组：β-TCP；E组：空白对照。术后4、8、12周行磁共振灌注成像检查并计算信号强度-时间(SI-T)曲线的最大线性斜率(SS_(max))和基线值(SI_(baseline)),拍摄恒河猴胫骨X线片并计算其透光度,行放射性核素骨显像检查并计算摄取比值,同时进行组织学检查。结果术后4、8、12周A组的SS_(max)值最高,术后8周与4周相比SS_(max)有较大幅度的提高(P=0．003)。术后12周A组5个样本的SS_(max)与X线片透光度呈负相关(rs=～0．892,P=0．042),与血管面积比值呈正相关(rs=0．894,P=0．041)。结论SI-T曲线的SS_(max)能够准确地反映组织工程骨的血管化情况,磁共振灌注成像检查具有无创、无辐射、高灵敏度和定量分析的优点。     

不同浓度5-溴脱氧尿嘧啶核苷标记山羊骨髓基质干细胞的体外研究

Objective To explore the feasibility of labeling and tracing in vitro goat bone marrow mesenchymal stem cells (BMSCs) by bromodeoxyuridine (BrdU) on the basis of investigation of its optimal concentration, incubating time and cytotoxicity. Methods A healthy goat, aged 10 months old, male, weighing 32 kg, was used in this study. Bone marrow was aspirated. BMSCs were isolated and cultured using the adherence method in vitro. The fourth passage of BMSCs (P4) were incubated with BrdU at 5, 10, 15, 20 μmol/L as 5, 10, 15, 20 μmol/L BMSC groups. Cells were not labeled by BrdU as negative control. The following parameters were measured: induction, differentiation and determination of goat BMSCs; the optimal mass concentration and incubation time of 5-BrdU labeling; cell positive rate at 12, 24, 48 and 72 hours in each group using immunofluoreseenee; the cell survival rate after various concentrations of BrdU ladling by trypan-blue exclusion. Results The morphology of the primary and passage goat BMSCs was fusiform in shape. Goat BMSCs could differentiate into osteoblasts and chondrocytes following induction. BMSC nucleus showed green fluorescence under fluorescence microscope after being labeled by BrdU. The mean labeling rate increased with the increase in the concentration and incubation time of BrdU, and reached to (93.32± 3.25)% after incubation in 15 μmol/L, BrdU for 48 hours. There were no significant differences between 15 μmol/L BrdU for 72 hours, 20 μmol/L BrdU for 48 hours and 72 hours (P > 0.05), or between the other groups or time points (P < 0.05). The labeling rate of the blank control group was 0. The cell survival rate was all above 90% (P > 0.05). Conclusions BrdU can be used as a labeling marker for goat BMSCs. When the concentration is 15 μmol/L and the incubation time is 48 hours, the optimal labeling effect can be achieved. Goat BMSCs labeled with BrdU is of high efficiency and safety.     

增强型生物活性玻璃-胶原复合支架材料的成骨效能研究

Objective To study the in vivo and vitro biocompatibility and osteogenetic capacity of enhanced bioactive glass/collagen composite scaffold. Methods Bone marrow stromal cells(BMSCs)were collected and induced to osteoblast-like cells.The growth rate of BMSCs was detected and compared progressively through Alamar Blue.The RNAs of the cells were collected and detected for bone morphogenetic protein-2(BMP-2),alkaline phosphatase(ALP),collagen Ⅰ(Col-Ⅰ)through qRT-PCR on the fourth and seventh days.Scaffolds with induced osteoblasts were embedded into 3 nude mice subcutaneously in vivo and detected after 6 weeks.X-ray,qRT-PCR and tissue staining were used to detect the mRNA expressions of BMP-2,Col Ⅰ,osteocalcin(OCN)and ostcopontin(OPN)and bone formation. Results SEM(scanning electronic microscopy)showed BMSCs attached to the scaffold tightly and viably and proliferated actively on the scaffold.The growth rate in the experimental group was significantly higher after 7 days(P<0.05)than in the control group.qRT-PCR showed that the mRNA expressions of BMP-2,ALP and Col-Ⅰ in the experimental group were significantly higher than in the control group on the seventh day(P<0.05).X-ray showed that the dense images of embedded scaffolds were locally similar to those of normal bone after 6 weeks.qRT-PCR showed that the mRNA expressions of BMP-2,Col Ⅰ,OCN and OPN in the experimental group were significantly higher than those of normal bone(P<0.05).HE and Massort staining of the paraffin sections showed the scaffolds degraded generally and osteoblasts and chondrocytes proliferated abundantly and distributed irregularly.Bone formation could be observed obviously. Conclusion Enhanced bioactive glass/collagen composite scaffolds have good biocompatibility and osteogenetic capacity in vitro and vivo.     

不同比例激活富血小板血浆对其生长因子浓度及人骨髓基质干细胞增殖的影响

目的 确定激活剂凝血酶与富血小板血浆(PRP)混合的最适比例,并探讨凝血酶与PRP中血小板衍生生长因子.AB(PDGF.AB)和转化生长因子-β(TGF-β)对人骨髓基质干细胞(hBMSCs)增殖的联合作用.方法 按1000 U凝血酶溶于1 mL质量百分比为10%的CaCl2配制激活剂.按PRP与激活剂混合比例为1:1、5:1、10:1、20:1、40:1分为A、B、C、D、E 5组.ELISA法测定各组0、1、8、24、120 h后PRP凝胶中PDGF-AB、TGF-β1的浓度;MTT法评价各组PRP凝胶上清对hBMSCs增殖的影响.结果 PRP与凝血酶混合后,PDGF-AB、TGF-β1浓度立即升高,在激活1 h后达到高峰.B、C、D组PRP凝胶中PDGF-AB、TGF-β1的浓度最高,并明显促进hBMSCs的增殖;而A组则抑制hBMSCs的增殖.结论 PRP对hBMSCs增殖的作用与其所含有PDGF-AB、TGF-β1存在浓度依赖性,而凝血酶可能在一定浓度范尉内主要通过对PRP中生长因子浓度的调控来影响hBMSCs的增殖,但是当凝血酶浓度过高时则抑制hBMSCs的增殖.     

单纯血管束、神经束分别植入组织工程骨修复兔骨缺损对降钙素基因相关肽和神经肽Y表达的影响

目的 比较单纯血管、神经束分别植入组织工程骨修复兔骨缺损对神经肽降钙素基因相关肽(calcitonin gene related peptide,CGRP)和神经肽Y(neuropeptide Y,NPY)表达影响. 方法 36只新西兰大白兔,随机平均分为3组:A组为组织工程骨+股血管束植入,B组为组织工程骨+感觉神经束植入,C组为单纯组织工程骨.每只动物均在右侧股骨制作长1.5 cm的段缺性骨与骨膜缺损,钢板固定后分别用以上方法修复大段骨缺损.术后3、6、12个月行免疫组化染色检测修复骨段内的CGRP和NPY的表达,并采用图像分析软件行半定量分析. 结果 CGRP和NPY的表达量在A、B、C 3组均随时间推移而增多(P=0.000),在3个月时A组较B组多(P=0.000),但在6个月和12个月时A组和B组的表达量无明显差异(P>0.05).A、B两组在3、6、12个月时的表达最均较C组多(P=0.000). 结论 与单纯神经束植入组相比,单纯血管束植入组织工程骨修复兔骨缺损在术后3个月可以明显促进CGRP和NPY表达,但后期两组对神经肽表达的影响作用无明显差异.