人骨髓基质干细胞与支架材料纳米羟基磷灰石／聚酰胺66的生物相容性研究北大核心CSCD

目的研究人骨髓基质干细胞（hBMSCs）与纳米羟基磷灰石／聚酰胺66（nHA／PA66）复合后的生物学特性。方法体外分离培养hBMSCs后将其分为A、B、C3组：A组为未经骨向诱导的hBMSCs,B组为经骨向诱导的hBMSCs,C组为与nHA／PA66复合并骨向诱导的hBMSCs。通过噻唑蓝（MTT）检测B、c组细胞在第1、2、3周的增殖情况;电镜扫描观察C组细胞在支架上的黏附情况;碱性磷酸酶（ALP）活性检测以及矿化结节染色验证hBMSCs的体外成骨分化能力,并通过检测第6、12天ALP的活性研究其与nHA／PA66复合后的骨生成作用。结果c组细胞在nHA／PA66上黏附良好;B组与C组细胞生长增殖活性差异无统计学意义（P〉0．05）,说明骨向诱导后的hBMSCs增殖活性不受支架材料nHA／PA66影响;B组矿化结节数量明显多于A组,第6、12天A组ALP活性值均低于B、c组,差异有统计学意义（P〈0．05）,而B、c组问差异无统计学意义（P〉0．05）,说明hBMSCs体外骨向诱导后ALP表达增强并骨向分化,nHA／PA66并没有影响hBMSCs的成骨潜能。B、c组细胞在第12天的ALP活性值显著高于第6天,差异均有统计学意义（P〈0．05）,说明AKP活性随骨向诱导时间增长而逐渐增强。结论hBMSCs适于在nHA／PA66上黏附、增殖与骨向分化,nHA／PA66可以作为hBMSCs的载体应用于骨组织工程。     

含锶羟基磷灰石对骨质疏松症大鼠的影响

目的研究含锶羟基磷灰石对骨质疏松症大鼠的影响。方法制备羟基磷灰石（HA)和10%含锶羟基磷灰石（10% SrHA,即HA中Sr与Ca的摩尔比例为1：9)种植体,以扫描电镜(SEM)、能量色散X射线能谱仪(EDX)、X射线衍射(XRD)检 测材料表面形貌特征。实验中将20只雌性SD大鼠均行双侧卵巢切除术,12周后将所有动物随机平均分为两组,并分别在 大鼠右侧股骨远端植人羟基磷灰石(HA)种植体和10%含锶羟基磷灰石（10% SrHA)种植体。植人术后8周,收集标本行显 微CT( micro-CT)和组织学分析。结果实验所得数据采用SPSS17. 0软件进行统计学分析。在micro~CT检验的结果中,相比 于HA种植体组,10% SrHA种植体组的相对骨体积提高了 42. 6%,骨结合率提高了 47. 1%,平均骨小梁数量提高了 27. 3%, 平均骨小梁厚度提高了 31. 5%,平均骨小梁连接密度提高了 37. 4%,而平均骨小梁间距降低了 20. 1%。在组织学检验结果 中,10%SrHA组的相对骨面积密度提高了 47. 6%。结论以HA种植体作为对照,10% SrHA种植体可以提高骨密度和骨结 合率,同时改善种植体周的骨小梁微结构。     

人骨髓基质干细胞与表面置换珊瑚羟基磷灰石培养的定向诱导分化

目的观察人骨髓基质干细胞（hMSC）与表面置换的珊瑚羟基磷灰石（SCHA）体外培养的细胞粘附、增殖和分化,寻找理想的骨修复材料。方法海南天然滨珊瑚在特定温度和压力下部分水热反应,制成表面置换的珊瑚羟基磷灰石,将SCHA薄片与人骨髓基质干细胞体外培养,经诱导后于4、8、12、16d分别用荧光显微镜和扫描电镜（SEM）观察细胞活性、粘附和分化过程。结果SCHA保留原有的珊瑚贯穿多孔的三维结构。荧光显微镜可见hMSC在SCHA的表面和孔道内生长良好,第16d达最高水平;电镜显示细胞粘附良好,分化为成骨细胞,分泌大量胶原纤维,可见合成的钙结节。结论hMSC在表面置换的珊瑚羟基磷灰石内粘附、增殖、分化良好,两者具有较好的生物相容性,SCHA是一种良好的骨组织工程支架材料。     

羟基磷灰石掺锶对成骨细胞增殖分化及矿化影响的实验研究

[目的]探讨羟基磷灰石(HA)中掺锶对成骨细胞增殖、分化和矿化的影响,以及HA材料中适宜的掺锶量。[方法]用掺锶量分别为1%,5%和10%的HA生物陶瓷粉末及纯HA生物陶瓷粉末制备的浸提液培养SD大鼠成骨细胞,在不同时间点检测成骨细胞增殖、碱性磷酸酶(ALP)活性、核心结合因子(cbfal)基因表达,以及矿化结节形成的情况。[结果]各组成骨细胞的增殖无明显差别(P0.05);细胞培养第14、21d,掺锶各组成骨细胞的ALP活性、cbfalmRNA表达,及矿化结节数量等方面均显著高于HA组(P0.01),其中以掺锶量5%组最高,但与10%组相比无显著差异。[结论]HA中掺锶能上调成骨细胞cbfalmRNA的表达,促进成骨细胞分泌碱性磷酸酶,从而促进成骨细胞的分化、矿化,促进骨形成。详细的作用机制及HA中最佳的掺锶量尚需进一步的研究。     

骨髓基质干细胞复合纳米羟基磷灰石/聚乳酸构建组织工程骨

目的：了解骨髓基质干细胞（MSCs）复合纳米羟基磷灰石／聚乳酸（n-HA／PLA）构建组织工程骨的异位成骨作用。方法：选择6只新西兰白兔，实验组于动物脊柱左侧肌肉内植入MSCs复合n-HA／PLA构建的组织工程骨，对照组于右侧植入n-HA／PLA生物材料。术后4、8周取材，行溴脱氧尿嘧啶（Brdu）标记细胞检测和组织学观察。结果：术后4周，实验组材料边缘均可检测到Brdu标记的MSCs。术后4、8周，实验组材料内部有新骨形成，随着时间推移，新骨量增多，编织骨向板层骨过渡。对照组材料未见新骨形成。结论：MSCs复合n-HA／PLA构建的组织工程骨具有很好的异位成骨作用。     

3D打印胶原/羟基磷灰石支架对骨髓间充质干细胞成骨分化的作用研究

目的探讨3D打印胶原/羟基磷灰石支架对骨髓间充质干细胞成骨分化的作用。 方法分离SPF级雄性SD大鼠骨髓间充质干细胞,实验分为：对照组、浸提组、诱导组及浸提诱导组,MTT法检测对照组,浸提组的细胞增殖情况,对比分析各组细胞的碱性磷酸酶（ALP）活性,对各组细胞诱导培养17天后进行茜素红染色,观察钙结节染色情况。 结果MTT结果显示,浸提组与对照组在不同时间点的OD值比较,差异无统计学意义（P>0.05）,ALP活性结果显示,不同时间点各组与对照组相比,差异均有统计学意义（P<0.05）;不同时间点浸提诱导组与浸提组相比,差异均有统计学意义（P<0.05）;诱导组在48 h及72 h与浸提组相比,差异均有统计学意义（P<0.05）。茜素红染色结果显示,对照组细胞无钙结节点,浸提组及诱导组镜下肉眼可明显观察到红色区域染色,镜下观察可见钙结节点,浸提诱导组所产生的钙结节点的数量、大小以及染色的颜色深度均明显优于其他各组。 结论3D打印胶原/羟基磷灰石支架具有生物相容性好,可促进BMSCs向成骨分化,对细胞毒性低等特点,适宜用作骨缺损的治疗。     

羟基磷灰石/二氧化锆复合材料颗粒对骨髓间充质干细胞增殖及成骨分化的影响

目的 采用分子生物学方法评价羟基磷灰石/二氧化锆(HA/ZrO2)复合材料颗粒对兔骨髓间充质干细胞(MSCs)增殖及成骨分化的影响.方法 按不同比例将纳米HA粉体与纳米ZrO2粉体混合在高温下烧结制备HA/ZrO2复合材料颗粒,分离和培养兔MSCs.MTT法检测复合材料颗粒悬液对兔MSCs增殖促进作用,ALP法测定碱性磷酸酶活性,RT-PCR检测细胞Ⅰ型胶原(collagen Ⅰ),骨钙蛋白(osteocalcin),骨桥蛋白(osteopontin)mRNA表达.结果 HA和含有HA的复合材料颗粒均对细胞增殖有一定促进作用.Vonkossa染色显示复合材料和纯HA颗粒可使细胞阳染程度降低.细胞在HA和含有HA的复合材料颗粒的成骨诱导培养液中的ALP活性均显著高于常规培养基质及纯ZrO2组(P＜0.05).RT-PCR结果显示复合材料颗粒能够促进collagen Ⅰ和osteocalcin基因的表达.结论 HA/ZrO2复合材料颗粒可促进骨髓间充质干细胞增殖及成骨分化.

Abstract：

Objective To evaluate the effect of the HA/ZrO2 composite particle on proliferation and osteogenesis of rabbit mesenchymal stem cells (MSCs) by using molecular biology methods.Methods The HA/ZrO2 composite particles were sintered at high temperature using the powder of HA and ZrO2 with different proportions.MSCs were isolated from rabbits and cultured.The effect of the composite particles on promoting cell proliferation of rabbit MSCs was detected using MTT method.Alkaline phosphatase activities were measured with ALP method.RT-PCR method was applied to measure the expression of collagen Ⅰ , osteocalcin and osteopontin mRNA.Results Pure HA pauicles and composite particles which contain HA promoted MSCs proliferation.Vonkossa staining showed that both pure HA particks and composite particles decreased the degree of positive stain of MSCs.ALP test showed that cell activity in the culture medium that contained pure HA particles or composite particles was significantly higher than that in the conventional culture medium and that contained pure ZrO2 particles (P ＜ 0.05＝.RT-PCR results showed that composite particles promoted the expression of collagen Ⅰ and osteocalcin gene.Conclusion The HA/ZrO2 composite particles promote proliferation and osteogenesis of MSCs.     

羟基磷灰石/二氧化锆复合材料颗粒对骨髓间充质干细胞增殖及成骨分化的影响

Objective To evaluate the effect of the HA/ZrO2 composite particle on proliferation and osteogenesis of rabbit mesenchymal stem cells (MSCs) by using molecular biology methods.Methods The HA/ZrO2 composite particles were sintered at high temperature using the powder of HA and ZrO2 with different proportions.MSCs were isolated from rabbits and cultured.The effect of the composite particles on promoting cell proliferation of rabbit MSCs was detected using MTT method.Alkaline phosphatase activities were measured with ALP method.RT-PCR method was applied to measure the expression of collagen Ⅰ , osteocalcin and osteopontin mRNA.Results Pure HA pauicles and composite particles which contain HA promoted MSCs proliferation.Vonkossa staining showed that both pure HA particks and composite particles decreased the degree of positive stain of MSCs.ALP test showed that cell activity in the culture medium that contained pure HA particles or composite particles was significantly higher than that in the conventional culture medium and that contained pure ZrO2 particles (P ＜ 0.05＝.RT-PCR results showed that composite particles promoted the expression of collagen Ⅰ and osteocalcin gene.Conclusion The HA/ZrO2 composite particles promote proliferation and osteogenesis of MSCs.     

羟基磷灰石/二氧化锆复合材料颗粒对骨髓间充质干细胞增殖及成骨分化的影响

Objective To evaluate the effect of the HA/ZrO2 composite particle on proliferation and osteogenesis of rabbit mesenchymal stem cells (MSCs) by using molecular biology methods.Methods The HA/ZrO2 composite particles were sintered at high temperature using the powder of HA and ZrO2 with different proportions.MSCs were isolated from rabbits and cultured.The effect of the composite particles on promoting cell proliferation of rabbit MSCs was detected using MTT method.Alkaline phosphatase activities were measured with ALP method.RT-PCR method was applied to measure the expression of collagen Ⅰ , osteocalcin and osteopontin mRNA.Results Pure HA pauicles and composite particles which contain HA promoted MSCs proliferation.Vonkossa staining showed that both pure HA particks and composite particles decreased the degree of positive stain of MSCs.ALP test showed that cell activity in the culture medium that contained pure HA particles or composite particles was significantly higher than that in the conventional culture medium and that contained pure ZrO2 particles (P ＜ 0.05＝.RT-PCR results showed that composite particles promoted the expression of collagen Ⅰ and osteocalcin gene.Conclusion The HA/ZrO2 composite particles promote proliferation and osteogenesis of MSCs.     

辛伐他汀促进大鼠骨髓基质细胞的成骨分化

目的观察辛伐他汀体内给药对去卵巢大鼠骨髓基质细胞(BMSC)增殖和分化的影响,探讨辛伐他汀的成骨作用机制.方法24只12周龄雌性SD大鼠随机分成4组,每组6只假手术组(G1)和去卵巢组(G2)每天蒸馏水灌胃;去卵巢大鼠10mg·kg-1·d-1辛伐他汀灌胃(G3);去卵巢大鼠20mg·kg-1·d-1辛伐他汀灌胃(G4).实验持续4周,第29天处死所有大鼠取骨髓细胞培养.用Cell Counting Kit-8(CCK-8)法检测成骨细胞的增殖能力;细胞培养第14天用半定量RT-PCR和western blot检测Runx2/Cbfal mRNA和蛋白的表达、第16天检测碱性磷酸酶活性(ALP)和第21天检测茜素红染色.结果辛伐他汀不能促进去卵巢大鼠成骨细胞增殖.G3组和G4组的Runx 2/Cbfa1 mRNA表达水平和ALP活性都显著高于G2组,给药两组之间无显著差别.G4组的Runx 2/Cbfal蛋白表达水平比G2组和G3组显著增加,G2组与G3组无明显差别.茜素红染色表明辛伐他汀能促进成骨细胞的矿化能力,两种剂量组之间无明显差别.结论辛伐他汀体内给药能够促进去卵巢大鼠BMSC向成骨细胞分化,但是不能促进细胞增殖.     

成骨生长肽对大鼠骨髓基质细胞增殖和成骨分化的作用

目的：观察成骨生长肽诱导体外培养的大鼠骨髓基质细胞增殖及向成骨分化的作用。方法：取6周龄SD大鼠，贴壁法分离培养骨髓基质细胞，在不同浓度成骨生长肽的诱导下，观察细胞形态变化，绘制骨髓基质细胞生长曲线，碱性磷酸酶和钙结节组织化学染色。结果：成骨生长肽对骨髓基质细胞增殖和成骨分化的作用呈剂量依赖性：成骨生长肽浓度在10^-10及10^-11mol／L时促进骨髓基质细胞增殖，而在10^-8及10^-9mol／L时对骨髓基质细胞的增殖略有抑制作用；成骨生长肽浓度在10^-10及10^-11mol／L时骨髓基质细胞碱性磷酸酶染色基本呈阴性，与对照组相比差异无统计学意义，而在10^-8及10^-9mol／L浓度下可以显著提高骨髓基质细胞碱性磷酸酶染色的阳性率。结论：成骨生长肽可以明显促进大鼠骨髓基质细胞增殖及向成骨细胞分化，其促成骨活性具有显著的浓度依赖性。     

Electrophysiological study on differentiation of rat bone marrow stromal stem cells into neuron-like cells in vitro by edaravone

To explore the electrophysiological proper-ties of differentiation of rat bone marrow-derived stromal stem cells (rBMSCs) to neuron-like cells in vitro by edaravone, a new type of free radical scavenger. Methods: Stromal stem cells were separated from rat bone marrow with Ficoll-Paque reagent and expanded in different culture medium in vitro, rBMSCs were induced by edaravone containing serum-free L-DMEM. Morphologic observation and Western blot analysis including the ex-pression of Nav1.6, Kv1.2, Kv1.3, Cav1.2 were performed, and whole patch-clamp technique was used. Results: Cyton contraction and long processes were shown in differentiated stromal stem cells. Nav1.6, Kv1.2, Kv1.3 and Cav1.2 were expressed in both differentiated and undifferentiated cells. However, the expression of channel proteins in differentiated cells was up-regulated. Consistently, their resting potential and outward currents were also enhanced in the differentiated cells, which was especially significant in the outward rectifier potassium current. Conclusion: In vitro, neuron-like cells derived from rBMSCs, induced by edaravone, possess electrophysiologi-cal properties of neurons.     

骨髓基质细胞体内成骨分化研究

目的探讨将自体骨髓基质干细胞（BMSCs）移植到股骨头缺损坏死部位后能否成活、增殖,并向成骨分化,促进骨修复。方法取狗犬髂骨骨髓,体外培养扩增BMSCs,经5-溴脱氧尿嘧啶核苷（BrdU）标记,移植到股骨头骨缺损模型。术后5周取材。行大体组织观察,组织化学染色,免疫组化检测骨钙素、骨桥素。结果BMSCs能增加骨缺损区内的成骨量、提高骨基质矿化的程度和骨成熟度,提高成骨细胞分化特异性标记物骨钙素、骨桥素的表达。新骨形成越活跃的地方,BrdU阳性细胞数目越多,很多成骨细胞BrdU阳性,新骨形成区的很多血管壁也分布着BrdU阳性细胞。结论移植的骨髓细胞在股骨头内能成活,并能停留在骨坏死、缺损部位生长增殖。移植的BMSCs能分化成为多种细胞,包括成骨细胞、血管壁细胞等。BMSCs不但能直接通过分化成成骨细胞参与成骨,也能通过参与血管形成而促进成骨。     

缺氧状态对骨形态发生蛋白-2诱导分化体外复合富血小板血浆凝胶的骨髓间充质干细胞的影响

目的研究缺氧状态对骨形态发生蛋白-2(BMP-2)诱导分化体外复合富血小板血浆(PRP)凝胶的骨髓间充质干细胞(BMSCs)的影响。方法取第三代培养的BMSCs细胞与PRP凝胶相混合,通过构建细胞缺氧模型,根据培养条件的不同确定实验分组:常氧BMSCs+PRP组(A组)、常氧BMP-2+BMSCs+PRP组(B组)、低氧BMSCs+PRP组(C组)、低氧BMP-2+BMSCs+PRP组(D组)。反转录-聚合酶链反应法(RT-PCR)及Western-blotting检测各组成软骨及成骨基因的表达。结果 RT-PCR检测表明C组和D组的Ⅱ型胶原、蛋白聚糖和低氧诱导因子-1α(HIF-1α)表达均较A组和B组明显升高;Ⅰ型胶原和碱性磷酸酶(ALP)在B组的表达最高;A组与B组runt相关转录因子2(Runx2)的表达较C组和D组明显升高。Western-blotting检测结果表明B组和D组的Ⅱ型胶原表达较A组和C组明显升高;C组与D组的HIF-1α表达较A组与B组显著升高;D组的SOX-9表达较其余3组显著升高。所有结果差异均有统计学意义(P0.05)。结论低氧条件能显著促进BMP-2对BMSCs的成软骨诱导分化,同时抑制BMSCs的成骨分化。HIF-1α可能是这一过程的重要调节因子,其可能通过调节SOX-9的表达来发挥促BMSCs成软骨细胞分化的作用。     

体外诱导猕猴骨髓基质干细胞向雪旺细胞分化

目的探讨体外诱导猕猴骨髓基质干细胞向雪旺细胞分化的实验方法。方法采用BME、b-FGF、ATRA、BDNF、heregulin、Forskolin、PDGF依次联合诱导,倒置相差显微镜观察细胞形态学变化,免疫细胞化学鉴定S-100蛋白、GFAP和P75受体的表达率。结果诱导后的猕猴骨髓基质干细胞具有类似雪旺细胞的形态,免疫细胞化学结果显示S-100蛋白、GFAP和P75受体的表达率分别为(66.8±4.6)%、(57.3±5.4)%和(72.4±5.9)%。结论采用BME、ATRA、BDNF、heregulin、Forskolin、b-FGF、PDGF依次联合诱导,可使猕猴骨髓基质干细胞在体外向雪旺样细胞分化。     

Lack of telomerase activity in rabbit bone marrow stromal cells during differentiation along neural pathway

Objective: To investigate telomerase activity in rabbit bone marrow stromal cells ( BMSCs ) during their committed differentiation in vitro along neural pathway and the effect of glial cell line-derived neurotrophic factor (GDNF) on the expression of telomerase. Methods: BMSCs were acquired from rabbit marrow and divided into control group, GDNF (10 ng/ml) group. Cytokine·NSCs medium (prepared by our lab, Patent No. ZL02134314. 4) supplemented with 10% fetal bovine serum ( FBS) was used to induce BMSCs differentiation along neural pathway. Fluorescent immunocytochemistry was employed to identify the expressions of Nestin, neuronspecific endase (NSE), and gial fibrillary acidic protein (GFAP). The growth curves of the cells and the status of cell cycles were analyzed, respectively. During the differentiation, telomerase activitys were detected using the telomeric repeat amplification protocol-enzyme-linked immunosorbent assay (TRAP-ELISA). Results; BMSCs were successfully induced to differentiate along neural pathway and expressed specific markers of fetal neural epithelium, mature neuron and glial cells. Telomerase activities were undetectable in BMSCs during differentiation along neural pathway. Similar changes of cell growth curves, cell cycle status and telomerase expression were observed in the two groups. Conclusions: Rabbit BMSCs do not display telomerase activity during differentiation along neural pathway. GDNF shows little impact on proliferation and telomerase activity of BMSCs.     

骨髓基质干细胞定向分化为神经细胞及分化后细胞功能特征的研究

目的 探讨骨髓基质干细胞(BMSCs)定向分化为神经细胞的潜能及分化后细胞的功能特征.方法 体外分离、扩增、纯化Sprague-Dawley大鼠BMSCs,取第6代细胞接种并随机分为4组,实验组A、B、C组开始每组加预诱导夜(含预诱导剂碱性成纤维细胞生长因子和表皮生长因子),6d后A组继续加预诱导夜,其余两组在预诱导后加定向诱导剂(音猬因子和维甲酸)时选择是否保留预诱导液分为B组(保留)和C组(去除);D组不做任何干预,为空白对照组.采用细胞免疫荧光化学法检测分化后神经样细胞的形态和功能标志.结果 接种后的细胞迅速贴壁,加预诱导液6 d细胞增殖迅速,且可以观察到有类似神经干细胞的克隆团形成.在诱导剂的特定组合和时序的诱导下,A、B、C组均出现胞体收缩和突起伸出,呈现神经元细胞样改变.D组细胞形态基本没有变化.6～12 d,B组微管相关蛋白-2(MAP-2)阳性细胞率为59.17%±1.84%,胶质酸性蛋白(GFAP)阳性细胞率为22.10%±1.03%;C组MAP-2阳性细胞率为29.35%±4.85%,GFAP为36.35%±4.85%,A组和D组均未检测到上述细胞标志物.12～18 d,A组仅出现少量GFAP阳性细胞;B组MAP-2、GFAP阳性细胞率分别为72.54%±1.02%、21.54%±1.02%;C组MAP-2、GFAP阳性细胞率分别为31.02%±2.37%、23.45%±3.74%.神经细胞的功能标志胆碱乙酰转移酶、囊泡乙酰胆碱通道只有B组表达,12～18 d后阳性细胞率分别为45.11%±3.04%、28.22%±1.72%.结论 BMSCs在体外能定向诱导为具有一定功能的神经细胞,为脊髓损伤后细胞移植治疗奠定了基础.     

体外诱导骨髓间充质干细胞向肝细胞分化的实验研究

目的：探讨HGF和EGF对骨髓间充质干细胞向肝细胞方向的诱导分化作用，探索出合适的诱导条件，为肝细胞移植、生物人工肝、组织工程构建等提供基础。方法：骨髓来自杂种犬的髂骨，采用全血贴壁法分离培养骨髓间充质干细胞，流式细胞仪检测细胞CD标记；用含HGF和EGF的α—MEM培养液进行诱导培养，并于诱导后7、14、21d留取细胞；用免疫组化检测AFP、CK18，免疫荧光检测ALB，PAS反应检测糖原；流式细胞仪检测细胞分化率。结果：分离的骨髓间充质干细胞表面CD13、CD34和CD45皆为阴性，CD29为阳性。诱导至7d出现AFP表达．14d表达增高，21d时表达下降。CK18、ALB和糖原14d时出现表达，并随时间延长表达逐渐增加：21d时ALB表达阳性率可达50％以上。结论：HGF和EGF有诱导骨髓间充质干细胞向肝细胞分化的作用。