根尖乳头干细胞向牙髓牙本质复合体分化能力的实验研究

Experimental study on SCAP's defferentiation capacity to Dental pulp dentin complex differentiation ability

目的:应用组织工程方法,探讨根尖乳头干细胞(SCAP)分别在炎性环境(肿瘤坏死因子-α)及正常环境下向牙髓牙本质复合分化的可能性。方法:分离大鼠根尖乳头组织,采用酶消化法结合组织块法获得SCAP,鉴定细胞来源,进行MTT生长曲线的测定,分正常组、炎症组(10 ng/m L TNF-α)及空白对照组行体外成骨能力茜素红及免疫组化实验,并将3个组分别移植到大鼠背部皮下观察8/12周后新生组织的形成情况。结果:MTT结果显示10 ng/m L TNF-α组显著高于正常SCAP组,差异有统计学意义(P<0.5);茜素红染色显示均有矿化结节生成,与炎性组(10 ng/m L TNF-α)相比,正常组SCAP染色显著,矿化结节形成的范围大且呈深橘色;免疫组化染色显示SCAP胞浆中DSP/BSP均有表达;正常、炎症组移植物可见牙髓牙本质样结构形成,且DSP/BSP均阳性表达,空白对照组未见矿化组织形成。结论:SCAP具有高增殖及成骨向分化能力,可进行牙髓牙本质复合体的再生,炎性因子TNF-α对SCAP的生长增殖有促进作用同时不同程度上对SCAP矿化及成牙成骨向分化有抑制作用。

Objective：To discuss the possibility that SCAP recombines pulpo-dentinal complex or differentiates under inflammatory environment（TNF-α）and normal circumstances respectively,by applying tissue engineering method. Method：Separate root nipple tissue in rats and extract SCAP by combining enzyme digestion method with tissue block method,detect cell resource,test MTT growth curve,do the research on ontogenesis in vitro ability with alizarin red and immunohistochemical examination by separate into three groups,namely normal group,inflammation group（10ng/ml TNF-α）and blank control group. Then transfer three groups under rats＇ back skins,observing 8/12 weeks for new tissue＇s formation. Result：According to the MTT detection,10 ng/m L TNF-α group is remarkable higher than normal SCAP group,the differences are statistically significant（P〈0.5）. Results of the alizarin red staining show that all generate the mineral node. Compared with inflammatory group（10 ng/m L TNF-α）,normal SCAP group stain darker and the mineral nodes are large-scaled formed and colored as deep orange. Results the immunofluorescence staining show that DSP/BSP in SCAP endochylema both express.The graft in normal and inflammatory group can find the draft pulp dentine structure. Otherwise,DSP/BSP are expressed as positive. In blank control group,no mineral nodes are found. Conclusion：SCAP has high capability to proliferate and transfer to bones. It can regenerate to be pulp dentin complex. Inflammatory TNF-α can stimulate SCAP＇s proliferation and to some extent to inhibit the mineralization of SCAP and its capability of turning bones.