【作者】 陈强；

【导师】 叶哲伟；

【作者基本信息】 华中科技大学 ， 外科学， 2013， 硕士

【摘要】 目的成骨细胞的原代培养是成骨细胞体外实验的重要来源之一，现主要有酶消化法和组织块法2种，但这两种方法各有利弊。本实验拟探索一种高效、简便、可靠的体外培养新生SD大鼠原代成骨细胞的方法。方法新生清洁级SD乳鼠(出生24h内)15只，随机分为三组，每组5只，经乙醇浸泡消毒后取出颅骨，分别采用传统组织块培养法(A组)、优化组织块法(B组)和酶消化法(C组)进行处理，分离和培养成骨细胞。从细胞的形态学、生长特点、碱性磷酸酶(ALP)染色、ALP活性检测以及Ι型胶原免疫荧光染色等多方面对培养出的成骨细胞予以鉴定和比较。结果三种方法48h后均培养出成骨细胞，以骨块为中心分布，A组数量最少，C组最多。A、B两组细胞从第四天开始增长迅速，第七天达高峰，C组生长趋势低于前两组。ALP染色阳性可见浅棕色至棕黑色色素颗粒，A、B、C组成骨细胞阳性率依次为85%、92%、81%。ALP活性检测可见A、B组成骨细胞活性相似，且明显高于C组。3组I型胶原免疫荧光染色结果均呈阳性，A、B组细胞胞浆中I型胶原表达明显多于C组。结论优化后组织块法结合了传统组织块法和酶消化法的特点，可短期内培养出大量纯度高的成骨细胞，具有成骨细胞活性及成骨能力，可为骨替代材料或者骨组织工程的研究提供大量细胞。同时也为初学者提供了一种切实可行、操作简单的体外原代成骨细胞培养方法。目的目前我国结核病疫情十分严重，且合并感染骨结核者较多，治疗复杂，但效果欠佳。病灶清除术后联合局部抗结核治疗是现行值得考虑的方法之一。异烟肼(INH)作为抗结核的一线药物，对细胞内外结核菌都具有极强的杀菌效果，但对成骨细胞是否也具有毒性作用尚不明确。本实验拟研究不同浓度INH对新生SD大鼠原代成骨细胞增殖、碱性磷酸酶(ALP)活性和Ⅰ型胶原表达的影响，探讨异烟肼对体外培养成骨细胞的毒性作用，为骨结核病灶清除术后局部应用缓释微球提供理论依据。方法新生清洁级SD乳鼠(出生24h内)15只，采用优化组织块法体外分离培养原代成骨细胞并鉴定，鉴定方法同第一部分。取第3代成骨细胞，分别采用CCK-8法、碱性磷酸酶活性检测法和免疫荧光染色法测定，观察不同浓度（10μg/ml、20μg/ml、30μg/ml、40μg/ml、50μg/ml、60μg/ml、100μg/ml）异烟肼对成骨细胞增值、ALP活性以及Ⅰ型胶原蛋白表达的影响。结果采用优化组织块法成功培养去原代成骨细胞，24h后逐渐游离出来，在48h细胞数增多明显，排列紧密，三种染色检测均阳性。给予不同浓度INH干预72h后，与对照组比较，异烟肼浓度在30μg/ml时，成骨细胞增长开始受到抑制，ALP活性开始减弱，Ⅰ型胶原合成开始减少。随着异烟肼浓度增高，这种抑制作用逐渐增强。当异烟肼浓度达60μg/ml时，细胞活性很弱，大部分细胞已经死亡。结论治疗骨结核时，要严格控制异烟肼剂量，INH局部药物浓度达到10μg/ml-20μg/ml范围时就可有效杀灭结核菌，且不影响成骨细胞的活性和Ⅰ型胶原蛋白的表达，不影响结核病灶清除术后的骨愈合，同时为异烟肼缓释体的研究提供了理论基础。更多还原

【Abstract】 Objective Primary cultured osteoblasts is an important source for the study of osteoblasts in vitro. In present, there are two main methods, the enzyme digestion and tissue block method, but both of them have their own advantages and disadvantages. To explore an efficient, simple and reliable method to isolate and culture osteoblasts from new SD neonatal rats in vitro, and use it to gain a lot of high purity osteoblasts within short time.Methods The skull got from fifteen of24-hour old SD rats was disinfected in the ethanol, then randomly divided into three groups to isolate and culture osteoblasts:the traditional tissue explant method (A), optimized tissue explant method(B) and repeated by enzyme digestion method(C). N=5. The cells were evaluated from the follow aspects:the cell morphology, the growth characteristic, alkalinity phosphatase (ALP) dyes, the ALP activity examination, type I collagen immune fluorescence and so on. Results After48h, three methods all cultured osteoblasts, which were bone-centric distributed. Group A was fewer than Group B and C, Group C was most. The cells of Group A and B had begun rapid growth from the fourth day, reached top on seventh day. The growth trend of Group C was lower than the first two groups. The positive rates of three groups were85%,92%,81%. Through the ALP activity detection, we found that the osteoblast activity of Group A and B were similarly, and significantly higher than Group C. The results of type I collagen immunofluorescence staining of three groups were positive. The expression in the cytoplasmic of Group A and B was significantly more than the C group.Conclusion The method of optimized tissue explant combined the characterizes of the traditional tissue explant method and enzymatic digestion method. It could culture a large number of high-purity osteoblasts in short-time, which have osteoblast activity and estrogenic potential. It could provide plenty of cells for the research of bone substitute materials or bone tissue engineering. What is more, it provides a practical, simple method to culture the bone cell in vitro for the beginners. Objective At present, the epidemic situation of tuberculosis in China is very serious. The incidence of co-infected bone tuberculosis presented an increasing trend. The treatment is complex and ineffective. After debridement, combining with local anti-TB injection is one of the methods worth considering during the current. As the first-line anti-TB drugs, Isoniazid (INH) has a strong bactericidal effect to the intracellular mycobacterium tuberculosis. But it has toxic effects on osteoblasts is not clear. The study is to research the effects of different concentrations of INH on primary osteoblasts from neonatal SD rats according to the following several aspects:cell proliferation, ALP activity and the expression of type I collagen. To explore the toxic effects of INH on cultured osteoblasts. To provide a theoretical basis for the topical application of sustained-release microspheres.Methods The skull got from fifteen of24-hour old SD rats was disinfected in the ethanol, then isolated and cultured by optimized tissue explant method. It is same as the part1. The cells, got from the third passaged osteoblasts, cultivated together with INH at different concentrations of10μg/ml,20μg/ml,30μg/ml,40μg/ml,50μg/ml,60μg/ml,100μg/ml for some days. Then evaluated from the follow aspects:the cell morphology, the growth characteristic, alkalinity phosphatase (ALP) dyes, the ALP activity examination, type I collagen immune fluorescence by CCK-8, ALP activity assay and immunofluorescence staining determination.Results Through the method of optimized tissue explant, the primary osteoblasts were cultured successfully, which were freed after24h, increased significantly at48h. The cells were closely arranged, all of the staining were positive. Given different concentrations of INH to intervene the cells after72h, compared with the control group, the osteoblast growth was inhibited with30μg/ml, ALP activity began to weaken, type I collagen synthesis started to decrease. With the higher concentration of INH, the inhibition was gradually increased. When the concentration until the60μg/ml, cell activity was very weak, most of the cells were died.Conclusion When treatment, it is necessary to strictly control the dose of INH. INH could effectively kill tuberculosis, when the local drug concentration reached the range of10μg/ml-20μg/ml. It does not affect the activity of the osteoblasts and the expression of type I collagen, does not affect the bone healing of tuberculosis clear postoperative. As well as, it provides a theoretical basis for the research of isoniazid mitigation.更多还原