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Study on the Toxicity of Isoniazide with Various Concentrations on Cultured Osteoblasts in Vitro

Author: ChenQiang
Tutor: YeZheWei
School: Huazhong University of Science and Technology
Course: Surgery
Keywords: osteoblast primary culture identificationbone tuberculosis isoniazid osteoblasts toxic effect
CLC: R96
Type: Master's thesis
Year: 2013
Downloads: 12
Quote: 0
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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.

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