小鼠前列腺癌细胞对骨髓间充质干细胞诱导分化作用的初步研究

发布时间：2018-03-29 03:29

本文选题：骨髓间充质干细胞　切入点：前列腺癌细胞　出处：《天津医科大学》2014年硕士论文

【摘要】：目的在体外共培养环境中小鼠前列腺癌细胞对骨髓间充质干细胞诱导分化其机制进行初步探讨,补充并完善骨髓间充质干细胞在前列腺癌发生发展中的作用的小鼠模型,从而为前列腺癌细胞通过自分泌/融合机制诱导骨髓间充质干细胞向前列腺癌细胞方向分化奠定了一定的理论基础,并提出了一种崭新的癌细胞来源理论,为当代肿瘤的临床治疗和科学研究提供了一个新的方向。方法解剖标记有绿色荧光蛋白的小鼠,获得双下肢股骨和腓骨,冲洗骨髓腔得到全骨髓,应用贴壁培养筛选方法,选取10%胎牛血清、适当的细胞接种密度等条件进行细胞培养,对小鼠骨髓间充质干细胞的鉴定,则通过倒置相差光学显微镜观察光镜下细胞形态,通过脂肪诱导分化试剂培养骨髓间充质干细胞,并使用油红O染色来鉴定其干细胞特性,最后采用流式细胞学检测CD90、CD105、CD34和CD45指标染色结果对骨髓间充质干细胞进行细胞表型鉴定,分离和扩增则待细胞分裂贴壁90%以上即进行细胞传代。在体外环境中进行MSC与小鼠前列腺癌细胞的非接触式共培养,共培养72小时后,在倒置相差光学显微镜下观察MSC的形态变化,后通过文献筛查选取特异性较高的前列腺干细胞抗原(PSCA)指标作为小鼠前列腺癌细胞的标记物,并对非接触式共培养的MSC进行免疫组化染色观察,进而对于前列腺癌细胞是否可以诱导MSC向其分化的结果进行初步的判断。结果通过贴壁筛选方法进行分离和扩增,得到稳定传代的成纤维细胞型、长梭型、星型等多型性细胞形态,并对T3传代细胞进行脂肪诱导分化后得到明显有脂肪分化倾向的MSC,另还通过流式细胞学检查进行细胞表型鉴定,结果提示CD90阳性,CD105阳性,CD34阴性,CD45阴性。在体外环境中进行MSC与小鼠前列腺癌细胞非接触式共培养后72小时,观察结果提示：1、共培养后观察光镜下MSC细胞形态依然为多形性,边界清晰,与单独培养结果无明显异常。2、T3传代细胞进行脂肪诱导分化结果显示油红O染色呈现粉红色阳性结果。3、选取小鼠前列腺癌细胞特异性高的PSCA指标对共培养后的MSC进行免疫组织化学染色,提示有弱阳性结果。结论 1、本实验通过贴壁筛选方法和适当的细胞培养条件,成功分离和扩增培养得到稳定传代的符合克隆样生长和成纤维样细胞等形态特征,并且通过油红0染色、流式细胞学检查验证了具有干细胞特性的贴壁生长的小鼠骨髓间充质干细胞。 2、本研究通过体外环境非接触式共培养小鼠前列腺癌细胞与MSC,初步证实了经过72小时共培养后,MSC中的部分细胞表型发生了改变,表达了具有特异性较高的PSCA标记物,以此可以得到一个初步推测,即小鼠前列腺癌细胞在共培养体系中分泌的多种细胞因子可以通过旁分泌机制而非融合来促进MSC向前列腺上皮分化的作用。因此,本实验可以推测在体外环境中MSC受到肿瘤分泌的多种细胞因子的诱导,分化成为前列腺癌上皮细胞的肿瘤诱导MSC分化理论,肿瘤诱导MSC分化理论的提出,让我们重新认识了MSC在肿瘤形成中的作用,为以后肿瘤的基因靶向治疗等多方面提供更加有力的理论支持。
[Abstract]:objective
Co culture of mouse prostate cancer cells in the environment of bone marrow mesenchymal stem cells differentiation and its mechanism was preliminarily studied in vitro, supplement and improvement of bone marrow mesenchymal stem cells in a mouse model of the progression of prostate cancer, and prostate cancer cells by inducing the differentiation of bone marrow mesenchymal stem cells to prostate cancer cells laid a theoretical foundation for the autocrine secretion / fusion mechanism, and proposes a new cancer cell source theory, provides a new direction for clinical treatment and scientific research of contemporary tumor.
Method
Anatomical markers of green fluorescent protein in mice, two limb femur and fibula, flushing the bone marrow cavity by whole bone marrow adherent culture method, application of screening, selection of 10% fetal bovine serum, proper cell seeding density of cell culture, identification of bone marrow mesenchymal stem cells, through the inverted cells morphological observation under light microscope, optical microscope, reagent induced differentiation of bone marrow mesenchymal stem cells were cultured by using fat and oil red O staining to identify the characteristics of stem cells, the flow cytometric detection of CD90, CD105, CD34 and CD45 index of staining of bone marrow mesenchymal stem cells for phenotypic identification, isolation and the cell wall was more than 90% of cells.
The non-contact co culture of mouse MSC and prostate cancer cells in vitro, were cultured for 72 hours, to observe the morphological changes of MSC in the inverted optical microscope, after screening through literature selected high specificity of prostate stem cell antigen (PSCA) index as a marker of mouse prostate cancer cells, and the non-contact co cultured MSC were observed by immunohistochemical staining, and whether prostate cancer cells can induce the differentiation of MSC to the preliminary judgment results.
Isolated and amplified by adherent screening method, fiber cell type into stable passage, long spindle type, star type and other types of cells, and on T3 cells after differentiation were fat fat differentiation tendency of MSC, the other is through the flow cytometry examination of cell phenotype identification. The results showed that CD90 positive, CD105 positive, CD34 negative, CD45 negative.
MSC and mouse prostate cancer cells were cultured for 72 hours non contact after in vitro environment, study results suggest: 1, after morphological observation of MSC cells under light microscope is pleomorphic, co culture and individual culture results with clear boundary, no obvious abnormal.2, T3 cell differentiation showed fatty oil red O staining showed positive results of pink.3, select the specific mouse prostate cancer cells with high PSCA index of immunohistochemical staining of MSC after CO cultivation, suggesting a weak positive results.
conclusion
1, through this experiment, adherence screening method and appropriate cell culture conditions, culture and amplification of stable passage with cloning growth and morphological characteristics of fibroblast like cells isolated by oil red staining, and 0, has proved the mouse bone marrow adherent stem cell growth characteristics of mesenchymal stem cells check the flow cytometry.
2, this study by in vitro environmental non mouse prostate cancer cells were co cultured with MSC contact, confirmed after 72 hours after incubation, the cells changed phenotype in the MSC section, the expression of the PSCA marker with high specificity, which can get a preliminary speculation, that prostate cancer cells in mice co culture of various cytokine secretion system by paracrine mechanism and non fusion promoting effect of MSC to prostate epithelial differentiation. Therefore, this experiment can be speculated that the in vitro environment MSC induced by some cytokines secreted by tumor, differentiate into prostate cancer epithelial cells induced by tumor MSC tumor induced differentiation theory, put forward MSC differentiation theory, let us re understand the role of MSC in tumor formation, as gene target after tumor treatment and other aspects to provide more powerful theoretical support.

【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.25

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