本文選題：骨髓間充質(zhì)干細胞　切入點：前列腺癌細胞　出處：《天津醫(yī)科大學(xué)》2014年碩士論文

【摘要】：目的 在體外共培養(yǎng)環(huán)境中小鼠前列腺癌細胞對骨髓間充質(zhì)干細胞誘導(dǎo)分化其機制進行初步探討,補充并完善骨髓間充質(zhì)干細胞在前列腺癌發(fā)生發(fā)展中的作用的小鼠模型,從而為前列腺癌細胞通過自分泌/融合機制誘導(dǎo)骨髓間充質(zhì)干細胞向前列腺癌細胞方向分化奠定了一定的理論基礎(chǔ),并提出了一種嶄新的癌細胞來源理論,為當代腫瘤的臨床治療和科學(xué)研究提供了一個新的方向。 方法 解剖標記有綠色熒光蛋白的小鼠,獲得雙下肢股骨和腓骨,沖洗骨髓腔得到全骨髓,應(yīng)用貼壁培養(yǎng)篩選方法,選取10%胎牛血清、適當?shù)募毎臃N密度等條件進行細胞培養(yǎng),對小鼠骨髓間充質(zhì)干細胞的鑒定,則通過倒置相差光學(xué)顯微鏡觀察光鏡下細胞形態(tài),通過脂肪誘導(dǎo)分化試劑培養(yǎng)骨髓間充質(zhì)干細胞,并使用油紅O染色來鑒定其干細胞特性,最后采用流式細胞學(xué)檢測CD90、CD105、CD34和CD45指標染色結(jié)果對骨髓間充質(zhì)干細胞進行細胞表型鑒定,分離和擴增則待細胞分裂貼壁90%以上即進行細胞傳代。 在體外環(huán)境中進行MSC與小鼠前列腺癌細胞的非接觸式共培養(yǎng),共培養(yǎng)72小時后,在倒置相差光學(xué)顯微鏡下觀察MSC的形態(tài)變化,后通過文獻篩查選取特異性較高的前列腺干細胞抗原(PSCA)指標作為小鼠前列腺癌細胞的標記物,并對非接觸式共培養(yǎng)的MSC進行免疫組化染色觀察,進而對于前列腺癌細胞是否可以誘導(dǎo)MSC向其分化的結(jié)果進行初步的判斷。結(jié)果 通過貼壁篩選方法進行分離和擴增,得到穩(wěn)定傳代的成纖維細胞型、長梭型、星型等多型性細胞形態(tài),并對T3傳代細胞進行脂肪誘導(dǎo)分化后得到明顯有脂肪分化傾向的MSC,另還通過流式細胞學(xué)檢查進行細胞表型鑒定,結(jié)果提示CD90陽性,CD105陽性,CD34陰性,CD45陰性。 在體外環(huán)境中進行MSC與小鼠前列腺癌細胞非接觸式共培養(yǎng)后72小時,觀察結(jié)果提示：1、共培養(yǎng)后觀察光鏡下MSC細胞形態(tài)依然為多形性,邊界清晰,與單獨培養(yǎng)結(jié)果無明顯異常。2、T3傳代細胞進行脂肪誘導(dǎo)分化結(jié)果顯示油紅O染色呈現(xiàn)粉紅色陽性結(jié)果。3、選取小鼠前列腺癌細胞特異性高的PSCA指標對共培養(yǎng)后的MSC進行免疫組織化學(xué)染色,提示有弱陽性結(jié)果。 結(jié)論 1、本實驗通過貼壁篩選方法和適當?shù)募毎囵B(yǎng)條件,成功分離和擴增培養(yǎng)得到穩(wěn)定傳代的符合克隆樣生長和成纖維樣細胞等形態(tài)特征,并且通過油紅0染色、流式細胞學(xué)檢查驗證了具有干細胞特性的貼壁生長的小鼠骨髓間充質(zhì)干細胞。 2、本研究通過體外環(huán)境非接觸式共培養(yǎng)小鼠前列腺癌細胞與MSC,初步證實了經(jīng)過72小時共培養(yǎng)后,MSC中的部分細胞表型發(fā)生了改變,表達了具有特異性較高的PSCA標記物,以此可以得到一個初步推測,即小鼠前列腺癌細胞在共培養(yǎng)體系中分泌的多種細胞因子可以通過旁分泌機制而非融合來促進MSC向前列腺上皮分化的作用。因此,本實驗可以推測在體外環(huán)境中MSC受到腫瘤分泌的多種細胞因子的誘導(dǎo),分化成為前列腺癌上皮細胞的腫瘤誘導(dǎo)MSC分化理論,腫瘤誘導(dǎo)MSC分化理論的提出,讓我們重新認識了MSC在腫瘤形成中的作用,為以后腫瘤的基因靶向治療等多方面提供更加有力的理論支持。
[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.

【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R737.25

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本文編號：1679369