本文關(guān)鍵詞：骨髓間充質(zhì)干細胞分離培養(yǎng)及其向腫瘤局部歸巢機制的初步探索　出處：《華中科技大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 骨髓間充質(zhì)干細胞 腫瘤血管內(nèi)皮細胞 細胞培養(yǎng)及鑒定 腫瘤 歸巢 Snail 宮頸癌 上皮細胞間質(zhì)化轉(zhuǎn)變(EMT) 上皮細胞鈣黏蛋白(E-cadherin)

【摘要】：【研究背景及目的】骨髓間充質(zhì)干細胞是一類具有多能分化潛能的細胞,可以向骨組織,心肌,軟骨組織,肌肉等分化,并且可以特異遷移到損傷部位及腫瘤局部,被認為是具有應(yīng)用前景的基因治療細胞載體。然而,間充質(zhì)干細胞靶向歸巢腫瘤局部的機制卻不太清楚。本研究在于分離人類骨髓間充質(zhì)干細胞(Mesenchymal stem cells,MSCs),并探討其體內(nèi)靶向歸巢腫瘤局部的機制。 【方法】(1)密度梯度離心法獲得骨髓間充質(zhì)干細胞,并體外培養(yǎng)擴增。(2)流式細胞儀及激光共聚焦顯微鏡檢測骨髓間充質(zhì)干細胞表面標志分子CD44、CD105、CD90、CD34、CD45、CD19;不同條件培養(yǎng)基誘導(dǎo)骨髓間充質(zhì)干細胞的成骨分化、軟骨分化及脂肪分化,倒置顯微鏡觀察。(3)建立小鼠原位乳腺腫瘤模型,尾靜脈注射菲力磁(SPIO)體外標記的骨髓間充質(zhì)干細胞,利用冰凍切片對骨髓間充質(zhì)干細胞在小鼠腫瘤局部的分布情況進行檢測。(4)取新鮮乳腺癌組織,剪碎,膠原酶消化成單細胞懸液,細胞標記抗CD31單克隆抗體,免疫磁柱分離抗CD31單克隆抗體陽性細胞,即腫瘤血管內(nèi)皮細胞,并且體外原代培養(yǎng)。(5)流式細胞儀及激光共聚焦顯微鏡檢測腫瘤血管內(nèi)皮細胞標志性分子CD31、CD34、vWF。(6)Transwell模型驗證腫瘤細胞對間充質(zhì)干細胞的體外趨化能力。(7)Transwell模型血管內(nèi)皮細胞對骨髓間充質(zhì)干細胞體外趨化能力,結(jié)晶紫染色,顯微鏡照相,每個濾膜隨機選取5個200倍視野計數(shù)穿膜細胞數(shù),穿膜細胞數(shù)表示骨髓間充質(zhì)干細胞的體外遷移能力。 【結(jié)果】(1)原代貼壁細胞呈長梭形。體外培養(yǎng)一周,細胞形態(tài)均一,擴增速度快,克隆形成能力強。(2)流式細胞儀檢測發(fā)現(xiàn)骨髓間充質(zhì)干細胞表面分子標志物CD44、CD105、CD90陽性率分別為99.79±3.35% , 99.62±3.78%,98.93±3.62%,陰性標志物CD34、CD45、CD19為4.32±1.01%,4.04±1.05%,6.04±0.95%;激光共聚焦顯微鏡下,可見骨髓間充質(zhì)干細胞陽性標志物在細胞內(nèi)存在較高表達。不同的條件培養(yǎng)液成功誘導(dǎo)骨髓間充質(zhì)干細胞向骨組織、脂肪組織及軟骨組織分化,驗證了骨髓間充質(zhì)干細胞多向分化潛能。(3)體外SPIO標記的骨髓間充質(zhì)干細胞通過尾靜脈注射到荷瘤小鼠體內(nèi),細胞主要聚集在腫瘤局部。(4)免疫磁珠方法分離的腫瘤血管內(nèi)皮細胞,6-12小時貼壁生長,早期細胞呈小多角、球形,逐漸生長成梭形,于3～4d后融合,為單層呈鋪路石狀排列。(5)流式細胞儀檢測發(fā)現(xiàn),CD31、CD34、vWF陽性率分別為92.8±2.32%,99.87±1.95%,86.26±2.57%(6)Boyden小室結(jié)果顯示:在相同時間點,下室接種MDA -MB-231及MDA -MB-435s細胞組與對照組相比,穿膜細胞數(shù)無明顯改變。(7)Boyden小室結(jié)果顯示:腫瘤血管內(nèi)皮細胞組,骨髓間充質(zhì)干細胞穿膜細胞數(shù)明顯多于對照組(p㩳0.05) 【結(jié)論】體外可獲得較高純度的骨髓間充質(zhì)干細胞,且易于培養(yǎng)擴增。骨髓間充質(zhì)干細胞體內(nèi)靶向歸巢腫瘤局部的能力與腫瘤局部血管內(nèi)皮細胞有關(guān)。 目的:研究Snail與宮頸癌侵襲轉(zhuǎn)移間的關(guān)系,并探討其分子機制。方法:構(gòu)建正義全長Snail真核表達載體并轉(zhuǎn)染宮頸癌細胞系Hela、Siha。采用Western blot方法分析相關(guān)基因表達,細胞傷痕愈合實驗,Transwell模型穿膜細胞計數(shù)檢測細胞侵襲轉(zhuǎn)移能力。結(jié)果:(1)在轉(zhuǎn)染PEGFPC1/Snail的宮頸癌細胞株Hela、SiHa中E-cadherin表達明顯下調(diào),Snail和Vimentin則表達上調(diào)(p0.05)。(2)宮頸癌細胞系Hela、Siha轉(zhuǎn)染正義全長Snail真核表達載體,12小時細胞傷痕愈合能力顯著提高(p0.05);轉(zhuǎn)染PEGFPC1/Snail后,Hela、SiHa細胞12小時穿膜細胞數(shù)明顯增多(p0.05)。Snail過表達可顯著促進宮頸癌細胞株Hela、SiHa的侵襲轉(zhuǎn)移潛能。結(jié)論:轉(zhuǎn)錄因子Snail促進宮頸癌上皮細胞間質(zhì)化轉(zhuǎn)變(EMT)并提高其侵襲轉(zhuǎn)移能力。
[Abstract]:[background and objective] bone marrow mesenchymal stem cell is a kind of multipotent differentiation potential of cells, to bone tissue, muscle, cartilage tissue, muscle specific differentiation, and can migrate to the injury site and local tumor, gene therapy is considered to be a promising cell carrier. However, MSCs mesenchymal stem cells targeting tumor homing mechanism was not clear. This study is the isolation of human bone marrow mesenchymal stem cells (Mesenchymal stem cells, MSCs), and investigate its in vivo targeting homing mechanism of local tumor.
[Methods] (1) bone marrow mesenchymal stem cells obtained by density gradient centrifugation and cultured in vitro. (2) by flow cytometry and confocal microscopy for detection of bone marrow mesenchymal stem cell surface marker CD44, CD105, CD90, CD34, CD45, CD19; group of bone marrow mesenchymal stem osteogenic differentiation of different cell culture conditions, cartilage differentiation and adipocyte differentiation, inverted microscope. (3) establish a mouse model of breast cancer in situ, intravenous injection of Feridex (SPIO) labeled bone marrow mesenchymal stem cells, bone marrow mesenchymal stem cells were detected in the distribution of tumor by frozen section. (4) from the fresh breast cancer tissue, cut into pieces, collagenase digested into single cell suspension cells labeled with anti CD31 monoclonal antibody, anti CD31 monoclonal antibody positive cells separation immunomagnetic column, namely tumor vascular endothelial cell, and cultured in vitro. (5) Flow cytometry and confocal microscopy for detection of tumor vascular endothelial cell markers CD31, CD34, vWF. (6) Transwell model verification of tumor cells in vitro on mesenchymal stem cell chemotaxis ability. (7) the Transwell model of vascular endothelial cells on bone marrow mesenchymal stem cells in vitro chemotaxis ability, crystal violet staining, microscopy, each membrane 5 randomly selected 200 times magnification counting transmembrane cell number, the number of penetrating cells expressed mesenchymal stem cells migration ability in vitro.
[results] (1) adherent cells were fusiform. In vitro cultured for one week, the cells were all, amplification speed, clone formation ability. (2) flow cytometry demonstrated that bone marrow mesenchymal stem cell markers CD44, surface molecules CD105, CD90 positive rate was 99.79. 3.35%, 99.62 + 3.78%, 98.93 + 3.62%, negative markers CD34, CD45, CD19 was 4.32 + 1.01%, 4.04 + 1.05%, 6.04 + 0.95%; laser confocal microscopy showed that bone marrow mesenchymal stem cell markers in positive cell memory in high expression. Different conditioned medium induced bone marrow mesenchymal mesenchymal stem cells into bone, adipose tissue and cartilage tissue differentiation, verified the bone marrow mesenchymal stem cell differentiation in vitro. (3) SPIO labeled bone marrow mesenchymal stem cells by intravenous injection into tumor bearing mice, tumor cells mainly gathered in the local. (4) by immunomagnetic beads method Tumor vascular endothelial cells from 6-12 hours, the adherent growth of cells in the early stage of a small multi angle, spherical, gradually grow into the spindle, in 3 ~ 4D after fusion, monolayer arranged like cobblestone. (5) flow cytometry showed that CD31, CD34, vWF positive rate was 92.8. 2.32%, 99.87 + 1.95%, 86.26 + 2.57% (6) Boyden assay results showed that: at the same time, MDA -MB-231 and MDA under room inoculation of -MB-435s cells compared with the control group, the number of penetrating cells had no obvious change. (7) Boyden assay results showed that the tumor vascular endothelial cells group, bone marrow mesenchymal stem cell penetrating cell number was higher than that of control group (P? 0.05)
[Conclusion] bone marrow mesenchymal stem cells with high purity can be obtained in vitro, and are easy to culture and amplify. The ability of bone marrow mesenchymal stem cells to target tumor locally is related to local vascular endothelial cells.
Objective: To study the relationship between the invasion and metastasis of Snail and cervical cancer, and to explore its molecular mechanism. Methods: to construct the sense of full-length Snail eukaryotic expression vector and transfected into cervical cancer cell lines Hela and Siha. by Western blot method for analysis of gene expression, cell wound healing assay, Transwell cell count model of transmembrane cell invasion and metastasis. Results: (1) in PEGFPC1/Snail transfected cervical cancer cell line Hela, down regulated the expression of E-cadherin in SiHa, Snail and Vimentin expression (P0.05). (2) cervical cancer cell lines Hela, Siha transfected with full-length Snail eukaryotic expression vector, 12 hours cell wound healing significantly increased (P0.05); transfection PEGFPC1/Snail, Hela, SiHa cells 12 hours transmembrane cell number increased significantly (P0.05) over expression of.Snail can significantly promote cervical cancer cell lines Hela, SiHa invasion and metastasis potential. Conclusion: the transcription factor Snail promotes The interstitial transformation (EMT) of cervical cancer epithelial cells and the ability to invasion and metastasis are improved.

【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R329

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