【摘要】：目的 鑒定和分離以CD105+為標志的人腎癌干細胞,并通過比較CD105-shRNA和不同microRNA在腎癌干細胞和普通腎癌細胞中的功能,探討CD105和干細胞相關(guān)microRNA通過殺滅腎癌干細胞來治療腎癌的可行性和有效性。 方法 采取ACHN腎癌細胞系在免疫缺陷小鼠BALB/c-nu皮下成瘤的方式獲得腎癌組織細胞,并通過外源性anti-hCD105-PE抗體標記和流式細胞術(shù)分離CD105陽性的人腎癌干細胞。分別采用成球?qū)嶒�、成上皮誘導(dǎo)分化實驗鑒定CD105(+)細胞的腫瘤干細胞特性,并通過MTS和EdU細胞增殖實驗、周期測定、集落形成和細胞衰老程度測定等手段鑒定以CD105(+)為標志的腎癌干細胞的增殖特性和細胞衰老程度。在此基礎(chǔ)上,采用microRNA數(shù)據(jù)庫檢索、文獻檢索和qRT-PCR驗證的方式篩選在干細胞和非干細胞中差異性表達的microRNA,并采用雙熒光素酶的方式確定microRNA和其靶蛋白mRNA3'端非編碼區(qū)之間的結(jié)合程度,最終將篩選得到的microRNA采用mimics或者慢病毒轉(zhuǎn)染的方式探討其在腎癌干細胞的體外成球能力、順鉑耐藥能力、遷移能力中的作用。 結(jié)果 CD105(+)腎癌細胞的成球能力明顯強于CD105(-)細胞系A(chǔ)CHN,且在RPMI-1640培養(yǎng)基中培養(yǎng)兩周后,干細胞標志CXCR4、OCT-4、NANOG、HIF1A、DLK-1和EZH2逐漸降低；在DMEM-HG+10%FBS培養(yǎng)基中培養(yǎng)兩周后,其干細胞標志CXCRH-4、NANOG、 DLK-1、OCT-4和KLF4表達明顯下降。在其他鑒定實驗中,雖然ACHN-CD105(+)細胞的增殖速度下降且存在G0期阻滯,但其形成集落和成球能力均強于ACHN-CD105(-)細胞,衰老程度也顯著低于ACHN-CD105(-)細胞。在此基礎(chǔ)上,我們鑒定出27個在CD105(+)腎癌干細胞中差異性表達的microRNA,并證實miR-34a、miR-155、miR-200a可不同程度結(jié)合CD105的mRNA從而下調(diào)其表達,使CD105(+)腎癌干細胞對順鉑耐藥性下降,而采用shRNA敲降CD105的方式,我們也發(fā)現(xiàn)CD105具有維持ACHN-CD105(+)細胞耐藥性并防止其衰老的作用；同時我們也證明miR-335可結(jié)合OCT-4蛋白的mRNA從而降低其表達,并產(chǎn)生抗遷移、降低成球率和抑制SOX-2、OCT-4、NANOG、KLF-4等干細胞因子表達的效應(yīng)。 結(jié)論 經(jīng)ACHN細胞系于BALB/c-nu裸鼠皮下移植瘤細胞分選獲得的CD105(+)細胞具有腫瘤干細胞特性,可初步認定為腎癌干細胞；miR-34a、miR-155、miR-200a可通過下調(diào)CD105降低CD105(+)腎癌干細胞對順鉑的耐藥性；miR-335可通過下調(diào)OCT-4抑制CD105(+)腎癌干細胞的遷移能力、成球能力。 第一部分人腎癌干細胞的分離、培養(yǎng)和鑒定 目的 分離和鑒定以CD105(+)為標志的人腎癌干細胞 方法 將腎癌細胞系A(chǔ)CHN接種于BALB/c-nu裸鼠的腋窩皮膚下,一月后將形成的腫瘤消化成單細胞懸液并采用anti-hCD105-PE流式抗體4-C孵育半小時,于流式分選儀上分離得到ACHN-CD105(+)細胞并采用腎癌干細胞培養(yǎng)基擴增和凍存細胞。采用體外成球?qū)嶒�、RPMI-1640+10%FBS和DMEM-HG+10%FBS培養(yǎng)基誘導(dǎo)分化實驗、順鉑耐藥性實驗、MTS和EdU細胞增殖實驗、周期測定、集落形成實驗和細胞衰老程度測定的方式鑒定ACHN-CD105(+)細胞的增殖、分化和成瘤特性。 結(jié)果 CD105(+)細胞僅占ACHN細胞系皮下移植瘤細胞的3.05%,符合腫瘤干細胞理論中“極低比例的細胞驅(qū)動腫瘤生長”的觀點。ACHN-CDl05(+)可在腎癌干細胞培養(yǎng)基中持續(xù)擴增并維持CD105和干細胞相關(guān)因子的表達水平。在后續(xù)功能鑒定中,ACHN-CD105(+)細胞的成球率明顯高于ACHN-CD105(-)細胞,且在RPMI-1640+10%FBS和DMEM-HG+10%FBS培養(yǎng)基中培養(yǎng)兩周后干細胞相關(guān)因子CXCR-4、OCT-4、NANOG表達下降,順鉑耐藥實驗顯示ACHN-CD105(+)細胞的耐藥性明顯強于ACHN-CD105(-)細胞,細胞增殖實驗和集落形成實驗顯示ACHN-CD105(+)細胞增殖速度低于ACHN-CD105(-)細胞且有GO期阻滯現(xiàn)象,但其集落形成能力顯著優(yōu)于后者。細胞衰老程度測定顯示,ACHN-CD105(+)細胞衰老程度低于ACHN-CD105(-)細胞。 結(jié)論 經(jīng)ACHN細胞系于BALB/c-nu裸鼠皮下移植瘤細胞分選獲得的CD105(+)細胞具有腫瘤干細胞特性,可初步認定為腎癌干細胞。 第二部分miRNA在人腎癌干細胞中的功能和機制研究 目的 篩選可抑制ACHN-CD105(+)干細胞特性的miRNA并探討其作用機制 方法 采用miRNA數(shù)據(jù)庫篩選、文獻檢索和qRT-PCR驗證的方式篩選具有抑制ACHN-CD105(+)干細胞特性的miRNA,以雙熒光素酶實驗確定miRNA和其靶蛋白mRNA之間的結(jié)合程度并采取順鉑耐藥性實驗、細胞衰老程度測定、細胞遷移實驗和成球?qū)嶒灆z測經(jīng)過篩選獲得的miRNA的有效性和用于在體實驗的可行性。 結(jié)果 我們篩選得到27個在ACHN-CD105(+)和ACHN-CD105(-)細胞間差異性表達的miRNA,后期又確定了miR-34a、miR-155、miR-200a的靶標為CD105, miR-335的靶標為OCT-4。在轉(zhuǎn)染了miR-34a、miR-155、miR-200a的化學合成mimics之后,ACHN-CD105(+)細胞對順鉑的耐藥性顯著下降；在轉(zhuǎn)染miR-335的過表達慢病毒后,ACHN-CD105(+)細胞的遷移遷移能力和成球能力下降,包括OCT-4、NANOG、SOX-2、KLF-4在內(nèi)的多種干細胞相關(guān)因子表達降低。 結(jié)論 miR-34a、miR-155、miR-200a可通過下調(diào)CD105降低CD105(+)腎癌干細胞對順鉑的耐藥性;miR-335可通過下調(diào)OCT-4抑制CD105(+)腎癌干細胞的遷移能力、成球能力。
[Abstract]:objective
To identify and isolate human renal cell carcinoma stem cells marked by CD105+ and to compare the functions of CD105-shRNA and different microRNAs in renal cell carcinoma stem cells and normal renal cell carcinoma cells, to explore the feasibility and effectiveness of CD105 and stem cell-related microRNAs in the treatment of renal cell carcinoma by killing renal cell carcinoma stem cells.
Method
ACHN renal carcinoma cell line was subcutaneously tumorigenic in BALB/c-nu immunodeficient mice. CD105-positive human renal carcinoma stem cells were isolated by anti-hCD105-PE antibody labeling and flow cytometry. The proliferation characteristics and senescence degree of renal cell carcinoma stem cells marked by CD105 (+) were identified by MTS and EdU cell proliferation test, cell cycle test, colony formation and senescence degree test. Differentially expressed microRNA was used to determine the binding degree between microRNA and its target protein mRNA 3'-terminal non-coding region by dual luciferase method. Finally, the screened microRNA was transfected with mimics or lentiviruses to explore its role in globular formation, cisplatin resistance and migration of renal cancer stem cells in vitro.
Result
The globular ability of CD105 (+) renal carcinoma cells was significantly stronger than that of CD105 (-) cell line ACHN, and the expression of stem cell markers CXCR4, OCT-4, NANOG, HIF1A, DLK-1 and EZH2 decreased after two weeks in RPMI-1640 medium. The expression of stem cell markers CXCRH-4, NANOG, DLK-1, T-4 and KLF4 decreased significantly after two weeks in DMEM-HG+10% FBS medium. He identified 27 differentially expressed microRNAs in CD105 (+) renal cancer stem cells and confirmed the presence of microRNAs in these cells. Mi-34a, Mi-155, and Mi-200a can down-regulate the expression of CD105 mRNA to some extent, thus reducing the resistance of CD105 (+) renal cancer stem cells to cisplatin. By knocking down CD105 with shRNA, we also found that CD105 can maintain the drug resistance of ACHN-CD105 (+) cells and prevent their aging. We also proved that Mi-335 can bind to OCT-4 eggs. White mRNA reduces its expression and produces anti-migration, decreases the rate of globulation and inhibits the expression of stem cell factors such as SOX-2, OCT-4, NANOG and KLF-4.
conclusion
CD105 (+) cells derived from ACHN cell line subcutaneously transplanted in BALB/c-nu nude mice have the characteristics of tumor stem cells and can be initially identified as renal cancer stem cells; microRNAs-34a, microRNAs-155, microRNAs-200a can reduce the resistance of CD105 (+) renal cancer stem cells to cisplatin by down-regulating CD105 (+); microRNAs-335 can inhibit CD105 (+) renal cancer by down-regulating OCT-4. Cell migration ability and ball forming ability.
Part 1 isolation, culture and identification of human renal cancer stem cells
objective
Isolation and identification of human renal cancer stem cells marked by CD105 (+)
Method
ACHN cell line was inoculated into the axillary skin of BALB/c-nu nude mice. After one month, the tumor was digested into a single cell suspension and incubated with anti-hCD105-PE flow cytometry antibody 4-C for half an hour. ACHN-CD105 (+) cells were isolated from the axillary skin of BALB/c-nu nude mice by flow sorter. The cells were amplified and cryopreserved in vitro. The proliferation, differentiation and tumorigenesis of ACHN-CD105(+) cells were identified by RPMI-1640+10% FBS and DMEM-HG+10% FBS medium, cisplatin resistance, MTS and EdU cell proliferation, cell cycle, colony formation and cell senescence.
Result
CD105 (+) cells accounted for only 3.05% of ACHN subcutaneously transplanted tumor cells, which accorded with the view of "very low proportion of cells driving tumor growth" in cancer stem cell theory. The rate of cell globulation was significantly higher than that of ACHN-CD105 (-) cells. After two weeks of culture in RPMI-1640+10% FBS and DMEM-HG+10% FBS medium, the expression of stem cell-related factors CXCR-4, OCT-4 and NANOG was decreased. Cisplatin resistance test showed that ACHN-CD105 (+) cells were more resistant than ACHN-CD105 (-) cells. Cell proliferation test and colony formation test were obvious. The results showed that the proliferation rate of ACHN-CD105 (+) cells was lower than that of ACHN-CD105 (-) cells and the colony forming ability of ACHN-CD105 (-) cells was significantly better than that of ACHN-CD105 (-) cells.
conclusion
CD105 (+) cells derived from ACHN cell line in BALB/c-nu nude mice have the characteristics of tumor stem cells and can be identified as renal cancer stem cells.
The second part is about the function and mechanism of miRNA in human renal cancer stem cells.
objective
Screening of miRNA that inhibits ACHN-CD105 (+) stem cell characteristics and its mechanism
Method
MicroRNAs that inhibit ACHN-CD105 (+) stem cells were screened by microNA database screening, literature retrieval and qRT-PCR. The binding degree between microRNAs and their target protein mRNA was determined by double luciferase assay. Cisplatin resistance test, cell senescence test, cell migration test and globular test were carried out. The validity of the selected miRNA and its feasibility for in vivo experiments are obtained.
Result
We screened 27 differentially expressed microRNAs between ACHN-CD105 (+) and ACHN-CD105 (-) cells, and later identified that the targets of microRNAs-34a, microRNAs-155, and microRNAs-200a were CD105 and that of microRNAs-335 were OCT-4. After transfection of microRNAs-34a, microRNAs-155, and microRNAs-200a, the resistance of ACHN-CD105 (+) cells to cisplatin decreased significantly. Migration, migration and globulation ability of ACHN-CD105 (+) cells decreased after exposure to lentiviruses overexpressing microRNA-335, and expression of many stem cell-related factors, including OCT-4, NANOG, SOX-2 and KLF-4, decreased.
conclusion
Mi-34a, Mi-155, and Mi-200a can reduce the resistance of CD105 (+) renal cancer stem cells to cisplatin by down-regulating CD105; Mi-335 can inhibit the migration and globulation of CD105 (+) renal cancer stem cells by down-regulating OCT-4.
【學位授予單位】：華中科技大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R737.11

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