【摘要】： 腫瘤的形成過(guò)程目前尚未在學(xué)術(shù)界獲得統(tǒng)一的認(rèn)識(shí)。以往的隨機(jī)理論認(rèn)為基因的變異以一種完全隨機(jī)的方式在細(xì)胞內(nèi)積累,并且細(xì)胞隨機(jī)的發(fā)生了惡性的轉(zhuǎn)變。該理論在試圖揭示臨床種種現(xiàn)象時(shí)遇到了困難,比如獲得性耐藥等。近年來(lái)腫瘤干細(xì)胞理論得到了越來(lái)越多的實(shí)驗(yàn)支持。研究發(fā)現(xiàn)急性髓細(xì)胞樣白血病細(xì)胞中能分離出與骨髓造血干細(xì)胞具有相同干細(xì)胞表面標(biāo)志的癌細(xì)胞,這些細(xì)胞能在免疫缺陷小鼠中誘導(dǎo)急性髓細(xì)胞樣白血病的發(fā)生;這表明腫瘤組織中可能存在既具有干細(xì)胞特征,又具有腫瘤特征的腫瘤干細(xì)胞。目前,有文獻(xiàn)報(bào)道,在多種實(shí)體瘤中發(fā)現(xiàn)有腫瘤干細(xì)胞的存在,例如乳腺癌、腦腫瘤等。原發(fā)性肝細(xì)胞肝癌是我國(guó)常見(jiàn)惡性腫瘤之一,由于預(yù)后差,死亡率較高。臨床觀察發(fā)現(xiàn),肝癌組織內(nèi)的細(xì)胞在耐藥性上存在差異,這種異質(zhì)性的表現(xiàn)間接暗示了腫瘤干細(xì)胞的存在。對(duì)這群細(xì)胞的分離和鑒定對(duì)闡明肝癌發(fā)病機(jī)理、揭示肝癌復(fù)發(fā)和轉(zhuǎn)移的機(jī)制具有重要的意義。 邊群細(xì)胞是最近干細(xì)胞研究領(lǐng)域最熱門(mén)的一個(gè)研究方向。由于該類(lèi)細(xì)胞表面表達(dá)人類(lèi)乳癌耐藥蛋白(BCRP),且該膜蛋白能夠排出Hoechst33342,從而使用流式細(xì)胞儀能夠?qū)ζ溥M(jìn)行分選。該方法首先在分離骨髓造血干細(xì)胞上獲得了成功,隨后的研究表明,利用該方法可以從多種組織中高效、快速地分離得到成體干細(xì)胞。 目的: 利用Hoechst33342染色、流式細(xì)胞儀分離技術(shù)分離人類(lèi)肝癌細(xì)胞系來(lái)源的邊群細(xì)胞。在體外培養(yǎng)的情況下,對(duì)邊群細(xì)胞的分化能力和抗凋亡能力進(jìn)行檢測(cè)。對(duì)人類(lèi)乳癌耐藥蛋白進(jìn)行克隆,對(duì)人類(lèi)Tg737基因在邊群細(xì)胞中的缺失突變情況進(jìn)行檢測(cè)。 方法: 1.對(duì)人類(lèi)肝癌細(xì)胞系MHCC97、hHCC、Bel-7402中的邊群細(xì)胞進(jìn)行分析,對(duì)MHCC97中的邊群細(xì)胞進(jìn)行分選; 1)細(xì)胞常規(guī)培養(yǎng); 2) 0.25%胰酶溫和消化細(xì)胞,常規(guī)離心并以HBSS液洗滌細(xì)胞; 3)細(xì)胞懸液分為兩組,一組單純用Hoechst33342進(jìn)行染色,另一組同時(shí)加入鈣通道拮抗劑和相同濃度的Hoechst染料,兩組細(xì)胞均染色90分鐘,然后以流式細(xì)胞儀檢測(cè)肝癌細(xì)胞中的Hoechst熒光,通過(guò)使用雙色性反射濾鏡和不同的組合濾片將每個(gè)肝癌細(xì)胞中熒光信號(hào)分為兩個(gè)部分—Hoechst藍(lán)光部分和Hoechst紅光部分,采集這些光信號(hào),并利用流式細(xì)胞軟件形成二維散點(diǎn)圖,分析兩組肝癌細(xì)胞在二維散點(diǎn)圖上的形態(tài),通過(guò)設(shè)定“門(mén)”找出肝癌細(xì)胞中的邊群細(xì)胞,并邊群細(xì)胞進(jìn)行分析和分選。 2.邊群細(xì)胞的分化能力的檢測(cè) 1)根據(jù)Genebank中提供的甲胎蛋白(AFP)和細(xì)胞角蛋白19(CK19)的基因序列,使用Primer5引物設(shè)計(jì)軟件分別對(duì)兩條基因進(jìn)行半定量引物的設(shè)計(jì),并送公司合成。分別以邊群細(xì)胞和非邊群細(xì)胞兩種細(xì)胞群的cDNA為模板,行半定量檢測(cè)。提取兩群細(xì)胞的總蛋白,對(duì)AFP和CK19行蛋白定量檢測(cè); 2)將兩群細(xì)胞接種蓋玻片,分別行AFP和CK19雙色免疫熒光檢測(cè),以及Bcrp的免疫熒光檢測(cè); 3)將兩群細(xì)胞分別接種裸鼠,待成瘤后對(duì)瘤體冰凍切片,對(duì)片子行AFP和CK19的雙色免疫熒光檢測(cè)。 3.邊群細(xì)胞的抗凋亡能力的檢測(cè) 1)分選后的兩群細(xì)胞進(jìn)行常規(guī)培養(yǎng)12小時(shí),待細(xì)胞全部貼壁后,以PBS緩沖液替換完全培養(yǎng)基,按培養(yǎng)3、6、9個(gè)小時(shí)分別分組; 2)根據(jù)Genebank中提供的p53、Bcl-2和Bax的基因序列,使用Primer5引物設(shè)計(jì)軟件分別對(duì)三條基因進(jìn)行半定量引物的設(shè)計(jì),并送公司合成。分別以經(jīng)上述處理并分組的細(xì)胞的cDNA為模板,行半定量檢測(cè)。提取細(xì)胞的總蛋白行蛋白定量檢測(cè); 3)分選后的細(xì)胞接種96孔板,常規(guī)培養(yǎng)貼壁后,以PBS緩沖液替換完全培養(yǎng)基,培養(yǎng)3、6、9個(gè)小時(shí),使用MTT法檢測(cè)細(xì)胞在營(yíng)養(yǎng)缺乏的情況下的增殖能力; 4)分選后的細(xì)胞分別接種蓋玻片,常規(guī)培養(yǎng)并按上述方法處理,并對(duì)細(xì)胞行Bcl-2和Bax的雙色免疫熒光檢測(cè)。 4.人類(lèi)乳癌耐藥蛋白(Bcrp)的克隆根據(jù)Genebank中提供的Bcrp的基因序列,使用Primer5引物設(shè)計(jì)軟件設(shè)計(jì)Bcrp的巢式引物,以邊群細(xì)胞的cDNA為模板行聚合酶鏈反應(yīng),將反應(yīng)產(chǎn)物進(jìn)行電泳,并回收電泳中的目的條帶,克隆入pMD18T載體,送生物公司測(cè)序。 5.人類(lèi)Tg737基因在邊群細(xì)胞中的缺失情況的檢測(cè)根據(jù)Genebank中提供的Tg737的基因序列,使用Primer5引物設(shè)計(jì)軟件設(shè)計(jì)Tg737的引物,以邊群細(xì)胞的cDNA為模板行聚合酶鏈反應(yīng),將反應(yīng)產(chǎn)物進(jìn)行電泳,并回收電泳中目的條帶,克隆入pMD18T載體,送生物公司測(cè)序。 結(jié)果: 1.流式細(xì)胞儀的雙波長(zhǎng)熒光分析結(jié)果顯示在二維散點(diǎn)圖上有邊群細(xì)胞存在。其中,MHCC97細(xì)胞系中邊群細(xì)胞的比例接近0.25%,hHCC細(xì)胞系中比例接近0.5%,Bel-7402細(xì)胞系中比例接近0.45%。加入鈣通道拮抗劑后該群細(xì)胞明顯減少,因?yàn)榈蜔晒獾奶卣飨ФM(jìn)入到了非邊群細(xì)胞群中,這表明三種肝癌細(xì)胞的邊群細(xì)胞的表型均與Bcrp載體的主動(dòng)外泵作用有關(guān); 2.通過(guò)半定量檢測(cè)、蛋白定量以及免疫熒光檢測(cè),發(fā)現(xiàn)MHCC97來(lái)源的邊群細(xì)胞中AFP和CK19均有表達(dá),而AFP表達(dá)水平明顯較非邊群細(xì)胞中為高。通過(guò)體外成瘤實(shí)驗(yàn),發(fā)現(xiàn)瘤體中僅有部分細(xì)胞同時(shí)表達(dá)AFP和CK19。而對(duì)體外培養(yǎng)的邊群及非邊群細(xì)胞行Bcrp的檢測(cè),發(fā)現(xiàn)高度表達(dá)Bcrp的細(xì)胞位于邊群細(xì)胞形成的克隆的中心周?chē)?呈環(huán)形排列,而非邊群細(xì)胞形成的克隆中也有表達(dá)Bcrp的細(xì)胞存在; 3.通過(guò)MTT法,發(fā)現(xiàn)在營(yíng)養(yǎng)缺乏情況下,邊群細(xì)胞較非邊群細(xì)胞的生長(zhǎng)增殖能力為強(qiáng)。通過(guò)半定量分析和蛋白定量分析,發(fā)現(xiàn)在邊群細(xì)胞中,Bcl-2的表達(dá)水平上調(diào),Bax的水平出現(xiàn)下調(diào),而非邊群細(xì)胞中剛好相反。免疫熒光的分析結(jié)果同上。 4.將測(cè)序結(jié)果與Genebank中人類(lèi)乳癌耐藥蛋白的序列進(jìn)行比對(duì),結(jié)果顯示,pMD18T載體中所含有的基因片斷與人類(lèi)乳癌耐藥蛋白的基因序列同源性達(dá)到99%。 5.將測(cè)序結(jié)果與Genebank中人類(lèi)Tg737基因的序列進(jìn)行比對(duì),結(jié)果顯示,pMD18T載體中所含有的基因片斷與人類(lèi)Tg737的基因序列相比,出現(xiàn)大片斷的缺失和點(diǎn)突變。缺失部分位于第345個(gè)至第401個(gè)堿基,第1048個(gè)至第1148個(gè)堿基,第1650個(gè)堿基至第1741個(gè)堿基以及第2436個(gè)堿基至第2546個(gè)堿基之間,共缺失359個(gè)堿基。點(diǎn)突變位于第793個(gè)堿基,由C突變?yōu)門(mén),蛋白產(chǎn)物由絲氨酸突變?yōu)楸奖彼帷?結(jié)論: 1.利用Hoechst33342染料通過(guò)流式細(xì)胞儀技術(shù)確認(rèn)人類(lèi)肝癌細(xì)胞系MHCC97、hHCC和Bel-7402中存在邊群細(xì)胞。 2. MHCC97來(lái)源的邊群細(xì)胞共同表達(dá)AFP和CK19,這提示了邊群細(xì)胞確實(shí)具有一定的雙向分化能力。這說(shuō)明邊群細(xì)胞具有一定的原始特征。 3.邊群細(xì)胞體外培養(yǎng)形成的克隆中,Bcrp強(qiáng)陽(yáng)性的細(xì)胞呈環(huán)形圍繞克隆中心分布,提示了邊群細(xì)胞在分裂增殖的過(guò)程中,以環(huán)形向外生長(zhǎng),外圍和中心的細(xì)胞都是由邊群細(xì)胞分裂而來(lái)的非邊群細(xì)胞,分裂后仍保持邊群細(xì)胞特征的細(xì)胞都處于中心和外圍細(xì)胞之間。非邊群細(xì)胞體外培養(yǎng)形成的克隆中,也有Bcrp強(qiáng)陽(yáng)性的細(xì)胞存在,但是,這些細(xì)胞數(shù)量較少,且在克隆中散在分布。這個(gè)發(fā)現(xiàn),與臨床上腫瘤中心常為壞死組織的現(xiàn)象一致,也提供了先化療后手術(shù)的實(shí)驗(yàn)基礎(chǔ)。非邊群細(xì)胞形成的克隆中有邊群細(xì)胞的存在,說(shuō)明流式細(xì)胞儀分選過(guò)程可能不夠完善,有少數(shù)邊群細(xì)胞漏網(wǎng),或者可能是有比邊群細(xì)胞更原始的細(xì)胞存在,而這些細(xì)胞的特征尚不為人所知。 4.在營(yíng)養(yǎng)缺乏的條件下,相比較非邊群細(xì)胞,邊群細(xì)胞具有更加明顯的抗凋亡能力。并且,與非邊群細(xì)胞相反,邊群細(xì)胞中的Bcl-2表達(dá)上調(diào),而B(niǎo)ax表達(dá)下調(diào),促進(jìn)了邊群細(xì)胞的存活。這可能是邊群細(xì)胞較非邊群細(xì)胞具有明顯的抗凋亡能力的機(jī)制所在。 5.人類(lèi)乳癌耐藥蛋白Bcrp被成功克隆入pMD18T載體,測(cè)序同源性99%,可以用于后續(xù)的實(shí)驗(yàn)。 6.人類(lèi)Tg737基因在邊群細(xì)胞內(nèi)出現(xiàn)大片斷的缺失和點(diǎn)突變。由于該基因位于13號(hào)染色體的著絲粒附近,乙肝病毒x蛋白或其它機(jī)制對(duì)該區(qū)具有破壞作用,可導(dǎo)致該區(qū)多種基因缺失變異。因此,該基因缺失的一種可能原因是病毒感染或其它機(jī)制所造成的一個(gè)副事件。然而,Tg737的蛋白產(chǎn)物構(gòu)成的復(fù)合體在EGFR軸、b連環(huán)蛋白、細(xì)胞骨架、細(xì)胞周期調(diào)節(jié)、細(xì)胞黏附等方面均有重要的作用。對(duì)于細(xì)胞的惡性轉(zhuǎn)化,這些方面的改變都是必須的。因此,另外一個(gè)可能原因是Tg737基因缺失變異屬于細(xì)胞惡性改變的早期的和必須的事件。
[Abstract]:Previous stochastic theories have suggested that gene mutations accumulate in cells in a completely random manner and that malignant changes occur randomly. The theory has encountered difficulties in trying to reveal clinical phenomena, such as acquired drug resistance. Cancer stem cell theory has been supported by more and more experiments. It has been found that cancer cells with the same stem cell surface markers as bone marrow hematopoietic stem cells can be isolated from acute myeloid leukemia cells. These cells can induce the occurrence of acute myeloid leukemia in immunodeficient mice, suggesting that tumor tissue can Primary hepatocellular carcinoma (PHC) is one of the most common malignant tumors in China. It has a high mortality rate due to poor prognosis. Clinical observation shows that hepatocellular carcinoma (HCC) is one of the most common malignant tumors in China. Differentiations in drug resistance of cells in tissues indirectly indicate the presence of cancer stem cells. Isolation and identification of these cells are of great significance in elucidating the pathogenesis of hepatocellular carcinoma and revealing the mechanism of recurrence and metastasis of hepatocellular carcinoma.
Borderline cells are one of the hottest research areas in stem cell research recently. Because these cells express human breast cancer resistance protein (BCRP) on their surface and the membrane protein can excrete Hoechst 33342, it can be sorted by flow cytometry. Studies have shown that this method can efficiently and rapidly isolate adult stem cells from various tissues.
Objective:
Hoechst 33342 staining and flow cytometry were used to isolate human hepatocellular carcinoma (HCC) cell line-derived marginal group cells. Differentiation ability and anti-apoptosis ability of marginal group cells were tested in vitro. Human breast cancer resistance protein was cloned and deletion mutation of human Tg737 gene in peripheral group cells was detected. Test.
Method:
1. The edge group cells of human hepatocellular carcinoma cell lines MHCC97, hHCC, Bel-7402 were analyzed, and the edge group cells of MHCC97 were sorted.
1) routine cell culture.
2) 0.25% trypsin was used to digest the cells gently, centrifugation and washing cells with HBSS solution.
3) Cell suspension was divided into two groups: one group was stained with Hoechst 33342 alone, the other group was stained with calcium channel antagonist and the same concentration of Hoechst dye. The cells in both groups were stained for 90 minutes, then the fluorescence of Hoechst in hepatocellular carcinoma cells was detected by flow cytometry, and each liver was stained with two-color reflectance filters and different combinations of filters. The fluorescence signals in cancer cells are divided into two parts-the Hoechst blue light part and the Hoechst red light part. These light signals are collected and two-dimensional scatter plots are formed by using flow cytometry software. The morphology of two groups of hepatocellular carcinoma cells on the two-dimensional scatter plots is analyzed. The edge group cells in hepatocellular carcinoma cells are found by setting the gate and the edge group cells are analyzed. And sorting.
Detection of differentiation ability of 2. side population cells
1) According to the gene sequences of alpha-fetoprotein (AFP) and cytokeratin 19 (CK19) provided by Genebank, primer 5 software was used to design semi-quantitative primers for the two genes and synthesized by the company. The total protein of AFP and CK19 were quantitatively detected.
2) The two groups of cells were inoculated into cover slides and detected by AFP and CK19 double-color immunofluorescence and Bcrp immunofluorescence respectively.
3) The two groups of cells were inoculated into the nude mice respectively, and then frozen sections of the tumors were prepared after tumor formation. The AFP and CK19 immunofluorescence assays were performed on the tumors.
Detection of anti apoptotic ability of 3. side population cells
1) The two groups of cells were cultured for 12 hours. After all the cells adhered to the wall, PBS buffer was used to replace the complete medium. The cells were grouped into three, six and nine hours.
2) According to the gene sequences of p53, Bcl-2 and Bax provided by Genebank, the primers of three genes were designed and synthesized by Primer5 software. The total proteins of the cells were extracted and detected quantitatively.
3) The cells were inoculated into 96-well plate after sorting, and then adhered to the wall of conventional culture. PBS buffer was used to replace the complete medium for 3,6,9 hours.
4) After sorting, the cells were inoculated with cover slides, cultured routinely and treated according to the above methods. The cells were detected by double-color immunofluorescence assay of Bcl-2 and Bax.
4. The cloning of human breast cancer resistance protein (Bcrp) was based on the Bcrp gene sequence provided by Genebank. The nested primers of Bcrp were designed by Primer5 primer design software. The PCR products were electrophoretized with the DNA of the peripheral group cells as template. The target bands of the electrophoresis were recovered and cloned into pMD18T vector and sent to the biological company for detection. Order.
5. Detection of the deletion of human Tg737 gene in peripheral group cells. According to the gene sequence of Tg737 provided by Genebank, the primers of Tg737 were designed by Primer5 primer design software. The PCR products were electrophoretized with the template of DNA of peripheral group cells. The target bands were recovered and cloned into pMD18T vector. Sequencing of biological companies.
Result:
1. Dual-wavelength fluorescence analysis of flow cytometry showed that there were edge groups in the two-dimensional scatter plot. The proportion of edge groups in MHCC97 cell line was close to 0.25%, that in hHCC cell line was close to 0.5%, and that in Bel-7402 cell line was close to 0.45%. The number of edge groups of cells decreased significantly after calcium channel antagonists were added because of the low fluorescence. The disappearance of the sign into the non-marginal group indicated that the phenotype of the marginal group cells of the three hepatocellular carcinoma cells was related to the active extracellular pumping of the Bcrp vector.
2. By semi-quantitative detection, protein quantification and immunofluorescence assay, we found that AFP and CK19 were expressed in MHCC97-derived marginal cells, while AFP expression was significantly higher than that in non-marginal cells. Bcrp was detected. The cells highly expressing Bcrp were found to be located around the center of the clone formed by the peripheral group cells and arranged in a circular pattern.
3. MTT assay showed that the growth and proliferation ability of marginal group cells was stronger than that of non-marginal group cells under nutritional deficiency. Semi-quantitative analysis and protein quantitative analysis showed that the expression of Bcl-2 was up-regulated and the level of Bax was down-regulated in marginal group cells, but not just in marginal group cells.
4. Sequencing results were compared with the sequence of human breast cancer resistance protein in Genebank. The results showed that the gene fragment contained in pMD18T vector had 99% homology with the gene sequence of human breast cancer resistance protein.
5. Sequencing results were compared with the sequence of human Tg737 gene in Genebank. The results showed that there were large deletions and point mutations in the pMD18T vector compared with the human Tg737 gene. The deletions were located in 345 to 401 bases, 1048 to 1148 bases, 1650 bases to 1741 bases. A total of 359 base pairs were deleted from each base and from 2436 to 2546. Point mutation was located at 793 base pairs, from C to T, and protein products from serine to phenylalanine.
Conclusion:
1. Hoechst 33342 dye was used to confirm the existence of marginal group cells in human hepatocellular carcinoma cell lines MHCC 97, hHCC and Bel-7402 by flow cytometry.
2. MHCC97-derived marginal group cells co-express AFP and CK19, suggesting that marginal group cells do have a certain ability of bi-directional differentiation.
3. Bcrp-positive cells were distributed around the center of the clone in vitro, suggesting that in the process of division and proliferation, the cells of the peripheral and central groups grew outward in a circular shape. Both the peripheral and central cells were non-peripheral cells derived from the peripheral group cells, and all the cells remained the characteristics of the peripheral group cells after division. There are also Bcrp-positive cells in the clones of non-marginal group cells cultured in vitro. However, these cells are small in number and scattered in the clones. This finding is consistent with the clinical phenomenon that tumor centers are often necrotic tissues, and provides an experimental basis for chemotherapy followed by surgery. The presence of marginal group cells in the clones of cell formation suggests that the process of flow cytometry sorting may be imperfect, with a few marginal group cells leaking out of the network, or perhaps with more primitive cells than marginal group cells, and the characteristics of these cells are unknown.
4. Under the condition of nutrition deficiency, edge group cells have more obvious anti-apoptosis ability than non-edge group cells. In contrast to non-edge group cells, the expression of Bcl-2 in edge group cells is up-regulated, while the expression of Bax is down-regulated, which promotes the survival of edge group cells. Where is the mechanism?
5. Human breast cancer resistance protein Bcrp was successfully cloned into pMD18T vector, and the sequence homology was 99%. It can be used in subsequent experiments.
6. The deletion and point mutation of human Tg737 gene occur in the peripheral population cells. Because the gene is located near the centromere of chromosome 13, the hepatitis B virus X protein or other mechanisms can destroy the region and lead to the deletion and mutation of many genes in the region. Therefore, one possible reason for the deletion of the gene is virus infection or other factors. However, the complex of Tg737 protein products plays an important role in EGFR axis, b-catenin, cytoskeleton, cell cycle regulation, cell adhesion and so on. These changes are necessary for malignant transformation of cells. Therefore, another possible reason is the deletion of Tg737 gene. It is an early and necessary event of malignant transformation of cells.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2007
【分類(lèi)號(hào)】：R735.7;R329

【引證文獻(xiàn)】

相關(guān)博士學(xué)位論文 前1條

1 宋志;抑癌基因Tg737在肝癌發(fā)病機(jī)制中作用的初步研究[D];第四軍醫(yī)大學(xué);2010年

，

本文編號(hào)：2230428