一、肝癌细胞来源的边群细胞的分化及抗凋亡能力的研究 二、人类Bcrp基因的克隆和Tg737基因在边群细胞中缺失突变情况的
发布时间：2018-09-08 11:49
【摘要】： 肿瘤的形成过程目前尚未在学术界获得统一的认识。以往的随机理论认为基因的变异以一种完全随机的方式在细胞内积累,并且细胞随机的发生了恶性的转变。该理论在试图揭示临床种种现象时遇到了困难,比如获得性耐药等。近年来肿瘤干细胞理论得到了越来越多的实验支持。研究发现急性髓细胞样白血病细胞中能分离出与骨髓造血干细胞具有相同干细胞表面标志的癌细胞,这些细胞能在免疫缺陷小鼠中诱导急性髓细胞样白血病的发生;这表明肿瘤组织中可能存在既具有干细胞特征,又具有肿瘤特征的肿瘤干细胞。目前,有文献报道,在多种实体瘤中发现有肿瘤干细胞的存在,例如乳腺癌、脑肿瘤等。原发性肝细胞肝癌是我国常见恶性肿瘤之一,由于预后差,死亡率较高。临床观察发现,肝癌组织内的细胞在耐药性上存在差异,这种异质性的表现间接暗示了肿瘤干细胞的存在。对这群细胞的分离和鉴定对阐明肝癌发病机理、揭示肝癌复发和转移的机制具有重要的意义。 边群细胞是最近干细胞研究领域最热门的一个研究方向。由于该类细胞表面表达人类乳癌耐药蛋白(BCRP),且该膜蛋白能够排出Hoechst33342,从而使用流式细胞仪能够对其进行分选。该方法首先在分离骨髓造血干细胞上获得了成功,随后的研究表明,利用该方法可以从多种组织中高效、快速地分离得到成体干细胞。 目的: 利用Hoechst33342染色、流式细胞仪分离技术分离人类肝癌细胞系来源的边群细胞。在体外培养的情况下,对边群细胞的分化能力和抗凋亡能力进行检测。对人类乳癌耐药蛋白进行克隆,对人类Tg737基因在边群细胞中的缺失突变情况进行检测。 方法: 1.对人类肝癌细胞系MHCC97、hHCC、Bel-7402中的边群细胞进行分析,对MHCC97中的边群细胞进行分选; 1)细胞常规培养; 2) 0.25%胰酶温和消化细胞,常规离心并以HBSS液洗涤细胞; 3)细胞悬液分为两组,一组单纯用Hoechst33342进行染色,另一组同时加入钙通道拮抗剂和相同浓度的Hoechst染料,两组细胞均染色90分钟,然后以流式细胞仪检测肝癌细胞中的Hoechst荧光,通过使用双色性反射滤镜和不同的组合滤片将每个肝癌细胞中荧光信号分为两个部分—Hoechst蓝光部分和Hoechst红光部分,采集这些光信号,并利用流式细胞软件形成二维散点图,分析两组肝癌细胞在二维散点图上的形态,通过设定“门”找出肝癌细胞中的边群细胞,并边群细胞进行分析和分选。 2.边群细胞的分化能力的检测 1)根据Genebank中提供的甲胎蛋白(AFP)和细胞角蛋白19(CK19)的基因序列,使用Primer5引物设计软件分别对两条基因进行半定量引物的设计,并送公司合成。分别以边群细胞和非边群细胞两种细胞群的cDNA为模板,行半定量检测。提取两群细胞的总蛋白,对AFP和CK19行蛋白定量检测; 2)将两群细胞接种盖玻片,分别行AFP和CK19双色免疫荧光检测,以及Bcrp的免疫荧光检测; 3)将两群细胞分别接种裸鼠,待成瘤后对瘤体冰冻切片,对片子行AFP和CK19的双色免疫荧光检测。 3.边群细胞的抗凋亡能力的检测 1)分选后的两群细胞进行常规培养12小时,待细胞全部贴壁后,以PBS缓冲液替换完全培养基,按培养3、6、9个小时分别分组; 2)根据Genebank中提供的p53、Bcl-2和Bax的基因序列,使用Primer5引物设计软件分别对三条基因进行半定量引物的设计,并送公司合成。分别以经上述处理并分组的细胞的cDNA为模板,行半定量检测。提取细胞的总蛋白行蛋白定量检测; 3)分选后的细胞接种96孔板,常规培养贴壁后,以PBS缓冲液替换完全培养基,培养3、6、9个小时,使用MTT法检测细胞在营养缺乏的情况下的增殖能力; 4)分选后的细胞分别接种盖玻片,常规培养并按上述方法处理,并对细胞行Bcl-2和Bax的双色免疫荧光检测。 4.人类乳癌耐药蛋白(Bcrp)的克隆根据Genebank中提供的Bcrp的基因序列,使用Primer5引物设计软件设计Bcrp的巢式引物,以边群细胞的cDNA为模板行聚合酶链反应,将反应产物进行电泳,并回收电泳中的目的条带,克隆入pMD18T载体,送生物公司测序。 5.人类Tg737基因在边群细胞中的缺失情况的检测根据Genebank中提供的Tg737的基因序列,使用Primer5引物设计软件设计Tg737的引物,以边群细胞的cDNA为模板行聚合酶链反应,将反应产物进行电泳,并回收电泳中目的条带,克隆入pMD18T载体,送生物公司测序。 结果: 1.流式细胞仪的双波长荧光分析结果显示在二维散点图上有边群细胞存在。其中,MHCC97细胞系中边群细胞的比例接近0.25%,hHCC细胞系中比例接近0.5%,Bel-7402细胞系中比例接近0.45%。加入钙通道拮抗剂后该群细胞明显减少,因为低荧光的特征消失而进入到了非边群细胞群中,这表明三种肝癌细胞的边群细胞的表型均与Bcrp载体的主动外泵作用有关; 2.通过半定量检测、蛋白定量以及免疫荧光检测,发现MHCC97来源的边群细胞中AFP和CK19均有表达,而AFP表达水平明显较非边群细胞中为高。通过体外成瘤实验,发现瘤体中仅有部分细胞同时表达AFP和CK19。而对体外培养的边群及非边群细胞行Bcrp的检测,发现高度表达Bcrp的细胞位于边群细胞形成的克隆的中心周围,呈环形排列,而非边群细胞形成的克隆中也有表达Bcrp的细胞存在; 3.通过MTT法,发现在营养缺乏情况下,边群细胞较非边群细胞的生长增殖能力为强。通过半定量分析和蛋白定量分析,发现在边群细胞中,Bcl-2的表达水平上调,Bax的水平出现下调,而非边群细胞中刚好相反。免疫荧光的分析结果同上。 4.将测序结果与Genebank中人类乳癌耐药蛋白的序列进行比对,结果显示,pMD18T载体中所含有的基因片断与人类乳癌耐药蛋白的基因序列同源性达到99%。 5.将测序结果与Genebank中人类Tg737基因的序列进行比对,结果显示,pMD18T载体中所含有的基因片断与人类Tg737的基因序列相比,出现大片断的缺失和点突变。缺失部分位于第345个至第401个碱基,第1048个至第1148个碱基,第1650个碱基至第1741个碱基以及第2436个碱基至第2546个碱基之间,共缺失359个碱基。点突变位于第793个碱基,由C突变为T,蛋白产物由丝氨酸突变为苯丙氨酸。 结论: 1.利用Hoechst33342染料通过流式细胞仪技术确认人类肝癌细胞系MHCC97、hHCC和Bel-7402中存在边群细胞。 2. MHCC97来源的边群细胞共同表达AFP和CK19,这提示了边群细胞确实具有一定的双向分化能力。这说明边群细胞具有一定的原始特征。 3.边群细胞体外培养形成的克隆中,Bcrp强阳性的细胞呈环形围绕克隆中心分布,提示了边群细胞在分裂增殖的过程中,以环形向外生长,外围和中心的细胞都是由边群细胞分裂而来的非边群细胞,分裂后仍保持边群细胞特征的细胞都处于中心和外围细胞之间。非边群细胞体外培养形成的克隆中,也有Bcrp强阳性的细胞存在,但是,这些细胞数量较少,且在克隆中散在分布。这个发现,与临床上肿瘤中心常为坏死组织的现象一致,也提供了先化疗后手术的实验基础。非边群细胞形成的克隆中有边群细胞的存在,说明流式细胞仪分选过程可能不够完善,有少数边群细胞漏网,或者可能是有比边群细胞更原始的细胞存在,而这些细胞的特征尚不为人所知。 4.在营养缺乏的条件下,相比较非边群细胞,边群细胞具有更加明显的抗凋亡能力。并且,与非边群细胞相反,边群细胞中的Bcl-2表达上调,而Bax表达下调,促进了边群细胞的存活。这可能是边群细胞较非边群细胞具有明显的抗凋亡能力的机制所在。 5.人类乳癌耐药蛋白Bcrp被成功克隆入pMD18T载体,测序同源性99%,可以用于后续的实验。 6.人类Tg737基因在边群细胞内出现大片断的缺失和点突变。由于该基因位于13号染色体的着丝粒附近,乙肝病毒x蛋白或其它机制对该区具有破坏作用,可导致该区多种基因缺失变异。因此,该基因缺失的一种可能原因是病毒感染或其它机制所造成的一个副事件。然而,Tg737的蛋白产物构成的复合体在EGFR轴、b连环蛋白、细胞骨架、细胞周期调节、细胞黏附等方面均有重要的作用。对于细胞的恶性转化,这些方面的改变都是必须的。因此,另外一个可能原因是Tg737基因缺失变异属于细胞恶性改变的早期的和必须的事件。
[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.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R735.7;R329
本文编号：2230428
[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.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R735.7;R329
【引证文献】
相关博士学位论文 前1条
1 宋志;抑癌基因Tg737在肝癌发病机制中作用的初步研究[D];第四军医大学;2010年
，本文编号：2230428
本文链接：https://www.wllwen.com/yixuelunwen/binglixuelunwen/2230428.html
最近更新
教材专著
