自噬基因Beclin1在人肺腺癌A549细胞系SP及非SP细胞亚群中表达的实验研究
摘要
背景
肺癌正成为当今世界各国最常见的恶性肿瘤之一,并逐渐成为大多数国家人口中导致死亡的恶性肿瘤之首,对目前世界范围内的人类生命和健康造成巨大威胁,给患者及其家庭增加了沉重的负担和极大的痛苦,也占去了较多的医疗卫生资源和社会财富。各个国家的临床医院、科研单位和学者都投入了大量的物力、财力和精力,从临床的手术及放化疗、实验的不同层面和新思路新方法等方面进行探索研究。虽然运用于临床的新的治疗方法仍进展缓慢,但在分子生物学、人类基因组学、肿瘤干细胞学说等方向的研究取得了一定的成果,如：肺癌早期诊断的分子标记物的筛选；在非小细胞肺癌中发现抑癌基因TSLCl/CADMl；利用基因序列和表达谱的不同,区分人肺腺癌和鳞癌；通过基因检测,预测肺癌患者术后的复发率；实验室研究支持肿瘤干细胞学说；肿瘤干细胞的分离和提纯；SP细胞及非SP细胞的成功分选；肿瘤细胞自噬方向研究的不断深入；等等,虽然多数尚未被运用于临床,但深入研究,必定可以找到新的预防、诊断和治疗靶点。SP细胞成功被分离,并已被实验室研究证实具有干细胞特性。自噬作为肿瘤研究的新方向,也已有实验室研究表明常用自噬基因Beclinl和LC3在癌组织、癌旁组织和正常组织中的表达具有差异性,并推测可能与其干细胞特性有关。在此背景下,研究自噬基因在SP细胞和非SP细胞中的表达有无差异,绕过干细胞不易分离提纯的难题,通过研究SP细胞来研究人肺癌干细胞,从干细胞自噬方向找到治疗的新靶点成为该研究的最初出发点
目的
探讨自噬基因Beclinl在人肺腺癌A549细胞系SP及非SP细胞中的表达。寻找两者间是否存在差异,为进一步的肿瘤细胞自噬作用调节的药物实验找到实验基础和依据。
材料和方法
对肺癌A549细胞系细胞进行实验室培养,待细胞生长进入对数期后,收集细胞,用流式细胞学方法分选人肺腺癌A549细胞系中SP细胞及非SP细胞亚群；成功分选后,采用Western blot方法分别检测Beclinl基因在SP细胞和非SP细胞亚群中的表达情况。
结果
流式细胞学方法成功分选得到SP细胞及非SP细胞亚群,Western blot检测Beclinl基因在SP细胞亚群中的相对表达量为2.795±0.090,在非SP细胞亚群中的相对表达量为3.048±0.124。两相对表达量比较：t=5.220.,P<0.05,差异有统计学意义。
结论
自噬基因Beclinl在人肺腺癌A549细胞系SP细胞亚群中的表达较非SP细胞亚群下降,这可能与SP细胞亚群具有的肿瘤干细胞特性有关。使通过对SP细胞的自噬进行调节达到消灭肿瘤细胞成为一种新的研究方向。
Backround
Lung cancer is one of the most common malignant tumors in the world.In many countries,lung cancer is on the top of the list about death causing by cancers. People's health is threatened by lung cancer, many patients are suffering it and lots of money is spent on it.Many hospitals and docors have paid much attention on this disease, so many new methods for treatment and diagnosis are under research.Although the step of progress for treatment in lung cancer is really slow, we still have some breakthrough in biochemistry,human genomics and cancer stem cell. For example:the screening of molecular markers about early diagnosis of lung cancer;found the suppressor genes TSLC1/CADM1in non-small cell lung cancer;use the difference between gene sequences and expression patterns,we can distinguish adenocarcinoma and squamous cell carcinoma;Through the genetic testing, predict the postoperative recurrence rate of lung cancer;the doctrine of cancer sten cells;separation and purification of tumor stem cells;SP cells and NSP cells were been separation successful;the progress in the research of cancer stem cell is improving, and so on. Although many research results are not used in clinic, they still have brilliant future in many ways. Some scientists have successfully separated SP cells from cancer cells, they also find that SP cells have traits of stem cells. Autophagy is becoming one of the most popular things in the cancer research. Some scientists have found differences in the expression of autophagy gene Beclinl and LC3between the tumor tissue and the normal tissue, they also believe the differences have some relationships with the stem cell traits. In this context,to explore the expression of Autophagy-related Genes Beclinl in SP cells and NSP cells of human lung adenocarcinoma cell line A549,around the difficult problem that the stem cells purification,though the SP cells, the purpose to the stem cells of lung cancer,stem cell autophagy is becoming one of the new methods for cancer treatment.
Objective
To explore the expression of Autophagy-related Genes Beclinl in SP cells and NSP cells of human lung adenocarcinoma cell line A549,to find the differences between SP and NSP cells,to provide some basis and foundation for the next drug experiments,about the adjustment of Autophagy-related Genes.
Materials and Methods
Human lung adrenocortical carcinoma cell line A549was cultured properly in vivo. SP and NSP cells in cell line A549were isolated by FACS respectively,the expression of autophagy-related genes Beclinl were detected by Western blot.
Result
SP and NSP cells were isolated by FACS,the expression of Autophagy-related Genes Beclinl in SP cells was2.795±0.090, and in NSP cells was3.048±0.124. t=5.220, P<0.05, there were significant diference.
Conclusions
The expression of Autophagy-related Genes Beclinl in SP cells was lower than in NSP cells.There may have some relationships with the trait of tumor stem cell in SP cells,the adjustment of Autophagy-related Genes will become a new methods to treatment tumors.
肺癌正成为当今世界各国最常见的恶性肿瘤之一,并逐渐成为大多数国家人口中导致死亡的恶性肿瘤之首,对目前世界范围内的人类生命和健康造成巨大威胁,给患者及其家庭增加了沉重的负担和极大的痛苦,也占去了较多的医疗卫生资源和社会财富。各个国家的临床医院、科研单位和学者都投入了大量的物力、财力和精力,从临床的手术及放化疗、实验的不同层面和新思路新方法等方面进行探索研究。虽然运用于临床的新的治疗方法仍进展缓慢,但在分子生物学、人类基因组学、肿瘤干细胞学说等方向的研究取得了一定的成果,如：肺癌早期诊断的分子标记物的筛选；在非小细胞肺癌中发现抑癌基因TSLCl/CADMl；利用基因序列和表达谱的不同,区分人肺腺癌和鳞癌；通过基因检测,预测肺癌患者术后的复发率；实验室研究支持肿瘤干细胞学说；肿瘤干细胞的分离和提纯；SP细胞及非SP细胞的成功分选；肿瘤细胞自噬方向研究的不断深入；等等,虽然多数尚未被运用于临床,但深入研究,必定可以找到新的预防、诊断和治疗靶点。SP细胞成功被分离,并已被实验室研究证实具有干细胞特性。自噬作为肿瘤研究的新方向,也已有实验室研究表明常用自噬基因Beclinl和LC3在癌组织、癌旁组织和正常组织中的表达具有差异性,并推测可能与其干细胞特性有关。在此背景下,研究自噬基因在SP细胞和非SP细胞中的表达有无差异,绕过干细胞不易分离提纯的难题,通过研究SP细胞来研究人肺癌干细胞,从干细胞自噬方向找到治疗的新靶点成为该研究的最初出发点
目的
探讨自噬基因Beclinl在人肺腺癌A549细胞系SP及非SP细胞中的表达。寻找两者间是否存在差异,为进一步的肿瘤细胞自噬作用调节的药物实验找到实验基础和依据。
材料和方法
对肺癌A549细胞系细胞进行实验室培养,待细胞生长进入对数期后,收集细胞,用流式细胞学方法分选人肺腺癌A549细胞系中SP细胞及非SP细胞亚群；成功分选后,采用Western blot方法分别检测Beclinl基因在SP细胞和非SP细胞亚群中的表达情况。
结果
流式细胞学方法成功分选得到SP细胞及非SP细胞亚群,Western blot检测Beclinl基因在SP细胞亚群中的相对表达量为2.795±0.090,在非SP细胞亚群中的相对表达量为3.048±0.124。两相对表达量比较：t=5.220.,P<0.05,差异有统计学意义。
结论
自噬基因Beclinl在人肺腺癌A549细胞系SP细胞亚群中的表达较非SP细胞亚群下降,这可能与SP细胞亚群具有的肿瘤干细胞特性有关。使通过对SP细胞的自噬进行调节达到消灭肿瘤细胞成为一种新的研究方向。
Backround
Lung cancer is one of the most common malignant tumors in the world.In many countries,lung cancer is on the top of the list about death causing by cancers. People's health is threatened by lung cancer, many patients are suffering it and lots of money is spent on it.Many hospitals and docors have paid much attention on this disease, so many new methods for treatment and diagnosis are under research.Although the step of progress for treatment in lung cancer is really slow, we still have some breakthrough in biochemistry,human genomics and cancer stem cell. For example:the screening of molecular markers about early diagnosis of lung cancer;found the suppressor genes TSLC1/CADM1in non-small cell lung cancer;use the difference between gene sequences and expression patterns,we can distinguish adenocarcinoma and squamous cell carcinoma;Through the genetic testing, predict the postoperative recurrence rate of lung cancer;the doctrine of cancer sten cells;separation and purification of tumor stem cells;SP cells and NSP cells were been separation successful;the progress in the research of cancer stem cell is improving, and so on. Although many research results are not used in clinic, they still have brilliant future in many ways. Some scientists have successfully separated SP cells from cancer cells, they also find that SP cells have traits of stem cells. Autophagy is becoming one of the most popular things in the cancer research. Some scientists have found differences in the expression of autophagy gene Beclinl and LC3between the tumor tissue and the normal tissue, they also believe the differences have some relationships with the stem cell traits. In this context,to explore the expression of Autophagy-related Genes Beclinl in SP cells and NSP cells of human lung adenocarcinoma cell line A549,around the difficult problem that the stem cells purification,though the SP cells, the purpose to the stem cells of lung cancer,stem cell autophagy is becoming one of the new methods for cancer treatment.
Objective
To explore the expression of Autophagy-related Genes Beclinl in SP cells and NSP cells of human lung adenocarcinoma cell line A549,to find the differences between SP and NSP cells,to provide some basis and foundation for the next drug experiments,about the adjustment of Autophagy-related Genes.
Materials and Methods
Human lung adrenocortical carcinoma cell line A549was cultured properly in vivo. SP and NSP cells in cell line A549were isolated by FACS respectively,the expression of autophagy-related genes Beclinl were detected by Western blot.
Result
SP and NSP cells were isolated by FACS,the expression of Autophagy-related Genes Beclinl in SP cells was2.795±0.090, and in NSP cells was3.048±0.124. t=5.220, P<0.05, there were significant diference.
Conclusions
The expression of Autophagy-related Genes Beclinl in SP cells was lower than in NSP cells.There may have some relationships with the trait of tumor stem cell in SP cells,the adjustment of Autophagy-related Genes will become a new methods to treatment tumors.
引文
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